Abstract
Humanized mice are limited in terms of modeling human immunity, particularly with regards to antibody responses. Here we constructed a humanized (THX) mouse by grafting non-γ-irradiated, genetically myeloablated KitW-41J mutant immunodeficient pups with human cord blood CD34+ cells, followed by 17β-estradiol conditioning to promote immune cell differentiation. THX mice reconstitute a human lymphoid and myeloid immune system, including marginal zone B cells, germinal center B cells, follicular helper T cells and neutrophils, and develop well-formed lymph nodes and intestinal lymphoid tissue, including Peyer’s patches, and human thymic epithelial cells. These mice have diverse human B cell and T cell antigen receptor repertoires and can mount mature T cell-dependent and T cell-independent antibody responses, entailing somatic hypermutation, class-switch recombination, and plasma cell and memory B cell differentiation. Upon flagellin or a Pfizer-BioNTech coronavirus disease 2019 (COVID-19) mRNA vaccination, THX mice mount neutralizing antibody responses to Salmonella or severe acute respiratory syndrome coronavirus 2 Spike S1 receptor-binding domain, with blood incretion of human cytokines, including APRIL, BAFF, TGF-β, IL-4 and IFN-γ, all at physiological levels. These mice can also develop lupus autoimmunity after pristane injection. By leveraging estrogen activity to support human immune cell differentiation and maturation of antibody responses, THX mice provide a platform to study the human immune system and to develop human vaccines and therapeutics.
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Main
Many of the more than the 1,600 immune response mouse genes are incongruent with their human equivalents, resulting in divergencies or deficiencies of mice as predictors of human immune responses1, making availability of a ‘humanized’ mouse model that faithfully reproduces human immune responses a high priority. The first humanized immune system mice were constructed by injecting human peripheral blood lymphocytes or (CD34+) human hematopoietic stem cells (huHSCs; hu prefix for human or humanized is used throughout) into severe combined immunodeficiency Prkdcscid (SCID) mice or Rag1/Rag2 knockout (KO) mice2,3,4,5,6. Subsequently, huHSC grafting of immunodeficient nonobese diabetic NOD.Cg-Prkdcscid Il2rgtm1Wjl/Sz or NOD.Cg-Prkdcscid Il2rgnull (NSG) mice7,8, in which Il2rg deletion results in defective cytokine signaling in multiple immune cell receptors, furthered the scope of humanized mice8. In huNSG mice, the NOD phagocytic cell SIRPα receptor variant cross-reacts with human CD47 to induce a ‘don’t eat me’ signal, thereby limiting human cell phagocytosis5,9. NSG mice, however, allow for poor huHSC accessibility to the bone marrow (BM) hematopoietic niche5,6,7,8, a limitation only partially obviated by mouse myeloablation through γ-radiation, which, however, increases risk of wasting, infection and mortality5. In addition, huNSG mice remain poor immune responders. Attempts to make them better responders have included knock-in or transgenic insertion of cytokine genes, generally resulting, however, in abnormal supraphysiological cytokine expression2,3,4,5,6.
Although mutated IgG to ovalbumin have been detected in γ-irradiated knock-in huIL6 Rag2−/−Il2rg−/−SIRPαh/m mice (RG SKI interleukin (IL)-6)10, a humanized mouse capable of mounting fully mature antibody responses has yet to be established. Maturation of the antibody response entails B cell somatic hypermutation (SHM), class-switch DNA recombination (CSR), differentiation of plasma cells (PCs) making high-affinity antibodies and generation of specific memory B cells (MBCs). The National Institute of Allergy and Infectious Diseases has emphasized the need for a novel and more advanced human immune system mouse model2, a recommendation that has gone essentially unheeded. Generation of homozygous KitW-41J mutant NSG mice has yielded genetically myeloablated NSGW41 (NOD.Cg-KitW-41JPrkdcscidIl2rgtm1Wjl/WaskJ) and NBSGW (NOD.Cg-KitW-41JTyr+PrkdcscidIl2rgtm1Wjl/ThomJ) mice, supporting huHSC engraftment without γ-radiation11,12. Mutated KitW-41J hampers mouse (mo)HSCs docking onto BM stromal cells and opens up an ample niche for huHSCs docking through binding of mouse stem cell factor11,12, which is engaged by huHSC c-Kit. Adult NBSGW and NSGW41 mice grafted intravenously with cord blood huCD34+ cells supported greater huCD45+ lymphoid and myeloid cell reconstitution than γ-irradiated NSG mice5,11,12. Despite their obvious potential, however, NBSGW and NSGW41 mice have not been leveraged to construct an advanced humanized mouse that faithfully replicates human immune responses2,3,4,5,6.
We created a humanized (THX) mouse by grafting NBSGW12 and NSGW41 (ref. 11) neonates with cord blood huCD34+ cells through intracardiac injection, followed by conditioning with 17β-estradiol (E2), the most potent and physiologically abundant estrogen. E2 supports differentiation of HSCs13,14,15, lymphoid and myeloid immune cells, including marginal zone (MZ) B cells, follicular helper T (TFH) cells, germinal center (GC) B cells, MBCs and granulocytes, all expressing estrogen receptors ERα and ERβ13,14,15,16,17,18,19,20,21,22,23,24,25,26. E2 also boosts B cell AID and BLIMP-1 expression, enabling SHM/CSR and PC differentiation27,28,29,30. THX mice reconstitute a human immune system, including peripheral lymph nodes (LNs), Peyer’s patches and human thymic epithelial cells (huTECs). They mount mature neutralizing antibody responses to Salmonella (S.) Typhimurium and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike S1 receptor-binding domain (RBD), together with B cell-related cytokines. Finally, THX mice are amenable to develop systemic lupus autoantibodies and immunopathology.
Results
THX mice support full and sustained development of human immune cells
To make huNBSGW and huNSG mice, we injected intracardially (left ventricle) NBSGW and γ-irradiated NSG neonates with cord blood huCD34+ cells. To make THX mice, we fed huNBSGW mice E2 ad libitum in drinking water starting at 14 to 18 weeks of age. After 4 weeks, THX mice were ready for experimental use or continued on E2 for use at a later time. Female and male THX mice showed comparable blood E2 levels (82.17 ± 10.36 pg ml−1 and 82.75 ± 5.72 pg ml−1, respectively, mean ± s.e.m.), higher than those in female and male huNBSGW mice (20.94 ± 1.88 and <5 pg ml−1) and within women’s physiological E2 level (35–500 pg ml−1; Extended Data Fig. 1 and Supplementary Table 1). THX and huNBSGW mice sustained human peripheral blood mononuclear cells (huPBMCs) at higher levels (up to 96.1% and 89.3% huCD45+ cells, respectively) than huNSG mice (Fig. 1a,b and Extended Data Fig. 2a). They showed more blood huB cells, huT cells, human dendritic cells (huDCs), human natural killer (huNK) cells and human monocytes, and more huB cells in spleen and LNs than huNSG mice (Fig. 1e and Supplementary Figs. 1 and 2). THX mice displayed higher levels of circulating huIgM, huIgD, huIgG, huIgA and huIgE, and had a longer lifespan than huNBSGW and huNSG mice (Fig. 1c,d). Their spleens contained a spectrum of huCD45+ lymphoid and myeloid cells, like spleens of humans who died from accidental death31 (Fig. 1f and Supplementary Tables 2–4a–g). THX mice showed blood huCD45−CD235a−CD61+ platelets and, as in other humanized mice, few huCD235a+ red blood cells5 (Supplementary Table 5a,b). THX and huNBSGW mice harbored more BM huCD34+ cells than huNSG mice (Fig. 1g). Thus, female and male THX mice reconstitute human lymphoid and myeloid cells, showed higher levels of huIgM, huIgD, huIgG, huIgA and huIgE than huNBSGW and huNSG mice, and extended survival.
THX mice BCR huV(D)J gene repertoire reflects that of humans
The THX mouse huBCR repertoire mirrored that of humans. Indeed, THX mouse huCD19+IgM+ B cells expressed huVHDJH-Cμ transcripts with probabilistic VH gene usage, that is, reflecting the genomic representation of human VH genes (huIgH locus haploid complement consists of 36–49 functional VH genes segregated in seven families32), with V3 family genes, particularly V3–V30, as the most frequently utilized, followed by V1 and V4 (Fig. 2a,b). Like humans, THX mouse huIgM+ B cells showed preponderant human D3 and JH3 utilization and dominant V3 to JH4 combination (Fig. 2c). Their huVHDJH-Cμ transcripts showed a pseudo-normal CDR3 length distribution, which peaked at 14 amino acids, mimicking huIgM+ B cells in humans (Fig. 2d). Discrete huIgM+ B cell clones identified by unique and identical huVHDJH-Cμ transcripts showed even greater diversity than in humans (Fig. 2e). THX mouse huIgM+ B cells displayed a Vκ gene utilization similar to that of humans32, albeit biased to Vκ4, and a Jλ-Cλ3 utilization versus human huIgM+ B cells Jλ-Cλ2 and Jλ-Cλ3 (Fig. 2f). Thus, the THX mouse huIgM+ BCR repertoire mirrors that of humans, with minor differences in VκJκ and VλJλ gene expression.
THX mice transition from a mouse to a human-like intestinal microbiome
The BCR repertoire underpins antibody diversity, which, in turn, conditions gut microbiome composition33. Non-intentionally immunized THX, huNBSGW and NBSGW mice showed distinct and shared gut bacteria families (Extended Data Fig. 3). Muribaculaceae together with other families contributing to the human gut microbiome34 made up for the THX mouse microbiome. This shared most bacteria families with huNBSGW mice and substantially differed from that of NBSGW mice, which was dominated by the characteristically ‘murine’ Rikenellaceae, still found in the ‘transitional’ microbiome of huNBSGW mice, but absent in THX mice. Thus, reflecting the impact of human immune cells and E2, the THX gut microbiome consists of bacteria all found in human gut microbiome and shows little similarity to that of (non-grafted) NBSGW mice.
THX mice huTCRa and huTCRb gene repertoires reflect those of humans
THX mouse spleen huTCRα and huTCRβ repertoire diversity largely reflected the human genomic representation of huVα, huJα, huVβ and huJβ genes32 (Fig. 3a), and broadly overlapped with huTCRα and huTCRβ gene expression in human blood, including huVβ and huJβ gene pair preferences (Fig. 3b,c). THX mice huVαJα−Cα and huVβDJβ−Cβ CDR3 lengths followed a pseudo-normal distribution, 4 to 21 amino acids, peaking at 11 and 13 amino acids, comparable to huT cells in humans (Fig. 3d). THX mouse huVβDJβ−Cβ transcripts identified discrete huT cell clones of a diversity comparable to humans (Fig. 3e). Thus, THX mouse huT cells express diverse huTCRα and huTCRβ repertoires, with huVα, huJα, huVβ and huJβ gene utilization reflecting huVα, huJα and huVβ, huJβ genomic representation and overlapping with that of huTCRα and huTCRβ in humans.
THX mice mount a T cell-dependent-specific and mature antibody response
Upon intraperitoneal (i.p.) immunization with T cell-dependent NP16-CGG conjugated hapten, THX mice showed serum huIgM at levels comparable to huNBSGW mice. They, however, made significantly greater amounts of total and high-affinity NP4-specific huIgG1, huIgG2, huIgG3, huIgA and huIgE than huNBSGW mice, with JAX NSG huCD34 mice making virtually no such antibodies (Fig. 4a). THX mice showed spleen huIgG+ and huIgA+ B cells, huCD27+CD38+ plasmablasts (PBs)/PCs, as accompanied by MZ huCD19+IgM+IgD+CD27+ B cells and including class-switched memory huCD19+huIgD−CD27+ and huCD19+IgG+CD27+ B cells35 at greater numbers than huNBSGW and JAX NSG huCD34 mice (Fig. 4b). Their B cells expressed higher levels of huAID and huBLIMP-1 than huNBSGW mice (Fig. 4c). THX 406, 407, 408 and 409 mouse huIgG+ and huIgA+ B cells accumulated more than 2.0 × 10−2 somatic point mutations per base, with a high ratio of replacement (R) to silent (S) mutations36 in huV1DJH-Cγ, huV3DJH-Cγ, huV1DJH-Cα1 and huV3DJH-Cα1 transcripts. In such THX mice, select huIgG+ B cell clones, expressing mainly V1 and V3 (including V3–V30) genes, expanded and intraclonally diversified, likely responding to NP16-CGG (Fig. 4d–f and Extended Data Fig. 4a,b)—NP16-specific huB cells sorted from THX mouse 406 included the two largest huV1DJH-Cγ1 clones. Thus, THX mice can mount a mature T cell-dependent response, entailing B cell huAID and huBLIMP-1 expression, SHM/CSR, BCR-driven clonal selection and intraclonal diversification, differentiation of specific huPCs and huMBCs, yielding high-affinity antibodies and as accompanied by huMZ B cells.
THX mice mount a T cell-independent specific and mature antibody response
Mature T cell-independent antibody responses are mounted by Tcrb−/−Tcrd−/− and NSG/B mice through B cell Toll-like receptors (TLRs)37,38,39. THX mice i.p. injected with T cell-independent TLR9 ligand DNP-CpG made greater amounts of total and high-affinity DNP5-specific huIgM, huIgG, huIgA and huIgE than huNBSGW mice, with JAX NSG huCD34 mice making few high-affinity antibodies (Fig. 4g). They showed greater numbers of spleen huIgG+ and huIgA+ B cells, class-switched memory huIgD−CD27+ B cells and huCD27+CD38+ PBs/PCs, together with more spleen and blood MZ huIgM+IgD+CD27+ B cells than huNBSGW or JAX NSG huCD34 mice (Fig. 4h–j). THX mice showed increased B cell huAID and huBLIMP-1 expression and DNP5-specific hugM, huIgG and huIgA antibody-secreting cells (ASCs) in spleen and BM than huNBSGW mice (Fig. 4k,l). They secreted huIgM, huIgD, huIgG and huIgA in the respiratory tract (bronchoalveolar lavage fluid, BALF) and showed huIgM-, huIgD- and huIgA-expressing B cells in intestinal lamina propria together with huCD3+ T cells (Fig. 4m,n). They also developed Peyer’s patches, not detected in huNBSGW mice, hosting MZ huCD19+IgM+IgD+CD27+ B cells, class-switched huCD19+IgG+ and huIgA+ B cells, GC huCD19+CD38+CD27−IgG+ and huCD19+CD38+CD27–IgA+ B cells, memory huCD19+CD27+IgD−B cells, huCD19+CD38+CD138+CD27+ PBs, huCD19−CD38+CD138+CD27+ PCs, huCD3+CD4+CD8− and huCD3+CD4−CD8+ T cells, and huCD3+CD4+CXCR5+PD-1+ TFH cells (Extended Data Fig. 2b). In THX mice, gut lymphoid cells were associated with high levels of fecal huIgD and huIgA (free and bound to fecal bacteria; Fig. 4o,p). THX mice huB cells accumulated somatic point mutations at more than 1.9 × 10−2 changes per base with high R:S mutation ratios, through select clonal expansion and intraclonal diversification of huV1DJH-Cμ-, huV3DJH-Cγ- and huV4DJH-Cγ-expressing huB cells (Extended Data Fig. 5a,b). Thus, THX mice can mount a mature T cell-independent antibody response, entailing B cell huAID and huBLIMP-1 expression, SHM/CSR, huPC and huMBC differentiation, huB cell clonal selection and intraclonal diversification, yielding high-affinity antibodies. Also, unlike huNBSGW mice, they develop gut-associated Peyer’s patches containing MZ huB cells, huT cells, GC huIgG+ and huIgA+ B cells, memory huB cells and huPBs/huPCs. THX mice also display MZ huB cells in blood and spleen, secrete BALF huIgM, huIgD, huIgG, huIgA and fecal antibacterial huIgD and huIgA.
huB cells from THX mice have full differentiation potential
Naive huIgM+IgD+ B cells from THX mice and from humans were cultured in vitro to compare their potential to undergo CSR, PC and memory-like B cell differentiation. Upon culture with T cell-dependent (CD154, IL-2, IL-4 and IL-21) or T cell-independent (CpG, IL-2, IL-21, transforming growth factor (TGF)-β and retinoic acid, or CpG, IL-2, IL-4 and IL-21) stimuli, THX mouse huB cells underwent CSR to IgG, IgA and IgE, differentiated to huCD27+CD38+ PBs and class-switched huIgD−CD27+ memory-like huB cells like huB cells from humans, expressing comparable AICDA, PRDM1 and post-recombination huVHDJH-Cγ, huVHDJH-Cα and huVHDJH-Cε transcripts (Extended Data Fig. 6a–d).
THX mice develop LNs, huTECs, huTFH cells, generate huMBCs and form GCs
Deficient peripheral lymphoid organ development, particularly LNs, has been an important limitation of humanized mice5. Unlike similarly immunized huNBSGW or JAX NSG huCD34 mice, NP16-CGG-immunized THX mice developed well-formed cervical, mediastinal, axillary and mesenteric LNs. They showed greater numbers of spleen huB cells, huT cells, huNK cells, huDCs and human monocytes (Fig. 5a,b). THX mice also showed an increased huCD5− to huCD5+ B cell (B2/B1) ratio as compared to huNBSGW or JAX NSG huCD34 mice and accumulated more class-switched LN GC huCD20+CD38+CD27− B cells and circulating memory huCD19+CD38−IgD−CD27+ B cells (Fig. 5c–e). Further, they developed GCs containing huCD20+ B cells, huCD3+ T cells, proliferating huKi67+ cells, huBCL6+ B cells, huAID+ B cells and huBLIMP-1+ PBs, while huNBSGW and JAX NSG huCD34 mice did not (Fig. 5f). In spleen and LNs, the proportions of huCD4+ T and huCD4+CD8+ T cells were comparable across the three humanized mouse models, while huCD8+ T cells were more numerous in JAX NSG huCD34 than THX or huNBSGW mice (Fig. 5g). Further, THX but not huNBSGW or JAX NSG huCD34 mice showed abundant huTFH cells in spleen and mesenteric LNs (Fig. 5h). Unlike huNBSGW mice, THX mouse thymi showed medullary and cortical organization and abundant huTECs (huEpCAM+CD45−). They also showed huCD19+ B cells and huCD14−CD11c+ DCs, which together with huTECs mediate T cell selection40,41,42, as well as huCD14+ monocytes, which like huB and huT cells did not decrease with age (Fig. 5i,j). Finally, THX mouse thymus huCD45+ cells broadly expressed human major histocompatibility complex (huMHC) class I and/or huMHC class II (Fig. 5k). Thus, THX mice develop peripheral LNs and thymus-containing huT cells, huTECs, huB cells, huDCs and human monocytes, differentiate huTFH and huB cells to form GCs, increase B2/B1 cell ratio and generate huMBCs.
Flagellin-vaccinated THX mice mount a neutralizing response to Salmonella
THX mice vaccinated with purified S. Typhimurium flagellin made anti-flagellin huIgM, huIgG and huIgA, a Salmonella-neutralizing response comparable to humans, and survived S. Typhimurium infection, while non-vaccinated THX mice did not (Fig. 6a–d). Their bactericidal antibody response was accompanied by blood and spleen MZ huCD19+IgM+IgD+CD27+ B cells, flagellin-specific huCD19+IgG+ and huCD19+IgA+ B cells, huCD19+CD38+CD138+CD27+ PBs, CD19−CD38+CD138+CD27+ PCs and specific memory huCD19+CD27+ B cells, at higher frequencies than similar cells in humans. Flagellin-specific spleen huB cells sorted from THX mice expressed huVHDJH-Cγ and huVHDJH-Cα1 transcripts involving V1, V3 and V4 genes, pseudo-normal huIgH CDR3 lengths distribution, peaking at 16 amino acids, and bearing substantial loads of point mutations. huIgM+, huIgG+ and huIgA+ B cells underwent select clonal expansion and intraclonal diversification, with the three largest huVHDJH-Cα1-expressing huB cell clones accounting for a greater proportion of huVHDJH-Cα1-huB cells than the three largest huVHDJH-Cγ-expressing huB cell clones did of huVHDJH-Cγ-huB cells (Fig. 6e–i and Extended Data Fig. 7a–e). Also, vaccinated THX mice showed blood incretion of huAPRIL, huBAFF, huTGF-β, human interferon gamma (huIFN-γ), huIL-2, huIL-4, huIL-6, huIL-10 and huIL-21 at human physiological concentrations (Extended Data Fig. 8 and Supplementary Table 6). Thus, flagellin-vaccinated THX mice mount a protective antibody response to Salmonella, entailing SHM/CSR, huB cell clonal selection and intraclonal diversification, huPC and huMBC differentiation, huMZ B cells and blood incretion of antibody response-related human cytokines.
COVID-19 mRNA-vaccinated THX mice mount an RBD-neutralizing response
THX mice mounted a mature antiviral response. THX mice vaccinated intramuscularly (i.m.) with Pfizer-BioNTech 162b2 coronavirus disease 2019 (COVID-19) mRNA, according to human vaccination schedule, made huIgM, huIgG and, to a moderate degree, huIgA to SARS-CoV-2 Spike S1 RBD (37 amino acid core peptide) as well as RBD-specific huASCs, huCD19+ B cells, memory huCD19+CD27+ B cells and huCD19+CD27+CD38+ PBs (Fig. 7a–c). They showed blood incretion of huAPRIL, huBAFF, huTGF-β, huIFN-γ, huIL-2, huIL-4, huIL-6, huIL-10 and huIL-21 at human physiological concentrations (Extended Data Fig. 8 and Supplementary Table 7). THX mice sera with high RBD-binding huIgG titers displayed SARS-CoV-2-neutralizing activity comparable to huIgG1 monoclonal antibodies, as assessed by two different Spike S1 RBD–ACE2 platforms (Cayman Chemical and EpigenTek; Fig. 7d). In vaccinated THX mice, huB cell huVHDJH-CH transcripts displayed huIgH CDR3 lengths peaking at 13 and 17 amino acids and substantial loads of V gene somatic point mutations, greater in huVHDJH-Cγ and huVHDJH-Cα1 than in huVHDJH-Cμ transcripts, all with high R:S mutation ratios (Fig. 7e and Extended Data Fig. 9a). huIgG+, huIgA+ and huIgM+ B cells underwent select clonal expansion and intraclonal diversification, with the three largest huVHDJH-Cγ- and huVHDJH-Cα1-expressing huB cell clones accounting for a greater proportion of huVHDJH-Cγ-huB and huVHDJH-Cα1-huB cells than the three largest huVHDJH-Cμ-expressing huB cell clones did of huVHDJH-Cμ-huB cells (Fig. 7f and Extended Data Fig. 9a). In fact, the latter comprised a multitude of huIgM+ B cell ‘microclones’, likely not participants in the anti-RBD response. The interplay of SHM and CSR in shaping B cell intraclonal diversification was exemplified by genealogical trees outlining the stepwise evolution of two clones, one developing from an unmutated huV3-53D1-26JH1-Cμ-B cell progenitor, the other from an unmutated huVκ3-11 Jκ1-Cκ-B cell progenitor (Fig. 7g). RBD-specific huB cells were sorted from spleens of additional mRNA-vaccinated THX mice, and paired huVHDJH-Cγ and huVκJκ or huVλJλ gene segments were amplified from single huB cells to make 100 recombinant human monoclonal antibodies. These showed predominant utilization of human V3, V4 and V1, reflecting the human haplotypic representation of these human VH genes, together with human Vκ3, Vκ1 and Vκ2 as well as Vλ1 and Vλ2 genes; as expected, somatic point mutations were more frequent in VH than in Vκ or Vλ gene segments (Extended Data Fig. 9b). Forty-five of the 100 human monoclonal antibody huB cell clones (27 huIgM, 5 huIgG1 and 13 huIgA1) were selected based on greater RBD-binding activity and characterized for paired huIgH and huIgL genes (Extended Data Fig. 9c). Thus, upon COVID-19 mRNA vaccination, THX mice mount a mature neutralizing antibody response to Spike S1 RBD, entailing SHM/CSR, huB cell select clonal expansion and intraclonal diversification, huPC differentiation, generation of huMBCs and blood incretion of antibody response-related human cytokines.
RBD–KLH-vaccinated THX mice mount a mature antibody response to RBD
THX mice mounted a mature antibody response to SARS-CoV-2 Spike S1 RBD, as elicited by RBD (47 amino acid peptide containing a core of 37 amino acids) conjugated to keyhole limpet hemocyanin (KLH; i.p. priming and boost). RBD–KLH-injected THX mice, not non-vaccinated controls, made specific huIgM, huIgG and, to a lesser extent, huIgA antibodies to RBD (37 amino acid core peptide) (Extended Data Fig. 10a,b). Their huB cell huVHDJH-Cμ and huVHDJH-Cγ transcripts displayed heterogeneous huIgH CDR3 lengths and heavy loads of somatic point mutations with high R:S mutation ratios (Extended Data Fig. 10c,d)—huVHDJH-Cγ-huB cells underwent greater select clonal expansion and intraclonal diversification than huVHDJH-Cμ-huB cells, which comprised a multitude of ‘microclones’, reflecting moderate to no clonal expansion (Extended Data Fig. 10e,f). Thus, upon vaccination with SARS-CoV-2 Spike S1 RBD–KLH, THX mice mount a specific mature antibody response to RBD, involving SHM/CSR, huB cell clonal selection and intraclonal diversification.
THX mice can model SLE autoimmunity
Pristane, a saturated terpenoid alkane with pro-inflammatory activity, can induce lupus-like autoimmunity in C57BL/6, BALB/c and γ-irradiated humanized NSG mice43. Male and female 18-week-old THX mice (generated from huNBSGW and huNSGW41 mice) were injected i.p. with pristane or PBS. As early as 3 weeks after pristane injection, THX mice developed a malar rash evocative of the ‘butterfly rash’ in individuals with systemic lupus erythematosus (SLE), concomitant with rising levels of serum huIgG and huIgA, including antinuclear, anti-dsDNA, anti-histone, anti-Sm/anti-RNP and anti-RNA huIgG autoantibodies, eventually leading to kidney glomerular huIgG deposition and immunopathology (Fig. 8a,b). As compared to THX mice, ‘Lupus THX’ mice showed fewer mesenteric LN huCD19+IgM+ and huCD19+IgD+ B cells, greater class-switched huCD19+IgG+ and huCD19+IgA+ B cells as well as spleen and BM huPCs (Fig. 8c). Lupus THX mouse huB cells accumulated somatic point mutations in huVHDJH-Cγ and huVHDJH-Cα1 transcripts and expressed higher levels of huAID and huBLIMP-1 than THX mice (Fig. 8d,e). Likely reflecting an ongoing antigen-driven process, huIgG+ and huIgA1+ B cells selectively expanded and intraclonally diversified, as exemplified by the largest huV3DJH-Cγ- and huV3DJH-Cα1-huB cell clones emerging from unmutated progenitors (Fig. 8f). Diversified huVβDJβ cells also underwent select clonal expansions (Fig. 8g). Finally, Lupus THX mice suffered 45% mortality at 12 weeks after pristane injection, contrasting with 100% survival of THX mice (Fig. 8a). Thus, THX mice are amenable to model human SLE, including huB and huT cell clonality, autoantibodies to nuclear components and kidney immunopathology leading to reduced lifespan.
Discussion
Humanized mice have been constructed using BM, fetal liver or umbilical cord blood huCD34+ cells or PBMCs, with cord blood being a highly enriched source of HSCs5. In THX mice, cord blood huCD34+ cell engraftment of genetically myeloablated KitW-41J mice enables human cell multilineage development and full immune tolerance. Intracardiac injection would maximize huCD34+ cell dissemination to multifocal BM sites, thereby facilitating huCD45+ cell colonization of peripheral lymphoid organs, such as LNs, gut-associated lymphoid tissue and Peyer’s patches. In THX mice, this is promoted by E2 and contrasts with underdeveloped peripheral lymphoid formations in huNBSGW, JAX NSG huCD34 and other humanized mice2,3,4,5,6. In these, LN development could be achieved only by supraphysiological expression of transgenic murine thymic stromal cell-derived lymphopoietin44. Thus, in addition to neonatal grafting of KitW-41J immunodeficient mice by intracardiac injection, the innovative estrogen conditioning is critical to the making of THX mice.
An important limitation of humanized mouse models is the failure to mount mature antibody responses2,3,4,5,6. E2 support of antibody response maturation is consistent with stronger antibody responses to viral vaccines, such as SARS-CoV-2 virus, influenza and hepatitis B virus, or bacterial vaccines, such as diphtheria, tetanus and pneumococcus, and greater incidence of autoantibody-mediated autoimmunity in female than male mice and humans21,22,45,46,47. Accordingly, E2 promotes differentiation of virtually all immune cells, including B cells, T cells and granulocytes, all of which express ERα and ERβ14,20,21,22,25,26. Although more information is needed on E2 impact on HSC differentiation, CD34+ HSCs express ERα and ERβ (encoded by Esr1 and Esr2 genes) and engraft more efficiently in immunodeficient female than male mice13,14,15,24,48.
The comparable blood E2 levels in male and female THX mice were higher than in huNBSGW mice, but well within women’s E2 physiological range. The critical role of E2 in promoting B cell differentiation in THX mice likely reflects an intrinsic B cell estrogen activity16,18,49, as revealed by the THX mouse mature antibody response to T cell-independent DNP-CpG. In THX mice, E2 is critical in promoting development of LNs, Peyer’s patches and GCs, supporting differentiation of huTECs, huTFH cells and huGC B cells, increasing B2:B1 cell ratio and generating huMBCs. E2 conditioning was also important for the appearance of huIgM, huIgD, huIgG and huIgA in BALF and feces, as well as the high baseline levels of huIgD, huIgG and huIgA in non-intentionally immunized THX mice. Additionally, E2 supported differentiation of huMZ B cells, which contribute antibodies that provide the first-line of defense against blood-borne microbial pathogens. Spleen huMZ B cells in THX mice, whether immunized with NP-CGG, DNP-CpG or Salmonella flagellin, were comparable, as a proportion of B cells, to spleen MZ B cells in humans and mice50. As in humans, THX mice huMZ B cells occurred at a greater proportion in circulating blood than spleen.
E2 induces a genetic program, including Ptpn6, Bcl2 and Vcam1 expression, that promotes B cell activation and survival, while dampening pro-apoptotic mediators, such as PD-1 (ref. 16). The direct impact of estrogen on B cell differentiation was reflected in the ability of huB cells from THX mice to undergo CSR, PC and memory-like B cell differentiation in vitro as efficiently as B cells from healthy humans, in response to T cell-dependent and T cell-independent stimuli. Indeed, E2 promotes B cell AID expression and SHM/CSR by upregulating HoxC4, a transcription factor that induces the Aicda promoter to activate this gene27,28,29. E2 also downregulates miR-26a, a most abundant microRNA in B cells and suppressor of Aicda transcription, thereby further promoting AID expression30. Additionally, estrogen response elements are clustered within IgH switch (S) regions51, potentially enabling E2 amplification of CSR. Once bound to estrogen response elements, ERα forms complexes with GATA3 and PBX1 co-transcription factors and other ERα immune cell function agonists, including NF-κB, AP-1 and C/EBPβ, leading to increased RNA polymerase II recruitment14. High estrogen:androgen ratios support differentiation of class-switched MBCs and PCs, as in human aromatase transgenic male mice52. By contrast, progesterone (P4), the most important progestogen, precursor of testosterone and potent agonist of nuclear progesterone receptor, exerts a negative activity on B cell proliferation, differentiation and Aicda expression, thereby dampening SHM/CSR53,54. P4 impact on B cells can reduce antibody-mediated defense and promote disease, such as in P4-treated female mice infected with influenza virus55. Like P4, testosterone would exert a negative impact on immune cell activities, thereby contributing to weaker antibody responses to bacterial and viral vaccines in men than women21,22,45,46,47.
THX mouse human antibody responses to T cell-dependent and T cell-independent conjugated haptens, Salmonella flagellin and viral SARS-CoV-2 Spike S1 RBD peptide, entailed SHM/CSR mediating intraclonal diversification of selectively expanded huIgG+ and huIgA+ B cell clones, whose sizes accounted for major proportions of their respective huIgG+ and huIgA+ B cell repertoires. This contrasted with the, generally, multitude of huIgM+ B cells with virtually no clonal expansion, possibly progenitors of expanded class-switched and somatically hypermutated huIgG+ and huIgA+ B cell clones. In COVID-19 mRNA- or flagellin-vaccinated THX mice, the lower level of circulating anti-RBD or anti-flagellin huIgA than huIgG was incongruous with the comparable huIgA+ and huIgG+ B cell clonal expansions, huIgA and huIgG mutational loads and huIgA and huIgG ASC numbers. It, however, is consistent with the lower level of anti-RBD huIgA than huIgG in blood and saliva of COVID-19 mRNA-vaccinated humans56,57 as well as the lower level of anti-flagellin huIgA than huIgG in humans infected with Salmonella58. The predominant V3, V4 and V1 gene utilization by the class-switched antibodies in COVID-19 mRNA-vaccinated THX mice is evocative of similar V gene utilization by the class-switched antibody response in COVID-19 mRNA-vaccinated humans59. The mutational load of greater than 10−2 changes per base in huB cell huVHDJH-Cγ transcripts in COVID-19 mRNA- and RBD–KLH-vaccinated THX mice is also evocative of the heavy mutational load of COVID-19 mRNA vaccine-induced huIgG response in humans59,60,61, possibly reflecting the high immunogenicity of Spike S1 RBD62,63.
Humanized mice generally lack thymic huMHCs, resulting in huT cells selected on mouse MHC, a shortcoming corrected by grafting human thymus fragments, as in BLT mice5. THX mouse mature antibody responses induced by NP16-CGG, Salmonella flagellin and Pfizer COVID-19 mRNA were presumably dependent on CD4+ T cells educated on huTECs or other human cells expressing MHC class II40,41,42, such as huB cells and huDCs, also present in THX mouse thymus. But how could THX mice populate their thymus with huTECs, which supposedly emerge from non-hematopoietic CD34− progenitors? In fact, epithelial cells can differentiate from CD34+ stem cells, including cord blood CD34+ cells64,65,66, possibly giving rise to huTECs. Interestingly, TECs express ERα and ERβ, consistent with an E2 role in promoting their differentiation67.
THX mouse maturation of antibody response involved blood incretion of huAPRIL and huBAFF at human physiological concentrations. APRIL supports B cell proliferation, CSR and PC differentiation, while BAFF supports immature B cell survival, B cell differentiation and antibody production68. Flagellin-vaccinated and Pfizer COVID-19 mRNA-vaccinated THX mice displayed comparable concentrations of blood huAPRIL. The former, however, showed higher levels of circulating huBAFF, likely reflecting flagellin induction of this B cell cytokine69. In THX mice, huAPRIL and huBAFF occurred together with huTGF-β, huIFN-γ, huIL-2, huIL-4, huIL-6 and huIL-10, all at human physiological levels and, possibly, as promoted by ERα signaling14,19,20,49. THX mouse physiological levels of human B cell growth factors and cytokines contrast with the generally dysregulated levels of knock-in or transgenic growth factors and cytokines in other immunized mice5, as exemplified by the supraphysiological expression of human granulocyte-macrophage colony-stimulating factor (huGM-CSF) and huIL-3 in huNSG-SGM3 mice, huGM-CSF, huIL-3 and huIL-6 in huMISTRG(6) mice or huBAFF (TNFS13B) in huBAFFKI mice70.
A shortcoming of humanized mice has been the lack of GCs, contributing to impaired antibody responses5. In THX mice, E2 supports differentiation of huTFH cells, which make cytokines, such as IL-4, IL-6, IL-10 and IL-21, and critically promote GC huB cell differentiation, GC formation, BCR affinity maturation and generation of PCs and MBCs71,72. E2 promotes expansion of TFH cells via PPARγ, thereby supporting the class-switched antibody response49,73. In activated huPBMCs, E2 increases not only PD-1+CXCR5+ TFH but also ICOS+ TFH cells, both important for GC formation49,72. In addition, E2 enhances expression of CXCR4 and CXCR5, which are central to GC dark and light zone organization as well as T cell homing by modulating expression of T cell chemokine receptors, such as CCR5 (refs. 49,74). Finally, E2 increases CD4+ T cell CD154 expression22 and upregulates EZH2 histone methyltransferase, which helps TFH cell differentiation75.
Another shortcoming of humanized mice is poor development of human myeloid cells, particularly neutrophils5. Expression of huGM-CSF and huIL-3 in γ-radiation myeloablated humanized NSG-SGM3 and MISTRG mice as well as additional expression of human granulocyte colony-stimulating factor (hG-CSF), as in humanized MISTRGGR mice, has partially corrected human myeloid cell underrepresentation5,76. Neutrophils express both ERα and ERβ20,25,26, and estrogen has been shown to increase neutrophils in women’s peripheral blood and in mouse blood, BM and spleen25. THX mice reconstituted human neutrophils, to almost one-fourth of spleen huCD45+ cells, a proportion comparable to neutrophils in spleen of humans31. Finally, human platelets in THX mice accounted for approximately one-third of total platelets, possibly also as a result of direct E2 impact on megakaryocytes, which express ERα and ERβ and whose maturation is boosted by estrogen77.
The THX mouse gut microbiome, which consisted of Muribaculaceae and other bacterial families found in humans, profoundly differed from NBSGW mice microbiome, which was dominated by the exquisite ‘murine’ Rikenellaceae. By contrast, it shared bacteria, including the dominant Muribaculaceae, with huNBSGW mice, which, possibly reflecting the lack of E2 conditioning, also harbored remnants of Rikenellaceae, not found in THX mice. The human-like gut microbiome together with free and bacteria-bound fecal huIgD and huIgA, likely induced by microbial stimulation of gut lymphoid cells’ TLRs38,39,78, suggests that THX mice are suited to model human intestinal mucosa antibody responses. Nevertheless, further investigation is needed to elucidate the mechanisms underpinning E2 contribution to shaping the THX mouse microbiome in gut and airways and, possibly, the potential E2 contribution to support huILCs and peripheral resident T cells, both important in mucosal homoeostasis and defense.
Lupus murine models, such as MRL/lpr and genetically modified Sle1, Sle2 and Sle3 mice, all share a nonhuman immune system, mediating an autoantibody response that does not faithfully reproduce that of individuals with SLE. Estrogen plays a role in accelerating mouse lupus autoimmunity and may play a role in the development of human lupus16,17,19,23,43,79. E2 enhances anti-dsDNA antibody production in lupus huB cells and ERα accelerates lupus development in autoimmune (NZBxNZW)F1 mice in a B cell-intrinsic fashion17,20,79. Consistent with B cell clonal expansion in individuals with lupus, Lupus THX mice expanded and intraclonally diversified select huIgG+ and huIgA+ B cells and made class-switched autoantibodies to cell nuclear components, eventually leading to lupus-like symptoms and immunopathology. By overcoming limitations posed by the differences between mouse and human lupus43, Lupus THX mice would lend themselves to testing novel therapeutic approaches with immediate translatability to individuals with lupus. They would also provide a first proof-of-concept of THX mice modeling human disease.
Thus, THX mice achieve sustained human immune system reconstitution and express huBCR and huTCR repertoires as diverse as those of humans. They unveil and leverage a critical estrogen activity to promote human immune cell differentiation as well as maturation of human antibody and autoantibody responses. The mechanisms by which E2 supports these processes and incretion of relevant human cytokines remain to be defined in further detail, as do potential E2 long-term side-effects16,52, which, however, were not observed in THX mice. Thus, by overcoming the limitations of current humanized mouse models, THX mice provide an advanced and powerful platform for in vivo studies of human immune responses, particularly, antibody and autoantibody responses, for development of human vaccines and immune therapeutics, including modulators of unwanted human antibody responses.
Methods
Mice
C57BL/6J (RRID: IMSR_JAX: 000664), NSG (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ, RRID: IMSR_JAX: 005557)8, NBSGW (NOD.Cg-KitW-41JTyr+PrkdcscidIl2rgtm1Wjl/ThomJ, RRID: IMSR_JAX:026622)12, NSGW41 (NOD.Cg-KitW-41JPrkdcscidIl2rgtm1Wjl/WaskJ, RRID: IMSR_JAX:026497)11 and JAX NSG huCD34 (RRID: IMSR_JAX: 005557) mice (Supplementary Table 8) were purchased from The Jackson Laboratory (JAX NSG huCD34 mice were constructed by grafting γ-irradiated 3-week-old female NSG mice with cord blood huCD34+ HSCs). In all experiments, male and female mice were used in virtually equal proportions.
huCD34+ HSCs to be used for construction of THX, huNBSGW, huNSGW41 and huNSG mice were isolated from human umbilical cord blood collected immediately after cesarean section from full-term, normally developed male and female newborns (in approximately equal numbers) upon informed consent from healthy puerperae (18–45 years old with no infectious disease or history of cancer) of different ages, races and ethnic backgrounds (Supplementary Table 9; Department of Obstetrics and Gynecology, The University of Texas Long School of Medicine). CD34+ cells were purified using EasySep Human CD34 Positive Selection Kit II (17856, STEMCELL Technologies) according to the manufacturer’s instructions, yielding at least 99% huCD34+ cell preparations. Freshly purified huCD34+ cells were resuspended in PBS supplemented with 2% FBS for immediate grafting or frozen in 10% dimethylsulfoxide, 72% FBS, 18% RPMI medium and kept in liquid nitrogen for later grafting.
huNSG mice (Supplementary Table 10) were constructed by myeloablative conditioning of NSG mice neonates (within 48 h of birth) with (1 Gy) γ-radiation, followed by intracardiac (left ventricle) injection with purified cord blood huCD34+ cells (1.5 × 105 freshly isolated or frozen-thawed huCD34+cells in 50 μl PBS supplemented with 2.0% FBS) using a 27-gauge needle. huNBSGW (Supplementary Table 11) and huNSGW41 mice were constructed by grafting non-γ-irradiated, genetically myeloablated NBSGW and NSGW41 mice neonates (within 48 h of birth) intracardially (left ventricle) with cord blood huCD34+ cells. THX mice were generated by feeding huNBSGW or huNSGW41 mice E2 (3301, Sigma-Aldrich; 1.5 μM in drinking water resulting in a dose of 6.1 × 10−4 mg per kg body weight per day) ad libitum starting at 14–18 weeks of age (18 weeks in most cases) and continuing thereafter. After 4 weeks of E2 conditioning, huNBSGW or NSGW41 mice (referred to as THX mice; Supplementary Table 12) were ready for experiments or continued E2 for later use. E2 conditioning of huNBSGW or huNSGW41 mice did not start before 14 weeks of age, as estrogen (albeit at high dose) might inhibit early thymus development by T cell proliferation.
Most THX mice were constructed using NBSGW mice as only a dozen NSGW41 mice were acquired in 2019 from The Jackson Laboratory before the sale of such mice was discontinued. NSGW41-based THX mice were used in the human antibody response to NP-CGG (n = 3), in the lupus studies as part of the healthy THX controls (n = 4 of 12) as well as for generation of Lupus THX mice (n = 5) as described in ‘Lupus THX mice, human autoantibodies, immunopathology and mortality’. huCD45+ cells in blood, spleen and BM of humanized mice were identified by flow cytometry using APC-anti-huCD45 monoclonal antibody (304011, BioLegend; 1:100 dilution) and Pacific Blue-anti-moCD45 monoclonal antibody (103125, BioLegend; 1:100 dilution). Generally, THX and huNBSGW mice displayed up to 96.1% and 89.3% huCD45+ cells in circulating blood, respectively. huNSG and JAX NSG huCD34 mice displayed, at peak, approximately 45% and 20% huCD45+ cells, respectively. Circulating huCD45+ mononuclear cell numbers (cells per ml of blood) were measured by complete blood count analysis, in which blood was collected in EDTA-coated microtubes and analyzed using a XT2000iV or XE-5000 blood analyzer (Sysmex). THX, huNBSGW and huNSG mice used in all experiments were 20 to 24 weeks of age, unless indicated otherwise. JAX NSG huCD34 mice were 23 weeks of age. Mice used in all experiments were housed in a pathogen-free barrier animal vivarium facility at The University of Texas Health Science Center at San Antonio and were free of infection or disease. Housing rooms were maintained at a 14-h light/10-h dark cycle and controlled temperature of approximately 22–23 °C with 40–60% humidity. Food (Teklad LM-485 Sterilizable Mouse/Rat Diet, 7912, Inotiv) and water were sterilized.
Estrogen
Serum estradiol concentrations in non-intentionally immunized THX and huNBSGW mice (18–24 weeks old) were measured using Cayman Estradiol ELISA Kit (501890, Cayman Chemical), according to the manufacturer’s instructions, and compared to mice and human physiological range80,81,82,83,84,85,86. This platform uses an estradiol acetylcholinesterase conjugate (estradiol acetylcholinesterase Tracer) in an inhibition/competition assay, measuring serum estradiol concentration by OD at 414 nm. High OD readings reflect low estradiol concentrations, while low OD readings reflect high concentrations. Sera were collected from equal numbers of male and female mice, with female mice sera collected generally during proestrus, metestrus and diestrus.
FACS and CyTOF
For the cell surface FACS analysis, cells from blood of healthy humans or blood, BM (tibia and femur), thymus, spleen, LNs (cervical, mediastinal, axillary, mesenteric) and/or Peyer’s patches of humanized mice (THX, huNBSGW, huNSG or JAX NSG huCD34 mice) were surface stained with fluorochrome-conjugated monoclonal antibodies (Supplementary Table 13) in Hank’s Buffered Salt Solution (HBSS, MT21022CM, Fisher Scientific) plus 0.1% bovine serum albumin (BSA, BP1600-100, Fisher Scientific; BSA-HBSS) for 20 min. After washing, cells were resuspended in BSA-HBSS for flow cytometry. In vitro-stimulated and/or ex vivo mononuclear cells were stained with FITC-anti-huCD45 monoclonal antibody (clone 30-F11, 368507, BioLegend; 1:100 dilution), PE-anti-huCD45 monoclonal antibody (clone 2D1, 368509, BioLegend; 1:100 dilution), Pacific Blue-anti-moCD45 monoclonal antibody (clone 2D1, 103125, BioLegend; 1:100 dilution), PE-anti-huCD19 monoclonal antibody (clone HIB19, 302208, BioLegend; 1:100 dilution), PE-Cyanine7-anti-huCD19 monoclonal antibody (clone HIB19, 302216, BioLegend; 1:100 dilution), FITC-anti-huCD20 monoclonal antibody (clone 2H7, 302303, BioLegend; 1:100 dilution), BV510-anti-huCD138 monoclonal antibody (clone MI15, 356517, BioLegend; 1:100 dilution), PE-anti-huIgM monoclonal antibody (clone MHM-88, 314507, BioLegend; 1:100 dilution), BV510-anti-huIgM monoclonal antibody (clone MHM-88, 314521, BioLegend; 1:100 dilution), BV650-anti-huIgM monoclonal antibody (clone MHM-88, 314525, BioLegend; 1:100 dilution), APC-Fire 750-anti-huIgM monoclonal antibody (clone MHM-88, 314545, BioLegend; 1:100 dilution), BV421-anti-huIgD monoclonal antibody (clone IA6-2, 348225, BioLegend; 1:100 dilution), BV785-anti-huIgD monoclonal antibody (clone IA6-2, 348241, BioLegend; 1:100 dilution), BV421-anti-huIgG monoclonal antibody (clone M1310G05, 410703, BioLegend; 1:100 dilution), FITC-anti-huIgG monoclonal antibody (clone G18-145, 555786, BD Pharmingen; 1:100 dilution), APC-anti-huIgA monoclonal antibody (clone IS11-8E10, 130-113-427, Miltenyi Biotec; 1:50 dilution), FITC-anti-huIgA (c31577, Invitrogen; 1:100 dilution), APC-Fire 750-anti-huIgE monoclonal antibody (clone MHE-18, 325515, BioLegend; 1:100 dilution), APC-Cyanine7-anti-huCD11c monoclonal antibody (clone Bu15, 337217, BioLegend; 1:100 dilution), APC-anti-huCD14 monoclonal antibody (clone 63D3, 367117, BioLegend; 1:100 dilution), BV786-anti-huCD56 monoclonal antibody (clone 5.1H11, 362549, Biolegend; 1:100 dilution), PE-anti-huCD27 monoclonal antibody (clone M-T271, 356405, BioLegend; 1:100 dilution), BV650-anti-huCD38 monoclonal antibody (clone HB-7, 356619, BioLegend; 1:100 dilution), PE-Cyanine7-anti-huCD5 monoclonal antibody (clone UCHT2, 300621, BioLegend; 1:100 dilution), Super Bright 600-anti-huCD3 monoclonal antibody (clone OKT3, 63003741, eBioscience; 1:100 dilution), APC-anti-huCD4 monoclonal antibody (clone A161A1, 357407, BioLegend; 1:100 dilution), BV421-anti-huCD4 monoclonal antibody (clone A161A1, 357423, BioLegend; 1:100 dilution), PE-anti-huCD8 monoclonal antibody (clone SK1, 344705, BioLegend; 1:100 dilution), Alexa Fluor 700-anti-huCD8 monoclonal antibody (clone SK1, 344723, BioLegend; 1:100 dilution), PE-anti-huCXCR5 monoclonal antibody (clone J252D4, 356903, BioLegend; 1:100 dilution), FITC-anti-huCXCR5 monoclonal antibody (clone J252D4, 356913, BioLegend; 1:100 dilution), FITC-anti-huPD-1 monoclonal antibody (clone NAT105, 367411, BioLegend; 1:100 dilution), PE-Cyanine7-anti-huPD-1 monoclonal antibody (clone A17188B, 621615, BioLegend; 1:100 dilution), Pacific Blue-anti-huICOS monoclonal antibody (clone C398.4A, 313521, BioLegend; 1:100 dilution), PE-anti-huEpCAM (clone EPR20532-225, ab237397, Abcam; 1:100 dilution), PE-Cyanine7-anti-moEpCAM (clone G8.8, 118216, BioLegend; 1:100 dilution), APC-anti-huHLA-A,B,C (MHC I) monoclonal antibody (clone W6/32, 311409, BioLegend; 1:100 dilution), FITC-anti-huHLA-DR, DP, DQ (MHC II) monoclonal antibody (clone Tü39, 361705, BioLegend; 1:100 dilution) or 7-AAD (A9400, Sigma-Aldrich). For analysis of human red blood cells and platelets, THX mice red blood cells were stained with APC-anti-moTER-119 monoclonal antibody (clone TER-119, 116211, BioLegend; 1:100 dilution) and FITC-anti-huCD235a monoclonal antibody (clone HI264, 349103, BioLegend; 1:100 dilution). THX mice (low forward scatter) platelets were stained with PE-Cyanine7-anti-moCD41 monoclonal antibody (clone MWReg30, 133915, BioLegend; 1:100 dilution) and PerCp-anti-huCD61 monoclonal antibody (clone VI-PL2, 336409, BioLegend; 1:100 dilution).
For the intracellular FACS analysis, AID-expressing and BLIMP-1-expressing huB cells and huPBs/PCs (2.0 × 106 cells) were surface stained with anti-huCD45, anti-huCD19, anti-huCD27, anti-huCD38 and anti-huCD138 monoclonal antibodies, as well as Fixable Viability Dye eFluor 780 (65-0865-14, Fisher Scientific). After washing, cells were fixed in Cytofix/Cytoperm buffer (554655, BD Biosciences, 250 µl) and incubated at 4 °C for 45 min. Cells were washed twice in BD Perm/Wash buffer (554723, BD Biosciences) for permeabilization and stained with Alexa Fluor 647–anti-huAID antibody (bs-7855R-FITC, Bioss; 1:50 dilution) or Alexa Fluor 488-anti-huBLIMP-1 monoclonal antibody (clone 646702, IC36081G, R&D Systems; 1:50 dilution) in BD Perm/Wash buffer for 30 min at 4 °C. Cells were washed again twice in BD Perm/Wash buffer and resuspended in BSA-HBSS for flow cytometry.
Flow cytometry analysis and sorting were performed using single-cell suspensions. Cells were gated by forward and side scattering to exclude dead cells and debris (Supplementary Fig. 1a–c). Cell analysis was performed on pre-gated huCD45+ cells using a BD LSR-II or FACS Celesta flow cytometer (BD Biosciences) with FACSDiva software v9.4 (BD Biosciences). Data were acquired and analyzed using FlowJo v10.9 (Tree Star).
To assess human immune lymphoid and myeloid cell reconstitution in THX mice, single-cell suspensions of splenic white cells from non-intentionally immunized THX mice (20–24 weeks old) were incubated for 30 min at 4 °C with a 50 μl cocktail of metal conjugated anti-human monoclonal antibodies (Supplementary Table 2) from the MaxPar Direct Immune Profiling Assay, 30 Marker Kit (201325, Fluidigm), followed by washing for 10 min at room temperature. Cell viability was measured by DNA intercalation (Cell-ID Intercalator-103Rh). Labeled cells were analyzed by Helios mass cytometer (CyTOF software v6.7, Fluidigm) using a flow rate of 0.045 ml min−1. Human immune lymphoid and myeloid cell population frequencies, quality-control metrics and data plot displays were acquired using Maxpar Pathsetter software v3.0 (401018, Fluidigm).
Bacteria-bound huIgD and huIgA in THX and huNBSGW mice were detected as we described38,78. Briefly, feces (10 mg) were suspended in 100 μl PBS, homogenized and centrifuged at 400g for 5 min to remove large particles. Supernatant was centrifuged at 8,000g for 10 min, then analyzed for free huIgD and huIgA by ELISA. To detect bacteria-bound huIgD and huIgA, the bacterial pellet was resuspended in 1 ml PBS containing 1.0% (wt/vol) BSA. After fixation with 7.2% formaldehyde for 10 min at room temperature, bacteria were washed with PBS, stained with FITC-anti-huIgD (clone IA6-2, 348205, BioLegend; 1:100 dilution) or APC-anti-huIgA (clone IS11-8E10, 130-113-427, Miltenyi Biotec; 1:50 dilution) monoclonal antibodies on ice for 30 min, washed again then resuspended in PBS containing 0.2 μg ml−1 DAPI for flow cytometry analysis. All events revealed by DAPI were considered as bacteria.
Human mononuclear cells
huPBMCs were isolated from buffy coats obtained from healthy male and female human donors of different ages (18–65 years old), races and ethnic backgrounds (South Texas Blood and Tissue Center; Supplementary Table 14). The buffy coat was diluted at a 1:2 ratio in sterile PBS (pH 7.4, BP3991, Fisher Scientific) and then applied to a Histopaque-1077 density gradient (10771, Sigma-Aldrich) in 50 ml SepMate tubes (85450, STEMCELL Technologies), which were spun at 1,000g for 10 min. Recovered huPBMCs were washed in PBS and resuspended in RPMI (10-040-CV, Corning RPMI-1640 medium) supplemented with 10% vol/vol Hyclone FBS (42Q7980K, Gibco) and 1% vol/vol antibiotic-antimycotic solution (penicillin–streptomycin and amphotericin B, SV30079.01, Cytiva Life Sciences; FBS-RPMI).
Human immune cells were isolated from humanized mouse blood, BM, thymus, spleen, LNs and/or Peyer’s patches, and suspended in ACK Lysis Buffer (BP10-548E, Lonza) to lyse erythrocytes. Peripheral blood (approximately 250 μl) was collected from the submandibular vein into microtubes containing heparin (H19, Fisher Scientific; 25 μl, 1,000 units per ml). After quenching with FBS-RPMI and centrifugation, erythrocyte-free cells were resuspended in FBS-RPMI for further preparation or analysis.
Differentiation of naive human B cells from humans and from THX mice
To analyze CSR, PC and MBC differentiation, naive huCD19+IgM+IgD+B cells were isolated from huPBMCs obtained from healthy participants by negative selection using EasySep Human Naive B Cell Isolation Kit (17254, STEMCELL Technologies), according to the manufacturer’s instructions, yielding at least 98% huCD19+IgM+IgD+B cells. After pelleting, huB cells were resuspended in FBS-RPMI before further analysis or stimulation. Naive huCD19+IgM+IgD+B cells were isolated from THX mouse spleens by negative selection using biotin-anti-huCD43 (9620-08, clone DF-T1, SouthernBiotech; 1:50 dilution) and biotin-anti-huCD3 monoclonal antibodies (300403, clone UCHT1, BioLegend; 1:50 dilution) followed by positive selection using biotin-anti-huIgD monoclonal antibody (348212, clone IA6-2, BioLegend; Supplementary Table 15) and MagniSort Streptavidin Positive Selection Beads (MSPB-6003-74, Thermo Fisher Scientific), yielding at least 98% huCD19+IgM+IgD+B cells. After pelleting, B cells were resuspended in FBS-RPMI. Naive huIgM+IgD+B cells from humans or THX mice were cultured in FBS-RPMI (5.0 × 105 cells per ml) for 72 h (for RNA transcript analysis) or up to 120 h (for flow cytometry analysis) upon stimulation with: membrane-CD154 (3.0 U ml−1)38,39,78 or CpG ODN 2395 (Eurofins Genomics, 2.5 μg ml−1) plus recombinant huIL-2 (589102, BioLegend, 100 ng ml−1), recombinant huIL-4 (574002, BioLegend, 20 ng ml−1) and recombinant huIL-21 (571202, BioLegend, 50 ng ml−1) for CSR to huIgG. For CSR to huIgA, naive huIgM+IgD+ B cells were cultured under similar conditions upon stimulation with membrane-CD154 or CpG ODN 2395 plus recombinant huIL-2, recombinant huIL-21, recombinant TGF-β (781802, BioLegend, 4.0 ng ml−1) and recombinant retinoic acid (11017, Cayman Chemicals, 4.0 ng ml−1). Pre-gated huCD45+huCD19+ cells were stained with specific human monoclonal antibodies (Supplementary Table 13) to detect huIgM+, huIgD+, huIgG+, huIgA+ or huIgE+ B cells, huCD27+CD38+ PBs and class-switched huCD27+IgD− memory-like B cells by flow cytometry.
huBCR IgM+ B cell and huTCR repertoires and huIgM+ B and T cell clonality
To analyze expressed huVHDJH-Cμ, huVκJκ-Cκ and huVλJλ-Cλ or huVαJα-Cα and huVβJβ-Cβ gene repertoires, huIgM+ B cells and huT cells were isolated from blood of healthy humans (Supplementary Table 14) and spleens of non-intentionally immunized THX mice (20–24 weeks old). RNA (2 µg) was extracted using RNeasy Mini Kit (74104, Qiagen). huVHDJH-Cμ, huVκJκ-Cκ and huVλJλ-Cλ or huVαJα-Cα and huVβJβ-Cβ mRNA transcripts were reverse transcribed from huIgM+ B or huT cell RNA by RT-5′ RACE PCR using SuperScript III First-Strand Synthesis System (18080051, Invitrogen) and a huCμ-, huCκ-, huCλ-, huCα- or huCβ-specific reverse primer (Supplementary Table 16). Single-strand cDNA was cleaned up using QIAquick PCR purification kit (28104, QIAGEN) and 3′ poly-dA tailed by TdT and dATP. The dA-tailed cDNA was then amplified by PCR using a forward oligo-dT primer together with a nested huCμ-, huCκ-, huCλ-, huCα- or huCβ-specific reverse primer. Both forward and reverse primers were tagged with Illumina overhang adaptors. PCR amplification conditions were 95 °C for 30 s, 55 °C for 30 s and 72 °C for 40 s for 35 cycles. cDNA amplicons were cleaned up using QIAquick PCR purification kit, further amplified by index PCR involving Illumina clustering adaptors and beads cleanup, quantified and then loaded onto the Illumina MiSeq system using the 300-bp pair-end sequencing module. huVHDJH-Cμ, huVκJκ-Cκ, huVλJλ-Cλ, huVαJα-Cα and huVβJβ-Cβ repertoires were analyzed using IMGT/HighV-QUEST v1.9.2 (The International ImMunoGeneTics Information System; http://www.imgt.org/HighV-QUEST/home.action/).
To identify individual huB and huT cell clones and analyze huB or huT cell clonal diversity, huB cell VHDJH-Cµ or huT cell VβDJβ-Cβ transcripts (up to 250,000 sequences) of healthy humans and THX mice were analyzed by Illumina MiSeq amplicon sequencing and segregated based on the same huVH or huVβ gene segment, the same and unique huIgH or huTCRβ CDR3 together with the same huJH or huVβ sequence87,88,89,90,91. Each discrete clone was depicted as an individual rectangle or square (unique color), whose area reflects huB or huT cell clone size, as inferred from the sum of identical huVHDJH-Cµ or huVβDJβ-Cβ transcripts (TreeMaps, Microsoft Excel v16.83 and IMGT/HighV-QUEST statistic data).
THX mice huB cell SHM/CSR, clonality and intraclonal diversification
To analyze SHM in the NP16-CGG-induced antibody response, RNA (2 µg) was extracted from THX mice total and sorted NP16-specific huB cells using the RNeasy Mini Kit (74104, Qiagen), and cDNA was synthesized using the SuperScript III First-Strand Synthesis System (18080051, Invitrogen) with oligo-dT primer. Rearranged huV1DJH-Cγ, huV3DJH-Cγ, huV1DJH-Cα1 and huV3DJH-Cα1 cDNA was amplified using a huV1 or huV3 leader-specific forward primer together with a nested huCγ- or huCα-specific reverse primer tagged with Illumina overhang adaptors (Supplementary Table 16) and Phusion high-fidelity DNA polymerase (M0530S, New England BioLabs)—amplification of huIgH V1 and V3 genes was chosen as these families include gene members of high sequence similarity to mouse V1-72 (V186.2/V3 gene), the gene encoding the most efficient ‘NP-binding’ mouse IgH V segment (https://www.imgt.org/ligmdb/view?id=J00239/)36,92. PCR amplification conditions were 98 °C for 10 s, 60 °C for 45 s and 72 °C for 1 min for 30 cycles. The cDNA amplicons were further amplified and sequenced as described in ‘huBCR IgM+ B cell and huTCR repertoires and huIgM+ B and T cell clonality’. Somatic point mutations in recombined transcripts were analyzed using IMGT/HighV-QUEST v1.9.2 (https://www.imgt.org/HighV-QUEST/login.action/) and corrected for polymerase and sequencing error rates (0.008) to calculate the frequency of somatic point mutations. To analyze huB cell clonality and SHM in the DNP-CpG-, S. Typhimurium flagellin-, Pfizer COVID-19 mRNA- and RBD–KLH-induced antibody responses, THX mouse huB cell VHDJH-Cμ, VHDJH-Cγ, VHDJH-Cα, VκJκ-Cκ or VλJλ-Cλ transcripts were reverse transcribed, amplified and sequenced as described in ‘huBCR IgM+ B cell and huTCR repertoires and huIgM+ B and T cell clonality’, then analyzed for point mutations as described above.
B cell clonal diversity in immunized THX mice was analyzed as described in ‘huBCR IgM+ B cell and huTCR repertoires and huIgM+ B and T cell clonality’. To analyze intraclonal diversification, shared and unique point mutations in huVHDJH-CH transcripts within each huB cell clone were used to construct genealogical trees (phylogenetic maps), revealing sequential multistep accumulation of point mutations from unmutated progenitors, and allowing for detailed intraclonal diversification analysis. Genealogical trees were constructed by uploading FASTA files of all segregated huVHDJH-CH transcripts onto PHYLOViZ Online v2.0 (http://www.phyloviz.net/), which uses a JAVA implementation of the Feil’s goeBURST algorithm rules for visualization of multiple phylogenetic inference trees.
To quantify AICDA, PRDM1, huVHDJH-Cμ, huVHDJH-Cγ1, huVHDJH-Cα1 and huVHDJH-Cε transcript expression in huB cells from THX mice in vitro and ex vivo and huB cells from humans in vitro, RNA extraction and cDNA synthesis were performed as described above. Transcript expression was analyzed by SYBR Green dye (IQ SYBR Green Supermix, 115010139, Bio-Rad) incorporation in PCR reactions involving specific forward and reverse primers (Supplementary Table 16). Reactions were performed in an iCycler (Bio-Rad) real-time qPCR system under the following amplification cycles: 95 °C for 15 s, 40 cycles at 94 °C for 10 s, 60 °C for 30 s and 72 °C for 30 s—data acquisition was performed during this 72 °C extension step (Bio-Rad CFX Manager Software v3.1). Melting curve analysis was performed from 72 to 95 °C. The 2−ΔCt method (2−ΔCt = 2-[Ct(HPRT1)-Ct(target gene)]) was used to determine levels of transcripts, and data were normalized to levels of human HPRT1.
Humanized mice antibody response to conjugated haptens
THX, huNBSGW (20–24-week-old) and JAX NSG huCD34 (23-week-old) mice were injected i.p. with 4-hydroxy-3-nitrophenylacetyl (NP) conjugated to chicken gamma globulin (NP16-CGG, 16 NP molecules conjugated with one CGG molecule; N-5055C-5, Biosearch Technologies) or dinitrophenyl conjugated to CpG ODN2395 (DNP-CpG, one DNP molecule conjugated with one CpG molecule, custom synthesized by Eurofins Scientific) on day 0 (100 μg in 100 μl alum, Imject Alum Adjuvant, 77161, Thermo Scientific or 50 μg in 100 μl PBS), boosted (100 μg in 100 μl PBS or 50 μg in 100 μl PBS) on day 14 and euthanized on day 28. Total, NP-specific and DNP-specific human antibodies were analyzed by specific ELISAs, as described in ‘ASCs and titration of human antibodies’. For cell sorting, NP-specific spleen huB cells from NP16-CGG-immunized THX mice were single-cell FACS sorted after staining with NP16-PE (16 NP molecules conjugated with one PE molecule, sc-396483, Santa Cruz Biotechnology; 1:100 dilution). VHDJH-CH transcripts from sorted huB cells were analyzed for SHM/CSR, B cell clonality and intraclonal diversification, as described in ‘huB cell SHM/CSR, clonality and intraclonal diversification’.
THX mice neutralizing response to Salmonella and in vivo protection
THX mice (20–24 weeks old) were injected i.p. with S. Typhimurium flagellin (CVD1925 FliC, University of Maryland School of Medicine Center for Vaccine Development, 50 μg in 100 μl alum) or nil (100 μl alum) on day 0, boosted (50 μg in 100 μl PBS or 100 μl PBS alone) on day 14 and euthanized on day 28 (ref. 39).
Total human immunoglobulin and flagellin-specific human antibodies were analyzed by specific ELISAs, as described in ‘ASCs and titration of human antibodies’. Bactericidal activity of flagellin-induced antibodies in sera from flagellin-vaccinated and non-vaccinated THX mice was measured by in vitro killing of S. Typhimurium39. S. Typhimurium IR715, a virulent nalidixic acid-resistant derivative of wild-type isolate ATCC 14028 (provided by M. Raffatellu, University of California, San Diego) was grown in LB broth (BP1426-2, Fisher Scientific) overnight at 37 °C. Log-phase cultures were prepared by diluting overnight cultures to an OD600 of 0.05 in fresh LB medium and incubating them at 37 °C, with shaking at 250 rpm until an OD600 of 0.7 or 0.8 was attained. Stock cultures were prepared by diluting 500 µl of log-phase cultures in 500 µl of 50% sterile filtered glycerol (G33-1, Fisher Scientific) then further diluted in PBS to a cell density of approximately 104 CFUs per ml. Sera from flagellin-vaccinated THX mice, non-vaccinated THX mice and healthy humans were serially twofold diluted in PBS in round-bottom 96-well plates. Diluted sera (50 µl) or PBS (50 µl, negative control) were mixed with 25 µl baby-rabbit complement (CL3441, CEDARLANE, 25% final concentration) and incubated with 25 µl diluted S. Typhimurium (250 CFUs). Each sample mixture was shaken (115 rpm) at 37 °C for 1 h and then struck onto LB-agar plates. These were incubated at 37 °C overnight, after which CFUs were enumerated. To assess the protective response induced by flagellin vaccination in vivo, flagellin-vaccinated and non-vaccinated THX mice were infected orally with S. Typhimurium (1 × 105 CFUs) by gavage on day 21. The effective dose of bacteria given to mice was verified by plating dilutions of S. Typhimurium on LB-agar plates supplemented with nalidixic acid (N8878-25G, Sigma-Aldrich, 0.05 mg ml−1). Mice were monitored for 10 days, and Kaplan–Meier survival plots were generated (GraphPad Prism v10.0.3). For cell sorting, flagellin-specific spleen huB cells from flagellin-vaccinated THX mice underwent single-cell FACS after staining with AF647-flagellin (synthesized using iLink Andy Fluor 647 Antibody Labeling Kit, L038, ABP Biosciences). VHDJH-CH transcripts from sorted huB cells were analyzed for SHM/CSR, B cell clonality and intraclonal diversification, as described in ‘huB cell SHM/CSR, clonality and intraclonal diversification’.
THX mice neutralizing antibody response to COVID-19 mRNA or RBD–KLH
THX mice (20–24 weeks old) were injected i.m. with Pfizer-BioNTech 162b2 COVID-19 vaccine (Pfizer COVID-19 mRNA, 5 µg in 50 µl PBS) or nil (50 µl PBS) on day 0, boosted (5 µg in 50 µl PBS or 50 µl PBS alone) on day 21, according to the human vaccination schedule, and euthanized on day 28. ‘Discarded’ vials of Pfizer COVID-19 mRNA vaccine were obtained from The University of Texas Health Science Center at San Antonio vaccination facility within 6 h of opening and contained less than one full vaccine dose, thereby not diverting any amount of vaccine from humans for the purpose of this study. THX mice were injected i.p. with SARS-CoV-2 Spike S1 RBD (47 amino acid peptide containing the core 37 amino acids: FRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNG, custom synthesized by ABI scientific) conjugated to KLH (RBD–KLH, 50 μg in 100 μl alum) or nil (100 μl alum) on day 0, boosted (50 μg in 100 μl PBS or 100 μl PBS alone) on day 21 and euthanized on day 28.
Total human immunoglobulin and RBD-specific human antibodies or ASCs were analyzed by specific ELISAs or ELISPOTs, as described in ‘ASCs and titration of human antibodies’. The SARS-CoV-2 neutralization power of antibodies induced by COVID-19 mRNA vaccine in THX mice was measured using two different platforms: SARS-CoV-2 Neutralizing Antibody Detection ELISA Kit (502070, Cayman Chemical) and SeroFlash SARS-CoV-2 Neutralizing Antibody Assay Fast Kit (D-1008-96, EpigenTek), according to the manufacturer’s instructions. Sera from COVID-19 mRNA-vaccinated THX mice were serially twofold diluted in PBS-Tween 20 in 96-well plates pre-coated with SARS-CoV-2 Spike S1 RBD peptide (EpigenTek platform), or a recombinant rabbit Fc-tagged SARS-CoV-2 Spike S1 RBD peptide bound to an anti-rabbit Fc-specific antibody (Cayman platform), followed by addition of recombinant His-tagged ACE2 protein to each well. These platforms use a horseradish peroxidase (HRP)-conjugated anti-His antibody in an inhibition/competition assay to measure serum neutralizing human antibody concentration by OD reading at 450 nm. High OD readings reflect a low concentration of neutralizing antibodies, while low OD readings reflect a high concentration. SARS-CoV-2-neutralizing human monoclonal antibodies were provided as a positive control by EpigenTek and Cayman. Extensive controls performed by both Cayman Chemical and EpigenTek have validated measurements of their RBD competition assays with actual virus neutralization in COVID-19-positive and COVID-19-negative human sera (https://www.caymanchem.com/product/502070/sars-cov-2-neutralizing-antibody-detection-elisa-kit; www.epigentek.com/docs/D-1008.pdf).
Sequencing and cloning of original paired heavy-chain VHDJH-CH and light-chain VκJκ-Cκ or VλJλ-Cλ gene segments for construction of human antibody-producing cell microcultures was performed by The University of Texas MD Anderson Cancer Center Recombinant Antibody Production Core. Briefly, RBD-specific spleen huB cells of three COVID-19 mRNA-vaccinated THX mice underwent single-cell FACS using biotinylated RBD peptide (47 amino acids) and FITC-streptavidin (405201, BioLegend). huVHDJH-CH and light-chain huVκJκ-Cκ or huVλJλ-Cλ gene segments from sorted huB cells were amplified as cDNAs by single-cell RT–PCR and then sequenced. The single B cell huIgH constant region and huIGκ or huIGλ constant regions were determined. The amplified huVHDJH and huVκJκ or huVλJλ cDNAs were sequenced and cloned into pcDNA3.4 vectors that included the coding sequence for either human heavy-chain (γ1) or light-chain (κ or λ) constant regions to transfect ExpiCHO cells (A29127, Thermo Fisher). Transfected ExpiCHO cells were cultured in ExpiCHO Expression Medium (A2910001, Thermo Fisher) in 100 single-cell microcultures to produce recombinant human monoclonal antibodies. After 5 days, media were collected and analyzed for RBD-specific recombinant human antibodies by specific ELISA.
ASCs and titration of human antibodies
To measure total or specific huIgM, huIgD, huIgG (huIgG1, huIgG2, huIgG3 and huIgG4), huIgA or huIgE in humanized mice, sera were diluted 400-fold or 20-fold in PBS containing 0.05% vol/vol Tween 20 (BP337-500, Fisher Scientific; PBS-Tween 20), followed by serial twofold dilution. Serially diluted samples were incubated at room temperature in 96-well plates pre-coated with goat anti-huIgM antibody (2020-01, SouthernBiotech, 1.0 µg ml−1), goat anti-huIgD antibody (2030-01, SouthernBiotech, 1.0 µg ml−1), goat anti-huIgG antibody (huIgG1, huIgG2, huIgG3 and huIgG4, 2015-01, SouthernBiotech, 1.0 µg ml−1), goat anti-huIgA antibody (2050-01, SouthernBiotech, 1.0 µg ml−1), goat anti-huIgE antibody (GE-80A, ICL Labs, 1.0 µg ml−1), NP4-BSA (four NP molecules per one BSA molecule, Biosearch Technologies, 1.0 µg ml−1), DNP5.6-BSA (average of 5.6 DNP molecules per one BSA molecule, Cosmo Bio USA, 1.0 µg ml−1, referred to as DNP5 in the Results and figure legends), BSA (Biosearch Technologies, 1.0 µg ml−1), S. Typhimurium flagellin (2.0 µg ml−1) or SARS-CoV-2 Spike S1 RBD peptide (37 amino acid core peptide, FRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPL, ABI Scientific, 2.0 µg ml−1) in 0.1 M sodium carbonate/bicarbonate buffer at pH 9.6. After washing plates with PBS-Tween 20, bound human antibodies were detected with biotinylated goat anti-huIgM antibody (2020-08, SouthernBiotech; 1:5,000 dilution), goat anti-huIgD antibody (2030-08, SouthernBiotech; 1:5,000 dilution), goat anti-huIgG antibody (2015-08, SouthernBiotech; 1:5,000 dilution), goat anti-huIgG1 monoclonal antibody (555869, BD Pharmingen; 1:5,000), goat anti-huIgG2 monoclonal antibody (555874, BD Pharmingen; 1:5,000 dilution), goat anti-huIgG3 monoclonal antibody (3853-6-250, MABTECH; 1:5,000 dilution), goat anti-huIgG4 monoclonal antibody (555882, BD Pharmingen; 1:5,000 dilution), goat anti-huIgA antibody (2050-08, SouthernBiotech; 1:5,000 dilution) or goat anti-huIgE antibody (9250-08, SouthernBiotech; 1:5,000 dilution; Supplementary Table 15), followed by reaction with HRP-labeled streptavidin (405210, BioLegend), development with O-phenylenediamine substrate (P8806-50TAB, Sigma-Aldrich) or 3, 3′, 5, 5′ tetramethyl benzidine substrate (421101, BioLegend), and measurement of converted substrate absorbance at 492 nm or 450 nm, respectively. Total human antibody concentrations or specific human antibody titers were calculated from OD readings (using a reference curve constructed with known antibody concentrations; BioTek Gen5 Software v2.07) and expressed as µg equivalent per ml (µg eq ml−1) or RUs (defined as the dilution factor needed to reach 50% saturation binding) using GraphPad Prism v10.0.3 software or Excel v16.83 (Microsoft) software. To measure BALF human immunoglobulin concentrations, DNP-CpG-immunized THX and huNBSGW mice were euthanized on day 28, and lungs were lavaged with 1 ml PBS containing 0.1 mM EDTA. Human immunoglobulin concentrations were measured from the recovered 1 ml lavage fluids by specific ELISA as described above.
To detect huASCs (huPBs/PCs) by ELISPOT, splenic or BM cells from DNP-CpG-immunized or COVID-19 mRNA-vaccinated THX mice were suspended in FBS-RPMI then cultured at 37 °C overnight in 96-well PVDF Multi-Screen filter plates (activated with 35% ethanol, MAIPS4510, Millipore) coated with goat anti-huIgM antibody, goat anti-huIgG antibody, goat anti-huIgA antibody, DNP5.6-BSA or SARS-CoV-2 RBD peptide (all 5 µg ml−1). Spleen and BM cells were plated at 1.25 × 105 and 2.5 × 105 cells per well to analyze total and specific huASCs, respectively. After removing supernatants, plates were incubated with biotinylated goat anti-huIgM antibody, goat anti-huIgG or goat anti-huIgA antibody for 2 h, and then, after washing, incubated with HRP-conjugated streptavidin, followed by Vectastain AEC peroxidase substrate (SK-4200, Vector Laboratories). Individual ASC spots were detected using a CTL Immunospot Analyzer and software (CTL ImmunoCapture Software v6.5.7, Cellular Technology).
Human cytokines
To measure circulating human cytokines, sera were collected from flagellin-vaccinated and COVID-19 mRNA-vaccinated THX mice and analyzed for huAPRIL, huBAFF, huIFN-γ, huIL-2, huIL-4, huIL-6, huIL-10 and huIL-21 by Luminex Human Discovery Assay 8-Plex (LXSAHM-08, R&D Systems). Analysis of huTGF-β1 was performed by TGF-β Premixed Magnetic Luminex Performance Assay (FCSTM17, R&D Systems). Samples and reagents were prepared according to the manufacturer’s instructions. Briefly, sera were diluted at a 1:2.5 (Luminex Human Discovery Assay) or 1:15 (TGF-β Luminex Performance Assay) ratio in Calibrator Diluent RD6-52 or Calibrator Diluent RD6-50, respectively. Next, 50 μl working standards and 50 μl diluted sera were each mixed with 50 μl Human Magnetic Premixed Microparticle Cocktail (color-coded magnetic beads coated with analyte-specific capture antibodies) and incubated in 96-well microplates at room temperature for 2 h with shaking at 800 rpm. After washing plates with 100 μl per well of wash buffer using a Luminex microplate magnet, human cytokines were detected by addition of 50 μl Human Premixed Biotin-Antibody cocktail (biotinylated detection monoclonal antibodies specific for analytes of interest) followed by reaction with 50 μl streptavidin–phycoerythrin and measurement using a dual-laser flow-based detection Luminex FLEXMAP 3D analyzer (Luminex). One laser classifies the beads and determines the analyte that is being detected. The second laser determines the magnitude of the PE-derived signal, which is proportional to the amount of analyte bound. Cytokine concentrations were calculated using Belysa Immunoassay Curve Fitting Software (40–122, MilliporeSigma) and compared to human physiological range93,94,95,96,97,98.
H&E, immunohistochemistry and immunofluorescence microscopy
H&E and immunohistochemistry
To identify GCs in humanized mice, NP16-CGG-immunized THX, huNBSGW and JAX NSG huCD34 mouse spleens were fixed in paraformaldehyde (4%) overnight. Spleens were embedded in paraffin, sectioned, then stained with H&E or anti-huCD20 monoclonal antibody (1:200 dilution), anti-huCD3 monoclonal antibody (1:200 dilution), anti-huKi67 monoclonal antibody (1:200 dilution), anti-huBCL6 monoclonal antibody (1:200 dilution), anti-huAID (1:200 dilution) or anti-huBLIMP-1 monoclonal antibody (1:200 dilution), followed by reaction with anti-mouse IgG-HRP and brown precipitating HRP substrate 3,3′-diaminobenzidine (DAB). Spleen sectioning and staining was performed at The University of Texas Health Science Center at San Antonio Histology and Immunohistochemistry Laboratory. Images were captured using a Zeiss Imager-V.1 (ZEN Microscopy Software v3.9, 1× and 20× objective).
Immunofluorescence microscopy
To detect gut huB cells, huT cells, and huIgM-, huIgD- and huIgA-producing cells, DNP-CpG-immunized THX mouse intestines were fixed in paraformaldehyde (4%) overnight. Intestines were sectioned, then heated at 80 °C to adhere to glass slides, washed four times in xylene (214736-1L, Millipore Sigma) for 2 min, dehydrated twice with 100% ethanol for 1 min, dehydrated twice with 95% ethanol for 1 min, and washed twice in water for 1 min. Antigens were unmasked using 2 mM EDTA (15-575-020, Fisher Scientific) in 100 °C for 40 min, followed by a cooling step at 25 °C, thrice washing with TBS (15-567-027, Fisher Scientific) and final blocking by 10% BSA (BP1600-100, Fisher Scientific) for 15 min. Slides were washed again thrice with TBS and then stained with PE-Cyanine7-anti-huCD19 monoclonal antibody (clone HIB19, 302216, TONBO; 1:100 dilution), Super Bright 600-anti-huCD3 monoclonal antibody (clone OKT3, 63003741, eBioscience; 1:100 dilution), BV510-anti-huIgM monoclonal antibody (clone MHM-88, 314521, BioLegend; 1:100 dilution), BV421-anti-huIgD monoclonal antibody (clone HB-7, 348225, BioLegend; 1:100 dilution) or APC-anti-huIgA monoclonal antibody (clone IS11-8E10, 130-113-427, Miltenyi Biotec; 1:100 dilution) for 2 h in a dark, moist chamber (Supplementary Table 13). After washing thrice with 0.1% Triton X-100 (T9284, Sigma-Aldrich) in TBS, slides were air-dried, and coverslips were mounted using ProLong Gold Antifade Reagent with DAPI (P36935, Thermo Fisher Scientific). To detect human and mouse TECs, THX and huNBSGW mice thymi were snap frozen in Tissue-Tek O.C.T. Compound (45583, Sakura), sectioned by cryostat, loaded onto positively charged slides, fixed in cold acetone and stained with PE-anti-huEpCAM (ab237397, Abcam; 1:100 dilution) and PE-Cyanine7-anti-moEpCAM (118216, BioLegend; 1:100 dilution) monoclonal antibodies for 2 h at 25 °C in a moist chamber. Cover slips were then mounted on slides using ProLong Gold Antifade Reagent with DAPI. Fluorescent images were captured using a Zeiss Imager-V.1 (ZEN Microscopy Software v3.9, 20x objective).
Intestinal microbiota
Microbial DNA was extracted from feces of non-intentionally immunized THX, huNBSGW and NBSGW mice (22 weeks old) using Quick-DNA Fecal/Soil Microbe Microprep Kit (Zymo Research) according to the manufacturer’s instructions. To analyze gut bacterial microbiome composition, microbial DNA was tagged and sequenced using the Illumina MiSeq platform. Briefly, the V3–V4 hypervariable region of the bacteria 16S rRNA gene was amplified by PCR using tagged bact-341F primer 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3′, bact-850R primer 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3′ and Phusion high-fidelity DNA polymerase (M0530S, New England BioLabs). Multiplexing indices and Illumina sequencing adaptors were then added to the amplicons by limited-cycle amplification using the Nextera XT Index Kit (Illumina). Libraries were normalized, pooled and sequenced using the Illumina MiSeq platform. Sequencing and quality assessment were performed by The University of Texas Health Science Center at San Antonio Genome Sequencing Facility. Bacterial taxonomy was assigned using the Ribosomal Database Project (RDP) classifier v2.14 (http://rdp.cme.msu.edu/classifier/). Principle component analysis of gut bacterial composition in THX, huNBSGW and NBSGW mice was performed by ClustVis v1.0 (biit.cs.ut.ee/clustvis/), which uses clustering algorithms to construct plots visualizing similarities and/or differences between groups of samples.
Lupus THX mice, human autoantibodies, immunopathology and mortality
Lupus THX mice were generated by i.p. injection of 11 male and female THX mice (18 weeks old), constructed by huCD34+ cell engraftment of 6 NBSGW (2 males and 4 females) mice and 5 NSGW41 (2 males and 3 females) mice once with pristane (2,6,10,14-tetramethylpentadecane, P2870, Millipore Sigma, 500 μl) and continuing E2 treatment (Supplementary Table 17). Healthy THX controls (18-week-old) were constructed by huCD34+ cell engraftment of 8 NBSGW and 4 NSGW41 mice. Three additional healthy THX controls (18 weeks old) constructed by huCD34+ cell engraftment of NBSGW mice were used for ex vivo immune cell analyses and immunopathology control experiments and staining.
To measure total human immunoglobulin levels or specific human antibodies, sera from Lupus THX and control THX mice (injected with 500 µl PBS) were collected 6 weeks after pristane or PBS injection, serially twofold diluted then incubated at room temperature in 96-well plates coated with pre-adsorbed goat anti-huIgM antibody (1 µg ml−1), goat anti-huIgG antibody (1 µg ml−1), goat anti-huIgA antibody (1 µg ml−1), dsDNA (15632011, Thermo Fisher Scientific, 10 µg ml−1), histone (16736, Cayman Chemicals, 1.0 µg ml−1), Sm/RNP (A11600, Surmodics, 1.0 µg ml−1) or mouse liver RNA (10 µg ml−1). Total human antibody concentrations or specific human autoantibody titers were measured by specific ELISAs, as described in ‘ASCs and titration of human antibodies’.
To detect human antinuclear antibodies, sera from Lupus THX and healthy control THX mice, collected at 6 weeks after pristane injection, were serially diluted (from 1:50 to 1:400) in PBS and incubated on Hep-2 cell-coated slides (ANK-120, MBL-BION). Bound huIgGs were detected with FITC-anti-huIgG monoclonal antibody (clone G18-145, 555786, BD Pharmingen). Analysis of SHM/CSR, huB/huT cell clonality and intraclonal diversification in Lupus THX mice (6 weeks after pristane injection) was performed, as described in ‘huBCR IgM+ B cell and huTCR repertoires and huIgM+ B and T cell clonality’ and ‘huB cell SHM/CSR, clonality and intraclonal diversification’, in Lupus THX mice euthanized when showing obvious signs of disease and the three ‘additional’ healthy controls at corresponding ages (THX mice). To detect kidney huIgG deposition, Lupus THX and THX mice kidneys were processed for H&E and immunofluorescence staining then imaged as described in ‘H&E, immunohistochemistry and immunofluorescence microscopy’. Mortality of Lupus THX mice and THX mice was analyzed and depicted by Kaplan–Meier survival plots (GraphPad Prism v10.0.3).
Mouse IACUC and human Institutional Review Board protocols
Buffy coats were obtained upon informed consent from healthy donors, per the protocol of the South Texas Blood and Tissue Center. Human umbilical cord blood was collected from full-term, normally developed male and female newborns from healthy puerperae at the Department of Obstetrics and Gynecology, The University of Texas Long School of Medicine, The University of Texas Health Science Center at San Antonio, and obtained upon informed consent, per Institutional Review Board Protocol 17-653H. All experiments involving mice were performed in compliance with the animal protocol approved by The University of Texas Health Science Center at San Antonio Institutional Animal Care and Use Committee (IACUC protocol 20200019AR).
Sample size, randomization and statistical analysis
The exact sample size of all experiments is reported in the figure legends. In each experiment, at least five mice per group (except for the experiment of Fig. 1g) were used to ensure proper biological replicates. Sample size calculations were performed using power analysis, which accounts for effect size, standard deviation, type 1 error and 80% power in a two-sample t-test with a 5% significance level (two-sided test). G power software version 3.1.9.7 was used for these calculations. To construct humanized mice, immunodeficient mice from one litter were grafted with huCD34+ cells from the same donor. In those cases, in which litter sizes were small, multiple litters were combined and grafted with the same donor huCD34+ cells, and pups cross-fostered by a single nursing mother.
Replication: biological replicates were used in all experiments.
Randomization: After matching for sex and age, THX, huNBSGW, huNSG and JAX NSG huCD34 mice were randomly assigned to appropriate groups.
Statistical analyses: statistical analyses were performed using Excel v16.83 (Microsoft) or GraphPad Prism v10.0.3. Differences in antibody concentrations, cell proportions or numbers and RNA transcript expression were analyzed by two-sided Student’s unpaired t-test. Differences in mouse survival were analyzed by log-rank (Mantel–Cox) test.
Experimenters were blinded to group allocation for both data collection and analysis whenever possible. Experimenters were not blinded to group allocation during experimental sample collection.
Generally, THX and huNBSGW mice used in all experiments displayed up to 96.1% and 89.3% huCD45+ cells, respectively, in circulating blood. Generally, 2–3% of the constructed THX and huNBSGW mice at age 20–24 weeks displayed less than 90% and 88% huCD45+ cells in circulating blood, respectively, and were excluded from the study. Around 60% of huNSG and JAX NSG huCD34 mice displayed at peak approximately 45% and 20% huCD45+ cells, respectively, in circulating blood. huNSG and JAX NSG huCD34 mice displaying lower proportions of peak circulating blood human CD45+ cells were excluded from study. No data were excluded from analysis in vivo and in vitro.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Data availability
MiSeq amplicon sequencing data have been deposited in NCBI’s Sequence Read Archive under the BioProject code PRJNA1047643. Source data are provided with this paper. All other data supporting the findings of this study are present in the article and Supplementary Information.
References
Zschaler, J., Schlorke, D. & Arnhold, J. Differences in innate immune response between man and mouse. Crit. Rev. Immunol. 34, 433–454 (2014).
Allen, T. M. et al. Humanized immune system mouse models: progress, challenges and opportunities. Nat. Immunol. 20, 770–774 (2019).
Shultz, L. D. et al. Humanized mouse models of immunological diseases and precision medicine. Mamm. Genome 30, 123–142 (2019).
Stripecke, R. et al. Innovations, challenges, and minimal information for standardization of humanized mice. EMBO Mol. Med. 12, e8662 (2020).
Martinov, T. et al. Building the next generation of humanized hemato-lymphoid system mice. Front. Immunol. 12, 643852 (2021).
Ye, W. & Chen, Q. Potential applications and perspectives of humanized mouse models. Annu. Rev. Anim. Biosci. 10, 395–417 (2022).
Ito, M. et al. NOD/SCID/γcnull mouse: an excellent recipient mouse model for engraftment of human cells. Blood 100, 3175–3182 (2002).
Shultz, L. D. et al. Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2Rγnull mice engrafted with mobilized human hemopoietic stem cells. J. Immunol. 174, 6477–6489 (2005).
Takenaka, K. et al. Polymorphism in Sirpa modulates engraftment of human hematopoietic stem cells. Nat. Immunol. 8, 1313–1323 (2007).
Yu, H. et al. A novel humanized mouse model with significant improvement of class-switched, antigen-specific antibody production. Blood 129, 959–969 (2017).
Cosgun, K. N. et al. Kit regulates HSC engraftment across the human-mouse species barrier. Cell Stem Cell 15, 227–238 (2014).
McIntosh, B. E. et al. Nonirradiated NOD,B6.SCID Il2rγ-/- KitW41/W41 (NBSGW) mice support multilineage engraftment of human hematopoietic cells. Stem Cell Rep. 4, 171–180 (2015).
Nakada, D. et al. Oestrogen increases haematopoietic stem-cell self-renewal in females and during pregnancy. Nature 505, 555–558 (2014).
Kovats, S. Estrogen receptors regulate innate immune cells and signaling pathways. Cell Immunol. 294, 63–69 (2015).
Kumar, R. S. & Goyal, N. Estrogens as regulator of hematopoietic stem cell, immune cells and bone biology. Life Sci. 269, 119091 (2021).
Grimaldi, C. M., Cleary, J., Dagtas, A. S., Moussai, D. & Diamond, B. Estrogen alters thresholds for B cell apoptosis and activation. J. Clin. Invest. 109, 1625–1633 (2002).
Venkatesh, J., Peeva, E., Xu, X. & Diamond, B. Cutting edge: hormonal milieu, not antigenic specificity, determines the mature phenotype of autoreactive B cells. J. Immunol. 176, 3311–3314 (2006).
Cohen-Solal, J. F. et al. Hormonal regulation of B-cell function and systemic lupus erythematosus. Lupus 17, 528–532 (2008).
Hill, L., Jeganathan, V., Chinnasamy, P., Grimaldi, C. & Diamond, B. Differential roles of estrogen receptors α and β in control of B-cell maturation and selection. Mol. Med 17, 211–220 (2011).
Khan, D. & Ansar Ahmed, S. The immune system is a natural target for estrogen action: opposing effects of estrogen in two prototypical autoimmune diseases. Front. Immunol. 6, 635 (2016).
Klein, S. L. & Flanagan, K. L. Sex differences in immune responses. Nat. Rev. Immunol. 16, 626–638 (2016).
Moulton, V. R. Sex hormones in acquired immunity and autoimmune disease. Front. Immunol. 9, 2279 (2018).
Graham, J. H., Yoachim, S. D. & Gould, K. A. Estrogen receptor alpha signaling is responsible for the female sex bias in the loss of tolerance and immune cell activation induced by the lupus susceptibility locus Sle1b. Front Immunol. 11, 582214 (2020).
Fananas-Baquero, S. et al. Natural estrogens enhance the engraftment of human hematopoietic stem and progenitor cells in immunodeficient mice. Haematologica 106, 1659–1670 (2021).
Chakraborty, B. et al. Estrogen receptor signaling in the immune system. Endocr. Rev. 44, 117–141 (2023).
Hoffmann, J. P., Liu, J. A., Seddu, K. & Klein, S. L. Sex hormone signaling and regulation of immune function. Immunity 56, 2472–2491 (2023).
Park, S. R. et al. HoxC4 binds to the promoter of the cytidine deaminase AID gene to induce AID expression, class-switch DNA recombination and somatic hypermutation. Nat. Immunol. 10, 540–550 (2009).
Pauklin, S., Sernandez, I. V., Bachmann, G., Ramiro, A. R. & Petersen-Mahrt, S. K. Estrogen directly activates AID transcription and function. J. Exp. Med. 206, 99–111 (2009).
Mai, T. et al. Estrogen receptors bind to and activate the HOXC4/HoxC4 promoter to potentiate HoxC4-mediated activation-induced cytosine deaminase induction, immunoglobulin class switch DNA recombination, and somatic hypermutation. J. Biol. Chem. 285, 37797–37810 (2010).
Casali, P. et al. Estrogen reverses HDAC inhibitor-mediated repression of Aicda and class-switching in antibody and autoantibody responses by downregulation of miR-26a. Front. Immunol. 11, 491 (2020).
Gualdron-Lopez, M. et al. Multiparameter flow cytometry analysis of the human spleen applied to studies of plasma-derived EVs from Plasmodium vivax patients. Front. Cell Infect. Microbiol. 11, 596104 (2021).
Lefranc, M. P. Immunoglobulin and T cell receptor genes: IMGTI and the birth and rise of immunoinformatics. Front. Immunol. 5, 22 (2014).
Kubinak, J. L. & Round, J. L. Do antibodies select a healthy microbiota? Nat. Rev. Immunol. 16, 767–774 (2016).
King, C. H. et al. Baseline human gut microbiota profile in healthy people and standard reporting template. PLoS ONE 14, e0206484 (2019).
Moroney, J. B., Vasudev, A., Pertsemlidis, A., Zan, H. & Casali, P. Integrative transcriptome and chromatin landscape analysis reveals distinct epigenetic regulations in human memory B cells. Nat. Commun. 11, 5435 (2020).
Chang, B. & Casali, P. The CDR1 sequences of a major proportion of human germline Ig VH genes are inherently susceptible to amino acid replacement. Immunol. Today 15, 367–373 (1994).
Pone, E. J. et al. BCR-signalling synergizes with TLR-signalling for induction of AID and immunoglobulin class-switching through the non-canonical NF-κB pathway. Nat. Commun. 3, 767 (2012).
Sanchez, H. N. et al. B cell-intrinsic epigenetic modulation of antibody responses by dietary fiber-derived short-chain fatty acids. Nat. Commun. 11, 60 (2020).
Rivera, C. E. et al. Intrinsic B cell TLR-BCR linked coengagement induces class-switched, hypermutated, neutralizing antibody responses in absence of T cells. Sci. Adv. 9, eade8928 (2023).
Takaba, H. & Takayanagi, H. The mechanisms of T cell selection in the thymus. Trends Immunol. 38, 805–816 (2017).
Wang, H. X. et al. Thymic epithelial cells contribute to thymopoiesis and T cell development. Front. Immunol. 10, 3099 (2019).
Castaneda, J. et al. The multifaceted roles of B cells in the thymus: from immune tolerance to autoimmunity. Front. Immunol. 12, 766698 (2021).
Richard, M. L. & Gilkeson, G. Mouse models of lupus: what they tell us and what they don’t. Lupus Sci. Med 5, e000199 (2018).
Li, Y. et al. A human immune system mouse model with robust lymph node development. Nat. Methods 15, 623–630 (2018).
Flanagan, K. L., Fink, A. L., Plebanski, M. & Klein, S. L. Sex and gender differences in the outcomes of vaccination over the life course. Annu. Rev. Cell Dev. Biol. 33, 577–599 (2017).
Fischinger, S., Boudreau, C. M., Butler, A. L., Streeck, H. & Alter, G. Sex differences in vaccine-induced humoral immunity. Semin. Immunopathol. 41, 239–249 (2019).
Wilkinson, N. M., Chen, H. C., Lechner, M. G. & Su, M. A. Sex differences in immunity. Annu. Rev. Immunol. 40, 75–94 (2022).
Notta, F., Doulatov, S. & Dick, J. E. Engraftment of human hematopoietic stem cells is more efficient in female NOD/SCID/IL-2Rgc-null recipients. Blood 115, 3704–3707 (2010).
Monteiro, C. et al. Human pregnancy levels of estrogen and progesterone contribute to humoral immunity by activating TFH/B cell axis. Eur. J. Immunol. 51, 167–179 (2021).
Weill, J. C., Weller, S. & Reynaud, C. A. Human marginal zone B cells. Annu. Rev. Immunol. 27, 267–285 (2009).
Jones, B. G. et al. Binding of estrogen receptors to switch sites and regulatory elements in the immunoglobulin heavy chain locus of activated B cells suggests a direct influence of estrogen on antibody expression. Mol. Immunol. 77, 97–102 (2016).
Aguilar-Pimentel, J. A. et al. Increased estrogen to androgen ratio enhances immunoglobulin levels and impairs B cell function in male mice. Sci. Rep. 10, 18334 (2020).
Pauklin, S. & Petersen-Mahrt, S. K. Progesterone inhibits activation-induced deaminase by binding to the promoter. J. Immunol. 183, 1238–1244 (2009).
Hall, O. J. & Klein, S. L. Progesterone-based compounds affect immune responses and susceptibility to infections at diverse mucosal sites. Mucosal Immunol. 10, 1097–1107 (2017).
Hall, O. J. et al. Progesterone-based contraceptives reduce adaptive immune responses and protection against sequential influenza A virus infections. J. Virol. 91, e02160-16 (2017).
Jalkanen, P. et al. COVID-19 mRNA vaccine induced antibody responses against three SARS-CoV-2 variants. Nat. Commun. 12, 3991 (2021).
Sheikh-Mohamed, S. et al. Systemic and mucosal IgA responses are variably induced in response to SARS-CoV-2 mRNA vaccination and are associated with protection against subsequent infection. Mucosal Immunol. 15, 799–808 (2022).
Mastroeni, P. & Rossi, O. Antibodies and protection in systemic Salmonella infections: do we still have more questions than answers? Infect. Immun. 88, e00219–e00220 (2020).
Fraley, E. R. et al. Effects of prior infection with SARS-CoV-2 on B cell receptor repertoire response during vaccination. Vaccines 10, 1477 (2022).
Turner, J. S. et al. SARS-CoV-2 mRNA vaccines induce persistent human germinal centre responses. Nature 596, 109–113 (2021).
Wang, Z. et al. mRNA vaccine-elicited antibodies to SARS-CoV-2 and circulating variants. Nature 592, 616–622 (2021).
Yang, J. et al. A vaccine targeting the RBD of the S protein of SARS-CoV-2 induces protective immunity. Nature 586, 572–577 (2020).
Dai, L. & Gao, G. F. Viral targets for vaccines against COVID-19. Nat. Rev. Immunol. 21, 73–82 (2021).
Sidney, L. E., Branch, M. J., Dunphy, S. E., Dua, H. S. & Hopkinson, A. Concise review: evidence for CD34 as a common marker for diverse progenitors. Stem Cells 32, 1380–1389 (2014).
Boisson-Vidal, C., Benslimane-Ahmim, Z., Lokajczyk, A., Heymann, D. & Smadja, D. M. Osteoprotegerin induces CD34+ differentiation in endothelial progenitor cells. Front. Med. 5, 331 (2018).
Hassanpour, M., Salybekov, A. A., Kobayashi, S. & Asahara, T. CD34 positive cells as endothelial progenitor cells in biology and medicine. Front. Cell Dev. Biol. 11, 1128134 (2023).
Lee, H., Kim, H., Chung, Y., Kim, J. & Yang, H. Thymocyte differentiation is regulated by a change in estradiol levels during the estrous cycle in mouse. Dev. Reprod. 17, 441–449 (2013).
Vincent, F. B., Saulep-Easton, D., Figgett, W. A., Fairfax, K. A. & Mackay, F. The BAFF/APRIL system: emerging functions beyond B cell biology and autoimmunity. Cytokine Growth Factor Rev. 24, 203–215 (2013).
Kuley, R. et al. B cell activating factor (BAFF) from neutrophils and dendritic cells is required for protective B cell responses against Salmonella Typhimurium infection. PLoS ONE 16, e0259158 (2021).
Lang, J. et al. Replacing mouse BAFF with human BAFF does not improve B-cell maturation in hematopoietic humanized mice. Blood Adv. 1, 2729–2741 (2017).
Crotty, S. T follicular helper cell biology: a decade of discovery and diseases. Immunity 50, 1132–1148 (2019).
Mintz, M. A. & Cyster, J. G. T follicular helper cells in germinal center B cell selection and lymphomagenesis. Immunol. Rev. 296, 48–61 (2020).
Park, H. J., Park, H. S., Lee, J. U., Bothwell, A. L. & Choi, J. M. Gender-specific differences in PPARγ regulation of follicular helper T cell responses with estrogen. Sci. Rep. 6, 28495 (2016).
Cyster, J. G. & Allen, C. D. C. B cell responses: cell interaction dynamics and decisions. Cell 177, 524–540 (2019).
Bhan, A. et al. Histone methyltransferase EZH2 is transcriptionally induced by estradiol as well as estrogenic endocrine disruptors bisphenol-A and diethylstilbestrol. J. Mol. Biol. 426, 3426–3441 (2014).
Zheng, Y. et al. Human neutrophil development and functionality are enabled in a humanized mouse model. Proc. Natl Acad. Sci. USA 119, e2121077119 (2022).
Dupuis, M. et al. Effects of estrogens on platelets and megakaryocytes. Int. J. Mol. Sci. 20, 3111 (2019).
Xu, Y., Zhou, H., Post, G., Zan, H. & Casali, P. Rad52 mediates class-switch DNA recombination to IgD. Nat. Commun. 13, 980 (2022).
Tabor, D. E. & Gould, K. A. Estrogen receptor alpha promotes lupus in (NZBxNZW)F1 mice in a B cell intrinsic manner. Clin. Immunol. 174, 41–52 (2017).
Soldin, O. P. et al. Steroid hormone levels in pregnancy and 1 year postpartum using isotope dilution tandem mass spectrometry. Fertil. Steril. 84, 701–710 (2005).
Stricker, R. et al. Establishment of detailed reference values for luteinizing hormone, follicle stimulating hormone, estradiol, and progesterone during different phases of the menstrual cycle on the Abbott ARCHITECT analyzer. Clin. Chem. Lab. Med. 44, 883–887 (2006).
Sluss, P. M. et al. Mass spectrometric and physiological validation of a sensitive, automated, direct immunoassay for serum estradiol using the Architect. Clin. Chim. Acta 388, 99–105 (2008).
Ingberg, E., Theodorsson, A., Theodorsson, E. & Strom, J. O. Methods for long-term 17β-estradiol administration to mice. Gen. Comp. Endocrinol. 175, 188–193 (2012).
Zenclussen, M. L., Casalis, P. A., Jensen, F., Woidacki, K. & Zenclussen, A. C. Hormonal fluctuations during the estrous cycle modulate heme oxygenase-1 expression in the uterus. Front. Endocrinol. 5, 32 (2014).
Verdonk, S. J. E. et al. Estradiol reference intervals in women during the menstrual cycle, postmenopausal women and men using an LC-MS/MS method. Clin. Chim. Acta 495, 198–204 (2019).
Varghese, M. et al. Sex hormones regulate metainflammation in diet-induced obesity in mice. J. Biol. Chem. 297, 101229 (2021).
Ueki, Y. et al. Clonal analysis of a human antibody response. Quantitation of precursors of antibody-producing cells and generation and characterization of monoclonal IgM, IgG, and IgA to rabies virus. J. Exp. Med. 171, 19–34 (1990).
Ikematsu, H., Harindranath, N., Ueki, Y., Notkins, A. L. & Casali, P. Clonal analysis of a human antibody response. II. Sequences of the VH genes of human IgM, IgG, and IgA to rabies virus reveal preferential utilization of VHIII segments and somatic hypermutation. J. Immunol. 150, 1325–1337 (1993).
Kasaian, M. T., Ikematsu, H., Balow, J. E. & Casali, P. Structure of the VH and VL segments of monoreactive and polyreactive IgA autoantibodies to DNA in patients with systemic lupus erythematosus. J. Immunol. 152, 3137–3151 (1994).
Ikematsu, H., Ichiyoshi, Y., Schettino, E. W., Nakamura, M. & Casali, P. VH and VL segment structure of anti-insulin IgG autoantibodies in patients with insulin-dependent diabetes mellitus. Evidence for somatic selection. J. Immunol. 152, 1430–1441 (1994).
Ichiyoshi, Y. & Casali, P. Analysis of the structural correlates for antibody polyreactivity by multiple reassortments of chimeric human immunoglobulin heavy and light chain V segments. J. Exp. Med. 180, 885–895 (1994).
Lefranc, M. P. Antibody Informatics: IMGT, the International ImMunoGeneTics Information System. Microbiol. Spectr. https://doi.org/10.1128/microbiolspec.AID-0001-2012 (2014).
Grainger, D. J. et al. The serum concentration of active transforming growth factor-β is severely depressed in advanced atherosclerosis. Nat. Med. 1, 74–79 (1995).
Koyama, T. et al. Raised serum APRIL levels in patients with systemic lupus erythematosus. Ann. Rheum. Dis. 64, 1065–1067 (2005).
Kim, H. O., Kim, H. S., Youn, J. C., Shin, E. C. & Park, S. Serum cytokine profiles in healthy young and elderly population assessed using multiplexed bead-based immunoassays. J. Transl. Med 9, 113 (2011).
Poorbaugh, J. et al. Measurement of IL-21 in human serum and plasma using ultrasensitive MSD S-PLEX(R) and Quanterix SiMoA methodologies. J. Immunol. Methods 466, 9–16 (2019).
Han, H. et al. Profiling serum cytokines in COVID-19 patients reveals IL-6 and IL-10 are disease severity predictors. Emerg. Microbes Infect. 9, 1123–1130 (2020).
Eslami, M. et al. BAFF 60-mer, and differential BAFF 60-mer dissociating activities in human serum, cord blood and cerebrospinal fluid. Front. Cell Dev. Biol. 8, 577662 (2020).
Acknowledgements
Paolo Casali is Professor Emeritus of Medicine, Molecular Biology & Biochemistry, University of California, Irvine, CA 92697, USA. We thank B. Lopez, A. Bible and O. Acosta (Department of Obstetrics & Gynecology, The University of Texas Long School of Medicine and University Hospital) for their invaluable help in collecting human umbilical cord blood. We thank T. Elhashim and B. P. Kandel (The University of Texas Long School of Medicine) for their help with cord blood huCD34+ cell purification and grafting of NBSGW mice. We thank S. McKeel, H. Etlinger and A. O’Shea (The University of Texas Health Science Center at San Antonio COVID-19 Vaccine Clinic) for coordinating collection of residual Pfizer COVID-19 mRNA vaccine. We thank A. V. Griffith and lab members for help in thymus analysis. We thank Z. Lai (The University of Texas Health Science Center at San Antonio Genome Sequencing Facility) for high-throughput sequencing, C. -M. Wang (The University of Texas Health Science Center at San Antonio Bioanalytics and Single-Cell Core Facility) for CyTOF and human cytokine analysis, and The University of Texas Long School of Medicine Histology and Immunohistochemistry Laboratory for H&E staining and immunohistochemistry. This work was supported by National Institutes of Health (NIH) grants R01 AI105813, R01 AI167416 and T32 AI138944, and Lupus Research Alliance grant 641363 to P.C. C.E.R. was supported by PHS grant K12 GM111726 San Antonio Biomedical Education and Research-Institutional Research and Academic Career Development Award (SABER-IRACDA). Y.Z. was recipient of a fellowship from the Department of Oncology, Xiangya Hospital, Xiangya School of Medicine, Central South University, China. The University of Texas Health Science Center at San Antonio Genome Sequencing Facility/Bioinformatics is supported by NIH-NCI P30 CA054174 (May MD Anderson Cancer Center, The University of Texas Health Science Center at San Antonio), NIH Shared Instrument S10 grant 1S10OD021805-01, CPRIT Core Facility Award RP160732 and NIH IIMS/CTSA grant UL1 TR002645. The University of Texas Health Science Center at San Antonio Flow Cytometry core is supported by NIH-NCI P30 CA054174-20, IIMS/CTSA grant UL1 TR001120 and Texas CPRIT Core Facility Award RP210126. The University of Texas Health Science Center at San Antonio Bioanalytic and Single-Cell Core is supported by Texas CPRIT Core Facility Award RP150600. The University of Texas MD Anderson Cancer Center Recombinant Antibody Production Core is supported by Texas CPRIT Core Facility Award RP190507.
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D.P.C. constructed THX mice, contributed to design and performance of experiments, analysis of primary data, creation of figures and reviewed the manuscript. C.E.R. constructed THX mice, contributed to performance of experiments, analysis of data, creation of figures, tables and writing. Y.Z. constructed THX mice, contributed to performance of experiments, analysis of primary data, creation of figures and writing. Y.X. performed select experiments and created related figures. P.S.R. coordinated the collection of human umbilical cord blood. Z.X. helped design select experiments. H.Z. helped design select experiments, analyzed related data and crafted related figures. P.C. conceptualized and designed this study, coordinated all work, planned experiments, analyzed primary data, edited figures and tables, wrote the manuscript and secured funds for performance of the work. All authors reviewed and approved the manuscript.
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Nature Immunology thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editor: N. Bernard, in collaboration with the Nature Immunology team.
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Extended data
Extended Data Fig. 1 Serum 17β-estradiol concentrations in humanized mice.
Serum estradiol concentrations (pg ml−1) in non-intentionally immunized female (n = 12) and male (n = 12) THX mice and non-intentionally immunized (non-E2-treated) female (n = 12) and male (n = 12) huNBSGW mice measured by specific ELISA (Cayman Chemical estradiol platform). Each dot in histograms depicts E2 concentration from an individual mouse and the bar depicts the mean with s.e.m. Estradiol concentrations in female and male THX mice were comparable and significantly greater than in huNBSGW mice (P < 0.0001, two-sided Student’s unpaired t-test). The normal blood estradiol concentration in mice can vary depending on factors such as age, sex and stage of the estrous cycle in females. In female C57BL/6 mice, blood estradiol concentration range is as follows: Proestrus (the stage just before estrus), 5–60 pg ml−1; Estrus (the stage when ovulation occurs), 15–200 pg ml−1; Metestrus (the stage just after estrus), 5–50 pg ml−1; Diestrus (the stage between metestrus and proestrus), 5–40 pg ml−1. In male mice, blood estradiol concentrations are lower ( < 5.0 pg ml−1). In women, blood estradiol concentration range is as follows: Follicular phase (days 1–14 of the menstrual cycle), 35–400 pg ml−1; Mid-cycle (around day 14 of the menstrual cycle), 100–500 pg ml−1; Luteal phase (days 14–28 of the menstrual cycle), 35–400 pg ml−1; Postmenopausal women, less than 10–30 pg ml−1. In pregnant women, blood estradiol concentration range is as follows: First trimester, 300–3,000 pg ml−1; Second trimester, 1,900–10,000 pg ml−1; Third trimester, 2,000–14,000 pg ml−1. In men, blood estradiol concentration range is 10–30 pg ml−1. It is important to note that estradiol concentration ranges may vary depending on the laboratory that performs the test and the assay used for measurement. Blood estradiol concentration ranges reported here were derived from multiple sources80,81,82,83,84,85,86.
Extended Data Fig. 2 THX mice huCD45+ cell reconstitution and THX mice but not huNBSGW mice develop Peyer’s patches, containing huB cells, huMZ B cells, huGC B cells, huMBCs, huPBs/PCs and huT cells.
a, Identification of circulating huCD45+ PBMCs in non-intentionally immunized THX mice (n = 7) by flow cytometry. huCD45+ cells account for 92−96% of total (human plus mouse) CD45+ cells in blood of THX mice. b, THX (n = 6 of the 7 as in Fig. 4g) and huNBSGW (n = 6 of the 7 as in Fig. 4g) mice were injected i.p. with DNP-CpG (50 μg in 100 µl PBS) at day 0, boosted on day 14 and euthanized on day 28. (Top left row) Peyer’s patches in THX mice and lack thereof in huNBSGW mice. (Top right row) huCD3+CD4—CD8—, huCD3+CD4+, huCD3+CD8+, huCD3+CD4+CD8+ T cells and huCD3+CD4+CXCR5+PD-1+ TFH cells. (Middle row) MZ huCD19+IgM+IgD+CD27+ B cells (20.4% ± 0.7% huB cells), huIgM+ and huIgG+ B cells, GC huCD19+CD38+CD27—IgG+ and GC huCD19+CD38+CD27—IgA+ B cells, class-switched memory huCD27+huIgD— B cells, huCD19+CD27+CD38+CD138+ PBs and huCD19—CD27+CD38+CD138+ PCs in Peyer’s patches of DNP-CpG-immunized THX and huNBSGW mice. Due to the extreme paucity of cells in barely detectable Peyer’s patches of huNBSGW mice, not enough events could be collected for a meaningful analysis. Flow cytometry plots are from one THX and one huNBSGW mouse, each representative of 6 mice. huCD45+ cells were pre-gated in all FACS analyses. Captions on top of FACS plots indicate pre-gating markers. (Bottom row) Quantification of huMZ B cells, class-switched huB cells, huGC B cells, huMBCs and huPBs/PCs in Peyer’s patches of THX and huNBSGW mice. Each dot represents an individual mouse, the bar depicts the mean with s.e.m. Statistical significance was assessed by two-sided Student’s unpaired t-test (NS, not significant; ***P < 0.001).
Extended Data Fig. 3 Gut microbiome composition in NBSGW, huNBSGW and THX mice.
Left, bacterial families identified in gut microbiome of non-intentionally immunized (non-huHSC-grafted, non-E2-conditioned) NBSGW (n = 6), (huHSC-grafted, non-E2-conditioned) huNBSGW (n = 6) and (huHSC-grafted, E2-conditioned) THX mice (n = 6 including the 3 mice as in Fig. 2a–f) by high-throughput 16 s rRNA gene MiSeq amplicon sequencing. In histograms, different colors denote different bacterial families, depicted as stacked columns. Each column depicts microbiome composition in an individual mouse. THX, huNBSGW and NBSGW mice developed distinct gut bacterial microbiomes (THX, 8; huNBSGW, 7-8; NBSGW, 6 families). THX mice gut was colonized by Lactobacillaceae, Lachnospiraceae, Erysipelotrichaceae and Clostridiaceae bacterial families (phylum: Firmicutes), Muribaculaceae (Bacteroidetes), Akkermansiaceae (Verrucomicrobia) and Enterobacteriaceae (Pseudomonadota). NBSGW mice gut harbored predominately (up to virtually 80%) Rikenellaceae (Bacteroidetes), which are characteristic of mouse gut microbiome and were not found in THX mice. Rikenellaceae contributed moderately to gut microbiome of 3 out of 6 huNBSGW mice, suggesting a human pseudo-normalization of the mouse microbiome by human immune system elements’ development and differentiation. Disappearance of Rikenellaceae in THX mice suggested an important impact of E2 conditioning on further ‘humanization’ of these mice gut microbiome. Right, principal component analysis (PCA) of gut bacterial composition in the same non-intentionally immunized NBSGW (blue), huNBSGW (green) and THX (red) mice. Each dot depicts an individual mouse; colors denote NBSGW, huNBSGW and THX mice. THX and huNBSGW mice, both hosting bacterial families contributing to gut microbiota in healthy humans, fully segregate from NBSGW mice, which host predominately ‘murine’ Rikenellaceae.
Extended Data Fig. 4 NP16-CGG-immunized THX mice mount a T-dependent class-switched antibody response to NP entailing select B cell oligoclonal expansion and SHM-mediated intraclonal diversification.
a, b, Spleen huB cell huV3DJH-Cγ and huV3DJH-Cα1 transcripts in NP16-CGG-immunized THX mice (n = 3, same mice as in Fig. 4d–f) were analyzed for SHM, B cell clonal expansion and intraclonal diversification. (a) In the SHM pie charts, slices depict proportions of transcripts carrying given numbers of point-mutations; slice gray gradients depict increasing numbers of point-mutations; the overall mutation frequency (change/base) is listed below each pie chart. Spectrum of point-mutations depicted as donut charts. Means of total, S and R huV3 mutation frequencies in FR1, CDR1, FR2, CDR2 and FR3 of recombined huV3DJH-Cγ and huV3DJH-Cα1 transcripts depicted as histograms. (b) huV3DJH-Cγ1 and huV3DJH-Cα1 huB cell clones and intraclonal diversification, as depicted by TreeMaps and phylogenetic trees. Individual rectangle or square (unique color) area reflects huB cell clone size. In THX 406, 407, 408 and 409 mice, the 3 largest huV3DJH-Cγ1 huB cell clones accounted for 3.5%, 6.9%, 8.3% and 4.6% of huV3DJH-Cγ1 huB cells, while the 3 largest huV3DJH-Cα1 huB cell clones accounted for 22.6%, 31.2% and 12.5% of huV3DJH-Cα1 huB cells in THX 406, 407 and 408 mice. Select huIgG+B cell clones expressed V3 with V3-30 overutilization (over 24% of V3DJH-Cγ1 transcripts). Intraclonal diversification is depicted for each of the three largest clones as a genealogical tree constructed based on shared and unique point-mutations in recombined huV3DJH-Cγ1 and huV3DJH-Cα1 transcripts.
Extended Data Fig. 5 DNP-CpG-immunized THX mice mount a T-independent class-switched antibody response to DNP entailing select B cell oligoclonal expansion and SHM-mediated intraclonal diversification.
a, Spleen huB cell huVHDJH-Cγ transcripts in a DNP-CpG-immunized THX mouse (n = 1, THX 425 as in Fig. 4g) were analyzed for SHM. Left, huVH mutation frequency (change/base) in recombined huVHDJH-Cγ transcripts, as depicted by scatter plots. Each dot depicts a single sequence and bar depicts mean with s.e.m. Middle, in the SHM pie charts, slices depict proportions of transcripts carrying given numbers of point-mutations; slice gray gradients depict increasing numbers of point-mutations; the overall mutation frequency (change/base) is listed below each pie chart. Spectrum of point-mutations depicted as donut charts. Right, means of total, S and R huVH, huV1, huV3, huV4 mutation frequencies in FR1, CDR1, FR2, CDR2 and FR3 of recombined huVHDJH-Cγ transcripts depicted as histograms. b, huVHDJH-Cμ and huVHDJH-Cγ huB cell clones and intraclonal diversification in a non-intentionally immunized THX mouse (n = 1, THX 437) and DNP-CpG-immunized THX mouse 425 as in (a), as depicted by TreeMaps and phylogenetic trees. Individual rectangle or square (unique color) area reflects huB cell clone size. In the DNP-CpG-immunized mouse (THX 425), the 3 largest huV1DJH-Cμ, huV3DJH-Cμ and huV4DJH-Cμ huB cell clones accounted for 7.2%, 7.7% and 4.5% of huV1DJH-Cμ, huV3DJH-Cμ and huV4DJH-Cμ huB cells, while only accounting for 2.1%, 1.4% and 1.4% of similar huB cells in the non-intentionally immunized THX mouse (THX 437). In the same DNP-CpG-immunized THX mouse, the 3 largest huV1DJH-Cγ, huV3DJH-Cγ and huV4DJH-Cγ huB cell clones accounted for 22.3%, 16.8% and 29.4% of huV1DJH-Cγ, huV3DJH-Cγ and huV4DJH-Cγ huB cells.
Extended Data Fig. 6 THX mice huB cells undergo CSR and differentiation as efficiently as huB cells from adult humans.
a–c, Naïve huIgM+IgD+ B cells isolated from blood of healthy humans (n = 3, HS 14, 15, 16) and spleens of non-intentionally immunized THX mice (n = 3, THX 442, 443, 444) were cultured for 120 h upon stimulation with: (a) CD154 (3 U/ml), huIL-2 (100 ng/ml), huIL-4 (20 ng/ml) and huIL-21 (50 ng/ml), (b) CpG ODN2395 (2.5 mg/ml), huIL-2, huIL-21, TGF-β (4 ng/ml) and retinoic acid (4 ng/ml), or (c) CpG ODN2395, huIL-2, huIL-4 and huIL-21. Identification of huIgM+, huIgD+, huIgG+, huIgA+ or huIgE+ B cells, huCD27+IgD– class-switched memory-like B cells (huMB) and huCD27+CD38+ PBs by flow cytometry. huCD45+CD19+ cells were pre-gated in all FACS analyses. d, AICDA, PRDM1, VHDJH-Cμ, VHDJH-Cγ1, VHDJH-Cα1 and VHDJH-Cε transcript expression in HS and THX mice huB cell microcultures (n = 3 biological replicates for each different microculture), as measured by qPCR and normalized to HPRT1 expression (2−ΔCt method). In histograms, each dot represents transcript expression from one human or one THX mouse huB cell microculture and the bar depicts the mean with s.e.m. Statistical significance (d) was assessed by two-sided Student’s unpaired t-test (NS, not significant).
Extended Data Fig. 7 Flagellin-vaccinated THX mice mount a class-switched antibody response to S. Typhimurium entailing select B cell oligoclonal expansion and SHM-mediated intraclonal diversification.
a–e, THX mice (n = 3, THX 450, 451, 452, same mice as in Fig. 6g, h) were injected i.p. with S. Typhimurium flagellin on day 0 (50 μg in 100 μl alum), boosted on day 14 (50 μg in 100 μl PBS) and euthanized on day 28. (a) Flagellin-specific huCD19+ B cells, huIgG+ B cells, huIgA+ B cells and class-switched memory huCD19+huCD27+ B cells in flagellin-vaccinated THX mice spleen and blood of a healthy human, as identified by binding of AF647-labeled flagellin (AF647 alone as negative control); identification of huCD19+CD138+ PBs and huCD19—CD138+ PCs among pre-gated huCD27+CD38+ cells. huCD45+ cells were pre-gated in all FACS analyses. b–e, Spleen huB cell huVHDJH-CH transcripts analyzed for CDR3 length, R:S mutations, huB cell clonal size and diversity, mutation frequency and evolution of a huB cell clone. (b) CDR3 length distribution in huIgM+, huIgG+ and huIgA+ B cells. Colors denote different antibody isotypes; color gradients denote different THX mice. (c) Means of total, S and R huVH mutation frequencies in FR1, CDR1, FR2, CDR2 and FR3 of recombined huVHDJH-CH transcripts depicted as histograms. Data are from one THX mouse representative of 3 THX mice. (d) huVHDJH-Cμ, huVHDJH-Cγ and huVHDJH-Cα1 huB cell clonal size and diversity in flagellin-vaccinated THX mice (n = 3, THX 450, 451, 452) depicted as scatter plots. Bars depict the mean with s.e.m. (e) Left, point-mutation frequency (change/base) in huB cell huVHDJH-CH transcripts of flagellin-vaccinated THX mice (n = 2, THX 450, 451) depicted as scatter plots. Each dot depicts a single sequence and the bar depicts the mean with s.e.m. Right, evolutive lineage of a huB cell clone that underwent SHM and CSR in a flagellin-vaccinated THX mouse (n = 1, THX 450). The root represents the rearranged, unmutated and unswitched recombined huV3-33D3-10J4-Cμ gene sequence of the huB cell progenitor and the leaves represent somatically hypermutated or class-switched and somatically hypermutated huB cell sub-mutants. Nodes represent huB cell sub-mutants that emerged during the clonal evolutionary process.
Extended Data Fig. 8 Serum human cytokine concentrations in flagellin- and COVID-19 mRNA-vaccinated THX mice.
Serum huAPRIL, huBAFF, huTGF-β1, huIFN-γ, huIL-2, huIL-4, huIL-6, huIL-10 and huIL-21 (pg ml−1) in flagellin-vaccinated (n = 16) and Pfizer COVID-19 mRNA-vaccinated (n = 24) THX mice measured by Luminex Human Discovery Assay 8-plex or TGF-β Premixed Magnetic Luminex Performance Assay (R&D Systems Luminex Platform). In the histograms, each dot represents human cytokine concentration from an individual mouse and the bar depicts the mean with s.e.m. No significant difference was found in huAPRIL, huTGF-β1, huIFN-γ, huIL-2, huIL-6, huIL-10 and huIL-21 concentrations between flagellin-vaccinated and COVID-19 mRNA-vaccinated THX mice. huBAFF and huIL-4 concentrations were significantly greater in flagellin-vaccinated than in COVID-19 mRNA-vaccinated THX mice (P = 0.0134 and P = 0.0229, two-sided Student’s unpaired t-test). Flagellin-vaccinated THX mice showed blood incretion of huAPRIL, huBAFF, huTGF-β, huIFN-γ, huIL-2, huIL-4, huIL-6, huIL-10 and huIL-21 (205 ± 15.20, 231 ± 16.50, 7351 ± 794, 5.03 ± 1.94, 0.91 ± 0.16, 6.29 ± 0.45, 3.11 ± 1.30, 1.85 ± 0.30 and < 0.1 pg ml−1) at human physiological concentrations. COVID-19 mRNA-vaccinated THX mice also showed blood incretion of huAPRIL, huBAFF, huTGF-β, huIFN-γ, huIL-2, huIL-4, huIL-6, huIL-10 and huIL-21 (164 ± 14.45, 172 ± 14.86, 7627 ± 610, 18.00 ± 7.66, 0.84 ± 0.12, 4.43 ± 0.60, 3.06 ± 1.31, 1.89 ± 0.45 and < 0.1 pg ml−1, mean ± s.e.m.) at human physiological concentrations. In healthy adult humans, the approximate concentrations (range) of circulating cytokines are as follows: huAPRIL, 100–400 pg ml−1; huBAFF, 50–400 pg ml−1; huTGF-β1, 1000–10,000 pg ml−1; huIFN-γ, 0.1–4.2 pg ml−1; huIL-2, 0.1–2.0 pg ml−1; huIL-4, 0.5–4.0 pg ml−1; huIL-6, 0.1–5.0 pg ml−1; huIL-10, 0.1–2.8 pg ml−1; huIL-21, < 0.1 pg ml−1. In healthy humans, circulating huIL-21 is below 100 fg ml−1, a concentration below the detection limit of Luminex Human Discovery Assay 8-plex. It is important to note that human cytokine concentration ranges may vary depending on the type of assay used for measurement. The human cytokine concentration ranges reported here were derived from multiple sources93,94,95,96,97,98.
Extended Data Fig. 9 THX mice vaccinated with Pfizer-BioNTech 162b2 COVID-19 mRNA mount a class-switched and somatically hypermutated antibody response to SARS-CoV-2 Spike S1 RBD.
a, Spleen huB cell huVHDJH-Cμ, huVHDJH-Cγ and huVHDJH-Cα1 transcripts in COVID-19 mRNA-vaccinated THX mice (n=3, same mice as in Fig. 7e, f) were analyzed for CDR3 length, R:S mutations and huB cell clonal size. Left, CDR3 length distribution in recombined huVHDJH-CH transcripts. Colors denote different antibody isotypes; color gradients denote different THX mice. Middle, huVHDJH-Cμ, huVHDJH-Cγ and huVHDJH-Cα1 huB cell clonal size depicted as scatter plots. Bars depict the mean with s.e.m. Right, means of total, S and R VH mutation frequencies in FR1, CDR1, FR2, CDR2 and FR3 depicted as histograms. R:S data are from one THX mouse representative of 3 THX mice. b–c, Spleen RBD-specific huB cell huVHDJH-CH, huVκJκ and huVλJλ transcripts in 3 additional COVID-19 mRNA-vaccinated THX mice were reverse transcribed and amplified by RT-PCR. Paired huVHDJH and huVκJκ or huVλJλ gene segments from 100 single cells were used to make recombinant human monoclonal antibodies. (b) Left, huVH, huVκ or huVλ gene family member expression in 100 recombinant human monoclonal antibodies, as depicted by pie charts. Colors depict different huVH, huVκ or huVλ gene families; color gradients denote individual gene family members. The 100 human monoclonal antibodies showed predominant utilization of V3, V4 and V1, together with Vκ3, Vκ1 and Vκ2 as well as Vλ1 and Vλ2 genes. Middle, mutation frequency (change/base) of recombined huIgH VHDJH and huIg VκJκ or VλJλ regions in recombinant human monoclonal antibodies, as depicted by scatter plots. Each dot represents a single sequence and the bar depicts the mean with s.e.m. Right, CDR3 length distribution in paired VHDJH and human immunoglobulin VκJκ or VλJλ human monoclonal antibodies. huIgH CDR3 lengths varied between 5 and 25 amino acids, peaking at 12, 13 and 15 amino acids; those of huVκ and huVλ varied between 5 and 13 amino acids, peaking at 9 amino acids. c, Forty-five expressed recombinant human monoclonal antibodies were selected for analysis of paired huIgH and huIgL genes based on their highest RBD-binding activity (> 1.0 OD by specific ELISA). Shown are 27 huIgM (blue), 5 huIgG (red) and 13 huIgA (green) monoclonal antibodies. Consistent with the higher haploid representation of V3, V4 and V1 gene families, V3 genes were the most frequently utilized (35 human monoclonal antibodies), particularly V3-23 (10 human monoclonal antibodies), V3-30 (8), V3-9 (4) and V3-7 (3), followed by V4 (7) and V1 (3) genes. Twenty-two human monoclonal antibodies utilized JH4, 9 JH6 and 6 JH3, with JH1, JH5 and JH2 accounting for the remaining 8 human monoclonal antibodies. Thirty-five human monoclonal antibodies utilized Vκ genes, with Vκ3-11 as the most frequently utilized (8 human monoclonal antibodies) followed by Vκ1-44 (4) and Vκ4-1 (4). Ten human monoclonal antibodies utilized Vλ genes, with Vλ1-44 and Vλ2-14 (2 and 2 human monoclonal antibodies) as the most frequently utilized.
Extended Data Fig. 10 RBD-KLH-vaccinated THX mice mount a class-switched and somatically hypermutated antibody response to SARS-CoV-2 Spike S1 RBD.
a, b, THX mice were injected i.p. with RBD-KLH (100 μg in 100 μl alum) or nil (100 μl alum) on day 0, boosted (100 μg in 100 µl PBS or 100 μl PBS alone) on day 21 and euthanized on day 28. Total serum human immunoglobulin and RBD-specific huIgM, huIgG and huIgA antibodies in RBD-KLH-immunized (n=6) and non-immunized (n=6) THX mice measured by specific ELISAs (total human immunoglobulin concentrations expressed as μg eq ml−1 and RBD-specific human antibody titers as OD readings at different dilutions or RUs). In the histograms, each dot represents an individual mouse and the bar depicts the mean with s.e.m. Statistical significance was assessed by two-sided Student’s unpaired t-test (NS, not significant; **P<0.01, ***P<0.001). c–f, Spleen huB cell huVHDJH-CH transcripts in RBD-KLH-immunized THX mice (n=3 of the 6 as in a–b, THX 488, 490, 492) were analyzed for CDR3 length, clonal expansion and intraclonal diversification. (c) CDR3 length distribution in huVHDJH-CH transcripts. Colors denote different antibody isotypes; color gradients denote different THX mice. (d) huVH mutation frequency (change/base) in huVHDJH-Cγ (2.7+0.08x10-2, mean+s.e.m.) transcripts depicted as scatter plots (left) and pie charts (middle). Each dot represents a single sequence and the bar depicts the mean with s.e.m. Right, means of total, S and R huVH mutation frequencies in FR1, CDR1, FR2, CDR2 and FR3 of huVHDJH-Cμ and huVHDJH-Cγ transcripts depicted as histograms. R:S data are from one THX mouse representative of 3 THX mice. (e) huVHDJH-Cμ and huVHDJH-Cγ huB cell clonal size and diversity in RBD-KLH-immunized THX mice (THX 488, 490, 492) depicted as scatter plots. Bars depict the mean with s.e.m. (f) huVHDJH-Cμ and huVHDJH-Cγ huB cell clones and intraclonal diversification, as depicted by TreeMaps and phylogenetic trees. Individual rectangle or square (unique color) area reflects huB cell clone size. In THX mice 488, 490 and 492, the 20 largest huVHDJH-Cμ and huVHDJH-Cγ huB cell clones accounted for about one-tenth of huVHDJH-Cμ huB cells and one-fourth of huVHDJH-Cγ huB cells.
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Chupp, D.P., Rivera, C.E., Zhou, Y. et al. A humanized mouse that mounts mature class-switched, hypermutated and neutralizing antibody responses. Nat Immunol 25, 1489–1506 (2024). https://doi.org/10.1038/s41590-024-01880-3
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DOI: https://doi.org/10.1038/s41590-024-01880-3
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