Skip to main content

Advertisement

SpringerLink
Log in

IL-12 induces a B cell-intrinsic IL-12/IFNγ feed-forward loop promoting extrafollicular B cell responses

  • Article
  • Published:

From Nature Immunology

View current issue Submit your manuscript

Abstract

While some infections elicit germinal centers, others produce only extrafollicular responses. The mechanisms controlling these dichotomous fates are poorly understood. We identify IL-12 as a cytokine switch, acting directly on B cells to promote extrafollicular and suppress germinal center responses. IL-12 initiates a B cell-intrinsic feed-forward loop between IL-12 and IFNγ, amplifying IFNγ production, which promotes proliferation and plasmablast differentiation from mouse and human B cells, in synergy with IL-12. IL-12 sustains the expression of a portion of IFNγ-inducible genes. Together, they also induce unique gene changes, reflecting both IFNγ amplification and cooperative effects between both cytokines. In vivo, cells lacking both IL-12 and IFNγ receptors are more impaired in plasmablast production than those lacking either receptor alone. Further, B cell-derived IL-12 enhances both plasmablast responses and T helper 1 cell commitment. Thus, B cell-derived IL-12, acting on T and B cells, determines the immune response mode, with implications for vaccines, pathogen protection and autoimmunity.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1: IL-12 signaling on either B cells or CD4 T cells is sufficient to suppress GCs.
Fig. 2: IL-12 signaling in B cells is necessary and sufficient to promote PB differentiation.
Fig. 3: IL-12 promotes both proliferation and differentiation of purified B cells in vitro.
Fig. 4: IL-12-induced production of IFNγ by B cells promotes PB differentiation via autocrine signaling.
Fig. 5: Effects of IFNγ responsiveness and T-bet expression on PB responses in vivo and in vitro.
Fig. 6: IL-12 and IFNγ synergize to induce proliferation and differentiation at low concentrations, dependent on autocrine cytokine secretion.
Fig. 7: IL-12 and IFNγ induce distinct changes in gene expression in cultured B cells.
Fig. 8: IL-12 secreted from B cells influences both B and T cell differentiation in vivo, synergizing with IFNγ signals.

Similar content being viewed by others

Data availability

All RNA-seq data are deposited in the NCBI’s Gene Expression Omnibus database (GEO) with accession ID GSE231931. Source data are provided with this paper.

References

  1. Elsner, R. A. & Shlomchik, M. J. Germinal center and extrafollicular B cell responses in vaccination, immunity, and autoimmunity. Immunity 53, 1136–1150 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Weisel, F. & Shlomchik, M. Memory B cells of mice and humans. Annu. Rev. Immunol. 35, 255–284 (2017).

    Article  CAS  PubMed  Google Scholar 

  3. Liu, X., Yao, J., Zhao, Y., Wang, J. & Qi, H. Heterogeneous plasma cells and long-lived subsets in response to immunization, autoantigen and microbiota. Nat. Immunol. 23, 1564–1576 (2022).

    Article  CAS  PubMed  Google Scholar 

  4. Trivedi, N. et al. Liver is a generative site for the B cell response to Ehrlichia muris. Immunity 51, 1088–1101.e5 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. William, J., Euler, C., Christensen, S. & Shlomchik, M. J. Evolution of autoantibody responses via somatic hypermutation outside of germinal centers. Science 297, 2066–2070 (2002).

    Article  CAS  PubMed  Google Scholar 

  6. Di Niro, R. et al. Salmonella infection drives promiscuous B cell activation followed by extrafollicular affinity maturation. Immunity 43, 120–131 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  7. Vaidehi Narayanan, H. & Hoffmann, A. From antibody repertoires to cell-cell interactions to molecular networks: bridging scales in the germinal center. Front. Immunol. 13, 898078 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  8. Victora, G. D. & Nussenzweig, M. C. Germinal centers. Annu. Rev. Immunol. 40, 413–442 (2022).

    Article  CAS  PubMed  Google Scholar 

  9. Cunningham, A. F. et al. Salmonella induces a switched antibody response without germinal centers that impedes the extracellular spread of infection. J. Immunol. 178, 6200–6207 (2007).

    Article  CAS  PubMed  Google Scholar 

  10. Kaneko, N. et al. Loss of Bcl-6-expressing T follicular helper cells and germinal centers in COVID-19. cell 183, 143–157.e13 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Racine, R. et al. Impaired germinal center responses and suppression of local IgG production during intracellular bacterial infection. J. Immunol. 184, 5085–5093 (2010).

    Article  CAS  PubMed  Google Scholar 

  12. Elsner, R. A., Hastey, C. J., Olsen, K. J. & Baumgarth, N. Suppression of long-lived humoral immunity following Borrelia burgdorferi infection. PLoS Pathog. 11, e1004976 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  13. Hastey, C. J., Elsner, R. A., Barthold, S. W. & Baumgarth, N. Delays and diversions mark the development of B cell responses to Borrelia burgdorferi infection. J. Immunol. 188, 5612–5622 (2012).

    Article  CAS  PubMed  Google Scholar 

  14. Jacobson, B. A. et al. Anatomy of autoantibody production: dominant localization of antibody-producing cells to T cell zones in Fas-deficient mice. Immunity 3, 509–519 (1995).

    Article  CAS  PubMed  Google Scholar 

  15. Sang, A. et al. Activation of rheumatoid factor-specific B cells is antigen dependent and occurs preferentially outside of germinal centers in the lupus-prone NZM2410 mouse model. J. Immunol. 193, 1609–1621 (2014).

    Article  CAS  PubMed  Google Scholar 

  16. Jenks, S. A. et al. Distinct effector B cells induced by unregulated Toll-like receptor 7 contribute to pathogenic responses in systemic lupus erythematosus. Immunity 49, 725–739.e6 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Jenks, S. A., Cashman, K. S., Woodruff, M. C., Lee, F. E. & Sanz, I. Extrafollicular responses in humans and SLE. Immunol. Rev. 288, 136–148 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Hoehn, K. B. et al. Cutting edge: distinct B cell repertoires characterize patients with mild and severe COVID-19. J. Immunol. 206, 2785–2790 (2021).

    Article  CAS  PubMed  Google Scholar 

  19. Elsner, R. A. & Shlomchik, M. J. IL-12 blocks Tfh cell differentiation during Salmonella infection, thereby contributing to germinal center suppression. Cell Rep. 29, 2796–2809.e5 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Baumgarth, N. How specific is too specific? B-cell responses to viral infections reveal the importance of breadth over depth. Immunol. Rev. 255, 82–94 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  21. Cirelli, K. M. & Crotty, S. Germinal center enhancement by extended antigen availability. Curr. Opin. Immunol. 47, 64–69 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Lee, J. L. & Linterman, M. A. Mechanisms underpinning poor antibody responses to vaccines in ageing. Immunol. Lett. 241, 1–14 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Schijns, V. et al. Modulation of immune responses using adjuvants to facilitate therapeutic vaccination. Immunol. Rev. 296, 169–190 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Cancro, M. P. & Tomayko, M. M. Memory B cells and plasma cells: the differentiative continuum of humoral immunity. Immunol. Rev. 303, 72–82 (2021).

    Article  CAS  PubMed  Google Scholar 

  25. Barnas, J. L., Looney, R. J. & Anolik, J. H. B cell targeted therapies in autoimmune disease. Curr. Opin. Immunol. 61, 92–99 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Woodruff, M. C. et al. Extrafollicular B cell responses correlate with neutralizing antibodies and morbidity in COVID-19. Nat. Immunol. 21, 1506–1516 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  27. Pillai, S. Suboptimal humoral immunity in severe acute respiratory syndrome coronavirus 2 infection and viral variant generation. Clin. Lab. Med. 42, 75–84 (2022).

    Article  PubMed  Google Scholar 

  28. Kim, S. J. et al. Increased IL-12 inhibits B cells’ differentiation to germinal center cells and promotes differentiation to short-lived plasmablasts. J. Exp. Med. 205, 2437–2448 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Dubois, B. et al. Critical role of IL-12 in dendritic cell-induced differentiation of naive B lymphocytes. J. Immunol. 161, 2223–2231 (1998).

    Article  CAS  PubMed  Google Scholar 

  30. Durali, D. et al. In human B cells, IL-12 triggers a cascade of molecular events similar to Th1 commitment. Blood 102, 4084–4089 (2003).

    Article  CAS  PubMed  Google Scholar 

  31. Liu, N., Ohnishi, N., Ni, L., Akira, S. & Bacon, K. B. CpG directly induces T-bet expression and inhibits IgG1 and IgE switching in B cells. Nat. Immunol. 4, 687–693 (2003).

    Article  CAS  PubMed  Google Scholar 

  32. Harris, D. P. et al. Reciprocal regulation of polarized cytokine production by effector B and T cells. Nat. Immunol. 1, 475–482 (2000).

    Article  CAS  PubMed  Google Scholar 

  33. Vogel, L. A. et al. Direct binding of IL-12 to human and murine B lymphocytes. Int. Immunol. 8, 1955–1962 (1996).

    Article  CAS  PubMed  Google Scholar 

  34. Airoldi, I. et al. Expression and function of IL-12 and IL-18 receptors on human tonsillar B cells. J. Immunol. 165, 6880–6888 (2000).

    Article  CAS  PubMed  Google Scholar 

  35. Szabo, S. J. et al. A novel transcription factor, T-bet, directs Th1 lineage commitment. Cell 100, 655–669 (2000).

    Article  CAS  PubMed  Google Scholar 

  36. Lund, F. E. & Randall, T. D. Effector and regulatory B cells: modulators of CD4+ T cell immunity. Nat. Rev. Immunol. 10, 236–247 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Lehmann, J. et al. IL-12p40-dependent agonistic effects on the development of protective innate and adaptive immunity against Salmonella enteritidis. J. Immunol. 167, 5304–5315 (2001).

    Article  CAS  PubMed  Google Scholar 

  38. Tait Wojno, E. D., Hunter, C. A. & Stumhofer, J. S. The immunobiology of the interleukin-12 family: room for discovery. Immunity 50, 851–870 (2019).

    Article  CAS  PubMed  Google Scholar 

  39. Hasbold, J., Lyons, A. B., Kehry, M. R. & Hodgkin, P. D. Cell division number regulates IgG1 and IgE switching of B cells following stimulation by CD40 ligand and IL-4. Eur. J. Immunol. 28, 1040–1051 (1998).

    Article  CAS  PubMed  Google Scholar 

  40. Hodgkin, P. D., Lee, J. H. & Lyons, A. B. B cell differentiation and isotype switching is related to division cycle number. J. Exp. Med. 184, 277–281 (1996).

    Article  CAS  PubMed  Google Scholar 

  41. Tangye, S. G., Ferguson, A., Avery, D. T., Ma, C. S. & Hodgkin, P. D. Isotype switching by human B cells is division-associated and regulated by cytokines. J. Immunol. 169, 4298–4306 (2002).

    Article  CAS  PubMed  Google Scholar 

  42. Nutt, S. L., Hodgkin, P. D., Tarlinton, D. M. & Corcoran, L. M. The generation of antibody-secreting plasma cells. Nat. Rev. Immunol. 15, 160–171 (2015).

    Article  CAS  PubMed  Google Scholar 

  43. Trezise, S. & Nutt, S. L. The gene regulatory network controlling plasma cell function. Immunol. Rev. 303, 23–34 (2021).

    Article  CAS  PubMed  Google Scholar 

  44. Peng, S. L., Szabo, S. J. & Glimcher, L. H. T-bet regulates IgG class switching and pathogenic autoantibody production. Proc. Natl Acad. Sci. USA 99, 5545–5550 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Hess, J., Ladel, C., Miko, D. & Kaufmann, S. H. Salmonella typhimurium aroA- infection in gene-targeted immunodeficient mice: major role of CD4+ TCR-alpha beta cells and IFN-gamma in bacterial clearance independent of intracellular location. J. Immunol. 156, 3321–3326 (1996).

    Article  CAS  PubMed  Google Scholar 

  46. Sobah, M. L., Liongue, C. & Ward, A. C. SOCS proteins in immunity, inflammatory diseases, and immune-related cancer. Front. Med. (Lausanne) 8, 727987 (2021).

    Article  PubMed  Google Scholar 

  47. Oestreich, K. J. & Weinmann, A. S. Transcriptional mechanisms that regulate T helper 1 cell differentiation. Curr. Opin. Immunol. 24, 191–195 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Shaw, L. A. et al. Id2 reinforces TH1 differentiation and inhibits E2A to repress TFH differentiation. Nat. Immunol. 17, 834–843 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Gloury, R. et al. Dynamic changes in Id3 and E-protein activity orchestrate germinal center and plasma cell development. J. Exp. Med. 213, 1095–1111 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Hauser, A. E. et al. Chemotactic responsiveness toward ligands for CXCR3 and CXCR4 is regulated on plasma blasts during the time course of a memory immune response. J. Immunol. 169, 1277–1282 (2002).

    Article  CAS  PubMed  Google Scholar 

  51. Muehlinghaus, G. et al. Regulation of CXCR3 and CXCR4 expression during terminal differentiation of memory B cells into plasma cells. Blood 105, 3965–3971 (2005).

    Article  CAS  PubMed  Google Scholar 

  52. Stone, S. L. et al. T-bet transcription factor promotes antibody-secreting cell differentiation by limiting the inflammatory effects of IFN-γ on B cells. Immunity 50, 1172–1187.e7 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Jackson, S. W. et al. B cell IFN-γ receptor signaling promotes autoimmune germinal centers via cell-intrinsic induction of BCL-6. J. Exp. Med. 213, 733–750 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Afkarian, M. et al. T-bet is a STAT1-induced regulator of IL-12R expression in naive CD4+ T cells. Nat. Immunol. 3, 549–557 (2002).

    Article  CAS  PubMed  Google Scholar 

  55. Usui, T. et al. T-bet regulates Th1 responses through essential effects on GATA-3 function rather than on IFNG gene acetylation and transcription. J. Exp. Med. 203, 755–766 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Kano, S. et al. The contribution of transcription factor IRF1 to the interferon-γ-interleukin 12 signaling axis and TH1 versus TH-17 differentiation of CD4+ T cells. Nat. Immunol. 9, 34–41 (2008).

    Article  CAS  PubMed  Google Scholar 

  57. Kobayashi, M. et al. Identification and purification of natural killer cell stimulatory factor (NKSF), a cytokine with multiple biologic effects on human lymphocytes. J. Exp. Med. 170, 827–845 (1989).

    Article  CAS  PubMed  Google Scholar 

  58. Schulz, E. G., Mariani, L., Radbruch, A. & Hofer, T. Sequential polarization and imprinting of type 1 T helper lymphocytes by interferon-γ and interleukin-12. Immunity 30, 673–683 (2009).

    Article  CAS  PubMed  Google Scholar 

  59. Hua, Z. & Hou, B. The role of B cell antigen presentation in the initiation of CD4+ T cell response. Immunol. Rev. 296, 24–35 (2020).

    Article  CAS  PubMed  Google Scholar 

  60. Sonoda, E. et al. B cell development under the condition of allelic inclusion. Immunity 6, 225–233 (1997).

    Article  CAS  PubMed  Google Scholar 

  61. Hoiseth, S. K. & Stocker, B. A. Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines. Nature 291, 238–239 (1981).

    Article  CAS  PubMed  Google Scholar 

  62. Weisel, N. M. et al. Surface phenotypes of naive and memory B cells in mouse and human tissues. Nat. Immunol. 23, 135–145 (2022).

    Article  CAS  PubMed  Google Scholar 

  63. Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).

    Article  CAS  PubMed  Google Scholar 

  64. Liao, Y., Smyth, G. K. & Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923–930 (2014).

    Article  CAS  PubMed  Google Scholar 

  65. Law, C. W., Chen, Y., Shi, W. & Smyth, G. K. voom: precision weights unlock linear model analysis tools for RNA-seq read counts. Genome Biol. 15, R29 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  66. Ritchie, M. E. et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 43, e47 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank C. Leibler, K. Nickerson, S. Roberts, A. Gocher-Demske and A. Marshak-Rothstein (University of Massachusetts School of Medicine) for helpful discussions and critical reading of the manuscript. We thank L. Conter, S. Joachim, M. Price, N. Weisel, M. Berkey and E. Kunes for technical support. We thank the transplant coordinators at LiveOnNY for tissues from organ donors and members of the laboratory of D. L. Farber at Columbia University for providing access to human organ donor tissue samples. cDNA generation, library preparation and sequencing were performed by the University of Pittsburgh Health Science Sequencing Core at the University of Pittsburgh Medical Center Children’s Hospital of Pittsburgh. This work was supported by NIH grants nos R01-AI105018 and R01-AI043603 (M.J.S.) and no. P01-AI106697 (M.J.S. and D. L. Farber), R.A.E was supported by NIHT32-AI089443 (M.J.S. principal investigator).

Author information

Authors and Affiliations

  1. Department of Immunology, University of Pittsburgh, Pittsburgh, PA, USA

    Rebecca A. Elsner, Shuchi Smita & Mark J. Shlomchik

Authors
  1. Rebecca A. Elsner
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shuchi Smita
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mark J. Shlomchik
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

R.A.E. and M.J.S. conceived the project and designed experiments. R.A.E. performed experiments and analyzed data. S.S. performed bulk RNA-seq analysis. R.A.E. and M.J.S. interpreted data and wrote the manuscript.

Corresponding authors

Correspondence to Rebecca A. Elsner or Mark J. Shlomchik.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Immunology thanks Ignacio Sanz and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Editor: L. A. Dempsey, in collaboration with the Nature Immunology team.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 IL-12 signaling on either B cells or CD4 T cells is sufficient to suppress GC.

a-b, C57Bl/6 mice were infected with STm (Inf, red) or injected with HK bacteria control (Ctl, blue), 3 days later immunized with NP-CGG, and spleens analyzed 12 days later. a, Gating to identify NP-specific (NIP+) germinal center B cells for Fig. 1a, example shown is from HK STm control group. b, Comparison of the percentage of NP-specific B cells that are GC phenotype 12 days post NP-CGG immunization. Data are combined from 2 experiments (n = 3 Ctl WT, n = 9 Ctl KO, n = 7 Inf WT, n = 10 Inf KO), symbols represent individual mice, bars display mean +/- SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, P values calculated by one-way ANOVA with Tukey’s multiple comparisons. c-d, Gating scheme used to identify donor derived B cells and germinal centers for Fig. 1e,f, example shown is from HK STm control group with WT B cell donor. d, Example upstream gating plots for Fig. 1e,f to show gating of donor B cells for CD45.1 and CD45.2 genotypes.

Source data

Extended Data Fig. 2 Example calculation for competitive advantage in mixed bone marrow chimeras.

a, Example gating scheme to identify resting T cells (CD4+ CD44low PD-1-), resting B cells (CD19+ NIP− CD38high CD95low), and plasmablasts (CD138+ CD44high). Example is from chimera treated with HK-STm and NP-CGG as described in Fig. 2. b, Example FACS plots to be used to demonstrate calculations, same as Fig. 2a. Data are summarized below, showing paired comparisons of the CD45.1/CD45.2 ratio for resting T cells or B cells from the same mouse. Data are combined from 2 experiments, n = 8 HK, n = 9 Inf, n = 2 naïve, symbols indicate individual mice, paired two-tailed t-test was used to compare resting T and B cells for each treatment, *p < 0.05, **p < 0.01, ***p < 0.001. c, Example calculation to normalize resting B cell reconstitution on a per mouse basis. Resting B cells show a disadvantage for WT cells (30% WT vs 69% KO) compared to T cells, which show equal competition (48% WT vs 45% KO) after reconstituting irradiated mice with 50%/50% mixture of WT and IL12RKO bone marrow donor cells. Normalization to the resting T ratio gives a value < 1 which indicates KO advantage. d, Example calculation normalizing PB to resting B cells to account for WT disadvantage in reconstitution. When accounting for the WT disadvantage of resting B cell reconstitution (30% WT vs 69% KO), PB show WT advantage (44% WT vs 45% KO) giving a normalized value > 1 (2.23).

Source data

Extended Data Fig. 3 IL-12 promotes both proliferation and differentiation from B cells in vitro.

a, Bead purified IL12p40 KO B cells were cultured for 4 days with LPS + recombinant IL-12 at the indicated concentrations, then analyzed by flow cytometry. Gating scheme used for analysis of proliferation and plasmablast (Blimp-1 + CD138+) differentiation among live B cells in Fig. 3a,b. An example of Blimp-1 and CD138 expression among total B cells is shown for reference, and that gate was applied to all VPD peaks individually with examples shown for cells in division peak 1 and 4. In green is an example of FlowJo Proliferation Platform analysis. b, Extended titration of IL-12 on LPS stimulated cells with analysis at 3 and 4 days without Blimp-1 staining. The frequency of live CD138+ VPDlow (excluding undivided cells) was determined by flow cytometry (left), the number of live cells per well were enumerated using trypan blue stain (center), and the two were used to calculate the number of live CD138+ VPDlow cells per well in each condition. Data are individual mice combined from two experiments for n = 2 for each culture time point, shown are mean +/- SD. P values calculated by mixed-effects analysis with Dunnett’s multiple comparisons. c-e, Bead purified IL12p40 KO B cells were cultured for 4 days with CpG ODN1826 + recombinant IL-12 and analyzed by flow cytometry. Data are individual mice combined from 2 experiments for n = 2, shown are mean +/- SD. c, Example of proliferation dye dilution by histogram overlay, and %Divided and Proliferation index as above, with P value calculated by one-way ANOVA. d, Analysis of differentiation per division number, as described for Fig. 3b, with P value calculated by two-way ANOVA and Dunnett’s multiple comparisons comparing each group to 0 pg/mL IL-12. e, example FACS plots of %Blimp-1 + CD138+ cells in indicated culture conditions. For all charts (b, c, d), NS = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Source data

Extended Data Fig. 4 IL-12 induces B cell production of IFNγ that promotes PB differentiation through autocrine signaling.

a, Comparison of WT and IL12p40 KO B cells for Blimp-1 + CD138+ cell differentiation among proliferation peaks after four days of stimulation in the presence of LPS +/- recombinant IL-12, or IL-12 blocking antibody. Data are combined from 3 experiments for a total of n = 4 per genotype, symbols indicate the mean +/- SD. Statistical analyses are two-way ANOVA. b-e, Four-day LPS stimulation of bead purified B cells comparing dose titrations of recombinant IL-12 and IFNγ on both IL12p40 KO and IFNγ KO B cells. Data are combined from 3 experiments for n = 3 per genotype. All symbols (c,d) represent mean +/− SD. b, Representative FACS plots of proliferation dye dilution between groups. c, Precursor frequency of cells that have divided at least once (%Divided), and d, the average number of divisions of dividing cells (Proliferation index) as calculated by FlowJo proliferation analysis. Statistical analysis (c,d) are two-way ANOVA with Dunnett’s multiple comparison comparing each concentration to controls of 0 pg/mL IL-12 or 0 ng/mL IFNγ and comparing IL12p40 KO cells to IFNγ KO cells. e, Analysis of Blimp-1 + CD138+ cell differentiation among divisions comparing titration of IL-12 (left) or IFNγ (right) on IL12p40 KO (square symbols solid lines) or IFNγ KO (triangle symbols dotted lines) B cells, colors indicate concentrations of indicated cytokines added to cultures. Statistical analysis is two-way ANOVA with Tukey’s multiple comparison. For all charts (a, c, d, e), NS = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Source data

Extended Data Fig. 5 IL-12 and IFNγ promote proliferation and differentiation of human B cells.

a-c, Primary B cells purified from spleens of n = 7 individual human organ donors were cultured for 8 days with CpG +/- cytokines or blocking antibodies as indicated. a, Flow cytometry plots demonstrate upstream gating for one donor example. b, Histograms comparing proliferation of treatment groups for each individual donor. c, Analysis of %PB (CD38hi IRF4hi) per division for each individual donor (n = 1 each). Statistical comparisons are multiple paired one-tailed t-tests each compared to block/block (gray line), NS = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Source data

Extended Data Fig. 6 B cell expression of IFNγ receptor, but not T-bet, is necessary for maximal PB response during Salmonella infection.

a-d, Additional data described in Fig. 5a, data combined from two experiments, n = 11 HK B-WT, n = 12 Inf B-WT, n = 13 HK B-KO and Inf B-KO. a, NP-specific IgG1 and IgG2b antibody forming cells (AFC), and the sum of IgM+IgG1 + IgG2b + IgG2c. Symbols represent individual mice and bars the mean +/−SD, P values calculated by two-tailed t-tests. b, Example gating for IFNγ producing T and B cells (summarized in Fig. 5c). For CD4 + T cells, examples are shown from both infected and HK control mice as different gates for CD44+ cells were necessary. c, Example upstream gating for intracellular IFNgR stain, CD19+ and/or CD138+ gate represents B cells + PB, which is then separated in to ‘PB’ or CD138- cells, and then ‘GC’ and the remainder are called ‘non-GC/PB’ which represents a majority naïve cells. d, Histograms showing selection of Cre-escape cells among PB populations from B-KO mice (filled histograms). e, Additional ELISPOT data as described in Fig. 5e. Data combined from 2 experiments, n = 14 (HK B-WT, HK B-KO, Inf B-WT), n = 16 Inf B-KO, symbols represent individual mice and bars the mean +/- SD, all P values calculated by two-tailed t-test. f, Histograms showing T-bet expression among cells gated as in panel (c) with CD19+ and/or CD138+ gate. g, Splenocytes from mice treated for 3 days with recombinant IL-12 in vivo (Fig. 2c), were stained for T-bet expression in B cells. A population of T-bet+ IRF4- cells was found to be expanded in IL-12 treated mice. This population (orange) was overlayed on all B cells (gray) to show their CD38 CD95 staining in relation to CD38low CD95hi early GC B cells. FACS plots are representative of n = 10 mice per group. h, Proliferation index of in vitro cultured CD19-Cre+/− T-bet flox B cells from Fig. 5i, n = 3 biological replicates; symbols are mean +/- SD, analyzed by two-way ANOVA. One of two independent experiments with similar results is shown. i, Representative FACS plots of T-bet staining among CD19-Cre+/− T-bet flox B cells in indicated conditions from Fig. 5i. For all charts (a, e, h), NS = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Source data

Extended Data Fig. 7 Curated list of DEGs induced by IL-12 and IFNγ treatment.

a-d, B cells from n = 3 mice were individually bead purified and stimulated with LPS and the indicated concentrations of IL-12, IFNγ, and blocking antibodies for 4 or 24 hours, and RNA was isolated for bulk sequencing. a, Experimental design; all DEG were compared to IL-12 block + IFNγ block condition for their respective time point. b, Venn diagrams comparing up (top) and down (bottom) DEG for 4 hour IFNγ 0.5 ng/mL versus 3 ng/mL both with IL-12 block. c, Venn diagrams comparing (top left) down DEG for 4 versus 24 hour IFNγ 0.5 ng/mL (block IL-12), (bottom left) those plus 24 hr IL-12 20 pg/mL + IFNγ 0.5 ng/mL, (right) or all 24 hr groups; IL-12 20 pg/mL (block IFNγ), IFNγ 0.5 ng/mL (block IL-12), or IL-12 + IFNγ. d, Heatmap of curated list of top DEG of interest clustered by gene names. Numbers in boxes indicate logFC mean of 3 samples, * indicates significance (FDR < 0.05) of differential regulation compared to control. e, Quantification of IL-12 and IFNγ in culture supernatants from 3 time points. Data are combined from 2 experiments, n = 6 biological replicates per condition. Symbols represent the mean +/- SD. Comparisons are one-way ANOVA with Tukey’s multiple comparisons for suboptimal cytokine concentrations, and two-tailed paired t-test comparing LPS only to either IL-12 or IFNγ at sufficient concentrations (200 pg/mL and 10 ng/mL respectively), NS = not significant, *p < 0.05.

Source data

Extended Data Fig. 8 IL-12 and IFNγ DEG are enriched for TH1, Treg, and B cell proliferation genes.

Gene set enrichment analysis (GSEA) plots illustrating significant upregulation of TH1 Treg, and B cell proliferation related genes at 24 hours of IL-12 and IFNγ samples (n = 3) compared with their control conditions. The FDR adjusted P values (FDR) indicated in each chart were calculated using rankSumTestWithCorrelation function from the R limma package.

Extended Data Fig. 9 In vivo B cell derived IL-12 influences both B and T cells, and synergizes with IFNγ signals for maximal PB differentiation.

a, Representative FACS plots of B cells from mice treated with PBS or recombinant IL-12 as in Fig. 2b, and b, summary data of B cells expressing T-bet and producing IFNγ after IL-12 treatment. Data combined from two experiments, n = 10 per group, analyzed by two-tailed t-test. c-i, Additional data related to experiment described in Fig. 8c. Data combined from two experiments, n = 11 (WT/WT), n = 9 (WT/KO), n = 11 (KO/WT), n = 10 (KO/KO). For e and i, all symbols represent individual mice and bars the mean +/- SD. P values calculated by two-tailed t-tests.c, Gating scheme used to identify donor derived B cells and PB for Fig. 8d. Example shown is from the group IL12RKO T cells + WT B cells. d, Example FACS plots of donor derived B cells (TCRb-, CD19+ or CD138+ from either CD45.1 WT or CD45.2 KO gate), donor genotype is indicated above each chart. e, Additional summary from Fig. 8d showing the number of CD138+ donor B cells per spleen, and the fold change of IRF4 MFI on PB compared to ‘naïve’ (CD138− CD19+) B cells of the same mouse. f, Gating scheme used to analyze CD4 T cells from donor cells, example mouse received WT T cells and WT B cells. g,h, Representative FACS plots of donor derived T cells showing expression of T-bet+ IFNγ + Th1 cells in g, and CXCR5high Bcl6high TFH in h and i, Example FACS plots (h), and summary data (i), of the percentage of TFH among donor T cells per mouse. j, Additional analysis of mixed bone marrow chimera mice described in Fig. 8h. Fold advantage was calculated for GC B cells compared to resting cells from the same mouse as described in Extended Data Fig. 2. Data shown are combined from 2 experiments, (HK: n = 12 for IL-12R, n = 13 for IFNgR and IL-12R+IFNgR, Inf: n = 15 IL-12R, n = 16 IFNgR, n = 14 IL-12R+IFNgR), symbols represent individual mice and bars the mean +/− SD. Individual groups are tested against the hypothesis of no competitive difference by one sample t-test (two-tailed) compared to a hypothetical value of 1, and compared to each other by two-tailed t-tests. k, Visual representation of the proposed model. Early (4 hours) IL-12 initiates IFNγ production and autocrine IFNγ signals induce expression of genes that both establish negative feedback of IFNγ signaling, and an overlapping positive feedback loop between IL-12 and IFNγ signaling. IFNγ promotes IL-12 signaling by upregulating IL-12 signaling components (IL-12R and STAT4) and STAT1 which IFNγ signals through. At 24 hours, if both IL-12 and IFNγ are present, positive feedback overcomes negative regulation to sustain IFNγ signaling. Additionally, IL-12 and IFNγ synergize to induce genes which are not expressed under either condition alone. For all charts (b, e, i, j), NS = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Source data

Extended Data Fig. 10 Hypothetical models of synergy.

a, Experimental rationale for design of mixed bone marrow chimeras in Fig. 8h, with the null hypothesis being no synergy. b, Proposed model of positive feedback loop, summarizing sequencing results. Upper case protein names refer to protein function, lower case gene names refer to RNA sequencing data. ‘PROTEIN (Gene)’ refers to hypothetical regulatory links between transcription factors and target genes based on existing literature.

Supplementary information

Source data

Source Data Fig. 1

Statistical source data.

Source Data Fig. 2

Statistical source data.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

Statistical source data.

Source Data Fig. 5

Statistical source data.

Source Data Fig. 6

Statistical source data.

Source Data Fig. 7

Statistical source data.

Source Data Fig. 8

Statistical source data.

Source Data Extended Data Fig. 1

Statistical source data.

Source Data Extended Data Fig. 2

Statistical source data.

Source Data Extended Data Fig. 3

Statistical source data.

Source Data Extended Data Fig. 4

Statistical source data.

Source Data Extended Data Fig. 5

Statistical source data.

Source Data Extended Data Fig. 6

Statistical source data.

Source Data Extended Data Fig. 7

Statistical source data.

Source Data Extended Data Fig. 9

Statistical source data.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Elsner, R.A., Smita, S. & Shlomchik, M.J. IL-12 induces a B cell-intrinsic IL-12/IFNγ feed-forward loop promoting extrafollicular B cell responses. Nat Immunol 25, 1283–1295 (2024). https://doi.org/10.1038/s41590-024-01858-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41590-024-01858-1

  • Springer Nature America, Inc.

This article is cited by

Search

Navigation