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1 Introduction

During the evolution of life, as the always predominant factor, the Earth’s gravitational field has shaped the architecture of all biological systems decisively. It is therefore not surprising that sudden changes in gravity lead to discrepancies in the normal functions of life and of our immune system [see Chap. 1].

According to current knowledge, residence in microgravity strongly influences the human body and leads to a variety of deconditioning symptoms such as bone demineralization, muscle atrophy, reduced performance of the cardiovascular system, altered neurovestibular perception, and a strong deterioration of the immune system (Moore et al. 1996) [see also Chap. 3]. In brief, astronauts showed immune system depression, reduced activation of T lymphocytes, and reactivation of latent viruses. (Kimzey 1977; Sonnenfeld and Shearer 2002; Stowe et al. 2001; Mehta et al. 2014).

However, the immune system is not only responsible to defend infections, it is also essential for wound healing, tissue reorganization, and repair. Thus, there are serious reasons to believe that astronauts are exposed to high risk by not only being more susceptible to infections, but also by having poor wound healing and tissue repair. As plans emerge for humans to embark on long-term spaceflights to Mars, Moon, and asteroids in the future, the health risks of a defective regulation of the immunity during spaceflight are important to comprehend. Therefore, the understanding of how gravity affects immune cell function is the key to maintain a proper immune system of astronauts. But which cellular and molecular structures may require gravity for proper function and are thus dependent on the gravity on Earth?

Since the pioneering discovery of Cogoli et al. during the first Spacelab-Mission in 1983, it is well known that proliferative response of lymphocytes to mitogenic stimulation is suppressed in microgravity (Cogoli et al. 1984; Cogoli 1996). Follow-up experiments performed to verify these results demonstrated clearly that factors other than microgravity can be excluded to be responsible for the depressed activation of lymphocytes. Whereas the phenomenological effect of reduced activation of T cells in microgravity is well described and verified (Grove et al. 1995), the fundamental molecular mechanisms remain to be discovered.

For more than 30 years, in vitro experiments with isolated T lymphocytes, the key cell type of the adaptive immune system, have been performed using different research platforms that provide real and simulated microgravity. These experiments have confirmed the effects of altered gravity on the cellular level. Thus, isolated lymphocytes prove to be a suitable biological model system for studying whether and how Earth’s gravity is mandatory for cellular and molecular processes in mammalian cells. Numerous experiments in real microgravity of different length have been carried out during manned space missions, on board of orbital and suborbital flights (sounding rockets), and during parabolic flights. Studies using ground-based facilities with the aim to simulate the state of microgravity have supported these experiments in real microgravity. These facilities include fast-rotating clinostats, rotating wall vessel (RWV) bioreactors, random positioning machines (RPMs), and high-aspect ratio vessels (HARVs). As their results are comparable with results of experiments in real microgravity, RWVs and clinostats are recognized and valuable tools for simulating microgravity in suspension cell cultures (Herranz et al. 2013).

These studies in real and simulated microgravity were able to achieve new insights into gravity-sensitive functions in nonactivated and activated T lymphocytes, for example, cell cycle regulation (Thiel et al. 2012), epigenetic regulation (Singh et al. 2010), chromatin modification (Paulsen et al. 2010), differential gene expression (Chang et al. 2012; Thiel et al. 2012), altered microRNA expression profile (Mangala et al. 2011; Girardi et al. 2014; Hughes-Fulford et al. 2015), cell motility (Pellis et al. 1997; Sundaresan et al. 2002), and regulation of programmed cell death (Cubano and Lewis 2000; Lewis et al. 1998; Battista et al. 2012). Also the secretion of cytokines such as interleukin-2 (IL-2) and interferon-gamma (IFNγ) is influenced by gravitational changes (Hashemi et al. 1999; Chapes et al. 1992).

This chapter provides an overview of the results obtained over the last 30 years in in vitro experiments using T lymphocytes performed in space and in ground-based facilities with special emphasis on the used cell culture conditions. These results contribute to our current knowledge of how gravitational changes affect human T lymphocytes in vitro.

1.1 Regulation of T Lymphocytes in Real and Simulated Microgravity Experiments In Vitro

Up to now, several in vitro studies have been carried out in order to investigate effects of gravitational changes on isolated T lymphocytes. Representative studies demonstrating cellular and molecular alterations that have been observed in real and simulated microgravity are summarized in Tables 5.1 and 5.2.

Table 5.1 Summary of experiments performed under real microgravity conditions
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Table 5.2 Summary of experiments performed in ground-based facilities in simulated microgravity
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1.2 T Cell Activation Is Diminished in Microgravity

The first experiments were carried out in terms of studying phenomenological effects of microgravity on isolated T lymphocytes. So far it is well known that microgravity influences T lymphocytes by diminishing the reactivity of T lymphocytes to mitogenic stimulation during spaceflight (Cogoli et al. 1984, 1988; Cogoli and Cogoli-Greuter 1997; Bechler et al. 1986). Experimental studies using the ground-based facilities RWV, clinostat, and RPM yielded the same results (Schwarzenberg et al. 1999; Cooper and Pellis 1998; Hashemi et al. 1999).

Next, the question arose whether binding of the mitogen Concanavalin A (ConA) to the T cell receptor might be changed due to the absence of gravity and therefore leads to reduced T cell activation. But this assumption was disproved in four different experiments on board of sounding rockets (Cogoli-Greuter et al. 1997; Sciola et al. 1999). By means of fluorescently labeled ConA, these experiments showed that binding of the mitogen on T cell receptors was in principal not affected; only a slight delay of patching and capping was observed.

1.3 T Cell Function Remains Unchanged Despite Reduced Cell-Cell and Cell-Substrate Interactions

Another factor that could lead to an inhibited T cell function may be the lack of sedimentation under microgravity conditions. This could lead to reduced cell-cell and cell-substrate interactions which in turn could be responsible for the reduced proliferative response to mitogenic stimuli. However, the cultivation of peripheral blood mononuclear cells (PBMCs) in Teflon bags, which show a reduced cell-substrate interaction, had no effect on the proliferation of phytohemagglutinin (PHA) stimulated cells compared with cultures in standard cell culture flask with existing cell-substrate interactions (Cooper and Pellis 1998).

Furthermore, in experiments with the ground-based facility RWV, the proliferation of T lymphocytes after stimulation of the receptors CD2/CD28 and CD3/CD28 was completely inhibited (Cooper and Pellis 1998). These stimuli activate the cells without required co-stimulatory signals from cell-cell interaction. Moreover, investigations of human PBMCs and Jurkat T cells in real microgravity showed that cell aggregates and thereby cellular interactions occur despite the absence of gravity (Cogoli-Greuter et al. 1996, 1997; Limouse et al. 1991). Therefore, it has been assumed that changes in the signal transduction are responsible for inhibition of T cell function rather than the absence of cell-cell interactions.

1.4 Cytokine Pattern Changes Under Microgravity Condition

In addition to the activation of the T cell receptor complex and the co-receptor CD28, also a third signal via the interleukin-2 receptor (IL-2R) is necessary to fully activate the T cell and therefore to trigger proliferation and differentiation into functional effector T cells. Thus, the reduced functionality of T cells in microgravity might also be due to alterations in cellular IL-2 secretion or IL-2R surface expression, resulting in a disability of the positive regulatory feedback loop.

And in fact, experiments with human PBMCs, which were conducted during several space missions, showed that both the IL-2 secretion and IL-2R expression were greatly reduced in microgravity (Cogoli et al. 1993; Pippia et al. 1996; Hashemi et al. 1999). Additionally, clinostat, RWV, and RPM experiments, where PBMCs and primary human T cells have been exposed to simulated microgravity, confirmed these results (Cooper and Pellis 1998; Hashemi et al. 1999; Risso et al. 2005). Further ground-based experiments demonstrated that already the gene expression of IL-2 and its receptor was inhibited in activated T cells (Walther et al. 1998; Boonyaratanakornkit et al. 2005). However, co-stimulation of the cells with submitogenic concentrations of phorbol-12-myristate-13-acetate (PMA) was able to restore the proliferative response and the expression of IL-2R at the cell’s surface (Cooper and Pellis 1998; Hashemi et al. 1999).

Activated T cells also produce interferon-gamma (IFNγ) which is a major proinflammatory and regulatory cytokine. IFNγ plays an essential role in relevant immunological processes such as inflammatory reactions, cell-mediated immunity, and autoimmunity. Studies concerning the IFNγ secretion demonstrated that stimulation of PBMCs with ConA during the spaceflight resulted in an increased IFNγ secretion compared to 1 g ground controls (Chapes et al. 1992), whereas a PMA/ionomycin stimulation of samples isolated from astronauts immediately after their landing led to a significant reduction of IFNγ secretion of CD4+ T lymphocytes. In CD8+ T lymphocytes, however, these remained unchanged (Crucian et al. 2000). In addition, comparison of whole blood analyses from astronauts of short-term (Space Shuttle) and long-term missions (International Space Station (ISS)) showed that a short-term stay in space led to a reduction in the percentage of IFNγ producing T cells, while after long-term missions the proportion of IFNγ producing T cells remained unchanged (Crucian et al. 2008). Lymphocytes in simulated microgravity provided by RWV exhibited an initial reduction of IFNγ secretion. After 3 days, however, normal levels were restored (Cooper and Pellis 1998).

1.5 Microgravity Affects Cytoskeletal Structures and Cell Motility

The cytoskeleton is an internal filamentous network of different types of cytosolic fibers: actin filaments, microtubules, and intermediate filaments. This cytoskeletal network is responsible for giving a cell its shape and for generating the required forces for cell motility. But also other biological functions such as cell proliferation, survival, and death are influenced by the cytoskeleton. Since the cytoskeleton participates also in transduction of signals from the receptor at the plasma membrane to the nucleus, these cytoskeletal structures play additionally essential roles in maintaining receptor signaling integrity. Thus, further studies set their focus on the cytoskeletal network.

Indeed, several independent experiments performed under real microgravity conditions demonstrated significant changes in the cytoskeletal network. Analyses of T lymphocytes flown aboard of sounding rockets reported altered tubulin and vimentin structures which appeared in thick bundles (Cogoli-Greuter et al. 1997; Sciola et al. 1999). Jurkat T cells flown on board of the space shuttle Atlantis also showed a modified microtubule network (Lewis et al. 1998). The microtubules were coalesced, did not extend to the cell membrane, and the microtubule organizing center was disorganized.

Cell communication and signal transduction processes are not only affected by cell-to-cell interactions, they are also highly influenced by cell motility. Therefore, the cell motility of lymphocytes was observed under microgravity conditions. These studies showed that the cells were motile; however, their motility did not decrease with increasing duration of stimulation (Cogoli-Greuter et al. 1996, 1997). Furthermore, experiments using the ground-based facility RWV exhibited that after 24 h exposure to simulated microgravity, cell motility of PBMCs was inhibited (Pellis et al. 1997; Sundaresan et al. 2002). However, addition of PMA to the cells could restore cell motility (Sundaresan et al. 2002).

1.6 Distribution of Protein Kinase C (PKC) Isoforms to Destined Cellular Fractions Is Dysregulated

Since the cytoskeleton also has an impact on proper functioning of signal transduction substantially, restructuring of the cytoskeleton induced by microgravity could also result in hampered intracellular localization of signaling molecules. Accordingly, different PKC isoforms are associated with several cytoskeletal fibers. Upon T cell activation, under normal circumstances, these PKC isoforms are allocated to destined cellular compartments. In two spaceflight experiments with Jurkat T cells and primary human T cells, it could be shown that the relative distribution of certain PKC isoforms to different cell fractions in the in-flight microgravity samples greatly differed from the 1 g ground controls (Hatton et al. 2002; Schmitt et al. 1996). Further, primary T cells exposed to simulated microgravity in an RWV confirmed these results (Galleri et al. 2002). In addition, another RWV experiment revealed that also mRNA expression and protein expression of specific calcium-independent PKC isoforms in PBMCs were inhibited (Sundaresan et al. 2004).

1.7 Lack of Gravity Increases the Rate of Controlled Cell Death (Apoptosis)

Another reason for the reduced proliferative response of T lymphocytes in microgravity might be the initiation of the intracellular death program called programmed cell death or apoptosis. In fact, it could be shown by means of biochemical and microscopic investigations that Jurkat T cells exposed to microgravity demonstrated an increased rate of apoptosis (Cubano and Lewis 2000; Lewis et al. 1998; Battista et al. 2012). This was reflected in the release of apoptosis-related factors such as Fas/APO1 in the cell culture medium after about 2 days aboard different space shuttle flights in cell culture medium (Cubano and Lewis 2000; Lewis et al. 1998). Moreover, exposure of lymphocytes to microgravity resulted in increased DNA fragmentation, poly (ADP-ribose) polymerase (PARP) protein expression, as well as multiplied p53 and calpain mRNA. These changes were associated with an early increase of 5-lipoxygenase (5-LOX) activity (Battista et al. 2012). During an experiment that we conducted during the 8th DLR (German Aerospace Center) parabolic flight campaign, we observed an increase in p53 phosphorylation after 20s of real microgravity (Paulsen et al. 2010). Experiments in simulated microgravity, however, did not confirm microgravity-induced stimulation of apoptosis but revealed that radiation and activation-induced programmed cell death in T lymphocytes was inhibited (Risin and Pellis 2001).

1.8 Microgravity Does Not Intervene at the Level of Membrane-Proximal Processes of T Cell Receptor Signaling Within the First Minutes

Up to date it has not yet been revealed whether and how gravitational changes affect the T cell signal transduction, in particular the membrane-proximal and cytoplasmic signal transduction cascades as well as the IL-2/IL-2R activation loop. Although some studies suggest that microgravity intervenes at the level of PKC (Hatton et al. 2002; Schmitt et al. 1996), the addition of PMA to cells exposed to simulated microgravity restored T cell activation (Cooper and Pellis 1998; Simons et al. 2009), surface receptor expression (Hashemi et al. 1999), and cell motility (Sundaresan et al. 2002). In addition, the first activation signals binding, patching, and capping of ConA on the T cell receptors proceed normally (Cogoli-Greuter et al. 1997). Therefore, it is believed that the gravity-sensitive cellular targets are located more likely upstream of the PKC and downstream of the T cell receptor/CD3 complex.

Thus, in recent studies, we investigated the effects of altered gravity on several key elements involved in the early T cell signaling (Tauber et al. 2013, 2015). For this purpose, primary human CD4+ T lymphocytes were examined under real microgravity conditions aboard the sounding rocket MASER-12 (Tauber et al. 2013) and during the 19th DLR parabolic flight campaign (Tauber et al. 2015). Subsequently, we carried out experiments with the same experimental set-up in simulated microgravity using a fast-rotating 2D-clinostat (Tauber et al. 2015). We analyzed the impact of gravitational changes on the key molecules of the early T cell signaling events in both resting and ConA/CD28-activated CD4+ T lymphocytes. We quantified following signaling components: T cell receptor, membrane-proximal signal proteins LAT and ZAP-70, MAPK, IL-2R, and histone acetylation. Table 5.3 gives an overview of the obtained results.

Table 5.3 Overview of qualitative changes of selected proteins involved in T cell activation of primary human CD4+ T lymphocytes induced by altered gravity during the sounding rocket flight of MASER-12, during the 19th DLR parabolic flight campaign, and by 2D-clinorotation
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In addition to the microgravity effects, we investigated the influence of hypergravity during the rocket launch and during the climb of the airplane, as well as the influence of the cultivation of the cells in the experimental hardware. The analyses of the protein level after 6 min of real microgravity during the sounding rocket flight showed no obvious effects on the early signal transduction pathway in CD4+ T lymphocytes. Surprisingly, strong effects of the rocket launch could be observed, which often resulted in a significant reduction of the signal molecules. During the parabolic flight experiment, the 20 s hypergravity phase led to a rapid decrease of CD3 and IL-2R surface expression and reduced p-LAT in nonactivated primary T lymphocytes. The subsequent clinostat experiments showed a decreased CD3 surface expression, reduced ZAP-70 abundance, as well as an increased histone H3-acetylation in activated T lymphocytes after 5 min of clinorotation and a transient downregulation of CD3 and, further, a stable downregulation of IL-2R during 60 min of clinorotation.

However, based on these results it can be assumed that gravitational changes do not intervene at the level of the membrane-proximal key proteins within the first 6 min. The initial primary dysregulation of functional T cell activation will probably occur at the level of regulation of gene expression.

1.9 Gravitational Changes Induce Alterations in the Gene Expression Profile

The phenomenological characteristics of reduced T cell activation caused by microgravity are now well described. The exact underlying molecular mechanisms, however, are still unknown. So, during the last decade, several studies focused on the investigation of the impact of altered gravity on gene transcription (Thiel et al. 2012; Chang et al. 2012; Boonyaratanakornkit et al. 2005; Ward et al. 2006; Sundaresan and Pellis 2009; Kumari et al. 2009; Lewis et al. 2001).

The evaluations of genome-wide gene expression analyses of T cells revealed that both in real and simulated microgravity, the expression of very early genes, which are primarily regulated by the transcription factors NF-kB, CREB, ELK, AP-1 and STAT, were downregulated in comparison to 1 g controls (Chang et al. 2012; Boonyaratanakornkit et al. 2005). The observed changes in gene expression induced by altered gravity include a number of genes which are associated with responses to cell stress (Sundaresan and Pellis 2009), cell proliferation and differentiation (Ward et al. 2006; Sundaresan and Pellis 2009; Boonyaratanakornkit et al. 2005), cell cycle regulation (Kumari et al. 2009; Thiel et al. 2012), protein folding (Ward et al. 2006), DNA repair (Kumari et al. 2009), transport and degradation (Ward et al. 2006), apoptosis (Ward et al. 2006; Kumari et al. 2009; Lewis et al. 2001), and the cytoskeleton (Lewis et al. 2001). These results show that modulation of gene expression in reduced gravity covers a wide spectrum.

In an RWV study, alterations in the microRNA (miRNA) profiles of human lymphocytes exposed to simulated microgravity for 1–3 days were observed (Girardi et al. 2014). The examinations identified 42 differentially expressed miRNA whereof the upregulated miR-9-5p, miR-9-3p, and miR-155-5p, and the downregulated miR-150-3p and miR-378a-3p were the most dysregulated ones. Further, miRNA-correlated genes whose expression level was also significantly altered by simulated microgravity were investigated. Thus, several miRNA-mRNA pairs, which are involved in biological processes such as immunity and inflammatory response, cell proliferation, and apoptosis, were determined.

In a most recent study, Millie Hughes-Fulford and her team discovered the suppressed expression of the miRNA miR-21 due to altered gravity after 1.5 h T cell activation during spaceflight (Hughes-Fulford et al. 2015). Furthermore, microarray analysis showed that 85 genes associated with T cell signaling were significantly downregulated under microgravity conditions compared to 1 g in-flight controls. Of these gravity-sensitive genes, 17 were defined as targets of miR-21 whereof 5 genes are biologically confirmed targets and are under normal circumstances upregulated in parallel with miR-21. Therefore, it can be assumed that altered gravity influences T cell activation not only by transcription promotion but also by repressing translation via noncoding RNA mechanisms.

Further experimental studies with primary human T cells disclosed microgravity-induced epigenetic changes in DNA methylation and chromatin histone modifications (Singh et al. 2010). Such epigenetic mechanisms regulate and modify the activation of certain genes and therefore lead to differential expression of mRNA. Experiments that we conducted during several parabolic flight experiments (9th, 10th, and 13th DLR and 45th ESA Parabolic Flight Campaign) have revealed an association between microgravity-induced differentially mRNA expression and altered histone acetylation (Thiel et al. 2012).

2 Humoral Immunity

Apart from the cellular components, the adaptive immune system also includes the humoral immunity [see also Chap. 1]. However, the humoral immunity has not been investigated to that extent of which the cell-mediated immunity has been investigated. Short-term spaceflight has resulted in no change in levels of plasma immunoglobulins (Voss 1984; Stowe et al. 1999; Rykova et al. 2008), whereas, long-term spaceflight led to different results. Studies of cosmonauts during spaceflight have shown that immunoglobulin G (IgG) levels were unchanged, whereas IgA and IgM levels were in some cases increased (Konstantinova et al. 1993). In another study, immunological investigations comparing the preflight with the postflight situation indicated that the total amounts of serum IgA, IgG, and IgM were unchanged after long-term missions (Rykova et al. 2008). Therefore, the humoral immune responses may not be as sensitive to altered gravity as are cell-mediated immune responses.

3 Conclusion

These numerous studies carried out with T lymphocytes in microgravity have clearly shown that already individual cells are sensitive to changes in gravity. In addition, these experiments conducted under real and simulated microgravity conditions contributed greatly to our current knowledge of how changes of the gravitational force affect basic cellular mechanisms. The influence of microgravity on the function of T lymphocytes is reflected in a variety of cellular responses, which can be grouped into different categories displayed in Table 5.4 and Fig. 5.1.

Table 5.4 Overview of the observed effects of altered gravity on human T cells cultured in vitro
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Fig. 5.1
figure 1

Schematic summary of the influence of microgravity on T lymphocyte function (Modified from Hauschild et al. 2014). 1 Thiel et al. (2012), 2 Singh et al. (2010), 3 Paulsen et al. (2010), 4 Chang et al. (2012), 5 Mangala et al. (2011), 6 Girardi et al. (2014), 7 Hughes-Fulford et al. (2015), 8 Cubano and Lewis (2000), 9 Lewis et al. (1998), 10 Battista et al. (2012), 11 Hashemi et al. (1999), 12 Cooper and Pellis (1998), 13 Cogoli-Greuter et al. (1997), 14 Sciola et al. (1999), 15 Limouse et al. (1991), 16 Risso et al. (2005), 17 Boonyaratanakornkit et al. (2005), 18 Crucian et al. (2000), 19 Crucian et al. (2008), 20 Hatton et al. (2002), 21 Schmitt et al. (1996), 22 Galleri et al. (2002), 23 Tauber et al. (2015), 24 Tauber et al. (2013), 25 Ward et al. (2006), 26 Sundaresan and Pellis (2009), 27 Kumari et al. (2009), 28 Lewis et al. (2001), 29 Cogoli et al. (1985a, b), 30 Simons et al. (2010)

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Since the simulation of the microgravity yielded comparable results to real microgravity experiments (Herranz et al. 2013), it was possible to perform a large number of simulation experiments which would not have been feasible in this scale only by space experiments. So far, however, it has not been possible to formulate a generally accepted hypothesis from these various effects and to further locate any possible primary mechanism that underlies the effects of altered gravity on immune cells.

To date, in vitro research has mainly focused on the impact of altered gravity of T helper cells. In a recent in vivo study (Chang et al. 2015), in which the influence of microgravity was examined for tolerance induction, transgenic mice were exposed to microgravity for 15 days during spaceflight. In this experiment, it could be shown for the first time that the immune tolerance is inhibited in space. Moreover, it provides indications of a potential key role of regulatory T cells.

On closer inspections of the studies reviewed in this chapter, the experimental conditions vary widely from study to study. For example, stimuli used for the T cell activation ranged from mitogens over calcium ionophores up to antibodies against surface receptors. Moreover, different basal media for culturing of the T cells were used which were supplemented in most cases also with different additives in varying concentrations. In nearly all present studies, fetal bovine serum (FBS) has been added to the T cell culture medium ranging from 10 to 20 %.

However, the chemical composition of serum is highly variable and ill defined. Since it contains a large number of constituents, including biomolecules with a variety of growth-promoting and growth-inhibiting activities, the use of serum in cell culture media has obviously considerable effects on phenotypic and genotypic cell stability. Furthermore, the concentrations of the components vary not only from manufacturer to manufacturer but also from batch to batch. Thus, cell signaling, cell proliferation, and differentiation and of course gene expression are influenced by the varying components in different serum used for different experiments.

Therefore, the comparability between the various studies that have been carried out, in order to obtain an overall picture and to locate possible fundamental primary microgravity-induced mechanisms, is not reliable. Nowadays, this lack of standardization has to be regarded as unacceptable. Maintaining high-level standards is of fundamental importance for ensuring good scientific practice in order to maximize reproducibility, reliability, acceptance, and successful implementation of results. Moreover, since scientific research in the area of gravitational science is extremely expensive and elaborate, their resources should be spent wisely. Therefore, in order to achieve the highest level of reliability and comparability of the results, gravitational-related immunobiological research should benefit to a large extent from the latest technology for the standardization of cell and tissue cultures and the development of chemically defined media.

The knowledge of the effects of gravitational changes on T cell regulation and the identification of gravity-sensitive cell responses will help to understand the molecular mechanisms of the inhibited immune cell function in altered gravity and thus new targets for therapeutic or preventive interventions with respect to the immune system of astronauts during long-term space missions may be developed (Ullrich and Thiel 2012).