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As the National Aeronautics and Space Administration (NASA), plans to return to the moon and send humans to Mars, crew health and safety concerns need to be examined with a focus on longer duration exploratory class missions. One of the transient changes that has been observed during the ISS missions is dysregulation of immune system. Latent viral reactivation and diminished cellular mediated immunity along with a TH2-cytokine shift being the consistently observed effects of spaceflight on immune system. However, most of the changes observed in spaceflight are a composite effect of stress, microgravity, radiation, circadian disruption, altered nutrition, and sleep disturbances, all of which have an immune-altering effect. Discerning precise effects of the various components of spaceflight become crucial to devise appropriate countermeasures. Ground-based simulated microgravity systems can be used to understand the specific effects of microgravity on human immune system. Rotary cell culture system (RCCS) is a NASA validated ground-based model to simulate microgravity. Using this quiescent low-shear stress environment, human immune cells can be exposed to simulated microgravity (SMG) for brief periods by randomizing the gravity vector and facilitating continuous free fall. In short-term spaceflight, latent viral reactivation along with lowered viral-specific T cell function has been recorded. Healthy functioning of viral-specific T cells is a prerequisite to controlling viral infections. So, it becomes imperative to examine if microgravity plays a role in this reduction of viral-specific T-cell function. There is also a paucity of data on effect of spaceflight and simulated microgravity on γδ-T cells. These crucial effector lymphocytes are considered a connecting link between innate and adaptive immune system. Their function in spaceflight, especially Vγ9Vδ2 T cell subset, becomes important to control hematological malignancies in early stages. Therefore, this dissertation examined the effect of simulated microgravity on expansion potential and function of viral specific T-cells and γδ-T cells. Exposure to SMG impaired in vitro expansion of CMV-specific T-cells (RM ANOVA, F(1.571, 6.283)= 8.367, p=0.0198). 10-million PBMCs at day1 of the expansion in SMG-exposed condition yielded 17.63±3.75 million (MEAN±SEM, N=5) CMV-specific T-cells at the end of the expansion. In comparison, STATIC-1G control cells expanded to 33.8±7.57 million, while 1G-rotational control exposed cells expanded to 28.32±7.21 million cells. However, exposure to SMG did not affect in vitro function of CMV-specific T-cells (RM ANOVA, F(1.357,12.21)=0.7434, p=0.4457). Hundred thousand cytotoxic (CD8+) CMV-specific T-cells exposed to SMG at the end of expansion killed 804.6±166.3 (MEAN±SEM, N=10) autologous PHA blasts pulsed with CMV peptides. In comparison, 1G-control and 1G-rotational control killed 909.2±160.6 and 669.7±125 PHA blasts respectively. Exposure to SMG also impaired in vitro expansion of γδ-T cells (Wilcoxon signed ranks test, p=0.039). 10 million PBMCs at day 1 of the expansion in SMG-exposed condition yielded 102.3±23.07 million (MEAN±SEM, N=9) γδ-T cells at the end of the expansion. In comparison, 1G-control PBMCs yielded 113.7±23.91 million γδ-T cells. Γδ-T cells that were exposed to SMG and later expanded in 1G showed downregulation of inhibitory receptor CD158b (paired t-test, p=0.03) and killed more U266 target cells (paired t-test, p=0.04) compared to γδ-T cells that were expanded in 1G. Exposure to SMG upregulated activating receptor NKG2D on γδ-T cells that were expanded in 1G (Wilcoxon matched-pairs signed rank test, p=0.0078), without concomitant increase in function. Exposure to SMG did not impair γδ-T cells’ ability to kill tumor target cells (K562: paired t-test, t(8) =0.5032, p=0.628; U266: paired t-test, t(8) =0.1479, p=0.886). Another limitation of spaceflight data is lack of in vivo functional data. Although we have observed decreased in vitro function of various immune cells in several flight and ground-based simulation studies, how this translates to a more physiologically relevant in vivo model remains to be explored. Ergo, this dissertation also improved upon the current in vitro data by examining how exposure to SMG using a RCCS, affects in vivo anti-leukemia activity of human effector lymphocytes. A humanized NSG-tg(hu-IL15) mice model, which is used in a pre-clinical setting for hematopoietic stem cell transplantation studies, was used to examine the in vivo effect of exposure to SMG on PBMCs. Furthermore, we examined the efficacy of Zoledronic acid+IL2 (ZOL+IL2) therapy as a possible spaceflight countermeasure to revive the in vivo anti-leukemia activity of SMG exposed PBMCs. ZOL+IL2 is a clinical therapeutic strategy to accelerate favorable immune reconstitution. This therapy also improves NK cell and γδ-T cell activity in vivo, after a hematopoietic stem cell transplantation. Therefore, we expected administration of ZOL+IL2 to abrogate the effect of exposure to SMG on human PBMCs, by stimulating NK cells and γδ-T cells. Exposure to SMG impaired anti-leukemia activity of human immune cells in vivo. Tumor growth control was compared between mice that were injected with PBMCs exposed to SMG (TUMOR+SMG PBMCs) or 1G-control (TUMOR+1G PBMCs) to evaluate the effect of SMG on anti-leukemia activity of human immune cells in vivo. Mice injected with tumor cells only was used as a reference for unrestrained tumor growth. Bioluminescent intensity (BLI) score was used as a measure of tumor burden. A mixed effects model was used to analyze BLI scores with ‘condition’ (TUMOR control, TUMOR+SMG PBMCs, TUMOR+1G PBMCs) and ‘time’ as main effects and an interaction term ‘condition*time’ in the model. This revealed that tumor grew differentially over time in different conditions. A pairwise comparison revealed that 1G-exposed PBMCs controlled tumor growth better than SMG-exposed PBMCs (p<0.001). Peak BLI reached during the experiment further understated the inability of SMG-exposed PBMCs to control tumor growth (Friedman test, p=0.0018, N=12, TUMOR+SMG PBMCs>TUMOR+1G PBMCs). Exposure to SMG did not alter engraftment, survival or graft-versus-host-disease (GVHD) dynamics. ZOL+IL2 therapy improved anti-leukemia activity of human immune cells in vivo. Mice that received SMG PBMCs and given ZOL+IL2 therapy controlled their tumor better compared to mice that received SMG PBMCs without ZOL+IL2 therapy (Mixed effects model, p=0.0004). There were no differences in tumor control between mice that received SMG PBMCs along with ZOL+IL2 therapy and mice that received 1G-exposed PBMCs. This showed that ZOL+IL2 therapy abrogated the loss of in vivo function after exposure to SMG. ZOL+IL2 therapy did not alter survival and GVHD dynamics. There were non-significant increases in NK cell and γδ-T cell engraftment throughout the experiment in blood of the animals, showing that ZOL+IL2 therapy improved anti-leukemia effector immune cell engraftment, which helped in tumor control. In summary, these experiments advance our understanding of the effect of simulated microgravity on immune cells. Exposure to SMG detrimentally affects immune cell function and expansion potential both in vitro and in vivo. These negative effects could impair crew health and performance during an exploratory class missions. These experiments also highlight the importance of microgravity as a contributor to the immune dysregulation observed in spaceflight. Future studies should explore the sustenance of this detrimental effect in confluence with other perturbations of spaceflight. Future investigations should also include relevant immunotherapeutic countermeasures to improve crew health and performance during long-duration exploration class missions.