Microgravity effect on murine T cells exposed to suborbital flight aboard Blue Origin’s New Shepard vehicle

Numerous scientific experiments have been conducted in space. However, the precise mechanisms mediating successful human body adaption to the hostile space environment are still not delineated. The cost and logistic challenges of sending biological payloads to International Space Station are forcing scientists to find alternative research platforms. In this study, we investigated whether brief exposure to microgravity during the suborbital flight aboard Blue Origin’s New Shepard rocket modulated the behavior of the gravity-sensitive murine T cells. We assessed the effect of suborbital environment on different T cell subsets, activation markers, functionality, and cytokine secretion capabilities. Thus, to optimize the potential response of T cells, we cultured them in interleukin IL-2 alone or combined with IL-12. We found that exposure to microgravity decreased the expression of T cells with CD4+ cells being more sensitive to suborbital flight as compared to CD8+ cells. Our data indicate that the functional capabilities of flown T cells were reduced. Also, our findings suggest that supplementing cells with IL-2 and IL-12 cytokines may restore microgravity-mediated cellular alterations. Finally, our study provides insights on the microgravity effect on the murine T cells by utilizing a novel suborbital research platform.

There has been an escalated interest over the past few years by NASA and other private 73 stakeholders to pursue human spaceflight missions to the moon (e.g., ARTEMIS mission) and cancerous developments leading to elimination and long-term protection [15]. At first, naive T 93 lymphocytes need to be activated so that they can differentiate into effector cells to perform their 94 immune functions. This requires engagement of TCR (T Cell Receptor) by the peptide/major 95 histocompatibility complex (MHC) and the costimulatory signal provided by the interaction between accessory molecules, such as CD28 and CD3 on the surface of T cells [16]. Upon    and IL-2/IL-12 added one hour before handing off the payload to the NanoRacks team or about 177 10 hours before the flight. Those conditions lacked ground controls and were referred as IL-2,

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The diagram below (diagram 1) illustrates the different timeline when cytokines were added.

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The NanoLab was recovered 6 hours after liftoff. The temperature inside the NanoLab was

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To investigate whether brief exposure to microgravity leads to alterations in CD8+ and CD4+ 270 subsets, we stained cells with fluorescently labelled antibodies for these markers.

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More specifically, we found a slight decrease in CD8+ and CD4+ populations in all flight 290 conditions with the most significant difference observed in cells cultured in both cytokines 291 (p<0.05) ( Fig 2B). Our data revealed that T cells exposed to suborbital flight had a decreased 292 CD4+/CD8+ ratio in all conditions with the most profound differences observed in cells cultured 293 in IL-2 (p<0.01) and IL-12 (p<0.05) (Fig 2C). This suggests that culturing T cells with cytokines 294 have influence on the distribution of CD4+ and CD8+ expression. There was almost no variation 295 in the ratios of CD4+ to CD8+ when no cytokines were added. The latter is consistent with some 296 previous studies [22] which reported no difference between the CD4+ and CD8+ ratios.

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However, there was a profound change (near 12.2. % decrease) of this ratio when the IL-2 298 cytokine was supplied from day zero between the ground and flight conditions. Given we did not have this condition in duplicate and only one tube of cells was exposed to flight, the difference Column 1 shows the cell population for CD4+, CD8+, total apoptosis, live cells, and dead cells. cultured in the same manner as ground conditions. One-way ANOVA test was used to compute 320 the SE and RE means.

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Microgravity decreases the expression of the T cell activation 322 marker CD71 and T cell exhaustion marker PD-1. 323 We next sought to determine whether T cells exposed to suborbital flight condition would have a 324 decreased expression of early (CD69) mid-late (CD71) and late (CD25) activation markers as 325 well as the alteration in the checkpoint programmed death-1 PD-1 marker, also known as T cell 326 exhaustion indicator (Fig 3).

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Our data indicate that for CD69, flight cells cultured in IL-2 had a 7.2 % decrease,

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whereas flight cells cultured in IL-12 had a significant (p<0.05) increase in CD69 expression ( Fig  346 when compared to IL-2 flight condition. CD69 is a classical early marker of lymphocyte 347 activation due to its rapid appearance on the surface of the plasma membrane after stimulation.

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Given that CD69 regulates the secretion of IFNɣ whose expression is also influenced by the IL-349 12 cytokine, it is not surprising to see elevation of this marker in IL-12 condition. This suggests 350 that culturing T cells in IL-12 might be a good strategy to increase the expression of CD69.

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However, it is interesting to observe this effect in flight condition only, but not in ground control.

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Previous studies indicated that the duration of the mission played a significant role in CD69 353 expression which was elevated in short duration shuttle crewmembers, but it was significantly 354 reduced in long duration mission crewmembers [24].

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CD71 expression across flight conditions were slightly reduced except for cells cultured 356 in IL-2 from day 0 onward (Fig 3B). Thus, adding extra cytokines before the flight increased the 357 expression of CD71 in IL-2 (7.5 %) and IL-12 (2.8 %) flight conditions (Fig 3B). This suggests 358 that supplementing cells with these cytokines prior to the flight may restore the expression of 359 CD71. CD25 expression remained similar among different flight and ground conditions with a 360 slight increase (3%) in cells supplemented with additional IL-2 dose before the flight (Fig 3C).

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Since CD71 expression was downregulated in most flight conditions, it may be that this marker 362 is more sensitive to microgravity than CD69 and CD25 markers. In addition, we observed that

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Finally, we assessed the effect of microgravity on PD-1 expression (Fig 3D). PD-1 374 mediated-inhibitory signals play a major role in T cell exhaustion during chronic infections and 375 cancers, which makes PD-1 a valuable target of checkpoint blockade in cancer immunotherapy.

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Given that T cells become exhausted in large tumors, we hypothesized that T cells could also with no cytokines. There is some evidence to draw parallels between T cells in microgravity and 387 in cancer. As such, our data demonstrated that T cells exposed to microgravity resulted in 388 decreased expression of PD-1 whose expression is proportional to the strength of TCR 389 signaling to compensate T cell activation and to control immune response [28]. Thus, it is also 390 well-established that PD-1 expression on antigen-specific T cells reflects the functional avidity 391 and anti-tumor reactivity of these cells [28]. Therefore, the importance of PD-1 on human 392 immunity makes this molecule a valuable marker to explore its role in microgravity for 393 immunotherapy purposes, especially given that slow immune response in space could put

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After observing that brief exposure to microgravity can affect T cell markers of activation, we 407 next investigated the levels of apoptosis (Table 2, Fig 4).  (Fig 4A). Although IL-12 is a promising candidate for cancer 430 immunotherapy, it is however, associated with some level of toxicity [29] which is reflected in 431 our study by an increased level of apoptosis. Given that IL-12 may induce toxicity, our data also 432 revealed that the highest apoptosis rate was observed in conditions with IL-12 alone or in 433 combination with IL-2 ( Fig 4A). Furthermore, our data showed that these conditions also had 434 higher expression of PD-1 (Fig 3D) suggesting the correlation between PD-1 and apoptosis [30].

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It has been reported in the literature [31] that up-regulation of PD-1 could promote T cell 436 apoptosis in certain types of cancer. In addition, our results indicate that adding IL-2 and IL-12 437 to flown T cell cultures increases the percentage of live cells (Fig 4B). Compared to ground 438 controls, flown cells had fewer dead cells in all flight conditions (Fig 4C). This suggests that 439 microgravity may drive T cells into more apoptotic phenotype but may save them from dying.

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Notably, adding cytokines before the flight did not decrease the apoptosis nor increased the 441 number of live cells, except in cells primed with IL-2 which resulted in decrease number of dead 442 cells (Fig 4D). This suggests that IL-2 cytokine may be a good candidate to increase the live IFNɣ expression (about 6%). Given that one of the roles of IL-12 cytokine is to induce IFNɣ, it is 477 not surprising that cells cultured in IL-12 had the highest expression of IFN in flight conditions.

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A central mediator of IL-12-induced responses is IFNγ, which is secreted upon IL-12 stimulation 479 alone or with synergizing factors such as IL-2 and IL-18. We stimulated cells with both (PMA/I)

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(Figs 5C, 5D) and peptide (data not shown). We did not observe a profound difference between 481 these two stimulants suggesting that using either of these methods yields consistent results.

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Exposure to microgravity alters the expression of multiple 484 cytokines detected in T cell culture supernatants. 485 We collected supernatants from cells flown aboard Blue Origin's New Shepard as well 486 as from ground controls to perform cytokine secretion analysis using Multiplex Immunoassay kit.

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This extremely sensitive assay was chosen to detect the expression of five analytes in our T cell 488 cultures including IL-2, IL-4, IL-6, IL-10, and IL-12 (Table 3).   Table 3 has 6 columns, the first column is the condition (control, IL-2, IL-12, IL-2 and IL-12 499 combined, IL-2 from day 0), the second and third columns display the means of the ground and

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Our findings indicate that adding cytokines to T cell cultures lead to altered expression of 510 many cytokines among different ground and flight conditions (Fig 6A).  Table 3. We observed a significantly enhanced expression of IL-12 in flown T cells that 527 were cultured in IL-12 alone (p < 0.01) or in combination with IL-2 (p < 0.01) as compared to ground controls ( Fig 6B). This increase in IL-12 is consistent with the functional data which 529 indicated the elevated expression of IFNɣ in T cells cultured in IL-12. Adding IL-12 before the 530 flight increased the expression of IL-12 in T cells cultured in IL-12 alone or in combination with 531 IL-2. However, cells exposed to suborbital flight and cultured in IL-2 from day 0 had a lower 532 expression of IL-12 ( Fig 6B). Analysis of IL-2 expression revealed that this cytokine was slightly 533 elevated in all flown T cell conditions, except for cells cultured in IL-2 from day 0 when 534 compared to ground controls ( Fig 6C). However, the difference in expression was not significant 535 among different ground and flight conditions. The same pattern was observed in IL-4 536 expression, except for T cells cultured with IL-2/IL-12, which when exposed to suborbital flight 537 had a lower expression of IL-4 as compared to ground controls ( Fig 6D). The expression of 538 cytokine IL-6 was reduced in all flight conditions except for the cells cultured in IL-2 ( Fig 6E).

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Interleukin-6 is a pleiotropic cytokine mediating and facilitating the inflammatory responses.

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Literature suggests that IL-6 might promote T cell survival and proliferation [33,34], although 541 the precise mechanisms accounting for these effects are not well-delineated. Our data indicate 542 that culturing T cells with IL-2/IL-12 and IL-2 from day 0 leads to the highest expression of IL-6 543 across all conditions, but with lower expression in flight conditions, relative to ground controls.

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Given IL-6 may drive T cell survival, our data is in alignment with our other findings suggesting 545 that microgravity may have a negative effect on T cell survival and proliferation. Finally, we saw 546 a significant higher expression (p < 0.05) of IL-10 in flown T cells cultured in IL-2, but 547 significantly reduced expression in flown cells supplemented with either IL-12 or both cytokines 548 (Fig 6F). IL-10 is classified as an anti-inflammation marker involved in antibody production and 549 regulation of inflammation. However, its role in modulating the immunological response might be 550 quite complicated [35]. Our data show that microgravity significantly reduced the expression of 551 IL-10, however culturing T cells in IL-12 may restore the expression of IL-10 to some degree, 552 but not fully. Together, our data demonstrate that multiplex immunoassay is a sensitive assay capable of detecting different analytes in T cell cultures, exposed to microgravity. Furthermore, adding cytokines to T cell cultures may restore some of the antagonizing effects of microgravity,

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In general, the current knowledge of microgravity induced alterations in the immune system 596 derives from multiple sources, such as leukocytes obtained from astronauts, cells obtained from 597 flown animals (mostly mice and rats), and from in-vitro cell cultures. Therefore, comparison 598 across these different model systems should be made with caution. Although it is quite difficult 599 to compare our study's results with other studies due to different microgravity subjects and 600 models used and different assessment time points, our data supports findings from previous 601 studies on microgravity induced alterations on the immune system. However, given that we

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However, more studies are needed to investigate the effect of microgravity on T cells behavior 618 during the suborbital flight so that acquired data could be used to design comprehensive orbital 619 space platform experiments. Our team hopes to replicate our findings on the next suborbital 620 flight onboard the PLD Space's Miura 1 rocket where we will extend our analysis to gene 621 expression and signal transduction assays before moving on to orbital research platform.