Signal transduction in primary human T lymphocytes in altered gravity - results of the MASER-12 suborbital space flight mission

Abstract

We investigated the influence of altered gravity on key proteins of T cell activation during the MASER-12 ballistic suborbital rocket mission of the European Space Agency (ESA) and the Swedish Space Cooperation (SSC) at ESRANGE Space Center (Kiruna, Sweden). We quantified components of the T cell receptor, the membrane proximal signaling, MAPK-signaling, IL-2R, histone modifications and the cytoskeleton in non-activated and in ConA/CD28-activated primary human T lymphocytes. The hypergravity phase during the launch resulted in a downregulation of the IL-2 and CD3 receptor and reduction of tyrosine phosphorylation, p44/42-MAPK phosphorylation and histone H3 acetylation, whereas LAT phosphorylation was increased. Compared to the baseline situation at the point of entry into the microgravity phase, CD3 and IL-2 receptor expression at the surface of non-activated T cells were reduced after 6 min microgravity. Importantly, p44/42-MAPK-phosphorylation was also reduced after 6 min microgravity compared to the 1g ground controls, but also in direct comparison between the in-flight μg and the 1g group. In activated T cells, the reduced CD3 and IL-2 receptor expression at the baseline situation recovered significantly during in-flight 1g conditions, but not during microgravity conditions. Beta-tubulin increased significantly after onset of microgravity until the end of the microgravity phase, but not in the in-flight 1g condition. This study suggests that key proteins of T cell signal modules are not severely disturbed in microgravity. Instead, it can be supposed that the strong T cell inhibiting signal occurs downstream from membrane proximal signaling, such as at the transcriptional level as described recently. However, the MASER-12 experiment could identify signal molecules, which are sensitive to altered gravity, and indicates that gravity is obviously not only a requirement for transcriptional processes as described before, but also for specific phosphorylation / dephosphorylation of signal molecules and surface receptor dynamics.

Figure 1

Payload configuation of MASER-12, launch and recovery. A. Payload configuration of the MASER-12 sounding rocket. MASER-12 consists of a VSB-30 motor (S-30 solid rocket stage engine with a S-31 second stage engine) and of the payload. MASER-12 hosted five experiments and provided six minutes microgravity. The experiment of the study was performed in the module “Biology In Microgravity“(BIM-2) where the LAU (“Late Access Unit“) containing the isolated cells was installed shortly before the launch. (Pictures were kindly provided by SSC). B. MASER-12 was launched on February 13th 2012, 09:32 am, from the ESRANGE Space Centre near Kiruna, Sweden, north of the Arctic circle). C. Work with BIM-2 (Biology In Microgravity-2) module at the landing spot. The recovery of the LAU was successful. The module was switched off, by access from the umbilical connector, at 1 hour 20 minutes after lift-off. D. The Late Access Unit (LAU) was transported back in a dedicated helicopter from the landing spot.

Figure 2

Experiment module BIM-2. A. “Mix Unit” flight hardware cassette used for the experiment on the sounding rocket MASER-12. As depicted in the schematic illustration one hardware cassette hosts two experimental units. One experimental unit contains three chambers made of a flexible membrane: one main chamber for cell suspension and two storage chambers, one being the activator solution and one for the fixative. Releasing the tension from the springs beneath the activator and the fixative chamber pushes the solutions into the main chamber (also called reaction chamber) via little tubes in the lid. The picture was kindly provided by CCM, Nuenen, Netherlands. B. Picture of one hardware cassette. The picture was kindly provided by CCM, Nuenen, Netherlands. C. Late Access Unit (LAU). The main components of the LAU are a reference centrifuge incorporated in the middle and two static racks (μg rack) along the edge. These two components carry the flight hardware cassettes containing cell suspensions and reaction solutions. D. Acceleration profile of MASER-12 during the launch. Almost 13 g were detected during MASER-12 launch. The picture was kindly provided by Swedish Space Corporation (SSC).

Figure 3

Experimental design during the MASER-12 mission. For the analysis of resting T cells three different gravity conditions were tested: μg samples which were installed on the μg Rack in the LAU on board of the rocket, 1g samples that were installed on the on-board reference centrifuge providing 1g, and H/W samples that were installed in the hardware ground module analogue to the flight hardware. With re-entry of the payload into the Earth’s atmosphere all samples were automatically fixed. The analysis of T cells activation involved five different conditions. In addition to the μg and the 1g samples, baseline (BL) samples were used, which were fixed before onset of microgravity (μg phase). At this stage, the μg samples and the 1g samples were activated. The fixation of those two conditions were executed parallel with the appropriate H/W samples, although these were not activated. The fifth condition consisted of culture control (CC) samples, where T cells were cultured under normal culture conditions. Those samples were fixed manually a few minutes after rocket launch due to safety reasons.

Figure 4

CD3 and IL-2 receptor surface-expression of T lymphocytes exposed to different gravity conditions measured by FACS-analysis. Non-activated and Concanavalin A (ConA)/CD28-activated CD4+ T lymphocytes were exposed to altered gravity. A and B. Samples were stained against surface-CD3 and analyzed by FACS; data are expressed as relative fluorescence intensity (RFI). A: Non-activated T lymphocytes exposed to microgravity (μg) showed a significant reduction of CD3 surface expression in comparison to the cells of the flight hardware samples on ground (H/W). B: ConA/CD28 activated (+) T lymphocytes at 1g (on-board reference centrifuge) showed a significantly higher expression of CD3 compared with the cells of the baseline (BL) samples, while the μg samples did not show a significant higher level compared to BL samples. In hardware (H/W) controls the CD3 signal was higher than in BL samples, while culture control (CC) samples showed the highest signal of all experimental groups. Medians and interquartile ranges are shown (* p < 0.1, ** p < 0.05, ns = not significantly different, two-tailed Mann–Whitney-U-Test). C and D. Samples were stained against surface-IL-2R and analysed by FACS; data are expressed as relative fluorescence intensity (RFI). C: Non-activated (−) T lymphocytes exposed to microgravity (μg) showed a significant reduction of IL-2R in comparison to the cells of the flight hardware on ground (H/W). D: ConA/CD28 activated (+) T lymphocytes at 1g conditions (on-board reference centrifuge) (1g) showed a significantly higher expression of IL-2R compared with the cells of the baseline (BL) samples. In hardware (H/W) samples the IL-2R signal was higher than in the BL samples. No significant difference was found between culture controls (CC) and H/W samples. Medians and interquartile ranges are shown (* p < 0.1, ** p < 0.05, ns = not significantly different, two-tailed Mann–Whitney-U-Test).

Figure 5

ZAP 70-expression of T lymphocytes exposed to different gravity conditions measured by FACS-analysis. Non-activated and Concanavalin A (ConA)/CD28-activated CD4+ T lymphocytes were exposed to altered gravity. Samples were stained against ZAP 70 and analyzed by FACS; data are expressed as relative fluorescence intensity (RFI). A: Non-activated (−) T lymphocytes exposed to microgravity (μg) and to an on-board reference centrifuge (1g) showed a significant reduction of ZAP 70 staining compared to the cells of the hardware ground control (H/W). ZAP 70 staining was not significantly different between 1g and μg samples. B: ZAP 70 staining of ConA/CD28 activated (+) T lymphocytes was neither significantly different between 1g and μg samples, nor between baseline (BL) samples and 1g or μg samples. The ZAP 70 staining was significantly decreased between culture controls (CC) and H/W samples, and between H/W samples and baseline (BL). Medians and interquartile ranges are shown (* p < 0.1, ** p < 0.05, ns = not significantly different, two-tailed Mann–Whitney-U-Test).

Figure 6

pY171-LAT and pY266-LAT in T lymphocytes exposed to different gravity conditions measured by FACS-analysis. Non-activated and Concanavalin A (ConA)/CD28-activated CD4+ T lymphocytes were exposed to altered gravity. A and B. Samples were stained against phosphorylated LAT Y171 (LAT pY171) and analyzed by FACS; data are expressed as relative fluorescence intensity (RFI). A: Non-activated (−) T lymphocytes exposed to microgravity (μg) and to an on-board reference centrifuge (1g) showed a significant increase of LAT pY171-staining compared to the cells of the hardware ground control (H/W). LAT pY171-staining was not significantly different between 1g and μg samples. B: In ConA/CD28 activated (+) T lymphocytes LAT pY171-staining was not significantly different between 1g and μg samples, although both sample types showed a higher staining than baseline (BL) samples. LAT pY171-staining staining did not differ between BL and H/W samples. Medians and interquartile ranges are shown (* p < 0.1, ** p < 0.05, ns = not significantly different, two-tailed Mann–Whitney-U-Test). C and D. Samples were stained against phosphorylated LAT Y226 (LAT pY226) and analyzed by FACS; data are expressed as relative fluorescence intensity (RFI). C: Non-activated (−) T lymphocytes exposed to microgravity (μg) and to an on-board reference centrifuge (1g) showed a significant increase of LAT pY226-staining compared to the cells of the hardware ground control (H/W). LAT pY226-staining was not significantly different between 1g and μg samples. D: In ConA/CD28 activated (+) T lymphocytes LAT pY226-staining was neither significantly different between 1g and μg samples, nor between baseline (BL) samples and 1g or μg samples. LAT pY226-staining increased from culture control samples (CC) to H/W samples and from H/W samples to BL samples. Medians and interquartile ranges are shown. (* p < 0.1, ** p < 0.05, ns = not significantly different, two-tailed Mann–Whitney-U-Test).

Figure 7

P-p42/44 MAPK and tyrosine phosphorylation in T lymphocytes exposed to different gravity conditions measured by FACS-analysis. Non-activated and Concanavalin A (ConA)/CD28-activated CD4+ T lymphocytes were exposed to altered gravity. A and B. Samples were stained against P-p42/44 MAPK and analyzed by FACS; data are expressed as relative fluorescence intensity (RFI). A: Analysis of non-activated (−) T cells exposed to microgravity (μg) showed a significant reduction in RFI in μg samples compared to 1g on board reference samples (1g). B: In the analysis of ConA/CD28 activated (+) T lymphocytes a significant reduction of baseline (BL) samples was observed compared to hardware (H/W) samples. The RFIs of 1g samples and μg samples were not significantly different from BL samples or from each other. The medians and individual interquartile ranges are shown. (* p < 0.1, ** p < 0.05, ns = not significantly different, two-tailed Mann–Whitney-U-Test). C and D. Samples were stained against phospho-tyrosine (p-tyrosine) and analyzed by FACS; data are expressed as relative fluorescence intensity (RFI). C: Analysis of non-activated (−) T cells revealed no difference between microgravity (μg)-samples, on-board reference centrifuge (1g)-samples and hardware ground controls (H/W) in p-tyrosine stainings. D: In the analysis of ConA/CD28 activated (+) T lymphocytes p-tyrosine-staining was not significantly different between 1g and μg samples, but both sample types showed a higher staining than baseline (BL) samples. BL samples were significantly less stained than H/W samples. The medians and individual interquartile ranges are shown. (* p < 0.1, ** p < 0.05, ns = not significantly different, two-tailed Mann–Whitney-U-Test).

Figure 8

Acetyl-Histone H3 in T lymphocytes exposed to different gravity conditions measured by FACS-analysis. Non-activated and Concanavalin A (ConA)/CD28-activated CD4+ T lymphocytes were exposed to altered gravity. Samples were stained against acetyl-histone H3 and analyzed by FACS; data are expressed as relative fluorescence intensity (RFI). A: Analysis of non-activated (−) T cells revealed that microgravity (μg)-samples and on-board reference centrifuge (1g)-samples were stained significantly less for acetyl-histone H3 than hardware ground control (H/W)-samples. μg-samples and on-board reference centrifuge (1g)-samples did not differ in their staining intensities. B: In ConA/CD28 activated (+) T lymphocytes acetyl-histone H3-staining was neither significantly different between 1g and μg samples, nor between baseline (BL) samples and 1g or μg samples. BL samples were stained significantly less than H/W samples, and H/W samples were stained significantly stronger than culture control (CC) samples. The medians and individual interquartile ranges are shown. (* p < 0.1, ** p < 0.05, ns = not significantly different, two-tailed Mann–Whitney-U-Test).

Figure 9

Vimentin and β-tubulin expression in T lymphocytes exposed to different gravity conditions measured by FACS-analysis. Non-activated and Concanavalin A (ConA)/CD28-activated CD4+ T lymphocytes were exposed to altered gravity. A and B. Samples were stained against vimentin and β-tubulin and analyzed by FACS; data are expressed as relative fluorescence intensity (RFI). A: Analysis of non-activated (−) T cells revealed that microgravity (μg)-samples and on-board reference centrifuge (1g)-samples were stained significantly less for vimentin than hardware ground control (H/W)-samples. μg-samples and 1g-samples did not differ significantly in their staining intensities. B: In ConA/CD28 activated (+) T lymphocytes vimentin-staining was not significantly different between 1g and μg samples, but both samples types are stained significantly stronger than baseline (BL)-samples. BL samples were stained significantly less than H/W samples, and H/W samples were stained significantly less than culture control (CC) samples. The medians and individual interquartile ranges are shown. (* p < 0.1, ** p < 0.05, ns = not significantly different, two-tailed Mann–Whitney-U-Test). C and D. Samples were stained against β-tubulin and analyzed by FACS; data are expressed as relative fluorescence intensity (RFI) C: Non-activated (−) T lymphocytes exposed to microgravity (μg) showed a significant increase of β-tubulin in comparison to the cells of the flight hardware on ground (H/W). D: In the analysis of T cell activation ConA/CD28 activated (+) T lymphocytes both the 1g on-board reference centrifuge (1g) and the microgravity (μg) samples had a significantly higher expression of β-tubulin compared with the cells of the baseline (BL) samples. BL and H/W samples had a lower β-tubulin signal than the culture controls.