1. NIH-1a T-Cell Activation in Aging (T-Cell Act in Aging) Millie Hughes-Fulford, PhD 415-847-4492 milliehf@gmail.com

2. NIH-1a First of the NIH peer reviewed ISS experiments to fly on SPX. Uses state of the art technology Analysis based on excellent statistics (n=8). Our previous ISS experiments based on this method was published and has a featured editorial in Dec. 2012 article in Journal of Leukocyte Biology.

3. Infection in Spaceflight Apollo 7 all three crew members had a cold. Apollo 13, one of the crew members became very ill with a common bacteria which usually does not affect people with normal immune systems. In all, 15 of the 29 Apollo Astronauts had an infection either during flight or within one week after flight. (Biomedical Results of Apollo, Johnston, Dietlein, and Berry, NASA Washington, D.C. 1975

4. Loss of T cell activation Kimzey et al. results from Skylab 1977

5. Spacelab 1, November 1983

7. Activation of T cells in the Immune System

8. STS 40 Crew

10. The 3 signals required for full T cell activation Antigen/MHC TCR/CD3 CD28 IL-2R B7 (Accessory cell) IL-2 (Autocrine signal) Anti-CD3 or Con A anti CD28

11. T-Cell Purpose and Research Objectives Purpose The main objective of T-Cell Activation in Aging research is to investigate the earliest key events of immune activation, specifically T-cell activation, during microgravity exposure to characterize the role of the candidate molecular regulatory factors and cellular factors in the inhibition of T-cell activation. Research Objectives The National Institutes of Health (NIH) and the National Aeronautics and Space Administration (NASA) are cooperating to facilitate cellular and molecular biology research in space for better understanding of human physiology and human health on Earth. This payload studies human T cell regulation and activation mechanism for immune system function. There is a correlation between the natural reduction in immune function that occurs over time in the elderly population and spaceflight- associated immune function repression observed in astronauts. The payload research will investigate the gene expression and molecular activities to identify the causal mechanism of the altered immune functions.

14. Hierarchical clustering of gene expression profiles in T-cells activated on ground or on the RPM Induction In g-vector

15. NIH 1-a Aim: –Determine the cause of immunosuppression in spaceflight. Specific goals: –(1) perform transcriptomics (gene array of human genome) –(2) Identify molecular pathways that are dysregulated –(3) Find new therapeutic targets for treating immune dysfunction –(4) To compare data from gene arrays in spaceflight with normal and aging patients –(5) Discover new potential therapeutic targets to treat immune disease. Parameters measured: –Expression of all human genome using microarray (Postflight analysis) –Expression of key genes (identified from microarray) using quantitative real- time rtPCR. –Measure and determine if microRNA small regulators regulate the changes in function –Analyze gene arrays to determine differences in normal conditions as well as spaceflight and aging populations.

17. Presentation of Antigen/MHC TCR/CD3 CD28 B7 (Accessor cell)

18. Dynalbead with activating antibodies Anti-CD3 Anti CD28 TCR/CD3 CD28

19. Factors Limiting Cell Lifetime 7 million cells/900ul media (limited food supply) If cells exposed to ~22 o C they become hypothermic and can start apoptosis program (cell death) over period of experiment. If cells are exposed to temperatures > 30 o C, they will start depleting the limited food supply before experiment starts.

20. T-Cell Act in Aging Timeline Constraints Nominal Timeline (SpX-3 Science) 6 4 6 Sample Prep Part A: Isolate T-Cells (4-6 hrs) Part B: Load EC (7.5 hrs) *T-Cell clock starts at the mid point (~ 4 hrs) Leak Check/ Inspect EC (~2 hrs in vacuum) T0T0 25 T 2-A =25 hrs Late Load for Time Sensitive Payloads (ambient – no cold soak required) (Based on Suzan Voss v17 Late Load File) T 2-A Launch 62 T5T5 Launch to HA3 = 55 4 Capture to Berth Install Jumpers HA3 to Capture 3 3 12 Crew Sleep Ingress 3 T6T6 8 T6 = Access to Late Load Hardware T-Cell Ops Capture is always planned at ~ noon GMT 1 2 1.5 2 2.5 Install in KUBIK Pre-Incubate Activate Fix #1 @ 1.5 hrs Fix #2 @ 4 hrs Transfer to MELFI for SpX Return Complete Ops Time PointHours T0T0 T-Cell Sample Prep End6 T 2-A Late Load for Cold Stowage/Time Sensitive Payload – PD to ISSPO 25 T5T5 Launch to ISS Berthing 62 Launch to HA3: 55 hrs Capture & Berth: 7 hrs T6T6 Crew Access to Late Load Payload19 OpsPayload Operations On-Orbit8 Total120 hrs AB B* Locate DCB 1 Total of 1hr for crew ops

21. T-Cell ESA Provided Hardware The payload consists of the payload hardware (ESA responsibility) containing payload samples (PI responsibility). Samples will be contained in ESA provided Experiment Containers and processed in the ESA KUBIK Facility. Experiment Containers (ECs) (Qty 10) Crew Uses Tool to Activate/Fixate Sample Processing ECs Loaded in KUBIK Incubator

22. EC and EU

24. Sample Preparation Timeline (10 LRS, 2 centrifuges) Ficoll isolation Pellet + rinses PBMC Count PBMCs T cell isolation 1H 2H 0.5H Count T cells 1H 0.5H Load Cassettes 4H Leak Test 2H n=10 independent samples up to cassette load, at which time the best 8 donors are selected for experiment for n=8

25. Scrub Launch Plans There are 3 sets of EU hardware and 2 sets of EC containers We would be able to prepare second set if there was a 24 hour scrub blood samples available If there is 48 hour scrub would be able to order more blood and load a third set of EUs

26. T-Cell Payload Operations  The PI, working in conjunction with ESA, is responsible for integrating and conducting the payload scientific research.  ESA is responsible for the payload development, integration of samples into the payload, hardware supply, integration of the payload research in the KUBIK incubator and Columbus, the on-orbit operations and the ground segment required to support on-orbit operations.  NASA is responsible for payload tactical planning, the integration of the payload research with launch/return capabilities, cold stowage accommodations, and launch and landing site support, including provision of lab equipment at launch site.

27. Crew Training  Crew training will be performed by ESA at EAC the by April 2013.  Crew Member will be trained on the KUBIK, Experiment Container, Experiment Unit and Activation/Fixation Tool.  ESA will provide written documentation to the POIC CTC that crew training was accomplished.

28. EST Completed: EST was successfully held on April, 2013 at ESTEC in the Netherlands

29. FTR Completed: FTR successfully completed in May, 2013 at Kennedy Space Center

30. Our Previous Experiment Was Just Named Top ISS Science Discovery for 2012 July 16, 2013

31. Upcoming: Flight experiment on ISS delevered by SpaceX

32. Hughes-Fulford Laboratory UCSF, NCIRE AND VAMC SAN FRANCISCO Supported by NASA grants: NCC-2-1086, NAG-2-1286 NIH grant UH3-AG037628 and in part by VA Merit