NOTE: changed end date to 9/30/2013 per B. Crucian (Ed., 2/4/2013)

NOTE: End date is 3/29/2013 per HRP Master Task List dtd 7/12/2011 (Ed., 8/9/2011)

NOTE: End date now 9/30/2013 per JSC (09/2010)

NOTE: End date changed to 9/30/2011 per B. Corbin/JSC (3/2009)

NOTE: End date changed to 5/31/2011 per PI ; original end date was 4/2010 (2/09)

Results: Some shifts in leukocyte distribution occurred during flight, including alterations within subsets of CD8+ T cells which indicated an increased maturation state. General T cell function (both CD4 and CD8+ subsets) was consistently reduced early in-flight. The percentage of CD4+ T cells capable of producing IL-2 was depressed early in-flight and after landing. Significant and persistent mitogen-specific reductions in culture-stimulated T cell production of IFNg, IL-10, IL-5, TNFa and IL-6 were observed during spaceflight. Monocyte production of IL-10 was reduced, whereas IL-8 production was increased. Plasma cortisol was increased at landing but not in-flight. No significant changes occurred in anti-viral antibodies, but EBV T-cell function was decreased in-flight and was accompanied by an increase in EBV-infected cells in peripheral blood.

Conclusions. The data indicate that some alterations in immunity persist during spaceflight and are not merely related to the transitional stress of launch or landing. Ongoing immune dysregulation, reduced immune cell function and/or Th1/Th2 shifts, in conjunction with elevated radiation exposure and limited clinical care may increase specific clinical risks for crewmembers during exploration-class space missions.

This study investigated the status of the immune system during short duration spaceflight by collecting blood samples from US crewmembers during spaceflight. Flight blood samples were returned for terrestrial laboratory analysis. To our knowledge, this was the first study that returned live, ambient storage blood samples for functional assays. This approach allows an in-flight data point to be achieved without flying complicated laboratory instruments. The L-180 data were considered baseline for comparison of in-flight and landing day data. An additional sample was collected from ISS subjects at L-45, and for the Shuttle subjects at L-10; however, the L-10 sample was dictated by operational constraints. Astronauts are under considerable stress by L-10, so data at this timepoint may possibly be influenced by pre-launch stress and is not considered the best baseline measurement. The second baseline for ISS subjects was at L-45. Summary data findings are as follows:

There were alterations in the peripheral leukocyte distribution during short duration missions that were largely absent during long-duration ISS missions. However, there was an elevated WBC count and a maturation shift in CD8+ T cells during ISS missions.

T cell function was depressed during Shuttle missions in a mitogen specific fashion, with the more physiological relevant stimulus showing a consistent defect in T cell function. During ISS missions this reduction in T cell function was more apparently early, and trending to resolve as 6-month missions progressed.

A more downstream measure of T cell function, cytokine secretion, was profoundly dysregulated in ISS subjects for the duration of a 6-month mission.

Plasma cytokine levels in ISS astronauts were dysregulated for the duration of a 6-month mission.

A significant increase in constitutively activated CD8+ T cells was observed during ISS missions.

Latent viral reactivation for three herpes viruses, EBV, VZV, and CMV was observed significantly in crewmembers during both short and long duration spaceflight. Their viral load increased during flight than both before and after flight as well as the healthy controls on the ground. This effect was more pronounced in long duration space flight as compared to short duration spaceflight. Also shedding of VZV in saliva and CMV in urine of the ISS crewmembers continued up to 30 days or more after the spaceflight as compared to 5-7 days in space shuttle crewmembers. Although not significant due to subject number and inter-subject variability, antiviral antibodies were generally elevated consistent with the increased viral load.

EBV- and CMV-specific CD8+ T-cell number did not significantly change in either the Shuttle or ISS crewmembers. However, there was a pronounced decrease in function in EBV- and CMV-specific T-cells in the Shuttle crewmembers during and after flight. For ISS crewmembers, there was a decrease in EBV-specific T-cell function during and after flight. However this effect, except for landing, was not observed for CMV in long-duration flight.

Even though some changes in salivary cortisol levels as well as their circadian rhythm were observed in both ISS and Space Shuttle crewmembers, the differences were not significantly different. Likewise, plasma cortisol was not significantly changed during short- or long-duration flights although elevated levels were found at landing for both Shuttle and ISS missions.

These data indicate that immune system dysregulation, a previously established post-flight phenomenon, actually occurs during spaceflight prior to any physiological stress associated with landing and readaptation. This means that flight-associated variables such as microgravity, radiation, or the unique stresses that occur during missions, do influence human immunity. These may include microgravity exposure, confinement, disrupted circadian rhythms, or the physiological stress associated with spaceflight itself. The extremely busy work schedules associated with a short duration Space Shuttle mission also have the potential to impact immunity. In fact, stress effects on crewmember immunity, ahead of any in-flight variables, have been documented during L-10 to L-3 pre-flight studies. During ISS missions, it is far more likely that the observations do reflect ‘space normal’ for immunity during prolonged spaceflight. No matter which variable, or combination of variables, caused the observed in-flight changes, we now consider it established that immunity is dysregulated during spaceflight. The clinical consequences of immune system dysregulation during short-duration space flight are likely to be low for short duration space missions. If immune dysregulation were found to persist for the duration of an exploration class deep space mission, consisting of elevated radiation exposure, limited clinical care, and other exploration-specific variables, clinical risk for adverse health events would likely be substantially elevated.

NOTE: changed end date to 9/30/2013 per B. Crucian (Ed., 2/4/2013)

NOTE: End date is 3/29/2013 per HRP Master Task List dtd 7/12/2011 (Ed., 8/9/2011)

NOTE: End date now 9/30/2013 per JSC (09/2010)

NOTE: End date changed to 9/30/2011 per B. Corbin/JSC (3/2009)

NOTE: End date changed to 5/31/2011 per PI ; original end date was 4/2010 (2/09)

Samples for Integrated Immune per crew time point are 18.5 ml blood, 1 ml liquid saliva a dry saliva sample (pre-, in- and post-flight) and 4.0 ml of a 24hr urine pool (pre- and post-flight only). In-flight urine will be obtained via sample sharing if any other in-flight study is making the collection. The assays included in the study and the responsible laboratory are as follows:

JSC Immunology Laboratory

Leukocyte subsets

T cell function

Intracellular/secreted cytokine profiles

Mercer University

Plasma cytokine balance

Leukocyte cytokine RNA

Microgen Laboratories

Virus specific T cell number

Virus specific T cell function

Plasma stress hormones

Antiviral antibody titers

JSC Microbiology Laboratory

Latent herpesvirus reactivation (saliva/urine)

Saliva/urine stress hormones

Circadian rhythm analysis

As this study has progressed, 2 formerly long duration subjects with mission durations of ~45 days were reclassified as short duration. This allowed the short duration component to be completed on schedule, thus saving program resources, and also provided additional subject slots for full 6-month duration crewmembers. In all cases, no adverse clinical events except some bruising related to venipuncture have been reported. Most all urine and saliva samples were collected as planned.

From a technical perspective, the Integrated Immune science continues according to plan. All in-flight samples have been collected within 24-48 hours of landing/undocking, and for all in-flight samples the cellular viability upon sample processing has been acceptable. To date, live cells are being returned to Earth within the required timeframe to allow an in-flight determination of immune cell functional capabilities. The investigator team is grateful to the mission planners, schedulers, and crewmembers for enabling this to occur.

The science team is satisfied with the data obtained thus far. The distribution of the peripheral leukocyte populations, T cell functional characteristics, viral-specific immune parameters, the status of latent herpesvirus reactivation, and physiological stress has been determined for all completed subjects. For ISS subjects, 2-3 data collections have occurred per mission, allowing a determination of the kinetics related to observed changes. In most cases, this is completely novel data and represents our first comprehensive observation of the in-flight status of the immune system.

The final completed short duration data was recently presented at the 2010 NASA HRP Investigators Workshop. The abstract for that presentation may be found here: http://www.dsls.usra.edu/meetings/hrp2010/pdf/Immunology/1151CrucianMehta-IntImm.pdf In general, the data show that alterations in the distribution and function of the peripheral leukocytes, as well as alterations in viral specific immunity, physiological stress, and latent viral reactivation occur during short-duration spaceflight.

Mid-point long-duration data was presented at the April 2011 Humans in Space meeting, Houston, Texas. This presentation is completed and the long duration data are intriguing. The abstract for the ISS presentation may be found here: http://www.dsls.usra.edu/meetings/IAA/pdf/2104.pdf .

Also, via collaboration with the NASA-JSC Nutrition Laboratory and the in-flight Nutrition flight study (SMO-016E), the assessment of plasma cytokines, an original component of the Immunology flight study, has been expanded to include additional in-flight timepoints. The additional samples were provided by the Nutrition Laboratory, and augment the timepoints originally baselined by the Immunology flight study. This is fortuitous, because Immunology samples are collected during high-stress docked-operations timeperiods, near to undocking. Nutrition samples are collected away from docked-operations, and frozen for later transport to Earth. The joint-analysis allows a more complete sampling for both studies, including both high-stress and lower-stress in-flight periods. The initial joint-study plasma cytokine data was recently presented at the February 2012 NASA Human Research Program Investigators' Meeting. The abstract may be found here: http://www.dsls.usra.edu/meetings/hrp2012/pdf/4165.pdf

This flight study is progressing well from a technical perspective, with robust crew participation and consistent positive feedback from the crewmembers. To date, the data indicate that some of the parameters that define spaceflight-associated immune dysregulation do indeed persist for the duration of a 6-month ISS mission. This may represent a clinical risk to crewmember during exploration-class missions. Risks could include hypersensitivities, autoimmunity, infection, and malignancies. The ISS data will become better defined from a statistical perspective as the study progresses and the ‘n’ increases. Following completion of this study, it is expected that a monitoring strategy may be defined, that focuses on the most relevant parameters that are altered in-flight.

NOTE: End date is 3/29/2013 per HRP Master Task List dtd 7/12/2011 (Ed., 8/9/2011)

NOTE: End date now 9/30/2013 per JSC (09/2010)

NOTE: End date changed to 9/30/2011 per B. Corbin/JSC (3/2009)

NOTE: End date changed to 5/31/2011 per PI ; original end date was 4/2010 (2/09)

Samples for Integrated Immune per crew time point are 18.5 ml blood, 1 ml liquid saliva a dry saliva sample (pre-, in- and post-flight) and 4.0 ml of a 24hr urine pool (pre- and post-flight only). In-flight urine will be obtained via sample sharing if any other in-flight study is making the collection. The assays included in the study and the responsible laboratory are as follows:

JSC Immunology Laboratory: Leukocyte subsets; T cell function; Intracellular/secreted cytokine profiles

Mercer University: Plasma cytokine balance; Leukocyte cytokine RNA

Microgen Laboratories: Virus specific T cell number; Virus specific T cell function; Plasma stress hormones; Antiviral antibody titers

JSC Microbiology Laboratory: Latent herpesvirus reactivation (saliva/urine); Saliva/urine stress hormones; Circadian rhythm analysis As this study has progressed, 2 formerly long duration subjects with mission durations of ~45 days were reclassified as short duration. This allowed the short duration component to be completed on schedule, thus saving program resources, and also provided additional subject slots for full 6-month duration crewmembers. In all cases, no adverse clinical events except some bruising related to venipuncture have been reported. Most all urine and saliva samples were collected as planned.

From a technical perspective, the Integrated Immune science continues according to plan. All in-flight samples have been collected within 24-48 hours of landing/undocking, and for all in-flight samples the cellular viability upon sample processing has been acceptable. To date, live cells are being returned to Earth within the required timeframe to allow an in-flight determination of immune cell functional capabilities. The investigator team is grateful to the mission planners, schedulers and crewmembers for enabling this to occur.

The science team is satisfied with the data obtained thus far. The distribution of the peripheral leukocyte populations, T cell functional characteristics, viral-specific immune parameters, the status of latent herpesvirus reactivation and physiological stress has been determined for all completed subjects. For ISS subjects, 2-3 data collections have occurred per mission, allowing a determination of the kinetics related to observed changes. In most cases, this is completely novel data and represents our first comprehensive observation of the in-flight status of the immune system.

The final completed short duration data was recently presented at the 2010 NASA HRP Investigators Workshop. The abstract for that presentation may be found here: http://www.dsls.usra.edu/meetings/hrp2010/pdf/Immunology/1151CrucianMehta-IntImm.pdf In general, the data show that alterations in the distribution and function of the peripheral leukocytes, as well as alterations in viral specific immunity, physiological stress and latent viral reactivation occur during short-duration spaceflight.

Mid-point long-duration data will be presented at the April 2011 Humans in Space meeting, Houston, Texas. This presentation is completed and the long duration data are intriguing. The abstract for the ISS presentation may be found here: http://www.dsls.usra.edu/meetings/IAA/pdf/2104.pdf .

This flight study is progressing well from a technical perspective, with robust crew participation and consistent positive feedback from the crewmembers. To date, the data indicate that some of the parameters that define spaceflight-associated immune dysregulation do indeed persist for the duration of a 6-month ISS mission. This may represent a clinical risk to crewmember during exploration-class missions. Risks could include hypersensitivities, autoimmunity, infection and malignancies. The ISS data will become better defined from a statistical perspective as the study progresses and the ‘n’ increases. Following completion of this study, it is expected that a monitoring strategy may be defined, that focuses on the most relevant parameters that are altered in-flight.

NOTE: End date now 9/30/2013 per JSC (09/2010)

NOTE: End date changed to 9/30/2011 per B. Corbin/JSC (3/2009)

NOTE: End date changed to 5/31/2011 per PI ; original end date was 4/2010 (2/09)

Samples for Integrated Immune per crew time point are 18.5 ml blood, 1 ml liquid saliva a dry saliva sample (pre-, in- and post-flight) and 4.0 ml of a 24hr urine pool (pre- and post-flight only). In-flight urine will be obtained via sample sharing if any other in-flight study is making the collection. The assays included in the study and the responsible laboratory are as follows:

JSC Immunology Laboratory: Leukocyte subsets, T cell function, Intracellular/secreted cytokine profiles

Mercer University: Plasma cytokine balance, Leukocyte cytokine RNA

Microgen Laboratories: Virus specific T cell number, Virus specific T cell function, Plasma stress hormones, Antiviral antibody titers

JSC Microbiology Laboratory: Latent herpesvirus reactivation (saliva/urine), Saliva/urine stress hormones, Circadian rhythm analysis The vast majority of the Integrated Immune in-flight blood collections have been successful. Only 3 short duration subjects have been dismissed, due to insufficient in-flight samples. To date, only 2 long duration subjects have been dismissed. One of these was due to insufficient samples at the late timepoint, and the other was due to compatibility reasons. In addition, 2 formerly long duration subjects with mission durations of ~45 days were reclassified as short duration. This allowed the short duration component to be completed on schedule, thus saving program resources, and also provided additional subject slots for full 6-month duration crewmembers. In all cases, no adverse clinical events except some bruising related to venipuncture have been reported. Most all urine and saliva samples were collected as planned.

From a technical perspective, the Integrated Immune science continues according to plan. All in-flight samples have been collected within 24-48 hours of landing/undocking, and for all in-flight samples the cellular viability upon sample processing has been acceptable. To date, live cells are being returned to Earth within the required timeframe to allow an in-flight determination of immune cell functional capabilities. The investigator team is grateful to the mission planners, schedulers and crewmembers for enabling this to occur.

The science team is satisfied with the data obtained thus far. The distribution of the peripheral leukocyte populations, T cell functional characteristics, viral-specific immune parameters, the status of latent herpesvirus reactivation and physiological stress has been determined for all completed subjects. For ISS subjects, 2-3 data collections have occurred per mission, allowing a determination of the kinetics related to observed changes. In most cases, this is completely novel data and represents our first comprehensive observation of the in-flight status of the immune system.

The final completed short duration data was recently presented at the 2010 NASA HRP Investigators Workshop. The abstract for that presentation may be found here:

http://www.dsls.usra.edu/meetings/hrp2010/pdf/Immunology/1151CrucianMehta-IntImm.pdf

In general, the data show that alterations in the distribution and function of the peripheral leukocytes, as well as alterations in viral specific immunity, physiological stress and latent viral reactivation occur during short-duration spaceflight. An analysis of the ISS data will be necessary to determine if these changes persist or resolve during prolonged space missions. The investigator team is currently analyzing the ISS data to date for an official mid-point assessment.

Conclusion

This flight study is progressing well from a technical perspective, with robust crew participation and consistent positive feedback from the crewmembers. To date, the data indicate that some of the parameters that define spaceflight-associated immune dysregulation from prior post-flight studies seem to be in-flight phenomenon as well. Although this does not indicate immediate illness, if these alterations were to persist for the duration of exploration-class missions there could be clinical risk. Risks could include hypersensitivities, autoimmunity, infection and malignancies. The data will become better defined from a statistical perspective as the study progresses and the ‘n’ increases. Following completion of this study, it is expected that a monitoring strategy may be defined, that focuses on the most relevant parameters that are altered in-flight.

NOTE: End date changed to 9/30/2011 per B. Corbin/JSC (3/2009)

NOTE: End date changed to 5/31/2011 per PI ; original end date was 4/2010 (2/09)

Samples for Integrated Immune per crew time point are 18.5 ml blood, 1 ml liquid saliva a dry saliva sample (pre-, in- and post-flight) and 4.0 ml of a 24hr urine pool (pre- and post-flight only). In-flight urine will be obtained via sample sharing if any other in-flight study is making the collection. The assays included in the study and the responsible laboratory are as follows:

JSC Immunology Laboratory: Leukocyte subsets ; T cell function ; Intracellular/secreted cytokine profiles

Mercer University: Plasma cytokine balance ; Leukocyte cytokine RNA

Microgen Laboratories: Virus specific T cell number ; Virus specific T cell function ; Plasma stress hormones ; Antiviral antibody titers

JSC Microbiology Laboratory: Latent herpesvirus reactivation (saliva/urine) ; Saliva/urine stress hormones

Circadian rhythm analysis

Of 12 planned in-flight blood collections for long-duration crewmembers to date, 11 were successful. Of 11 planned in-flight blood collections for short-duration crewmembers, 9 were successful and 1 was completed partially. The combined success rate for in-flight blood collections is 20 out of 23. In all cases, no adverse clinical events except some bruising related to venipuncture have been reported. All urine and saliva samples were collected as planned.

From a technical perspective, the Integrated Immune science continues according to plan. All in-flight samples have been collected within 24-48 hours of landing/undocking, and for all in-flight samples the cellular viability upon sample processing has been acceptable. To date, live cells are being returned to Earth within the required timeframe to allow an in-flight determination of immune cell functional capabilities. The investigator team is grateful to the mission planners, schedulers and crewmembers for enabling this to occur.

The science team is satisfied with the data obtained thus far. The distribution of the peripheral leukocyte populations, T cell functional characteristics, viral-specific immune parameters, the status of latent herpesvirus reactivation and physiological stress has been determined for all completed subjects. For ISS subjects, 2-3 data collections have occurred per mission, allowing a determination of the kinetics related to observed changes. In most cases, this is completely novel data and represents our first comprehensive observation of the in-flight status of the immune system.

Although the ‘n’ completed to date is too low to allow statistical calculations, a summation of the data through the indicated 'n' follows:

Immunophenotype:

• No in-flight changes in bulk leukocyte subsets • Postflight granulocytosis • Late in-flight/postflight elevated B cells, reduced NK cells • In-flight, postflight trend toward elevated CD4:CD8 ratio, elevated memory T cell subsets • In-flight elevated effector memory, central memory T cells • No change in peripheral constitutively activated T cells

T cell function:

• Reduced T cell function consistently observed early in-flight (shuttle + ISS early), with at some indication of partial recovery (note: number of long-duration subjects at later in-flight points is reduced). • Dysregulated cytokine production profiles observed in-flight. The most consistently observed alteration is an in-flight reduction in IFNg production following activation, at levels similar to post-flight observations.

Viral specific immunity:

• Reduced function of EBV specific T cells in-flight and post-flight.

Latent herpesvirus reactivation:

• CMV reactivation via urine DNA analysis elevated post-flight. In-flight determination in-progress. • VZV reactivation via salivary DNA analysis elevated in-flight and post-flight. In-flight data indicate no resolution as long-duration missions progress.

Conclusion

This flight study is progressing well from a technical perspective, with robust crew participation and consistent positive feedback from the crewmembers. To date, the data indicate that some of the parameters that define spaceflight-associated immune dysregulation from prior post-flight studies seem to be in-flight phenomenon as well. Although this does not indicate immediate illness, if these alterations were to persist for the duration of exploration-class missions there could be clinical risk. Risks could include hypersensitivities, autoimmunity, infection and malignancies. The data will become better defined from a statistical perspective as the study progresses and the ‘n’ increases. Following completion of this study, it is expected that a monitoring strategy may be defined, that focuses on the most relevant parameters that are altered in-flight.

NOTE: End date changed to 9/30/2011 per B. Corbin/JSC (3/2009)

NOTE: End date changed to 5/31/2011 per PI ; original end date was 4/30/2010 (2/09)

SMO 015. The objective of this experiment is to understand the effects of space flight on the human immune system, and determine any clinical risk for exploration related to immune dysregulation. Numerous investigations have demonstrated a decrease in specific immune cell functions following space flights of varied duration. Should it persist for extended durations, this decrease in host defense may increase the potential for illness in crewmembers. To assess this, crewmember white blood cells collected during flight will be tested for changes in function or response to stimulation. The concentrations of factors that regulate immune function will also be determined. These data will be correlated with reactivation and shedding of latent herpes viruses and measurements of stress hormones. This information is needed to determine the crewmembers’ risk of adverse clinical events related to immunology that may occur during space flight, and in particular for exploration-class missions.