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European Journal of Applied Sciences – Vol. 12, No. 2
Publication Date: April 25, 2024
DOI:10.14738/aivp.122.16867
Llanos, P. J., Andrijauskaite, K., Gangadharan, S., Morris, J., & Wargovich, M. J. (2024). Murine T Cells Exposure to Suborbital Space
Flight Aboard Blue Origin’s New Shepard Vehicle. European Journal of Applied Sciences, Vol - 12(2). 382-404.
Services for Science and Education – United Kingdom
Murine T Cells Exposure to Suborbital Space Flight Aboard Blue
Origin’s New Shepard Vehicle
Pedro J. Llanos
Applied Aviation Sciences Department, Embry-Riddle
Aeronautical University, Daytona Beach, Florida, United States of America
Kristina Andrijauskaite
Department of Molecular Medicine, UT Health
San Antonio, San Antonio, Texas, United States of America
Sathya Gangadharan
Department of Mechanical Engineering, Embry-Riddle
Aeronautical University, Daytona Beach, Florida, United States of America
Jay Morris
Department of Molecular Medicine, UT Health
San Antonio, San Antonio, Texas, United States of America
Michael J. Wargovich
Department of Molecular Medicine, UT Health
San Antonio, San Antonio, Texas, United States of America
ABSTRACT
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 the International Space Station are forcing scientists to find
alternative research platforms. In this study, we investigated whether 3.2 min
exposure to microgravity using a novel research platform, 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 the 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 in combination with IL-12. Our results indicate that
exposure to suborbital flight decreased the expression of T cells with CD4+ cells
being more sensitive to suborbital flight as compared to CD8+ cells. Although our
data indicate that the functional capabilities of flown T cells were reduced, our
findings suggest that supplementing cells with IL-2 and IL-12 cytokines may restore
suborbital flight-mediated cellular alterations. Finally, this study is an example of a
multidisciplinary team effort with expertise in science, microgravity, and
technology.
Keywords: microgravity, suborbital flight, T cells, cytokines, Blue Origin, suborbital space.
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Llanos, P. J., Andrijauskaite, K., Gangadharan, S., Morris, J., & Wargovich, M. J. (2024). Murine T Cells Exposure to Suborbital Space Flight Aboard
Blue Origin’s New Shepard Vehicle. European Journal of Applied Sciences, Vol - 12(2). 382-404.
URL: http://dx.doi.org/10.14738/aivp.122.16867
INTRODUCTION
There has been an escalated interest over the past few years by NASA and other private
stakeholders to pursue human spaceflight missions to the moon (e.g., ARTEMIS mission) and
beyond [1], [2], [3]. However, space is a hostile environment associated with numerous health
hazards, including dysregulation of the immune system [2], [4], [5], [6]. The body of research
conducted in this field is quite substantial, but with conflicting findings and unclear
mechanisms on how spaceflight induces immune system alterations. Therefore, more research
is needed to facilitate more definite conclusions and to set up basis for interventions [7].
Research findings derived from sampling astronauts and conducting in-vitro and in-vivo
experiments reveal that spaceflight affects several immune parameters including reduced
production of cytokines, decreased proliferation rates, and suppressed functional capabilities
of different types of immune cells [8]. However, several studies conducted in the 90s showed
that spaceflight enhanced the production of certain cytokines [9], [10] and had conflicting
effects on T cell subpopulations with several studies demonstrating a decreased expression of
CD4+ (helper cytokine production) and CD8+ (cytotoxic activity) cells [11], [12] while others
showing an increased expression of CD4+ cells post flight [13]. This may be attributed to
methodological alterations, such as the use of different stimulants (e.g., ConA,
phytohemagglutinin, PMA/I), diverse animal models, and the specific flight profiles of different
space missions. One of the mechanisms leading to dysregulated immune system during or after
space travel is susceptibility to infections and viruses [14]. The main defense machinery to fight
infections is through T lymphocytes (T cells). They coordinate the host response against
microbial and cancerous developments leading to elimination and long-term protection [15].
At first, naive T lymphocytes need to be activated so that they can differentiate into effector
cells to perform their immune functions. This requires engagement of TCR (T Cell Receptor) by
the peptide/major 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 activation, T cells proliferate and differentiate into effector T cells driven by
autocrine interleukin-2 (IL-2) and other cytokines. T cells treated with various concentrations
of interleukins IL-2 and IL-12 can have different responses for mediating inflammation and
other infectious events. The ideal environment to study microgravity induced alterations on
the immune system is the International Space Station (ISS). However, because of the cost and
long launch preparation times, novel research platforms exist to enhance our understanding of
the impact of microgravity on immunity. Those platforms include terrestrial spaceflight
simulations, parabolic flights, balloons, clinostats, rotating wall vessels, random position
machines, etc. However, it is necessary to distinguish the extent to which these platforms mimic
microgravity. Currently, there are a very few accessible U.S. national space research platforms
(Blue Origin’s New Shepard, Virgin Galactic’s Space Ship Two, Exos’ SARGE-1 rocket from
Aerospace Systems and Technologies) providing yearly launches with a quick turnaround
payload recovery and high quality continuous microgravity time of about 3-5 minutes
depending of the flight provider and respective rocket flight profile (~100-120 km) as well as
other European vehicles (MASER 14 –suborbital express) that can reach apogee altitude of
about 240 km providing approximately 6 minutes of microgravity. These different companies
and their vehicles give scientists opportunities to fly payloads at various apogees and diverse
microgravity exposure times [17]. See Table 1.
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European Journal of Applied Sciences (EJAS) Vol. 12, Issue 2, April-2024
Table 1: Different microgravity research platforms.
One such example of a novel research platform to study microgravity is suborbital flight which
provides researchers the opportunity to design experiments, refine hypotheses and collect
preliminary data, which could be further tested using orbital spaceflights. Furthermore, this
unique research platform allows for a more controlled experimental design because of its larger
flexibility and shorter sample retrieval time. In this study, we investigated the influence of an
altered gravity on murine T cells during the suborbital mission aboard Blue Origin’s New
Shepard rocket. Given T cells are sensitive to environmental changes and acknowledging
microgravity induced immune changes demonstrated by other researchers, we hypothesized
that even a brief exposure to microgravity (3.2 min) during the suborbital space flight would
lead to alterations of T cell behavior and function. Further, we added IL-2 and IL-12 alone or
combined at different time-points to the T cell cultures. Our findings demonstrate that even a
brief exposure of about 3.2 minutes to microgravity modulated the activity of T cells as
measured by the alterations in different T cell subsets, cell surface markers’ expression,
functional, and cytokine secretion capabilities. However, we do acknowledge that other
suborbital flight stressors, such as changes in temperature or vibrations, may have had an
impact on the study findings.
METHODS
Splenocytes Isolation and T cell Activation
Spleens were dissected from euthanized C57BL/6 healthy 6-8 months old female mice (n=3)
derived from the breeding colony which was maintained in UT Health Science Center
(UTHSCSA) at specific pathogen-free facility according to the Institutional Animal Use and Care
Committee (IACUC) standards under approved IACUC protocol (#2013044AR). Euthanasia was
administered in a bell jar with isoflurane as the inhalation for induction or as a route of
isoflurane administration. Single cell suspensions of lymphocytes were achieved by sterile
Platform Microgravity
level (g)
Duration Volume
(m3)
Waiting
Time
Cost/Experiment
($)
Free fall
towers
10-3 – 10-6 < 5 s < 1 months 5 k$
Parabolic
flights
10-2– 10-3 ~ 20-25 s > 10 Months -1
year
125 k$
Sounding
rockets
10-4 – 10-5 5-13 min < 1 > 2 years > 400 k$
ISS 10-2 – 10-5 weeks, months
to years
> 1 > 5 years 1-5 M$
Blue Origin’s
New Shepard
10-2– 10-3 3.2-3.4 min NanoLab (0.5 kg)
(10cmx10cmx20cm)
6 months
to 1year
~8,000-16,000
Blue Origin’s
New Shepard
10-2– 10-3 3.2-3.4 min 0.0489 (11.34 kg) 6 months
to 1year
≥100 k$
Exos’ SARGE
-1
~0.005 3-4 min 1U
(10cmx10cmx10cm)
to 2U
6 months
to 1 year
~ 5-10 k$
MASER-14 ≤ 10-4 ~ 6 min <1
(10cmx10cmx20cm)
1 year to 2
years
≥ 25 k$