Academic Editor: Josef Jampílek
Background: We have previously shown that the anti-tumor activity of
human lymphocytes is diminished in vitro after 12-hours pre-exposure to
simulated microgravity (SMG). Here we used an immunocompromised mouse model to
determine if this loss of function would extend in vivo, and to also
test the efficacy of IL-2 and zoledronic acid (ZOL) therapy as a potential
countermeasure against SMG-induced immune dysfunction. We adoptively transferred
human lymphocytes that were exposed to either SMG or 1G-control into NSG-Tg
(Hu-IL15) mice 1-week after they were injected with a luciferase-tagged human
chronic myeloid leukemia (K562) cell line. Tumor growth was monitored 2x weekly
with bioluminescence imaging (BLI) for up to 6-weeks. Results: Mice that
received lymphocytes exposed to SMG showed greater tumor burden compared to those
receiving lymphocytes exposed to 1G (week 6 BLI: 1.8e
Immune dysregulation has been reported in astronauts following both short (14–17 days) [1, 2, 3, 4] and long (5–6 months) [5, 6] duration spaceflight. This includes diminished cell mediated immunity with a Th-2 cytokine shift, with latent viral reactivation being a frequent manifestation of immune dysregulation in astronauts. Many factors including microgravity, space radiation, isolation and confinement stress, altered circadian rhythms, nutritional deficiencies, and the gut microbiome are believed to play a role in altering astronaut immunity during extended space voyages [7].
A particular concern for astronauts is that space travel might impair anti-tumor
immune surveillance, increasing cancer risk in future exploration class mission
crew. We showed previously that the anti-tumor activity of natural killer (NK)
cells was diminished in crewmembers onboard the International Space Station [8].
We further demonstrated that pre-exposing human lymphocytes to 12 h of simulated
microgravity (SMG), impaired NK cell killing against a range of hematological
cancer cell lines in vitro when returned to the 1G environment [9]. The
SMG exposed NK cells expressed lower levels of the cytolytic granules perforin
and granzyme b, and were less capable of degranulation (CD107a+) and secreting
effector cytokines (e.g., TNF-
A limitation of our previous work was the reliance on in vitro assays
to assess the anti-tumor activity of human lymphocytes collected from astronauts
in space or following exposure to SMG [8, 9]. Unfortunately, these assays fail to
capture other key elements of the anti-tumor response unique to in vivo
setting including extravasation, homing, tumor infiltration, and killing.
Humanized mouse models offer an excellent platform to determine the anti-tumor
activity of human immune cells in vivo. NSG-Tg (Hu-IL15) animals harbor
Prkdc
Administration of subcutaneous interleukin-2 (IL-2) has shown to improve
anti-tumor activity of NK cells in pre-clinical mouse models [16, 17] and in
various clinical trials [18, 19, 20]. Zoledronic acid is an amino-bisphosphonate with
clinical therapeutic applications including improving bone mineral density [21, 22] and anti-tumor immunity through suppression of T
The aim of this study was to determine if pre-exposure to SMG would impair the anti-leukemic function of human lymphocytes in vivo using the NSG-Tg (Hu-IL15) mouse model (Fig. 1, Ref. [28, 29]). K562 leukemia bearing mice were engrafted with human immune cells exposed to either SMG or 1G and monitored for tumor growth over 6-weeks via bioluminescent imaging. Additionally, we tested if ZOL+IL-2 administered in vivo could revive SMG-induced loss of anti-leukemic function.
Experimental design.
Healthy adults between the age of 18–49 years volunteered for the study. Each participant provided written informed consent and had a single blood sample drawn by standard phlebotomy. Blood was collected in acid citrate dextrose (ACD) tubes and peripheral blood mononuclear cells (PBMCs) were isolated using density gradient separation method with Ficoll (Sigma-Aldrich, St. Louis, MO, USA) and processed immediately. All study procedures were approved by the Institutional Review Board (IRB) at University of Arizona.
We exposed human PBMCs to simulated microgravity (SMG) or 1-G control as previously described [9]. Briefly, a rotary cell culture system (RCCS) (Synthecon, Houston, TX, USA) was used to simulate microgravity. PBMCs were resuspended in 10% FBS+RPMI media in 10ml high-aspect ratio vessels (HARVs) and exposed to simulated microgravity for 12-hours while rotating on a horizontal axis at 10RPM. A 1G-control HARV was rotated on a vertical axis exposing PBMCs to similar shear stress while experiencing 1G gravitational force.
All mouse experiments were done in compliance with Institutional Animal Care and Use Committee (IACUC) guidelines at the University of Arizona under an approved protocol. NSG-Tg (Hu-IL15) animals were purchased from Jackson laboratory (Bar Harbor, ME, USA) and breeding colonies were maintained at the University Animal Care facility. Animals were housed for the duration of the experiments at the University Animal Care facility.
K562 (chronic myeloid leukemia) cell line was used to induce a wide-spread tumor
to evaluate systemic immune control of tumor growth and luciferase-tagged k562
made tumor growth monitoring viable. K562 cells also lack MHC1 molecules on their
surface making them susceptible to NK-cell killing. Animals were irradiated with
100 cGy in a Cesium
Subgroups of mice received an intraperitoneal injection of zoledronic acid (50
K-562-luc2 (ATCC® CCL-243-LUC2™) (American Type Culture Collection, Manassas, VA, USA), which is a K562 chronic myeloid leukemia cell line that has been transfected with luciferase, was used for the in vivo experiments [28, 29]. Tumor burden was measured immediately after D-luciferin (GoldBio, St Louis, MO) was injected intraperitoneally. A LagoX spectral imager (Spectral Instruments Imaging, Tucson, AZ) was used to quantify bioluminescent intensity (BLI) scores.
Antibodies (CD8 Vioblue, CD3 Viogreen, v
Statistical analysis was performed on Graphpad Prism 8.4.3 software (Graphpad,
San Diego, CA, USA). For tumor progression, bioluminescent intensity data was
log-transformed for normality as previously indicated [32]. A mixed effects model
was used to analyze differences between tumor growth with either 3 (e.g., SMG,
1G, vehicle) or 5 (e.g., SMG, SMG+ZOL+IL-2, 1G, vehicle, vehicle+ZOL+IL-2)
‘condition’ levels included in the model, along with a main effect for ‘time’ and
‘condition*time’ included as an interaction effect. When the interaction effect
was significant (indicating that tumor growth differed between conditions as the
experiment progressed), systematic pairwise comparisons were conducted with
Bonferroni correction to identify which conditions were significantly different
from each other over time. Peak BLI was analyzed using Friedman test for paired
comparisons. GVHD scores and engraftment data were analyzed using a linear mixed
model or a two-way RM ANOVA. A log-rank (Mantel-Cox) test was used for survival
analysis. Statistical significance was set at p
To evaluate the effect of SMG on anti-leukemia activity of human immune cells
in vivo we compared tumor growth between mice that were injected with
PBMCs exposed to SMG (TUMOR+SMG PBMCs) or 1G-control (TUMOR+1G PBMCs).
TUMOR+vehicle (PBS) was used as a control reference for unrestrained tumor
growth. Representative BLI images showing tumor dynamics in vivo are
shown in Fig. 2. For tumor growth, statistically significant effects were found
for time, condition, and time*condition (p
Bioluminescent intensity (BLI) images. Days 1, 12, 19, 22 and 28 of the experiment. Lane 1: tumor control, lanes 3, 5: tumor+ 1G PBMCs, lane 6: tumor+ SMG PBMCs, lane 8: PBMC control.
Peak tumor growth (as determined by highest BLI score) was significantly higher in the mice receiving SMG exposed PBMCs and vehicle compared to mice receiving 1G exposed PBMCs.
There were no significant differences for peak tumor growth between the SMG
exposed PBMC and vehicle conditions (p
Effect of SMG on effector immune cell function in
vivo. (a) shows effect of SMG exposure of immune cells on tumor growth
in vivo. (b) shows peak BLI scores during the experiment. (c) and (d)
show survival and GVHD incidence. N = 12, MEAN
To verify that exposure to SMG did not impair the PBMCs’ ability to engraft and
expand in a xenograft; mice were injected with PBMCs only and not tumor.
Engraftment was measured as a proportion of CD45human+ (CD45h+) cells in total
immune cells in mice blood collected at weekly intervals from week 2–5. The
engraftment dynamics of PBMCs in the mice were not statistically different
between SMG and 1G conditions (Fig. 4). This was true for total PBMCs as
defined by human CD45 expression as a percentage of the total human + mouse CD45
expressing cells (% p = 0.175; cells/uL, p = 0.628, Fig. 4a,c)
and for NK-cells (CD3-/CD56+) expressed as a percentage of CD45h+ cells (%
p = 0.219; cells/
Effect of exposure to SMG on human cell engraftment dynamics
in vivo in the absence of tumor. (a) and (c) show total PBMC
engraftment. (b) and (d) show NK cells engraftment. N = 10, MEAN
Effect of SMG on human cells engraftment
in vivo in the presence of tumor. (a) and (c) show total PBMC
engraftment. (b) and (d) show NK cells engraftment. N = 5, MEAN
In an attempt to ‘rescue’ the anti-leukemic effects of PBMCs pre-exposed to SMG
prior to adoptive transfer, we injected mice with ZOL+IL-2 on the day of PBMC
transfer and once weekly thereafter. For tumor growth, statistically significant
effects were found for time, condition, and time*condition (p
Effect of zoledronic acid+IL2 (ZOL+IL2) therapy on SMG induced
suppression of in vivo anti-leukemia activity. (a) shows effect of
ZOL+IL2 therapy on tumor growth in vivo. (b) shows peak BLI scores
during the experiment. (c) and (d) show survival and GVHD incidence. N = 5,
MEAN
To determine if ZOL+IL2 therapy increased the number and proportion of NK cells
and
Effect of SMG and ZOL+IL2 therapy on human cells
engraftment in vivo in the presence of tumor. (a) and (b) show total
PBMC. (c) and (d) show NK cell. (e) and (f) show
The overarching aims of this study were to determine if the loss of NK-cell function against a leukemic cell line in vitro due to exposure to simulated microgravity (SMG) would extend to an in vivo model, and to also test the efficacy of zoledronic acid and IL-2 (ZOL+IL-2) therapy as a countermeasure to SMG-induced immune dysfunction.
Using a human K562 tumor bearing immunocompromised mouse model, we found that
pre-exposing human PBMCs to SMG for 12 h inhibits their ability to control
leukemic growth in vivo after adoptive transfer, but systemic
administration of ZOL+IL-2 ‘rescued’ this SMG induced impairment of anti-leukemic
activity. These findings indicate that microgravity likely plays a contributory
role to immune system impairment during long duration space travel. Further,
ZOL+IL-2 (a treatment that is currently used to expand and increase the
anti-leukemic activity of NK-cells and
This is the first study, to our knowledge, to show that pre-exposing primary
human lymphocytes to SMG inhibits their ability to control leukemic growth
in vivo. Not only were the SMG exposed PBMCs less effective at
controlling leukemic growth in comparison to their 1G exposed counterparts, tumor
growth kinetics were identical between the SMG exposed and vehicle control
conditions indicating that SMG completely incapacitates the anti-leukemic
activity of human lymphocytes. This corroborates our previous findings that
pre-exposing human lymphocytes to SMG impairs their ability to kill various
hematologic cancer cells lines in vitro [34]. While the mechanisms
underpinning this response are not fully known, our prior in vitro
studies showed that SMG impairs several anti-tumor properties of NK-cells,
including their ability to deliver ‘lethal hits’ through cytotoxic degranulation
and secretion of effector cytokines [34]. Although it is possible that SMG could
have impaired human cell engraftment in the mouse, we somewhat alleviated this
potential confounder by showing similar levels of human leukocyte engraftment
between the SMG and 1G conditions. While blood levels of immune cells might not
be reflective of engraftment levels in tissues or in mice with tumor, this showed
that human immune cells (including NK cells) thrived similarly in mice after
exposure to 1G and SMG. In vivo expansion of (CD45h+CD3-CD56+) human NK
cells in the initial weeks was to be expected in an NSG-Tg (Hu-IL15) mouse model.
NSG-Tg (Hu-IL15) mice sustain IL-15 blood levels of 7.1 +/- 0.3 pg/mL [35]. These
levels are considerably higher than the usual undetectable levels in human blood
(
Hematological tumor lysis in a 4-hour in vitro killing assay using
mixed PBMCs is majorly attributed to the anti-tumor activity of NK cells;
however, the ability to control tumor growth in vivo over a 6-week
period might be a composite effect of all effector lymphocytes. While other
effector lymphocytes like CD8+ T cells could have played a role, we deem this
unlikely as GvHD scores (a manifestation of the human CD3+
There is a critical need to identify effective countermeasures that can mitigate
immune dysregulation during long duration spaceflight. Subcutaneous IL-2
injections are commonly used as an immunotherapeutic strategy to enhance
graft-versus-leukemia effects in bone marrow transplant recipients by
accelerating NK-cell reconstitution [38]. Additionally, zoledronic acid, an
amino-bisphosphonate that is used to prevent osteoporosis [21] and currently
being considered as a countermeasure to prevent decreases in bone mineral density
due to weightlessness [39], has also been shown to expand
In summary, we have demonstrated that exposure to SMG impairs the anti-leukemia activity of human effector immune cells in vivo as well as in vitro, and that ZOL+IL2 therapy improved the anti-leukemia activity of human effector immune cells after exposure to SMG. Limitations of the present study include the short-term exposure to microgravity, not identifying the lymphocyte subtypes affected by SMG, and the fact that all functional experiments were conducted in 1G. We conclude that microgravity plays a contributory role in the loss of tumor control and should be considered a risk factor for impaired anti-tumor immune responses during prolonged space voyages. Immuno-stimulating agents like ZOL+IL-2 may help mitigate clinical risks associated with immune dysregulation during prolonged spaceflight and its effectiveness as an immune system countermeasure in humans warrants investigation.
SMG, simulated microgravity; PBMC, peripheral blood mononuclear cells; NK cell,
Natural Killer cell; MHC, major histocompatibility complex; GVHD,
graft-versus-host-disease; ZOL, zoledronic acid; IL-2, interleukin-2; IL-15,
interleukin-15; TNF
RJS, PLM and EK designed the research. PLM, FLB, DMD and GMN performed the research. PLM, RJS, EK, MMM and BEC analyzed and/or interpreted the data. PLM and RJS wrote the manuscript. All co-authors edited the manuscript and approved its submission.
All study procedures were approved by the Institutional Review Board (IRB) at University of Arizona. All mouse experiments were done in compliance with Institutional Animal Care and Use Committee (IACUC) guidelines at the University of Arizona under an approved protocol.
The authors thank Emely Hoffman and Jessica Stokes for assistance with the animal work.
These studies were supported by NASA grants NNX16AB29G to RJS and 80NSSC19K1059 to RJS, EK and PLM.
The authors declare no conflict of interest.
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