B cell homeostasis is maintained during long-duration spaceflight

Abstract

Long-duration spaceflights reportedly induce immune dysregulation, which is considered a risk to astronaut safety and mission success. Recent studies have examined the impact of spaceflight on markers of adaptive and innate immunity, but no study, to date, has comprehensively evaluated humoral immunity and serological markers of B cell function. The aim of this study was to characterize changes in B cell numbers and phenotypes, along with plasma Igs and polyclonal free light chains (FLCs)—near-“real-time” biomarkers of Ig synthesis—in response to an ~6-mo mission to the International Space Station (ISS). Whole-blood samples were collected before flight, during flight (“Early flight,” “Mid-flight,” and “Late flight”), immediately upon return, and during a recovery period (R + 18, R + 30/R + 33, and R + 60/R + 66) from 23 ISS crew members. B Cell counts and phenotypes were measured throughout the duration of the mission, along with total plasma Ig and FLC levels. There was no effect of spaceflight on the number and proportion of the different B cell subsets. There was no difference in kappa FLC between preflight samples and either in-flight or recovery samples (P > 0.05), and only a marginal reduction was observed in lambda FLC levels upon return to Earth (P < 0.05). Furthermore, IgG and IgM remained unchanged during and after spaceflight compared with preflight values (P > 0.05). Of note, plasma IgA concentrations were elevated in-flight compared with baseline and recovery values (P < 0.05). These results indicate that B cell homeostasis is maintained during long-duration spaceflight, advocating for potential in-flight vaccination as viable countermeasures against viral reactivation during exploration-class missions.

INTRODUCTION

Long-duration spaceflights are associated with alterations to both innate and adaptive immunity (11, 24), including impaired cytokine response to antigenic stimuli (8, 49), natural killer (NK) cell cytotoxicity (46), neutrophil and monocyte oxidative burst capacity (30, 31), and lymphocyte distribution and proliferative capacity (40). As exposure to pathogens (45) and rate of latent viral reactivations (35, 36) are known to increase in space, profound immune dysregulation would likely have great clinical and operational significance during perennial missions. Logistical constraints unique to spaceflight have compelled the majority of space immunology research to be collected using short-duration missions or by comparing pre- to postflight measures of immune function. Whereas these studies provided valuable insights on the immune status of astronauts upon return on Earth, the magnitude of immune alterations during spaceflight is less clear. Furthermore, few studies have attempted to characterize comprehensively B cell and plasma cell homeostasis during orbital missions.

Effective humoral immunity is of fundamental importance to ensure adequate destruction of extracellular pathogens and the control of intracellular viral infections. As such, humoral immunity relies on the induction of different effector functions to antibody production, and any alteration in B cell production, ability to differentiate, and Ig output from plasma cells could lead to profound immune dysregulation and potentially endanger crew safety. The use of ground-based analogs to spaceflight, including hind-limb unloading, has highlighted significant reductions in B cell progenitor cells in the bone marrow of mice that led to reduced B lymphopoiesis (32). Furthermore, short-duration spaceflight missions have been shown to impact negatively cell phenotypes in the bone marrow (41) and splenic B cell counts in mice (21), whereas simulated microgravity reduces the hematopoietic stem cell proliferation rate (42). Although these changes appear to promote reductions in Ig output in animals following spaceflight (33), without affecting the breadth of the Ig repertoire (58), they may not translate to humans, as preflight Ig levels remain unchanged in cosmonauts following 6 mo in the International Space Station (ISS) (46). With the suggestion that exploration crew members receive in-flight vaccination against certain latent herpesviruses, to maintain optimal protection throughout the duration of an ~3-yr mission (13), optimal B cell homeostasis is likely to be of paramount importance to preserve vaccine response and crew member health. Unfortunately, however, those studies failed to measure in-flight changes in B cell phenotypes and Ig concentrations.

A limitation of the measurement of intact, circulating Igs, such as total plasma IgG, IgA, and IgM concentrations, is their slow clearance via cellular catabolism that confers them a relatively long biological half-life (1–3 wk) (16). As this limits assessment of shorter-term changes to Ig production, plasma free light chains (FLCs), with a short half-life of 2–6 h (16), are conventionally used for the diagnosis, prognostication, and monitoring of plasma cell dyscrasias (43, 44). In this context, plasma Ig FLCs are considered to be a sensitive barometer of plasma cell activation and immune competency (28, 39). Ig FLCs are produced by activated plasma cells during antibody synthesis at a rate influenced by the magnitude of immune activation (28, 57). Elevated FLC levels are indicative of inflammation and have been associated with metabolic disorders and type 2 diabetes (4, 27), chronic low-grade inflammation (6), myeloma (53), and mortality in the general population (17), whereas low levels identify immune suppression (28). Consequently, plasma Ig FLCs are likely the ideal candidates to detect early immune suppression in astronauts, and the characterization of the effects of long-duration spaceflight on plasma Ig FLC is of paramount importance.

The successful implementation of exploration-class missions to Mars or other near-Earth objects requires a better understanding of the impact of long-duration spaceflight on the immune system to evaluate the risks of crew adverse health events associated with immune dysregulations. The aim of this study was to assess the impact of long-duration spaceflight on B cell and plasma cell homeostasis by comprehensively analyzing changes in B cell number, phenotype, and soluble markers of humoral function during a 6-mo mission in the ISS.

MATERIALS AND METHODS

Subjects and Study Design

Data for this study were collected from two independent [National Aeronautics and Space Administration (NASA)]-funded studies: The “Integrated Immune” study, conducted at NASA–Johnson Space Center (JSC; B.C.) and the “Salivary Markers” study conducted at the University of Houston (R.J.S.). A total of 23 ISS crew members (15 from Integrated Immune and eight from Salivary Markers), ranging in age from 37 to 57 yr old (three women, age 47.1 ± 5.6 yr) were enrolled in this study. The ISS crew was affiliated with NASA, European Space Agency, Japanese Aerospace Exploration Agency, or Canadian Space Agency and participated in a 6-mo mission to the ISS. Data were collected over 18 separate ISS missions. Additionally, six ground-based controls (one woman, age 33.0 ± 7.1 yr) were enrolled in this study, as part of Salivary Markers, to ensure assay validity. The Institutional Review Board at Louisiana State University, the University of Houston, and NASA–JSC approved the study, and informed consent was obtained from all subjects.

Sample Collection

For both studies, plasma samples were collected in lithium heparin tubes, and whole-blood samples were collected in acid-citrate dextrose (ACD) tubes for cellular phenotyping during the Salivary Markers study (Vacutainer; Becton Dickinson, Franklin Lakes, NJ), before, during, and after the 6-mo mission in the ISS.

Specifically, plasma samples from the Integrated Immune study were collected at 180 and 45 days before launch (L − 180 and L − 45), 10 days after launch [“Early”; flight day (FD)-10], “Mid” (FD-90), last day in space [“Late”; FD-180/return (R) − 1], upon return on Earth (R + 0), and R + 30 from 15 ISS crew members. Plasma samples collected during the Salivary Markers study were drawn at L − 180, L − 60, FD-10, FD-90, FD-180/R − 1, R + 0, R + 18, R + 33, and R + 66 from eight astronauts and six ground-based controls. Blood samples used for B cell phenotyping were collected during Salivary Markers on eight ISS crew members in a Vacutainer, supplemented with an acidified glucose nutrient solution (ACD) to maintain optimal cellular viability during sample return on Earth. Technical constraints did not allow for timely return of whole blood on Earth at FD-10, and consequently, no B cell phenotype data were measured at that time point.

A temperature-controlled environment (temperature range: 6–24°C) was maintained during the 24–36 h of sample storage/transport to the laboratories at NASA–JSC and the University of Houston. Upon arrival to the respective laboratories, the plasma samples were centrifugally separated from whole blood and stored at −80°C, and B cell phenotype was characterized using whole-blood samples from the ACD tubes. Cryopreserved plasma samples were transported to the ImmunoEnergetics Laboratory at Louisiana State University, and freshly isolated B cell phenotypes were characterized on a BD Accuri C6 flow cytometer (BD Biosciences, San Jose, CA) at the University of Houston. Lymphocyte counts were determined using a BC-3200 auto hematology analyzer (Mindray, Mahwah, NJ).

Urine and saliva samples were collected during the Integrated Immune study to determine cytomegalovirus (CMV), Epstein-Barr virus (EBV), and varicella zoster virus (VZV) viral load in astronauts throughout the mission; results are reported in Mehta et al. (35, 36). In brief, aliquots of urine were sampled from a 24-h urine pool at each of the aforementioned time points, and fasting saliva samples were collected using sterile Salivette cotton rolls (Sardstedt, Newton, NC) immediately upon awakening. The urine samples were frozen until return to Earth, whereas the Salivette was stored in stability buffer (0.5% SDS, 10 mM Tris-Cl, and 1 mM EDTA) at room temperature for up to 2 wk before return to Earth for subsequent analysis (37). Viral DNA was extracted and quantified by Mehta et al. (38) at NASA–JSC by PCR, as described previously.

B Cell Phenotyping

B Cells were labeled with directly conjugated MAb and analyzed using four-color flow cytometry, as previously described (54). Briefly, aliquots of 50 µl whole blood were incubated with 5 µl each MAb for 30 min at room temperature in the dark. The following MAb were used to stain the cells: anti-CD20-FITC Clone #LT20 and anti-CD43-phycoerythrin (PE) Clone #84-3C1 (eBioscience, San Diego, CA); anti-IgD-PE Clone #IA6-2 and anti-IgM-PE Clone #G20-127 (BD PharMingen, San Diego, CA); anti-IgG-PE Clone #H2 (SouthernBiotech, Birmingham, AL); and anti-CD27-peridinin-chlorophyll Clone #0323, anti-CD25-allophycocyanin (APC) Clone #CD25-4E3, anti-CD5-APC Clone #L17F12, and anti-CD38-APC Clone #HIT2 (eBioscience). With the use of these MAb, CD20⁺ B cells were classified into immature (CD20⁺/CD43⁻/CD27⁻/IgD⁻), naive/transitional (CD20⁺/CD43⁻/CD27⁻/IgD⁺), and memory B cells (CD20⁺/CD43⁺/CD27⁺/CD38⁻); plasmablasts/plasma cells (CD20⁺/CD43⁺/CD27⁺/CD38^hi); B1 cells (CD20⁺/CD43⁺/CD27⁺/CD5⁻); regulatory B cells (CD20⁺/CD43⁺/CD27⁻/CD5⁺); IgG⁺ memory B cells (CD20⁺/CD27⁺/IgG⁺); and IgM⁺ B cells (CD20⁺/CD27⁺/IgM⁺; Table 1). Following incubation, erythrocytes were lysed by increased osmotic pressure, induced by the addition of 500 µl red blood cell lysis buffer (eBioscience) for 20 min at room temperature. Samples were then washed twice with PBS and resuspended in 250 µl PBS to be analyzed on a BD Accuri C6 flow cytometer. Flow cytometry analysis was conducted on the BD Accuri proprietary flow cytometry analysis software. Total cell numbers of each B cell subset were determined by the multiplication of the percentages of cells expressing each marker of interest by the total lymphocyte count.

Table 1.

Phenotypic characterization of the different B cell subsets

Cell Type	Phenotype	Reference
B Cells	CD20+	31a
Immature B cells	CD20+/CD43−/CD27−/IgD−	28a, 47a, 59a
Naïve/transitional B cells	CD20+/CD43−/CD27−/IgD+	28a, 47a, 59a
IgM+ B cells	CD20+/CD27+/IgM+	4a
Memory B cells	CD20+/CD43+/CD27+/CD38−	42a
IgG+ memory B cells	CD20+/CD27+/IgG+	4a
B1 cells	CD20+/CD43+/CD27+/CD5−	22a
Regulatory B cells	CD20+/CD43+/CD27−/CD5+	34a
Plasmablasts/plasma cells	CD20+/CD43+/CD27+/CD38hi	42a

Ig Analyses

Plasma samples from the 23 crew members (Integrated Immune, n = 15; Salivary Markers, n = 8) were thawed, and a total volume of 100 μl was analyzed for kappa (κ) and lambda (λ) FLCs using commercially available ELISAs (Seralite; Abingdon Health, Oxford, UK), using previously published methods (7). Briefly, diluted plasma samples were incubated in 96-well plates, precoated with either anti-κ or anti-λ FLC MAb at room temperature for 60 min. Following initial incubation, the wells were washed four consecutive times and incubated with horseradish peroxidase-labeled anti-κ or anti-λ detection antibody for 30 min at room temperature. After another washing step, the presence of κ or λ FLC in the plasma samples was detected using a colorimetric reaction and read on a SpectraMax i3× plate reader (Molecular Devices, San Jose, CA). The color intensity was directly correlated with the κ and λ FLC concentration in the samples. Plasma cystatin C was measured with a commercially available ELISA (R&D Systems, Minneapolis, MN) and used to calculate estimated glomerular filtration rate (eGFR) throughout the missions, based on an established algorithm (47). This estimate of renal function was used to account for changes in renal clearance of FLC in response to spaceflight.

Total IgA, IgM, and IgG were measured in a total of 150 μl thawed plasma sample from all astronauts and corresponding controls using commercially available ELISA kits (eBioscience).

Statistical Analysis

A longitudinal, repeated-measures design was used to determine the effects of long-term exposure to microgravity on proportions and numbers of the different B cell subsets, κ and λ FLCs, and Ig levels. Linear mixed models were used to evaluate potential differences in the main and interaction effects of time (L − 180, L − 45 to 60, early flight, mid-flight, late flight, R + 0, R + 1, R + 18, R + 30 to 33, and R + 66) after controlling for a potential change in eGFR. When a significant time effect was observed, post hoc tests were performed with Bonferroni correction. Data are presented as means ± SE. All statistical analyses were performed using SPSS version 24 (IBM, Armonk, NY), and significance was set at P < 0.05.

RESULTS

Effect of Long-Duration Spaceflight on Circulating B Cell Subsets

Cell counts for the different B cell subsets isolated from crew members throughout the Salivary Markers study are presented in Table 2. There was no change in the percentages of total B cells within the lymphocyte population or in the total number of B cells during the 6-mo mission (F_{B cell frequency} = 1.603, p_{B cell frequency} = 0.142; F_{B cell count} = 0.248, p_{B cell count} = 0.972), where F is the F-statistic, and p is the P value. Furthermore, long-duration spaceflight was not associated with any statistically significant change in plasma cells, immature, naïve/transitional, memory, and regulatory B cells; and B1 cells (P > 0.05) in crew members.

Table 2.

Changes in the numbers (cells/100 µl) of B lymphocyte lineage cells in 6-mo ISS crew members (n = 8)

	Sample Time Point
	L − 180	L − 60	FD-90/Mid-Flight	R − 1/Late Flight	R + 0	R + 18	R + 33	R + 66	Main Effect of Time F-Statistic (P value)
Total B cells, cells/100 µl
ISS crew members (n = 8)	12,128 ± 4,080	15,316 ± 3,036	21,892 ± 12,747	24,920 ± 14,955	19,865 ± 13,955	14,279 ± 6,157	13,831 ± 3,272	11,856 ± 4,376	0.248 (0.972)
% Lymphocytes
ISS crew members (n = 8)	10 ± 4	12 ± 3	13 ± 4	11 ± 4	13 ± 7	11 ± 5	11 ± 4	9 ± 4	1.603 (0.142)
Immature B cells
ISS crew members (n = 8)	482 ± 271	625 ± 568	2,618 ± 2,936	2,508 ± 3,785	2,322 ± 3,534	1,022 ± 1,233	866 ± 768	602 ± 645	0.665 (0.702)
Naïve/transitional B cells
ISS crew members (n = 8)	7,091 ± 3,751	9,787 ± 4,250	11,083 ± 5,605	12,896 ± 6,237	11,196 ± 7,842	7,855 ± 3,706	8,045 ± 3,292	7,044 ± 3,447	0.626 (0.733)
IgM+ B cells
ISS crew members (n = 8)	53 ± 155	11 ± 13	16 ± 22	29 ± 45	34 ± 46	6 ± 9	10 ± 11	11 ± 11	0.702 (0.670)
Memory B cells
ISS crew members (n = 8)	179 ± 158	208 ± 133	327 ± 243	409 ± 310	381 ± 327	202 ± 141	394 ± 477	196 ± 122	1.878 (0.080)
IgG+ memory B cells
ISS crew members (n = 8)	74 ± 177	38 ± 29	67 ± 63	41 ± 46	42 ± 43	57 ± 95	61 ± 73	28 ± 21	1.004 (0.432)
B1 cells
ISS crew members (n = 8)	123 ± 117	211 ± 256	225 ± 177	283 ± 357	242 ± 216	211 ± 171	167 ± 221	306 ± 383	0.932 (0.485)
Regulatory B cells
ISS crew members (n = 8)	129 ± 104	177 ± 64	288 ± 240	269 ± 151	312 ± 494	152 ± 51	252 ± 206	202 ± 124	0.943 (0.476)
Plasmablasts/plasma cells
ISS crew members (n = 8)	104 ± 86	178 ± 155	169 ± 102	197 ± 177	185 ± 202	161 ± 157	99 ± 93	173 ± 233	0.225 (0.979)

Average values are presented ± SD. FD-90, 90 days after launch (flight day); ISS, International Space Station; L − 180/L − 60, 180 and 60 days before launch; R − 1, last day in space before return (R).

Long-Duration Spaceflight and Blood Igs

Plasma intact Ig concentration during a 6-mo mission in the ISS.

There was no change in plasma IgG and IgM concentrations in astronauts throughout the mission (P > 0.05). The impact of long-duration spaceflight on total plasma Ig concentrations is presented in Table 3. Astronauts exhibited an increase in plasma IgA during flight compared with baseline values (L − 60/45; F = 7.077; P < 0.001). Upon return on Earth, plasma IgA concentrations decreased from in-flight levels and were back to preflight values (L − 60/L − 45) during recovery (R + 30; P = 0.047). All changes withstood adjustment for latent viral reactivation status and DNA load, along with eGFR.

Table 3.

Changes in plasma IgA, IgM, and IgG in astronauts before, during, and following 6 mo in the ISS (n = 23)

		L − 180	L − 60/L − 45	Early Flight	FD-90/Mid-Flight	R − 1/Late Flight	R + 0	R + 18	R + 30/R + 33	R + 66
IgA (mg/dl) ± SD	Crew members (n = 23)	131.51 ± 63.73	112.73 ± 49.64	126.60 ± 58.05*	136.38 ± 68.12*	140.58 ± 75.08*	126.64 ± 59.56	103.14 ± 32.67	112.46 ± 55.11	101.73 ± 27.51
IgG (mg/dl) ± SD	Crew members (n = 23)	1,218.41 ± 231.66	1,190.41 ± 253.88	1,181.47 ± 310.80	1,235.41 ± 277.07	1,249.98 ± 298.99	1,184.38 ± 209.64	1,359.33 ± 304.05	1,226.67 ± 399.88	1,267.50 ± 255.92
IgM (mg/dl) ± SD	Crew members (n = 23)	373.51 ± 546.67	408.84 ± 530.97	476.08 ± 665.62	422.57 ± 476.36	410.57 ± 491.51	346.95 ± 360.77	651.04 ± 751.93	295.78 ± 268.07	471.20 ± 324.15

Average values are presented ± SD. FD-90, 90 days after launch (flight day); ISS, International Space Station; L − 180/L − 60/L− 45, 180 and 60/45 days before launch; R − 1, last day in space before return (R).

^*P < 0.05, significant differences from baseline preflight values (L − 180 and L − 60/L − 45).

Plasma FLC concentration during a 6-mo mission in the ISS.

The effects of long-duration spaceflight on κ and λ FLCs, along with the ratio of κ/λ and total FLCs, are presented in Fig. 1. There was no effect of spaceflight on plasma κ FLC (P > 0.05), and only a minor decrease in the concentration of plasma λ FLC was observed immediately upon return on Earth (R + 0) in crew members compared with in-flight plasma λ FLC concentrations (Early: P = 0.03; Mid: P = 0.005; Late/R − 1: P = 0.012). The preferential reduction in plasma λ FLC at landing without any change in plasma κ FLC concentration led to a minor decrease in the κ/λ ratio at the mid-flight time point compared with baseline L − 60/L − 45 and return R + 0 and R + 30 κ/λ ratio (p_{L − 45} = 0.029; p_{R + 0} = 0.037; p_{R + 18} = 0.053 ; p_{R + 33} = 0.037). As plasma FLC levels can be impacted by altered production from plasma cells and/or impaired clearance from renal metabolism, cystatin C was measured to calculate eGFR (1) and account for variation in renal function during spaceflight. There was no change in kidney function during flight (P > 0.05); however, postflight eGFR values (R + 30) were significantly lower than preflight values (L − 60/L − 45; P = 0.015). Subtle changes in plasma FLC withstood adjustment for eGFR.

Fig. 1.

Changes in kappa (A) and lambda (B) free light chains (FLC) in 6-mo International Space Station astronauts (n = 23). The ratio of kappa/lambda and total FLC are presented in C and D, respectively. Mean values are presented ± SE. Significant differences between immediately postflight values (R + 0; *P < 0.05) and baseline values (L − 45; ^aP < 0.05). L, days before launch; Early, 10 days after launch; Mid, 90 days after launch; Late, last day in space; R, days after return on Earth. Horizontal lines represent where the significant differences are located.

In related analyses, we explored whether latent viral reactivation was associated with FLC levels in blood. CMV, EBV, and VZV reactivation status and viral DNA load from a subset of samples (Integrated Immune study) were included in the model as covariates. The rate and magnitude of latent viral reactivation observed in the Integrated Immune cohort are described elsewhere (51). In brief, 47% (7/15) of astronauts exhibited CMV reactivation at any point during the mission, 73% (11/15) of astronauts exhibited EBV reactivation at any point during the mission, and 60% (9/15) of astronauts exhibited VZV reactivation at any point during the mission. There was no difference in plasma κ and λ FLC concentrations in astronauts who exhibited CMV and EBV reactivation at any point during the mission (P > 0.05), and there was no association between CMV and EBV DNA load and plasma FLC at any time point (P > 0.05). When VZV DNA load was included in the model, the greater plasma λ FLC concentration observed in-flight compared with postflight values was associated with the magnitude of VZV reactivation (F_{VZV DNA load} = 4.937; p_{VZV DNA load} = 0.029).

DISCUSSION

The rapid progress made in aerospace technologies over the past 50 yr has dramatically increased the scope and duration of manned space-exploration missions. In addition to the novel technological challenges, safe implementation of exploration-class missions requires a profound understanding of the impact that prolonged exposure to space environments has on astronauts’ biology and health. In particular, numerous studies have raised concerns about the potential clinical risks associated with reported immune impairments observed during spaceflight (10, 11, 14, 50), such as reduced T cell (25, 26, 34) and NK cell functions (46), sustained production of proinflammatory cytokines (15, 35), altered neutrophil and monocyte microbicidal activity (30, 31), diminished antimicrobial protein concentration, and increased rate and magnitude of latent viral reactivation (36, 51). However, no study, to date, has attempted to characterize comprehensively the impact of long-duration spaceflight on humoral immunity in humans. The goal of this study was to identify changes in a broad range of phenotypically distinct B cell subsets, along with secreted Igs and FLCs in astronauts who completed 6 mo on the ISS. We found no change in the number of total B cells in response to spaceflight; however, there was a trend for increased memory B cells during spaceflight compared with baseline values. Contrary to supposition, only marginal changes were observed in soluble biomarkers of B cell homeostasis during spaceflight. Indeed, we found no effect of spaceflight on plasma κ FLC, IgG, and IgM levels and only modest changes to plasma λ FLC concentrations. Interestingly, long-duration spaceflight increased plasma IgA levels, which with the consideration of the correlation between mucosal and plasma IgA (55), may represent alterations in mucosal immunity.

Spaceflight has been shown to alter human and animal leukocyte distribution (12, 48), but the majority of studies was focused on T cells and NK cells (22, 46) and was limited to either short-duration missions (8–15 days) (23, 52) or to comparisons between pre- and postflight. Our data are in accordance with previous findings showing that total B cell counts and proportion within the lymphocyte compartment are not affected by long-duration spaceflight (46). However, the more advanced and detailed phenotypic analysis performed in this study highlighted for the first time that spaceflight does not have a detrimental impact on the phenotypic composition of the B cell compartment. Indeed, memory B cell counts changed modestly in-flight, whereas the number of naive/transitional and regulatory B cells remained unchanged during the mission. Animal studies have shown that short-duration spaceflight (41) and ground-based analogs to spaceflight, such as hind-limb unloading (32), had deleterious impacts on immune cell phenotypes in the bone marrow and led to a reduction in de novo generation of B cells (32) and total splenic B cell numbers (21). As both bone marrow progenitor cells (18) and B cells are known to respond to acute stressors, such as intense exercise (54), it can be hypothesized that the changes in B cell lymphopoiesis, observed in animals immediately after short-duration spaceflight, may be due to a variety of factors other than microgravity, including landing-associated stressors, rather than exposure to the space exposome.

Effective humoral responses rely on B cell activation, differentiation, and antibody output. Although there was no effect of spaceflight on the number of circulating plasma cells in astronauts, it is interesting to note that they had a greater amount of B1 cells than ground-based controls (data not shown). As B1 cells have recently been characterized as preplasmablasts (9), a greater circulating number could be associated with the increased capability to produce antibodies following activation. Conflicting data exist on the effect of spaceflight on Ig outputs, with some studies showing a reduction in in vitro IgG production in response to altered gravity (19), whereas others showed no change from baseline in IgG production after a 10- to 15-day flight (52, 56) and no alteration in the B cell repertoire of unimmunized animals (58). Our data are in partial agreement with the current literature, as we found no difference in total plasma IgG and IgM at any point in the mission, even after controlling for latent viral reactivation and DNA load. Interestingly, however, astronauts exhibited an increase in total plasma IgA during flight. Elevated plasma IgA has been observed in the urodele amphibian Pleurodeles waltl, exposed to microgravity for 6 mo (5), but also in rodents and human subjects under chronic psychological and physiological stress (20, 60), similar to those experienced by the astronauts on board the ISS. Furthermore, although the in-flight increases in plasma IgA concentrations were statistically significant compared with baseline values, astronauts’ plasma IgA levels did not exceed the normal clinical range observed on Earth (59). Consequently, the fluctuations in plasma IgA observed in this study are likely attributable to long-duration exposure to spaceflight-associated psychological and operative stressors rather than to alterations in B cell function.

The immune impairments associated with prolonged exposure to myriad stressors specific to the spaceflight environment are known to result in increased latent CMV, EBV, and VZV reactivation (35, 36, 51). However, no study, to date, has attempted to determine whether potential alterations in B cell activation and function could play a role in latent viral reactivations in space. Whereas our data did not show any biologically relevant change in total plasma Ig concentrations in response to spaceflight, it could be argued that the relatively long half-life of intact plasma Ig (?21 days) curtails their sensitivity at detecting changes in B cell function (16). As such, we sought to determine changes in κ and λ Ig FLCs, a more sensitive barometer of immune competency with short half-life (κ: 2–4 h; λ: 3–6 h), due to rapid renal clearance (16). Plasma κ FLC levels remained unchanged during the missions; however, there was a slight decrease in the concentration of plasma λ FLC immediately upon return on Earth (R + 0) in crew members compared with in-flight plasma λ FLC concentrations. Whereas this reduction in λ FLC at R + 0 compared with in-flight values reached statistical significance, no crew member reached clinically significant low levels of λ FLC (29). As there was no change in kidney function in-flight, this preferential reduction in plasma λ FLC is not due to increased renal clearance. However, whereas there was no effect of CMV and EBV reactivation on λ FLC concentrations, the magnitude of VZV reactivation during flight was associated with the greater levels of λ FLC observed at the same time points. This discrepancy, in response, between plasma κ and λ FLC could be explained by the difference in size between both light chains. Indeed, although κ FLCs are produced in greater quantities than λ FLCs during antibody synthesis (57), the larger size of the dimeric λ FLCs impedes their clearance by the kidney (16). Our results suggest that the B cell ability to produce FLCs and consequently, Igs in response to a viral or vaccine challenge in space remains intact. It should, however, be noted that whereas we measured the quantity of plasma κ and λ FLCs and total Igs in crew members, we did not characterize the quality of these antibodies. Animal studies have indeed shown that whereas similar quantities of antibodies could be produced in response to antigenic challenge in space (3), the quality of the produced antibodies remained inferior to those produced on Earth (2). Consequently, although the present study shows that B cell homeostasis appears to be preserved during long-duration spaceflight, it could be hypothesized that suboptimal immune responses could still be observed upon antigenic challenge in space.

One limitation of our study lies in its retrospective nature. These results were obtained from samples collected during two previously completed NASA studies, and all plasma Ig and FLC analyses were performed on previously archived samples. As such, we were unable to measure the antibody response to a specific vaccine but rather, measured B cell homeostasis during long-duration spaceflight. Furthermore, the changes in B cell number and phenotype during spaceflight were only characterized on the Salivary Markers cohort (n = 8). Technical constraints unique to spaceflight research prevented the immediate analysis of the isolated B cell populations. To mitigate this limitation, we performed validation work in our laboratory before this study and found that B cell number and phenotype remained unaltered for 48 h when collected from a Vacutainer supplemented with an acidified glucose nutrient solution. Finally, we also collected blood samples from ground-based controls to parallel samples collected on the ISS, thus controlling for any potential ex vivo aging of the blood samples. Unfortunately however, as the ground-based control subjects were recruited to ensure assay validity, rather than to serve as paired controls with each astronaut, they were not age or sex matched with the different crew members. Future studies should attempt to recruit populations of ground-based controls that closely match the astronaut population to reduce further the effects of various confounding factors (age, sex, fitness level, etc.) on the observed changes in immune function during spaceflight.

In conclusion, this is the first study to show comprehensively that long-duration spaceflight in human astronauts has no—or very limited—effect on B cell number, phenotype, and antibody output. These important results suggest that plasma immune competency is maintained in microgravity and that future in-flight vaccine-based countermeasures are likely to be efficient at further protecting astronauts from immune dysregulation and symptomatic latent viral reactivations during prolonged exploration-class missions.

GRANTS

Funding for this study was provided by grants from NASA (Omnibus Grant NNX17AB16G to G. Spielmann and Grants SMO 015 to B. Crucian and NNX12AB48G to R. J. Simpson).

DISCLOSURES

J. Campbell reports minor shares in Abingdon Health, the company that produces one of the assay (FLC ELISA Kits) used in this project.

AUTHOR CONTRIBUTIONS

G.S. and J.C. conceived and designed research; G.S., N.A., H.K., R.J.S., B.C., S.M., M.L., and J.C. performed experiments; G.S., N.A., H.K., R.J.S., S.M., M.L., and J.C. analyzed data; G.S., N.A., H.K., and J.C. interpreted results of experiments; G.S. prepared figures; G.S. and B.C. drafted manuscript; G.S., N.A., H.K., R.J.S., B.C., S.M., M.L., and J.C. edited and revised manuscript; G.S., N.A., H.K., R.J.S., B.C., S.M., M.L., and J.C. approved final version of manuscript.