The cytokine profile of human NKT cells and PBMCs is dependent on donor sex and stimulus
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Sex-related variations in natural killer T (NKT) cells may influence immunoregulation and outcome of infectious and autoimmune diseases. We analyzed sex-specific differences in peripheral blood NKTs and peripheral blood mononuclear cells (PBMCs) from men and women and determined the frequencies of NKT cells and their subpopulations [CD4+; CD8+; double negative (DN)] and the levels of cytokine production following stimulation with the NKT cell ligands α-Galactosylceramide (αGalCer) and Entamoeba histolytica lipopeptidephosphoglycan (Lotter et al. in PLoS Pathog 5(5):e1000434, 2009). Total and DN NKT cells were more abundant in women than in men. In women, αGalCer induced higher production of intracellular IFNγ, IL-4, IL-17 and TNF by CD4+ and DN+NKT cells. Both ligands induced expression of multiple cytokines in PBMCs and influenced the ratio of NKT cell subpopulations during long-term culture. Although the sex-specific differences in frequencies of NKT cells and their subpopulations were marginal, the significant sex-specific differences in cytokine production might influence disease outcomes.
KeywordsSex difference Immune response NKT cells Human αGalCer Entamoeba histolytica Cytokines Glycolipid
Entamoeba histolytica lipopeptidephosphoglycan
Immune responses differ between the sexes. In addition to behavioral, genetic, and hormonal factors, differences in the abundance and activation of various types of immune cells could explain some of the observed sexual dimorphisms in infectious diseases [2, 3]. Sex-specific differences in immune responses of women and men could underlie the higher susceptibility of men to infectious diseases caused by bacteria, viruses and parasites, e.g., tuberculosis , influenza A  and amebiasis [6, 7, 8]. By contrast, women exhibit more vigorous humoral and cellular immune responses and are more susceptible to cell-mediated autoimmune diseases [9, 10].
The relative proportions of certain immune cell populations differ between men and women. Men have higher monocyte counts  but a lower percentage of T lymphocytes within the total lymphocyte population . A rare T cell subset, the invariant natural killer T (NKT) cells, also exhibits sex-related differences in frequency and is more abundant in women than in men [11, 12, 13]. NKT cells have immunomodulatory properties, bridge the innate and adaptive immune response and play crucial roles in a variety of infectious diseases, autoimmune disorders and cancers [14, 15, 16]. They express an invariant T cell receptor (TCR) that consists of the Vα24-Jα18/Vβ11 chains in humans and Vα14-Jα18/Vβ8.2 chains in mice. Based on their expression of CD4 and CD8, NKT cells can be divided into three subpopulations: CD4+, CD8+ and CD4−CD8− (double negative; DN) . Whereas conventional T cells recognize protein antigens in the context of MHC class I and II molecules, NKT cells recognize exclusively glycolipid and lipid antigens presented by the MHC I-like molecule CD1d . Activated NKT cells can produce different pro- and anti-inflammatory cytokines, including interferon (IFN) γ, tumor necrosis factor α (TNF), interleukin (IL)-2, IL-4, IL-17, and tumor growth factor (TGF) β [18, 19, 20]. The strongest NKT cell activator identified to date is α-Galactosylceramide (αGalCer), a lipid molecule originally isolated from a marine sponge [21, 22]. Although NKT cells are attractive targets for immunotherapies, the very strong activation by αGalCer limits the clinical utility of this compound. Therefore, more moderate ligands provided by other microorganisms should be considered .
We recently isolated a glycolipid molecule from the membrane of a protozoan parasite, the Entamoeba histolytica lipopeptidephosphoglycan (EhLPPG). EhLPPG induces substantial IFNγ production in murine NKT cells in a CD1d-dependent manner, and treatment of mice with EhLPPG considerably reduces abscesses in a murine model of hepatic amebiasis .
NKT cells are modulated by sex hormones such as 17β-estradiol and testosterone, leading to sex-specific differences in NKT-mediated immune responses. For example, treatment of mice with αGalCer increases the serum IFNγ levels in females, but not in males. This difference is absent in estrogen receptor-deficient mice or ovariectomized mice . By contrast, testosterone substitution lowers the female resistance to hepatic amebiasis by decreasing IFNγ production in NKT cells .
In this study, we measured the frequencies of NKT cell and their subpopulations in men and women and analyzed the cytokines by stimulation with αGalCer and EhLPPG. We also analyze the cytokine induction also in bystander cells and investigated the influence of αGalCer and EhLPPG on NKT cell subpopulations upon enrichment.
Materials and methods
Human subjects and isolation of PBMCs
Peripheral blood mononuclear cells (PBMCs) from buffy coats or fresh blood samples from blood donors [23 men (31–53; 43.7 years ±5.8] and 22 women [33–51; 42.7 years ±6.1)] were used for cytokine analysis of NKT cells following stimulation with αGalCer and EhLPPG. The average age of the blood donors ranged between 31 and 54 years (43.3 ± 5.8). Samples were subjected to flow cytometer, ELISA or used to generate APCs from CD14+ monocytes. Buffy coats for isolation of PBMCs were kindly provided by the Department of Transfusion Medicine of the University Clinic Hamburg-Eppendorf. The cohort size is indicated individually for each experiment. All experiments were approved by the ethical review committee of the medical council of Hamburg (PV3551).
Briefly, PBMCs were isolated by density-gradient centrifugation in Biocoll (Biochrom AG). The leukocyte ring was removed and washed twice with PBS. The resultant pellet was resuspended in 1 ml of X-VIVO™ 15 (LONZA) supplemented with 1 % Pen/Strep (AppliChem Panreac) or in RPMI 1640 (10 % FCS, 1 % l-Glutamine, 1 % Pen/Strep). PBMCs were then used in human NKT cell or stimulation assays or for generation of APCs.
Intracellular cytokine production in human NKT cells
The NKT cell cytokine assay was a modification of a protocol described by Sandberg et al. . In brief, 1 × 106 human PBMCs/well were cultured in quadruplicates in 96-well round-bottom plates in X-VIVO™ 15 (LONZA) supplemented with 1 % Pen/Strep (AppliChem Panreac). Cells were stimulated with 1 µg/ml αGalCer or 10 µg/ml purified EhLPPG. To achieve optimal NKT cell activation without additional APCs, 3 µg/ml purified αCD28 was added to each well as a co-stimulant. Cells were incubated for 15 h at 37 °C in a humidified atmosphere containing 5 % CO2. After 1 h, 10 µg/ml brefeldin A was added to the culture to stop Golgi transport. After incubation, cells were harvested and NKT cells were assayed for cytokine production by staining with anti-IFNγ-PE/Cy7, anti-TNFα-FITC, anti-IL-4-PE and anti-IL-17A-BV421 antibodies. NKT cells were stained with anti-CD3-PerCP and anti-TCR Vα24-Jα18-APC. All antibodies were obtained from BioLegend. Flow cytometry was performed on a FACS LSRII instrument (BD Biosciences).
Supernatants of an intracellular cytokine assay from a representative donor were tested for 12 analytes (IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-17A, IL-17F, IL-21, IL-22, IFNγ and TNFα) using the multi-LEGENDplex™ analyte flow assay kit (BioLegend). Briefly, antibodies specific for the 12 analytes were conjugated to 12 different fluorescence-encoded beads. The beads were mixed with serum samples (diluted twofold), incubated with shaking for 2 h at room temperature, washed, and incubated for 1 h with a cocktail of 12 different biotinylated detection antibodies. Finally, streptavidin-PE was added, the samples were incubated for 30 min, and the beads were washed and analyzed.
Generation of APCs from CD14+ monocytes
To generate APCs, PBMCs were labeled with BD IMag™ Anti-Human CD14 Magnetic Particles (BD Biosciences). Next, 1 × 106 CD14+ monocytes were plated in 6-well plates in 5 ml of RPMI (10 % FCS, 1 % l-Glutamine, 1 % Pen/Strep) with 500 U/ml recombinant human IL-4 (Sigma) and 500 U/ml human GM-CSF (MACS Miltenyi Biotec). On days 3 and 6, half of the media was replaced with fresh media containing IL-4 and GM-CSF. Immature APCs were harvested on day 7 or 8 and used in in vitro NKT cell stimulation assays. APC purity was determined by flow cytometry after staining with anti-CD11c-BV421, anti-HLA-DR-PE and anti-CD14-AF700 antibodies. All antibodies were obtained from BioLegend. The negative PBMC fraction lacking CD14+ monocytes was frozen in liquid nitrogen prior to use.
NKT/PBMC cell activation without or with the addition of separately generated autologous APCs
For the NKT/PBMC cell assay without APCs, total 5 × 106 PBMCs/well were stimulated with 10 µg/ml αGalCer or 20 µg/ml EhLPPG in 100 µl of RPMI 1640 (10 % FCS, 1 % l-Glutamine, 1 % Pen/Strep) and incubated for 48 h at 37 °C under 5 % CO2. For the NKT/PBMC cell assay using the resultant APCs, 1 × 105 APCs/well were stimulated with 10 µg/ml αGalCer or 20 µg/ml EhLPPG in 50 µl of RPMI 1640 (10 % FCS, 1 % l-Glutamine, 1 % Pen/Strep) and incubated for 3–4 h at 37 °C under 5 % CO2. Next, 5 × 106 thawed PBMCs from the negative (CD14+ monocyte-depleted) PBMC fraction described above was added in 50 µl of RPMI 1640 to the stimulated APCs, and the mixed samples were incubated for 48 h at 37 °C under 5 % CO2. Both assays were run in duplicate. After 48 h, supernatants were collected and assayed for IFNγ using ELISA MAX™ Standard SET Human IFNγ (BioLegend).
Enrichment of human NKT cells
Enrichment of NKT cells was performed as described previously by Watarai et al. . In brief, 2 × 106 PBMCs were plated in 24-well plates in 1 ml of RPMI (10 % FCS, 1 % l-Glutamine, 1 % Pen/Strep) and specifically stimulated with either 100 ng/ml αGalCer or 400 ng/ml EhLPPG to expand NKT cells at 37 °C, 5 % CO2. To encourage proliferation and maturation of T cells, 20 U/ml recombinant human IL-2 (rh-IL-2) (Cell Sciences) was added to all wells. rh-IL-2 alone was used as a negative control. Every 3–4 days, half of the media was replaced with fresh media containing 20 U/ml rh-IL-2. On day 9, 5 × 104 APCs stimulated for 24 h with recombinant human IL-4, GM-CSF and αGalCer or EhLPPG, respectively, were added to the expanding cells. Success of expansion was determined on day 0, 8 and 16 via flow cytometry to detect total NKT cells and NKT cell subpopulations.
NKT cell frequencies and cytokine production were compared between women and men by Mann–Whitney U test. Comparisons of the cytokine production in stimulant-responding men and women (responders) and of NKT cell subsets following expansion were performed by unpaired Student’s t test. Differences were considered to be significant if p values were as follows: *p < 0.05; **p < 0.005; ***p < 0.0005.
Women tend to have higher total and DN NKT cell frequencies than men
Comparison of intracellular cytokine production by total NKT cells from men and women
CD4+ NKT and DN NKT cells are the major cytokine-producing NKT subpopulations in both sexes
IL-17A production was observed primarily following αGalCer stimulation of CD4+ and DN NKT cells, and little IL-17A was produced following stimulation with EhLPPG of DN NKT cells. The proportion of CD4+ NKT cells was significantly higher in women than in men (p = 0.03; Fig. 3d).
CD4+ NKT cells were the main cell producers of IL-4 following αGalCer and EhLPPG stimulation, and these cells were more abundant in women following αGalCer stimulation (women: 17.9 %; men: 9.5 %; Fig. 3e). EhLPPG induced a higher percentage of CD4+IL-4+ NKT cells in men (men: 22.9 %; women: 14.5 %). Women had a significantly higher percentage of DN IL-4+ NKT cells (8.1 %; *p = 0.03) than men, whereas EhLPPG stimulation induced no sex-specific difference in the percentage of DN IL-4+ NKT cells.
In summary, these data revealed significant sex-specific differences in IL-17A and IL-4 production in CD4+ and DN NKT cells, respectively. The CD4+ and DN NKT subpopulations were the main cytokine producers in women and men.
Sex-specific IFNγ production by bystander cells following αGalCer stimulation and addition of autologous APCs
Expression of multiple cytokines is induced in PBMCs following αGalCer and EhLPPG stimulation
In summary, stimulation with αGalCer and EhLPPG resulted in the expression of a variety of Th1- and Th2 cytokines in a reverse dose-dependent manner.
The ratio of NKT cell subpopulations in enriched NKT cells is sex specific and stimulus dependent
In summary, we found that αGalCer, but not EhLPPG, increased NKT cell numbers during long-term culture of PBMCs and both stimuli induced a sex-specific shift in the relative proportions of the NKT cell subpopulations.
Immune responses differ between the sexes, and differences in the frequency and activation of immune cells could contribute to the observed sex-specific differences in infectious and autoimmune diseases. NKT cells are important immune modulators involved in early control of an infection [14, 27]. As shown recently, the cytokine profile of these cells is modulated by sex hormones; specifically, estrogens increase and testosterone decrease the secretion of the proinflammatory cytokine IFNγ [24, 25]. In this study, we characterized the sex-specific differences in NKT cell frequency and cytokine production in response to the strong NKT cell activator αGalCer and the moderate NKT cell activator EhLPPG.
Several studies report that the frequency of NKT cells is higher in women than in men [11, 12, 13]. We also observed higher NKT cell numbers in women, although the differences were not statistically significant. By contrast, other studies observed no correlation between sex and NKT cell frequency [19, 28]. Various studies report different ratios of NKT cell subpopulations in each sex. We observed slightly higher percentages of CD4+ and CD8+ NKT cells in men and higher percentages of DN NKT cells in women. Two other studies also observed higher CD4+ NKT cell numbers in men [11, 13], whereas another study observed the opposite distribution in this subpopulation and more CD8+ and DN NKT cells in men . Consistent with our results, Kee et al.  also observed a higher frequency of DN NKT cells in the peripheral blood of women. These differences in the observed frequencies of NKT cells and their subpopulations may be due to the quantity or age of the subjects in each study. Consistent with this idea, several studies showed that NKT cell frequency significantly decreases with age [28, 29, 30, 31]. Moreover, the sex-specific difference in the abundance of NKT cells may have an impact on diseases with sexual dimorphism. Human DN NKT cells mainly produce Th1 cytokines . Therefore, the elevated percentage of DN NKT cells in women suggests that production of protective IFNγ during infection will be higher in women than in men. Parasite-induced amebic liver abscess (ALA) has a strong sex bias toward men, and a mouse model of the disease exhibits a similar preference toward males; however, the resistance of female mice is abolished in mice lacking NKT cells and IFNγ .
Sex-specific differences in cytokine secretion were documented for several immune cell populations. Phytohemagglutinin-stimulated lymphocytes from male blood donors produced higher levels of IFNγ and IL-2 and lower levels of IL-4 and IL-10 than those from females . Additionally, following LPS stimulation monocytes from men produce more TNFα, IL-1β and IL-12 than those from women . In the mouse model of ALA, NKT cells from female mice produce more IFNγ than NKT cells from male mice  and female mice have higher serum IFNγ levels following αGalCer treatment . In part due to the extremely low number of circulating NKT cells in men (0.001–0.1 %), little is known about sex-specific differences in the cytokine production by human NKT cells. In this study, however, we were able to analyze intracellular cytokine production by peripheral NKT cells by flow cytometry. We found that stimulation with the strong NKT cell activator αGalCer resulted in significantly more TNFα and IL-17A-positive NKT cells and a trend toward more IFNγ and IL-4-positive NKT cells in female blood donors. By contrast, EhLPPG stimulation induced no sex difference in the production of IFNγ, TNFα or IL-4 by NKT cells and a non-statistically significant increase in the percentage of IL-17A-positive NKT cells in women. Among the three NKT cell subpopulations, CD4+ and DN NKT cells were the major cytokine producers. In particular, CD4+ and DN NKT cells from female blood donors produced significantly more IL-17A after αGalCer stimulation. In addition, more IL-4+DN NKT cells were present in samples from female blood donors. EhLPPG induced no significant sex difference in the cytokine production by NKT cells, although a tendency toward sex-specific differences could be observed for IL-17A+CD4+, IL-17A+DN and IL-4+CD4+ NKT cells. By contrast, mitogen stimulation of sorted human NKTs resulted in higher proportions of IFNγ-, TNFα- and MIP-1-α-producing NKT cells in men than in women, but no difference in IL-4 production by NKT cells .
In another study using in vitro expanded human NKT cells, αGalCer stimulation induced Th1 and Th2 cytokines in all three NKT subpopulations, but cytokine production was higher in CD8+ NKT cells than in CD4+ and DN NKT cells . Analysis of cytokine production by PBMCs following specific NKT cell activation by αGalCer or EhLPPG revealed that non-NKT cells also contribute to cytokine production, putatively via indirect activation, which might reflect the immunoregulatory properties of NKT cells. In this study, we observed induction of a variety of cytokines, including the Th1 cytokines IFNγ, TNF, IL-17 A/F and IL-2 and the Th2 cytokines IL-4 and IL-10, as well as a strong induction of the pluripotent cytokine IL-6.
This observation might indicate that NKT cells express a sex-specific cytokine repertoire that differs depending on the used stimulant. A specific ligand like αGalCer may induce a gene expression pattern that involves sex hormone receptors, leading to a sex-dependent cytokine production. By contrast, a less specific mitogenic ligand like phytohaemagglutinin may induce more general regulation of immunity-related gene expression and might reflect the chromosomal repertoire of possible cytokine production.
The nature of the stimulus may also be important for the sex specificity of APCs such as plasmacytoid DCs (pDCs), which influence the subsequent cytokine production in responding immune cells. For example, pDCs from women produce higher levels of IFNα following ligation of the TLR7-agonist imiquimod, a heterocyclic amine, than pDCs from men . Moreover, immune cells from female mice and rats express higher levels of pathogen recognition receptors, such as TLR2, 3 and 4, and mount a more efficient immune response against specific pathogens than those from male rats . In our assays, we also observed a sex-specific influence of APCs on immune activation. Although sex-specific differences in cytokine production were also observed in the absence of separately generated autologous APCs, addition of these cells revealed more pronounced differences between the sexes.
The stimulus could also modulate the ratio of NKT cell subpopulations in a sex-dependent manner. Along these lines, we found that αGalCer induced strong NKT cell enrichment and favored the development of higher proportions of CD4+ NKT cells in PBMC cultures from female blood donors and higher proportions of CD8+ and DN NKT cells in the cultures from male blood donors. EhLPPG induced no enrichment of NKT cells, but still influenced the ratio of the NKT cell subpopulation over the course of long-term culture. Under these conditions, we observed a shift toward the CD4+ NKT cell subpopulation in men and CD4+ and DN NKT cell subpopulation in women.
Therefore, because EhLPPG facilitates NKT cell activation through engagement of TLR2, TLR4 and TLR6 , we hypothesize that EhLPPG provides a survival signal that is sufficient to alter the ratio of the NKT cell subpopulations.
In conclusion, we showed that women tend to have higher NKT cell frequencies than men and that their NKT cells produce significantly more TNFα and IL-17A following αGalCer stimulation. Moreover, a strong stimulus like αGalCer is more likely to induce distinct and significant sex-specific differences in the cytokine profile of human NKT cells than a more moderate or weaker stimulus such EhLPPG. Some stimuli may induce sex differences and a sex-specific outcome of a disease, whereas others may induce similar immune responses in both men and women. Sex hormones and X-chromosome-linked immune regulatory genes are thought to be major players in sex-specific immune responses, but the precise details of their regulatory biology and interactions remain unclear. Although some immune cell subsets exhibit sex-specific differences in frequency, the quality of the mounted immune response is likely to be far more important for sexual dimorphism in disease than the quantities of immune cells involved.
This work was supported by grants of the Collaborative Research Center 841, Bonn, Germany; the Bernhard Nocht Institute for Tropical Medicine, the Werner-Otto Stiftung and the “Vereinigung der Freunde des Tropeninstituts Hamburg, E.V.,” Hamburg, Germany.
Compliance with ethical standards
Conflict of interest
The authors do not have a commercial or other association that might pose a conflict of interest (e.g., pharmaceutical stock ownership, consultancy, advisory board membership, relevant patents, or research funding). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No information within the manuscript beyond the cited literature has been published by another journal.
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