Progesterone modulates the T cell response via glucocorticoid receptor-dependent pathways

Abstract

Problem:

Steroid hormones such as progesterone and glucocorticoids rise during pregnancy and are accountable for the adaptation of the maternal immune system to pregnancy. How steroid hormones induce fetal tolerance is not fully understood. We hypothesized that steroid hormones selectively regulate the T cell response by promoting T cell death.

Method of Study:

We incubated murine spleen cells isolated from non-pregnant and pregnant mice with physiological concentrations of steroid hormones in vitro and analyzed T cell subsets after 48 h of incubation.

Results:

We found that progesterone and the synthetic glucocorticoid dexamethasone induce T cell death. CD4⁺ regulatory T cells (T_reg) were refractory towards progesterone-induced cell death, in contrast to conventional CD4⁺ T cells, which resulted in a preferential enrichment of CD4⁺ T_reg cells in culture. T cells isolated from pregnant mice at early and late gestation showed comparable sensitivity to steroid-induced cell death. The target receptor for progesterone in immune cells is controversially discussed. We provide here support of progesterone binding to the glucocorticoid receptor as only T cells lacking the glucocorticoid but not the progesterone receptor showed resistance against progesterone-induced death.

Conclusions:

Our results indicate that high levels of progesterone during pregnancy can induce selective T cell death by binding the glucocorticoid receptor. Although physiological hormone concentrations were used, due to different bioavailability of steroid hormones in vivo these results have to be validated in an in vivo model. This mechanism might ensure immunological tolerance at the feto-maternal interface at gestation.

Introduction

Pregnancy is a unique immunological situation, as the maternal immune system has to tolerate the semi-allogenic fetus. The mother mounts an intricate endocrine and immune adaptation to pregnancy, and successful pregnancy maintenance has been attributed to the cross talk between hormones and the immune system. Steroid hormones such as progesterone and endogenous glucocorticoids (GCs) increase during pregnancy and are essential for pregnancy maintenance, as low levels of progesterone have been associated with spontaneous miscarriage ¹ and preterm labor ². Progesterone and also progestins such as dydrogesterone can shift the cytokine balance of immune responses towards an anti-inflammatory profile ^3–5 and induce the expansion of CD4⁺ and CD8⁺ regulatory T (T_reg) cells at the feto-maternal interface ^6–8. T_reg cells can inhibit effector T (T_eff) cells via different mechanisms such as the secretion of the immune-suppressive cytokine IL-10. T_reg suppress fetal rejection ⁹, but also account for the amelioration of autoimmune diseases observed during pregnancy, such as rheumatoid arthritis ^10,11, autoimmune hepatitis ¹² and multiple sclerosis ¹³.

A direct action of progesterone on T cells has long been postulated ^14–19. However, recent findings suggest that the conventional nuclear progesterone receptor (PR) might not be expressed or might be expressed at a very low level in T cells ^3,13. These contradictory observations on PR expression need further clarification. It has also been suggested that progesterone could modulate T cell function via binding to a membrane-bound PR expressed in T cells ^3,20,21 or exerts its effects on T cells via binding to the glucocorticoid receptor (GR). The GR is widely expressed in mouse and human leukocytes ²², belongs to the nuclear receptor superfamily and regulates gene transcription of inflammation related genes, ultimately leading to immune suppression ²². Via GR-dependent mechanisms, apoptosis of cells can be induced, a pathway through which GC e.g. eliminate thymocytes during positive and negative selection ^23,24. GC-induced apoptosis also affects mature T cells, albeit sensitivity is reduced compared to immature thymocytes ^23,25. Moreover, the mechanism of mature peripheral T cell apoptosis triggered via the GR is still unresolved ^25–27.

Emerging evidence indicates that progesterone can induce apoptosis of conventional CD4⁺ T cells, whereas T_reg are refractory to progesterone-induced cell death ¹³. However, it remains elusive if T cell sensitivity to steroids such as progesterone, progestins or (synthetic) GC is altered during gestation, when steroid hormone concentrations increase substantially. In the present study, we aimed to elucidate the effects of progesterone, the progestin dydrogesterone and the synthetic GC dexamethasone (DEX) on the induction of T cell death, using cells from pregnant and non-pregnant mice. Furthermore, we investigated the sensitivity of different T cell subpopulations to progesterone-induced death induction and to which extend the GR or PR are involved in such processes.

Materials and Methods:

Mice:

C57BL/6J wild type male and female mice were purchased from Charles River, GR^fl/flLck^cre (Nr3c1^tm2GSc; Tg^{(Lck-cre)1Cwi}) mice were previously described ^28–30 and PR^fl/flLck^cre were generated in our laboratory by intercrossing PR^fl/fl (Pgr^flox/flox) ³¹ with Lck^cre (Tg^{(Lck-cre)1Cwi}) ^28,29 mice. BALB/c males for timed pregnancies were obtained from our breeding facilities at the University Medical Center Hamburg-Eppendorf. Mice were kept under specific pathogen-free conditions at the central animal facility of the University Medical Center Hamburg-Eppendorf at a 12 hour light/dark cycle. For experiments age- and sex-matched mice (8-20 weeks) were used. Experimental procedures were followed in accordance with institutional guidelines. All experimental protocols were approved by the institutional animal care and use committee.

Allogenic mating:

Female C57BL/6 wild type or GR^fl/flLck^cre mice were mated to male BALB/c mice. The presence of a vaginal plug in the morning was considered as gestational day (gd) 0.5. Successful course of pregnancy after sighting the vaginal plug was confirmed by documentation of gestational weight gain. At gd 7.5 and 18.5, mice were sacrificed for in vitro T cell cultures.

In vitro T cell culture:

Spleens were either isolated from male, non-pregnant or BALB/c-mated pregnant C57BL/6 female mice. Single cell suspensions were prepared by passing the tissue through a 40 μm cell strainer. Lysis of erythrocytes was done in RBC Lysis Buffer (eBioscience/ThermoFischer Scientific, Waltham, MA) for 5 min. After centrifugation, cells were resuspended in PBS. 1×10⁶ cells were cultured in each well of a 24 well plate in 1 ml IMDM culture media (Gibco/ThermoFischer Scientific, Waltham, MA) containing 10 % FBS (Gibco), 2 mM L-glutamine (Gibco), 50 μM β-mercaptoethanol (Gibco) and penicillin/streptomycin (Sigma-Aldrich, Darmstadt, Germany). Progesterone (10⁻⁶ M) (Sigma-Aldrich), dydrogesterone (10⁻⁶ M) (Abbott Laboratories, Chicago, IL), corticosterone (10⁻⁷ M) (Sigma-Aldrich) and dexamethasone (10⁻⁸ M) (Sigma-Aldrich) diluted in DMSO (Sigma-Aldrich, Darmstadt, Germany) or DMSO (0.2%) alone were added and cells were cultured at 37°C and 5% CO₂ for 48 h.

Flow cytometric analysis:

Single cell suspensions were analyzed with flow cytometry. First, unspecific antibody staining was reduced by incubation with CD16/32 block (TueStain fcX™, BioLegend, San Diego, CA) and rat serum (Jackson Immuno Research, Bar Harbor, ME). Monoclonal antibodies specific for CD3 (clone 145-2C11), CD8 (53-6.7), CD4 (RM4-5), CD44 (IM7) and CD62L (MEL-14) were purchased from BioLegend and eBioscience. Pacific Orange (Life Technologies, Carlsbad, CA) was used for discrimination of dead cells. For intracellular staining, cells were fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience/ThermoFischer Scientific, Waltham, MA) following the manufacturer’s instructions. Subsequently, staining of the transcription factor Foxp3 (FJK-16s, eBioscience/ThermoFischer Scientific, Waltham, MA) was performed. After washing, cells were reconstituted in 2% BSA/2 mM EDTA PBS before multicolor acquisition at the LSR II flow cytometer (BD Bioscience, Heidelberg, Germany). For each condition 2-12 biological replicates were measured in duplicates. Data analysis was done using FlowJo software (Tree Star, Ashland, OR).

Statistics:

For the experimental data mean ± SEM and p-values were calculated. Levels of significance between groups were tested using two-way ANOVA and Bonferroni’s multiple comparison post-test. Level of statistical significance was defined as p<0.05 (*equals p<0.05, **equals p<0.01, ***equals p<0.001).

Results

Progesterone and glucocorticoids induce T cell death

To determine the capacity of steroid hormones to induce T cell death in vitro, we isolated spleen cells from C57BL/6 female non-pregnant mice and cultured them in the presence of progesterone and the synthetic glucocorticoid dexamethasone (DEX) (Figure 1A). After 48h, we assessed T cell subsets following the gating strategy depicted in Figure 1B and Figure 1C. DEX led to a significant increase of dead T cells within the CD4⁺ and CD8⁺ T cell subsets, reaching almost up to 100 % lethality. Similarly, in the presence of progesterone, CD4⁺ and CD8⁺ T cell death increased significantly, albeit not reaching levels induced by DEX (Figure 2A,B). Dydrogesterone, a progestin with high affinity for the PR did not induce such increased T cell death in vitro (Figure 2A,B). When compared to cells from non-pregnant mice and pregnant mice at gd 7.5, CD4⁺ and CD8⁺ T cells from mice at gd 18.5 showed reduced baseline cell death. However, CD4⁺ and CD8⁺ T cells from all three groups of mice displayed a similar increase in T cell death upon steroid stimulation in vitro (Figure 2A,B). In relation to baseline levels (DMSO treatment), we detected the strongest induction of cell death upon steroid stimulation in CD4⁺ and CD8⁺ T cells from pregnant mice at gd 18.5 (Suppl. Figure 1A,B).

Figure 1: In vitro stimulation of spleen cells.

(A) Spleen cells were isolated from non-pregnant and BALB/c-mated pregnant C57BL/6 females at gestational day (gd) 7.5 and 18.5. Progesterone (10⁻⁶ M), dydrogesterone (10⁻⁶ M) and DEX (10⁻⁸ M) were added and after 48 h of incubation at 37°C, cell subsets were analyzed by flow cytometry as depicted in (B) and (C).

Figure 2: Progesterone and DEX induce T cell death in vitro.

Spleen cells were isolated from non-pregnant (n=6) and BALB/c-mated pregnant C57BL/6 females at gestational day (gd) 7.5 (n=6) and 18.5 (n=6). Progesterone (P4, 10⁻⁶ M), dydrogesterone (Dydro, 10⁻⁶ M) and DEX (10⁻⁸ M) were added to the culture. After 48 h, dead CD4⁺ (A) and CD8⁺ (B) T cells were identified by staining with Pacific Orange. (C) Surviving regulatory T cells (T_reg) are shown as percentage of all surviving CD4⁺ T cells. Surviving naïve (CD62L⁺ CD44^low) (D) and effector (CD62L^neg CD44⁺) CD4⁺ T cells (T_eff) (E) are shown among surviving conventional CD4⁺ T cells (T_con). (F) The ratio of T_reg/T_eff was calculated. (G) Representative dot plots gated on CD4⁺ T cells. (A-F) Shown are means ± SEM. These results are pooled from two independently conducted experiments. Statistical significance was calculated using two-way ANOVA and Bonferroni’s multiple comparison post-test comparing the different conditions to DMSO with ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

Steroid hormone treatment, in particular DEX treatment, led to T cell death in all analyzed T cell populations. However, among the small fraction of surviving cells, we observed that progesterone and DEX, but not dydrogesterone, stimulation resulted in a relative increase of CD4⁺ T_reg cell frequencies (Figure 2C). Surviving conventional CD4⁺ T cells (T_con) were further divided into effector T cells (T_eff) and naïve T cells, because differential activities of glucocorticoids on activated and naïve T cells have been described ²³. In the presence of DEX, the frequencies of CD62L⁺ CD44^low naïve CD4⁺ T cells were significantly reduced among surviving conventional CD4⁺ T cells (T_con) (Figure 2D), whereas the frequencies of activated CD62L^neg CD44⁺ effector CD4⁺ T (T_eff) cells were significantly increased, compared to all other culture conditions (Figure 2E). Progesterone stimulation resulted in a slight increase of CD4⁺ T_eff cell frequencies, which did not reach levels induced by DEX. However, in contrast to incubation with DEX, frequencies of naïve CD4⁺ T cells remained largely unchanged in cultures with progesterone (Figure 2D,E). Since the rate of cell death differed between the various steroids in the cell culture (Figure 2A), we also calculated the ratio of CD4⁺ T_reg/T_eff cells. Progesterone stimulation led to a selective enrichment of T_reg over T_eff cells, mirrored by a ratio >1, whereas DEX showed the inverse effect (T_eff over T_reg). An increase of the T_reg/T_eff cell ratio upon progesterone stimulation could be observed especially in samples from late gestation (Figure. 2F). Representative dot plots supporting data of Figure 2A, C–E are shown in Figure 2G. As DEX is a synthetic glucocorticoid, we also cultured T cells in the presence of corticosterone, the endogenous glucocorticoid in rodents. We observed comparable T cell death induction and selective enrichment of CD4⁺ T cell subpopulations as after cultivation with DEX (Suppl. Figure 2A–F).

Progesterone induces T cell death via the GR

Since the absence of the PR in T cells has recently been described ^3,13 and we observed that the progestin dydrogesterone, which specifically binds to the PR, did not induce T cell death, we next aimed to test the role of the PR and GR in facilitating the steroid effects in vitro. We isolated cells from mutant mice, which express the cre recombinase under the promoter of the lymphocyte-specific protein tyrosine kinase Lck (Lck^cre) in combination with floxed alleles of the progesterone receptor (PR^fl/fl) or glucocorticoid receptor gene (GR^fl/fl). Because Lck expression is largely restricted to T cells, this approach allows us to evaluate the steroid effects on T cells lacking the PR or GR. Since our experiments shown in Figure 2 revealed comparable death induction by steroids between cells derived from non-pregnant and pregnant mice, we initially focused on the analysis of cells from non-pregnant PR^fl/flLck^cre and GR^fl/flLck^cre mice. Incubation of T cells lacking the PR with progesterone and DEX induced CD4⁺ and CD8⁺ T cell death comparably to T cells from wild type mice. However, T cells lacking the GR were refractory to progesterone- and DEX-induced CD4⁺ and CD8⁺ T cell death (Figure 3A,B). Among the surviving CD4⁺ T cells from GR^fl/flLck^cre mice, the progesterone-mediated effect on T_reg and T_eff cell subset composition was absent (Figure 3C-E). Representative dot plots supporting the data in Figure 3A and 3C are shown in Figure 3F and 3G, respectively. Corticosterone stimulation of T cells lacking the PR or GR revealed comparable effects on death of T cells and modulation of T cell subsets as DEX stimulation (Suppl. Figure 2G–K).

Figure 3: Influence of progesterone and DEX on T cells from PR^fl/flLck^cre and GR^fl/flLck^cre mice.

Spleen cells were isolated from non-pregnant C57BL/6 wild type mice (n=5), mice lacking the progesterone receptor (PR) in T cells (PR^fl/flLck^cre, n=2) and mice lacking the glucocorticoid receptor in T cells (GR^fl/flLck^cre, n=3). Spleen cells were cultured with progesterone (P4, 10⁻⁶ M), dydrogesterone (Dydro, 10⁻⁶ M) and DEX (10⁻⁸ M) for 48 h. Dead CD4⁺ (A) and CD8⁺ (B) T cells were identified by staining with Pacific Orange. (C) Surviving regulatory T cells (T_reg) are shown as percentage of all surviving CD4⁺ T cells. Surviving naïve (CD62L⁺ CD44^low) (D) and effector (CD62L^neg CD44⁺) T cells (T_eff) (E) are shown among surviving conventional CD4⁺ T cells (T_con). (F,G) Representative dot pots gated on CD4⁺ T cells. (A-E) Shown are means ± SEM. These results are pooled from two independently conducted experiments. Statistical significance was calculated using two-way ANOVA and Bonferroni’s multiple comparison post-test comparing the different conditions to DMSO with ^*p < 0.05, ^**p < 0.01, ^***p < 0.001. Levels of significance are demonstrated for cultures from wild type mice. Steroid treatment did not induce significant differences in GR^fl/flLck^cre. In cultures from PR^fl/flLck^cre mice due to the low sample size no statistical test could be applied.

We next tested whether the GR-dependent induction of cell death by steroids is altered during pregnancy. Similar to the results from cells of non-pregnant mice, we observed a complete abrogation of the effects of progesterone and DEX on CD4⁺ and CD8⁺ T cell death in cells from pregnant GR^fl/flLck^cre mice when compared to pregnant wild type mice (Figure 4A,B). Steroid hormone induced changes in the distribution of surviving CD4⁺ T cell subsets were also suppressed in cells from pregnant GR^fl/flLck^cre mice (Figure 4C-E). T cells from pregnant GR^fl/flLck^cre mice also failed to respond to corticosterone (Suppl. Figure 2L–P). Of note, reproductive outcome of allogenic pregnant GR^fl/flLck^cre mice was comparable to wild type C57BL/6 mice (data not shown) ¹³.

Figure 4: Influence of progesterone and DEX on T cells from pregnant GR^fl/flLck^cre mice.

Spleen cells were isolated from pregnant C57BL/6 wild type (n=4), and pregnant GR^fl/flLck^cre (n=3) mice at gestational day 7.5 and cultured for 48 h with progesterone (P4, 10⁻⁶ M), dydrogesterone (Dydro, 10⁻⁶ M) and DEX (10⁻⁸ M). Dead CD4⁺ (A) and CD8⁺ (B) T cells were identified by staining with Pacific Orange. (C) Surviving regulatory T cells (T_reg) are shown as percentage of all surviving CD4⁺ T cells. Surviving naïve (CD62L⁺ CD44^low) (D) and effector (CD62L^neg CD44⁺) T cells (T_eff) (E) are shown among surviving conventional CD4⁺ T cells (T_con). Shown are means ± SEM. These results are pooled from two independently conducted experiments. Statistical significance was calculated using two-way ANOVA and Bonferroni’s multiple comparison post-test comparing the different conditions to DMSO with ^*p < 0.05, ^**p < 0.01, ^***p < 0.001. Levels of significance are demonstrated for cultures from wild type mice. Steroid treatment did not induce significant differences in GR^fl/flLck^cre.

Progesterone-mediated T cell death induction is also observed in male T cells

Since sex-specific immune responses are well-known ^32–35, we next assessed whether cells derived from male and female animals are differentially affected by steroid hormones. We isolated spleen cells from male and female C57BL/6 wild type mice and cultured them with hormones as described before. Progesterone and DEX, but not the PR-specific dydrogesterone, induced CD4⁺ and CD8⁺ T cell death (Figure 5A,B) and caused changes in the surviving CD4⁺ T cell subsets without any significant sex-specific effect (Figure 5C–E). There was also no difference in the response of male and female T cells to corticosterone stimulation (Suppl. Figure 2Q–U).

Figure 5: Progesterone and DEX induce death in female and male T cells.

Spleen cells were isolated from non-pregnant female (n=4) and male C57BL/6 mice (n=4). Progesterone (P4, 10⁻⁶ M), dydrogesterone (Dydro, 10⁻⁶ M) and DEX (10⁻⁸ M) were added and after 48 h of incubation at 37°C, cells were analyzed by flow cytometry. Dead CD4⁺ (A) and CD8⁺ (B) T cells were identified by staining with Pacific Orange. (C) Surviving regulatory T cells (T_reg) are shown as percentage of all surviving CD4⁺ T cells. Surviving naïve (CD62L⁺ CD44^low) (D) and effector (CD62L^neg CD44⁺) T cells (T_eff) (E) are shown among surviving conventional CD4⁺ T cells (T_con). Shown are means ± SEM. These results are pooled from two independently conducted experiments. Statistical significance was calculated using two-way ANOVA and Bonferroni’s multiple comparison post-test comparing the different conditions to DMSO with ^*p < 0.05, ^**p < 0.01, ^***p < 0.001. Levels of significance are demonstrated for cultures from female and male mice.

Discussion

In the present study, we provide evidence that pregnancy-like levels of progesterone and GCs induce death in CD4⁺ and CD8⁺ T cells isolated from non-pregnant and – to a similar extent – pregnant mice. Moreover, we observed that CD4⁺ T_con cells were prone to such steroid-induced cell death, whereas CD4⁺ T_reg cells were rather refractory. This resulted in a relative increase of T_reg over T_eff cell frequencies in response to progesterone, but interestingly not in response to GCs. In contrast to GCs, progesterone was less active on the frequency of naïve CD4⁺ T cells. Progesterone has been proposed to modulate immunological tolerance towards the semi-allogenic fetus during pregnancy. We here provide insights into how such progesterone-induced effects might be mediated: via the GR-dependent selective enrichment of CD4⁺ T_reg over T_eff cells. Hence, the elimination of CD4⁺ T_con cells and subsequent increase in T_reg cell frequencies may account for the prevention of fetal loss. A similar T_reg cell accumulation and overall T cell death after progesterone treatment in vitro was described in a recent study of the role of T_con and T_reg cells in experimental autoimmune encephalomyelitis in pregnant mice¹³ as well as progesterone inducing death in Jurkat cells²¹. However, so far only non-pregnant mice were included in the in vitro analyses by Engler and colleagues. Surprisingly, when including T cells isolated from pregnant mice early and late during gestation, similar T cell death induction and relative CD4⁺ T_reg cell enrichment as compared to T cells isolated from non-pregnant mice were observed. T cell death upon steroid treatment was actually increased in cultures from pregnant mice at gd 18.5 in comparison to cultures from non-pregnant mice. In addition, the overall ratio of T_reg/T_eff cells was in favor of T_reg cell enrichment upon progesterone stimulation particularly in cells harvested from pregnant mice, since we observed an increased ratio of T_reg over T_eff cells in cultures from gd 18.5 pregnant mice.

We observed relatively high frequencies of dead cells, even without hormone treatment. This can be explained by the length of cell culture we chose. We refrained from the addition of survival cytokines such as IL-7 to our culture conditions, as this may have interfered with the sensitivity of T cells to hormones. Moreover, it is worth mentioning that published data on assessment of cell death show a great deal of ambiguity. We here related the frequency of dead cells to a life-dead staining assessment using the dye Pacific Orange, as this allowed for simultaneous intracellular staining. The commonly used Annexin V detects only early apoptosis, which underestimates the proportion of dead cells and may explain lower frequencies published by others¹³. We also included Annexin V in some of our experiments, which revealed approx. 10% Annexin V-positive cells in DMSO cultures (data not shown).

The mechanisms underlying increased resistance of T_reg cells to steroid hormone-induced death induction are still elusive. Reduced expression of the GR in T_reg cells could account for this effect. Albeit it was shown recently that the GR expression is comparable in T_reg and T_con cells, the expression of Gilz, a GR response gene, was increased in T_con compared to T_reg cells¹³. This could indicate increased activity of the GR in T_con cells. Further, increased expression of enzymes that inactivate progesterone (20α-hydroxysteroid dehydrogenase, 20α-HSD) or glucocorticoids (11β-HSD2) in T_reg cells could contribute to increased resistance of those cells from hormone-induced death induction. Weinstein et al. observed progesterone conversion to the metabolite 20α-dihydroprogesterone up to a progesterone concentration of approx. 5×10⁻⁷ M in splenic T lymphocytes before saturation of the enzyme³⁶. Endogenous expression of 11β-HSD2 in lymphocytes is generally low³⁷ (and unpublished data from our group) and thus, requires improvement of detection strategies.

The nuclear PR is discussed to show very low expression in T cells from mice and human ^3,13, which was also reported by the Immunological Genome Project ³⁷. However, it cannot be excluded that low PR expression in T cells could still have functional relevance. It had also been proposed that the effect of progesterone on T cells is mediated via a membrane bound PR ^3,20,21, the GR ¹³, or both. We here used a progestin with high affinity and – at these concentrations – selectivity to the PR, along with T cells lacking the PR (PR^fl/flLck^cre). Both approaches independently confirmed that progesterone or progestins do not induce T cell death via PR-dependent pathways. In contrast, T cells lacking the GR (GR^fl/flLck^cre) were resistant to progesterone induced cell death. This strongly implicates the interaction between progesterone and the nuclear GR in modulating the fate of T cell subsets. As the steroid receptors are closely related, we cannot entirely exclude functional interactions of the PR with GR on protein levels and in regulatory networks which might impair PR function in the absence of GR. However, we are not aware of any functional interaction of the PR and GR. There was reduced overall death in T cell cultures from GR^fl/flLck^cre mice in comparison to wild type mice, even without hormone stimulation. This can be explained by resistance of GR^fl/flLck^cre cells to progesterone and glucocorticoids present in FBS in the culture media³⁸. Progesterone has a low affinity to the GR. Thus, only high concentrations of progesterone lead to sufficient GR activation with functional relevance. Using 10⁻⁸ M of progesterone, we could not detect T cell death or T_reg enrichment in previous studies (data not shown). As our study focused on T cells, we can only provide limited insights in PR and GR expression in other immune cell populations. Previous experiments from our group could demonstrate a functional role of the PR expression in dendritic cells (DC) in maternal immune adaptation during pregnancy as mice lacking the PR in DCs showed increased DC activation in the uterus as well as impaired fetal outcome³⁹.

Glucocorticoid-induced T cell death has been shown in a number of scenarios, first and foremost in the context of positive and negative thymocyte selection ^23,24. Our results extend these observations and confirm that progesterone induces T cell death via the GR. In addition to activation by progesterone, the GR might also be a target of GC during pregnancy since GC serum concentration increases substantially during pregnancy⁴⁰. Similar to high sensitivity of immature thymocytes towards glucocorticoid-mediated killing, we observed increased sensitivity to glucocorticoid-mediated death in naïve CD4⁺ T cells in comparison to activated cells. Similar observations were made by Wang et al., who detected increased T cell death in thymocytes and splenic T cells in vitro²⁵ as well as Gieras et al. who observed Treg enrichment in thymus and spleen of offspring prenatally challenged with the synthetic glucocorticoid betamethasone⁴¹. Progesterone was less active on naïve CD4⁺ T cells, as no significant reduction in naïve T cell frequencies was observed. Although physiologically relevant concentrations of progesterone and GCs were used, it has to be kept in mind that in vivo the majority of steroid hormones are bound to plasma proteins. This results in a low bioavailability of progesterone and GCs that are active in serum. Despite the addition of FBS in the culture medium that also contains proteins, but to a far smaller amount, the high percentage of cell death observed in vitro by addition of GCs and progesterone might not occur in this magnitude in vivo.

Despite sex-specificity in immune responses is frequently reported^33–35, we observed a similar response of spleen cells isolated from female and male mice to steroid hormones. Lu and colleagues demonstrated increased GR expression in various leukocyte populations in healthy male in comparison to female adults⁴² and this observation is confirmed by data from the Immunological Genome Project³⁷. However, these observations on mRNA level do not necessarily reflect the protein expression of the GR or its functionality. Although De León-Nava et al. demonstrated similar PR expression in lymphocytes from male and female mice, progesterone limited lymphocyte proliferation only in cells from females³². As we analyzed cell death and not proliferation, their results do not contradict our observations.

Surprisingly, similar effects of steroid hormones were observed in T cells isolated from non-pregnant and pregnant mice early (gd 7.5) and late (gd 18.5) of gestation. We used 10⁻⁶ M of progesterone for in vitro cultures, which represents approximately three times the serum concentration in pregnant mice at gd 16.5 – the peak of progesterone concentrations in the serum ⁴³. However, this is considered to be within the physiological range of progesterone concentrations found at the feto-maternal interface. In experiments, using 10⁻⁸ M of progesterone, we did not observe an effect on T cells in vitro (data not shown). In addition, T cells are able to metabolize progesterone via the enzyme 20α-HSD³⁶, probably resulting in progesterone resistance. At concentrations that exceed the enzyme’s capacity to inactivate progesterone, T cells could become sensitive to progesterone-mediated effects. T cell death without hormone treatment was reduced in cultures from mice at late gestation. In addition, we observed slightly lower frequencies of dead cells in ex vivo analysis of spleen cells isolated from pregnant mice when compared to non-pregnant mice (Suppl. Figure3). Nevertheless, the effects of progesterone stimulation were comparable to those in cultures from non-pregnant mice or mice during early gestation. It could be hypothesized that 20α-HSD expression increases during late gestation resulting in partial resistance to progesterone at low concentrations potentially derived from FBS of the culture media³⁸. However, stimulation with high progesterone concentrations – as present at the feto-maternal interface – exceeds the enzymes capacity to inactivate progesterone and thus, results in T cell death and modulation of T cell subsets in vitro.

Our recent findings improve the understanding of immunological tolerance induction by progesterone during pregnancy. As progesterone and progestins such as dydrogesterone are frequently used as therapeutics to treat e.g. recurrent pregnancy loss⁴⁴, it is essential to clarify their exact modes of action. We here propose a mechanism of progesterone-induced immunological tolerance, the selective killing of CD4⁺ T_con cells in favor of T_reg cell survival.