Dendritic cell activation and memory cell development are impaired among mice administered medroxyprogesterone acetate prior to mucosal herpes simplex virus type 1 infection

Abstract

Epidemiological studies indicate that the exogenous sex steroid medroxyprogesterone acetate (MPA) can impair cell-mediated immunity, but mechanisms responsible for this observation are not well defined. Here, MPA administered to mice 1 week prior to herpes simplex virus type 1 (HSV-1) infection of their corneal mucosa impaired initial expansion of viral-specific effector and memory precursor T cells and reduced the number of viral-specific memory T cells found in latently infected mice. MPA treatment also dampened expression of the costimulatory molecules CD40, CD70, and CD80 by dendritic cells (DC) in lymph nodes draining acute infection, while co-culture of such DC with T cells from uninfected mice dramatically impaired ex vivo T cell proliferation compared to the use of DC from mice that did not receive MPA prior to HSV-1 infection. In addition, T cell expansion was comparable to that seen in untreated controls if MPA-treated mice were administered recombinant soluble CD154 (CD40 ligand) concomitant with their mucosal infection. On the other hand, the immunomodulatory effects of MPA were infection site-dependent, as MPA-treated mice exhibited normal expansion of virus-specific T cells when infection was systemic rather than mucosal. Taken together, our results reveal that the administration of MPA prior to viral infection of mucosal tissue impairs DC activation, virus-specific T cell expansion, and development of virus-specific immunological memory.

Introduction

At least 30 million women worldwide choose the progestin-only injectable contraceptive depot-medroxyprogesterone acetate (DMPA) for prevention of undesired pregnancy [1]. DMPA use is particularly common in areas with high prevalence of sexually transmitted infections [2]; a potential concern as DMPA may alter susceptibility to these infections. As examples, DMPA users were more likely to acquire human immunodeficiency virus (HIV) or Chlamydia trachomatis [3–6], and more likely to shed HIV or herpes simplex virus type 2 (HSV-2) in their lower genital tract [7, 8]. Most recently, a large-scale study conducted in seven African countries found that use of DMPA doubled the risk of HIV acquisition and transmission among HIV-serodiscordant couples [9]. However, linkages between hormonal contraceptive use and sexual practice, including increased frequency of unprotected intercourse, impedes a more certain identification of any causal relationship between DMPA and altered host immunity [10].

As unmeasured behavioral variables may confound the interpretation of epidemiological research, experimental models have been used to explore the immunomodulatory effects of exogenous sex steroids. In a murine model, progesterone treatment prior to intravaginal infection with an attenuated HSV-2 strain increased susceptibility of mice to challenge with wild-type HSV-2 [11]. Similarly, rhesus macaques administered DMPA prior to intravaginal immunization with an attenuated strain of simian immunodeficiency virus (SIV) were less protected than untreated controls from intravaginal challenge with a more pathogenic SIV strain [12]. On the other hand, protection was unaffected when systemic routes of infection were used to immunize and challenge DMPA-treated macaques [13], indicating that virus-specific adaptive immune responses at mucosal sites of infection may be preferentially susceptible to the effects of progestin-containing compounds. Currently lacking, however, is explicit demonstration that such compounds impair the development of immunologic memory elicited in response to viral infection of mucosal tissue.

Materials and Methods

Ethics statement

Experiments were conducted from a protocol approved by the University of Pittsburgh’s Institutional Animal Care and Use Committee, and in accordance with National Institutes of Health guidelines.

Mice

Wild-type (WT) and ovariectomized (ovx) female C57BL/6J (B6) mice (CD90.2⁺) and female BALB/cJ mice were purchased from the Jackson Laboratory (Bar Harbor, ME). gBT-I.1 mice on a B6.PL background (CD90.1⁺) were a gift from Francis Carbone (University of Melbourne, Australia), and CD154^−/− mice on a BALB/c background were a gift from Richard Flavell (Yale University, New Haven, CT).

In vivo procedures

The HSV-1 corneal infection model we utilized is a well-accepted model of mucosal tissue infection [14]. Mice were anesthetized by intraperitoneal (i.p.) injection of 1.8 mg ketamine hydrochloride (Fort Dodge Animal Health, Fort Dodge, IA) and 0.18 mg xylazine (Lloyd Laboratories, Shenandoah, IA), and implanted with 21-day sustained release pellets containing 50 mg MPA or matching placebo (Innovative Research of America, Sarasota, FL) in suprascapular subcutaneous tissue. Elsewhere, 1 or 4 mg of DMPA (Pfizer, New York, NY) was injected subcutaneously. These doses were selected to approximate serum progesterone levels detected in human pregnancy and serum MPA levels achieved by Depo-Provera® injection for prevention of undesired pregnancy. One week after pellet placement or DMPA injection, mouse corneas were scarified and infected bilaterally with 10⁵ plaque-forming units (PFU) of wild-type HSV-1 (RE strain). Other mice were infected corneally with 10³ PFU or intravenously (i.v.) or i.p. with 10⁶ PFU HSV-1. Draining lymph nodes (DLN), spleens, and trigeminal ganglia (TG) were excised at various days post infection (dpi) to assess the effects of pretreatment on expansion or function of various leukocyte subpopulations. As indicated, pellets were removed 14 dpi and DLN, spleens, and TG were excised ≥ 35 dpi to measure the effects of pretreatment on memory cell development. To assess in vivo T cell proliferation, mice were injected i.v. with 1 mg BrdU (BD Biosciences, San Diego, CA) 4 h before euthanasia. Effects of MPA on HSV-1 replication were assessed in TG, corneal tissue, and tear film collected with WECK-CEL® Surgical Spears (Medtronic Xomed, Jacksonville, FL). Where indicated, pretreated mice were administered 5 μg of mouse recombinant soluble CD154 (rsCD154) i.p. (eBioscience, San Diego, CA) concomitant with infection, and untreated mice were administered 250 μg of blocking anti-CD154 antibody i.p. (clone MR1), both 1 day before infection and 2 dpi. As indicated, 0.7 mg of acyclovir was administered i.v. 12 h and 24 h post-infection. In other experiments, mice 3 dpi were treated i.p. with the indicated doses of anti-CD8 mAb (2.43, BioXcell). As indicated, intact mice that had received 50 mg MPA pellets or 1 or 4 mg DMPA were sacrificed 7 days later to measure serum MPA levels with a radioimmunoassay (RIA) that was used in accordance with manufacturer’s instructions (Immunometrics, UK).

Ex vivo and in vitro procedures

For T cell stimulation assays, TG were excised 8 or 35 dpi, and dissociated into single-cell suspensions with collagenase type I (Sigma-Aldrich, St. Louis, MO). Cells were stimulated for 6 h with B6WT3 fibroblast targets (previously incubated 12 h with HSV-1) in the presence of FITC-conjugated anti-CD107a (1D4B, BD Biosciences) and GolgiPlug^™ (BD Biosciences) [15]. Cells were stained as indicated in the flow cytometric analysis section. For cell proliferation assays, BrdU (BD Biosciences) staining of DLN and TG single-cell suspensions were done according to manufacturer’s instructions. To characterize TG and DLN lymphocytic infiltrates or DC subpopulations in DLN of mice, single-cell suspensions were stained as indicated in the flow cytometric analysis section. To measure apoptosis levels in T cells from DLN, CaspaTag pan-caspase assay kits (Millipore, Billerica, MA) were used according to manufacturer’s instructions. For T cell proliferation assays, DLN 2 dpi were digested with collagenase D and DNase I (both Sigma-Aldrich), and resultant single-cell suspensions enriched for CD11c⁺ cells by magnetic-activated cell sorting (MACS) (Miltenyi Biotec, Auburn, CA). Splenocytes from gBT-I.1 mice were processed into single-cell suspensions, enriched for CD8⁺ cells using MACS (Miltenyi Biotec), and labeled using CellTrace^™ Violet Cell Proliferation Kit (Invitrogen). 2.5 × 10⁴ CD11c⁺ cells were co-cultured with 5 × 10⁴ labeled gBT-I.1 cells for 72 h in 200 μl RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 μM 2β-mercaptoethanol, gentamycin, penicillin, and streptomycin. Proliferation of the CD8⁺CD90.1⁺Vα2⁺ splenocytes was determined using flow cytometry.

Flow cytometric analysis

In ex vivo T cell stimulation assays, cells were stained with LIVE/DEAD® fixable aqua dead cell stain (Invitrogen, Carlsbad, CA), PerCP-conjugated anti-CD45 (30-F11, BD Biosciences) and Pacific Blue-conjugated anti-CD8α (53-6.7, BD Biosciences). Following cell fixation and permeabilization with Cytofix/Cytoperm^™ (BD Biosciences) and Perm/Wash^™ buffer (BD Biosciences), intracellular staining with allophycocyanin-conjugated anti-IFN-γ (XMG1.2, BD Biosciences) and phycoerythrin (PE)-conjugated anti-TNF (MP6-XT22, BD Biosciences) was performed. To characterize TG and DLN lymphocytic infiltrates at various dpi, single-cell suspensions were stained with LIVE/DEAD® fixable aqua dead cell stain, blocked with anti-CD16/32 mAb (2.4G2, BD Biosciences), and incubated with combinations of the following mAb as indicated: FITC-, PE-, PE-Cy7-, PerCP-, or allophycocyanin-conjugated mAb against CD11b (M1/70), CD25 (PC61), CD44 (IM7), CD45 (30-F11), CD62L (MEL-14), CD90.1 (OX-7), CD122 (TM-β1), CD127 (SB/199), CD183 (CXCR3-173), NK1.1 (PK136), Vα2 TCR (B20.1) (BD Biosciences); or PE-, PE-Cy7-, PerCP-Cy5.5, allophycocyanin-, allophycocyanin-eF750-, AF700-, eF450-conjugated mAbs against CD3ε (17A2 or eBio500A2), CD4 (GK1.5 or RM4-5), CD8α (53-6.7), CD8β (eBioH-35-17.2), CD11b (M1/70), CD11c (N418), CD28 (37.51), CD45 (30-F11), CD69 (H1.2F3), CD154 (MR1), MHC II (M5/114,15,2) (eBioscience); or allophycocyanin-conjugated anti-CD27 (LG.3A10), FITC- or PerCP-conjugated CD43 (1B11), PE-Cy7-conjugated CD90.2 (53-2.1) (BioLegend, San Diego, CA); or allophycocyanin-conjugated F4/80 (BM8) (Invitrogen); or PE-conjugated H-2K^b tetramers (NIAID tetramer facility) that contained gB_498-505 (SSIEFARL) peptide. After incubation with surface mAbs, cells were fixed with Cytofix or Cytofix/Cytoperm reagent (BD Biosciences). As indicated, cells were intracellularly stained with allophycocyanin-conjugated anti-human GzmB (GB12) (Invitrogen). To examine DC subpopulations in DLN of mice 2 dpi, single-cell suspensions were stained with FITC-, PE-, PE-Cy7-, or allophycocyanin-conjugated mAbs against CD40 (3/23), CD45R (RA3-GB2), CD80 (16-10A1), MHC-I (H-2K^b, AF6-88.5) (BD Biosciences); or PE-, PE-Cy7-, PerCP-Cy5.5, allophycocyanin-, eF450-conjugated mAbs against CD11b (M1/70), CD11c (N418), CD8α (53-6.7), CD70 (FR70), CD86 (GL1), CD317 (eBio129c), MHC-II (I-A/I-E, M5/111.15.2) (eBioscience). Samples processed for flow cytometric analysis were run on a FACSAria flow cytometer (BD Biosciences), and data were analyzed using FACS Diva (BD Biosciences) and FlowJo software (Tree Star, Ashland, OR). As previously described, iMFI (integrated median fluorescence intensity) was calculated from (percentage of positive cells) × (MFI) [16].

HSV replication assays

To determine multi-step in vitro growth kinetics, Vero cells infected with HSV-1 at MOI of 0.01 were incubated for 4, 12, 24, and 48 h, and cells and supernatants harvested for quantification of replicating virus by standard plaque assay. To quantify HSV-1 titers in tear film, surgical spear contents were transferred to PBS, and replicating virus titrated by standard plaque assay. For quantification of corneal HSV-1 transcripts, whole corneas were excised and total RNA was extracted using RNeasy plus mini kit (QIAGEN, Germantown, MD), and cDNA generated with high capacity cDNA reverse transcription kit (Applied Biosystems, Carlsbad, CA), both according to manufacturers’ instructions. Quantitative real-time PCR assays (Applied Biosystems) for the murine housekeeping gene encoding pyruvate carboxylase (PCX) and HSV-1 α (immediate early) genes ICP0 and ICP4 and γ2 (late) gene gH, and quantification of genomic copies of HSV-1 in infected TG were performed as previously described [17, 18].

Statistical considerations

Statistical analyses were performed using Prism 5 software (GraphPad, La Jolla, CA). Normality of the data was tested using D’Agostino–Pearson omnibus test. Differences between two groups were compared by unpaired Student t-test or unpaired Mann-Whitney U test, depending on distribution of the data. For comparison of multiple groups, depending on distribution of the data, one-way ANOVA and Tukey’s multiple comparison post hoc test or Kruskal-Wallis test on ranks and Dunn’s multiple comparison post hoc test were used. Two-way ANOVA was used for comparisons of viral growth over time (p < 0.05 were considered statistically significant). SPICE (version 5.2) was used for analyses of CD8⁺ T cells effector function [19].

Results

Antecedent MPA treatment reduced the size of effector and memory CD8⁺ T cell populations elicited by HSV-1 infection of corneal mucosa

To examine the effects of MPA treatment prior to HSV-1 infection (termed pretreatment) on anti-viral immunity, ovx B6 mice were subcutaneously implanted with 21-day sustained release pellets containing 50 mg MPA or matching placebo pellets 7 days prior to corneal infection. Mice were euthanized 8 dpi, the time point coinciding with peak HSV-specific CD8⁺ T cell infiltration into trigeminal ganglia (TG) [20] (in naïve mice, sensory ganglia effectively contain no CD8⁺ T cells [21]). Flow cytometry analyses that compared absolute numbers of CD4⁺ and CD8⁺ T cells, CD127⁺ T cells (memory precursors), and CD8⁺ T cells specific for the HSV-1 immunodominant peptide (gB_498-505) [22, 23], saw that pretreatment was associated with significant reductions in all measured T cell subpopulations (Fig. 1A). While relative frequencies of the memory precursors and gB_498-505-specific CD8⁺ T cells were similar (Fig. 1B), pretreatment mice was also associated with decreased numbers of gB_498-505-specific CD8⁺ T cell numbers in the lymph nodes draining infection (DLN) (Fig. 1C). The diminished virus-specific T cell infiltrates were also accompanied by reduced numbers of macrophages, NK cells, and NKT cells in TG 8 dpi, which lowered overall numbers of CD45⁺ cells in TG of pretreated mice (Fig. 1D–E). Although impaired expansion and infiltration of virus-specific T cells may have resulted from MPA-mediated defects in T cell activation, no differences in relative expression of the activation markers CD25, CD27, CD28, CD44, CD62L, CD69 and CD154 were seen among gB_498-505-specific CD8⁺ T cells in TG (8 dpi) or in DLN (5 and 8 dpi) (data not shown). Reduced numbers of memory cell precursors in TG of pretreated mice 8 dpi, on the other hand, correlated with reduced numbers of memory T cells in TG of latently infected mice (Fig. 1F). Similar to results seen in acute infection, however, pretreatment did not affect gB_498-505-specific CD8⁺ T cell percentages or relative expression of the activation markers CD27, CD43, CD69, CD62L, CD122, CD127 and CXCR3 in latently infected mice (data not shown).

Fig. 1.

Pretreatment reduces T cell expansion elicited by HSV-1 corneal infection. Ovx B6 mice were corneally infected with 10⁵ PFU HSV-1 seven days after the insertion of 21-day sustained release pellets containing 50 mg MPA or matching placebo. Mice were euthanized at indicated time points, and various tissues excised to enumerate infiltrating virus-specific T cells by flow cytometry. (A) Absolute numbers of CD4⁺, CD8⁺, gB_498-505-specific CD8⁺, and CD127⁺ (memory precursors) T cell subsets in TG 8 dpi. (B) Relative frequency of gB_498-505-specific CD8⁺ and CD127⁺ T cells in TG 8 dpi. Data in (A) and (B) were pooled from 3 independent experiments (n = 18 per group). (C) Absolute numbers of gB_498-505-specific CD8⁺ T cells in DLN at 5 and 8 dpi (n = 8–13 per group). (D) Absolute numbers of NK cells, NKT cells, and macrophages in TG 8 dpi (n = 10 per group). (E) Total number of live CD45⁺ cells (LIVE/DEAD⁻) in TG at 8, 15, and 35 dpi (n = 6–18 per group). (F) Absolute numbers of CD4⁺, CD8⁺, and gB_498-505-specific CD8⁺ T cells in TG 15 and 35 dpi (pellets removed 14 dpi) (n = 6–18 per group). Data in (C–F) were pooled from 2 independent experiments for each panel. Comparisons were made using unpaired two-tailed Student t-test, except for comparisons of the number of gB_498-505-specific CD8⁺ and CD4⁺ T cells per TG in (A), which were made using unpaired Mann-Whitney U test; (horizontal bars accordingly indicate mean and median values).

As MPA is administered to women with conserved ovarian function as an injectable formulation (DMPA), we determined if pretreatment of intact WT female B6 mice similarly inhibited T cell expansion. By comparing the effects of sustained release pellets containing 50 mg MPA, 4 mg DMPA, or 1 mg DMPA in intact mice, we assessed if MPA suppressed T cell expansion in a dose-dependent manner. Moreover, as differences in MPA metabolism between humans and mice are possible [24, 25], we also measured MPA serum concentrations 7 d after the administration of these treatments. As expected, pelleted and injectable MPA treatments suppressed CD8⁺ T cell expansion, and RIA revealed this effect to be dose-dependent (Fig. 2A–B). While sustained release pellets produced mean MPA serum levels of 64.12 ng/ml (a concentration similar to the levels seen in the second trimester of human pregnancy), the 4 and 1 mg injections of DMPA produced mean serum concentration of 21.37 ng/ml and 10.14 ng/ml, respectively (levels that approximate the peak and maintenance serum concentrations of MPA in women using Depo-Provera® for prevention of undesired pregnancy) [26, 27]. To exclude the possibility that MPA-mediated inhibition of T cell expansion was mouse strain specific, WT female BALB/cJ mice were administered 4 mg DMPA 7 d prior to corneal infection, and such mice showed reduced TG T cell infiltrates comparable to those seen among pretreated ovx and intact B6 mice (Fig. 8D). Taken together, these initial sets of experiments established that MPA inhibits T cell expansion elicited by mucosal HSV-1 infection, and greatly reduces the number of memory T cells found in the TG of latently infected mice.

Fig. 2.

Pretreatment impairs expansion of virus-specific CD8⁺ T cells induced by HSV-1 corneal infection in a dose-response manner. (A) Intact B6 mice were implanted with 21-day sustained release pellets containing 50 mg MPA, administered 1 or 4 mg DMPA, or were left untreated. 7 days later, all groups were corneally infected with 10⁵ PFU HSV-1. Mice were euthanized 8 dpi, and TG excised to enumerate infiltrating HSV-1 specific T cells by flow cytometry. Absolute numbers of CD8⁺ T cells per TG are shown (n = 20–25 per group) (data shown pooled from 3 independent experiments). Of note, the numbers of CD4⁺ and gB_498-505-specific CD8⁺ T cells were similarly reduced by pretreatment (data not shown). (B) Groups of mice administered MPA in exactly the same fashion as in (A) were sacrificed 7 days later to determine serum levels of MPA by radioimmunoassay (n = 5–8 per group) (** p < 0.01; *** p < 0.001 by one-way ANOVA and Tukey’s multiple comparison post test; horizontal bars indicate mean values).

Fig. 8.

Reduced CD40 expression by pretreated mice inhibits T cell expansion. Untreated B6 mice, untreated B6 mice administered α-CD154 mAb, and pretreated (4 mg DMPA) B6 mice were corneally infected with 10⁵ PFU HSV-1 (another group of pretreated mice were also administered rsCD154 concomitant with infection as indicated). Mice were euthanized 8 dpi, and TG excised to interrogate T cell infiltrates by flow cytometry. (A) Absolute numbers of total CD45⁺, CD8⁺, and CD4⁺ T cells are shown (n = 13–15 per group). (B) Panel depicts percentages of CD8⁺ T cells that expressed GzmB (n = 10 per group). (C) Relative expression of CD69 by CD4⁺ and CD8⁺ T cells (n = 10 per group) (data shown are pooled from 2 independent experiments). (D–G) To confirm results above using α-CD154 mAb, we corneally infected untreated BALB/cJ controls, pretreated BALB/cJ mice, and CD154^−/− BALB/cJ mice with 10⁵ PFU HSV-1, and harvested TG 10 dpi (n = 10 per group). (D) Absolute numbers of TG-resident CD45⁺ cells and CD8⁺ and CD4⁺ T cells. (E) Percentage of CD8⁺ T cells that express GzmB in TG. (F) Relative expression of CD69 by CD8⁺ and CD4⁺ T cells in TG 10 dpi (data are pooled from 2 independent experiments) (* p < 0.05; ** p < 0.01; *** p < 0.001 by one-way ANOVA and Tukey’s multiple comparison post test; horizontal bars indicate means). (G) Contour plots show CD69 and GzmB expression by CD8⁺ T cells infiltrating TG during acute infection of untreated BALB/cJ, pretreated BALB/cJ, and CD154^−/− BALB/cJ mice (representative data; numbers indicate percentages in each quadrant).

MPA decreased virus-specific CD8⁺ T cell effector function during acute infection

As MPA administered to mice after HSV latency was established (≥ 35 dpi) was shown to impair TG-resident CD8⁺ T cell effector function [15], we also explored the effects of pretreatment on CD8⁺ T cell virus-specific effector function. At 8 dpi, relative effector molecule expression (MFI) by TG-resident T cells that responded to stimulation with HSV-infected fibroblasts was similar among treated and untreated mice (data not shown), but pretreatment nearly doubled the number of T cells unresponsive to stimulation (Fig. 3). CD8⁺ T cells from pretreated mice also showed reduced levels of the cytolytic effector molecule granzyme B (GzmB) (Fig. 8B). CD8⁺ T cell effector function was restored in pretreated mice that had undetectable serum levels of MPA by 35 dpi (data not shown), indicating that MPA directly impaired virus-specific CD8⁺ T cell effector function during acute infection or that T cells less responsive to antigen stimulation had been eliminated during formation of the memory T cell repertoire.

Fig. 3.

Pretreatment diminishes virus-specific CD8⁺ T cell effector function. Intact B6 mice given 4 mg DMPA 7 days prior to corneal infection with 10⁵ PFU HSV-1 were euthanized 8 dpi. Excised TG were dispersed into single-cell suspensions and stimulated with HSV-1 infected targets to interrogate T cell effector function by flow cytometry. (A) Depiction of response profiles using Boolean analysis of 3 canonical CD8⁺ T cell effector functions (IFN-γ and TNF production and lytic activity as measured by CD107a surface expression) (n = 5 per group). (B) Pie charts compiled from bar graph data in (A) illustrate percentages of CD8⁺ cells that expressed 3, 2, 1 or 0 effector function markers (beginning at “12 o-clock” and moving clock-wise). Data shown in (A) and (B) are representative of 2 independent experiments (comparisons made using SPICE 5.2 [59]; horizontal bars and pie fractions indicate means). (C) Contour plots display expression of IFN-γ, TNF and CD107a by TG-resident CD8⁺ T cells stimulated ex vivo with infected targets. Data shown are representative results from each experimental group, and numbers indicate percentages within each quadrant.

MPA did not alter HSV-1 replication

We determined if the inhibition of T cell expansion and memory cell development among pretreated mice resulted from MPA-mediated suppression of HSV replication (an effect seen with in vitro HIV-1 replication) [28, 29]. Consistent with prior reports showing that size of the antigen load regulates T cell expansion and memory cell differentiation [30, 31], we saw that corneal infection with lower HSV-1 titers or treatment with the antiviral drug acyclovir (ACV) reduced HSV-specific T cell expansion (data not shown). But MPA did not affect in vitro HSV-1 growth (Fig. 4A), and pretreatment did not alter HSV-1 titers in the cornea or TG of acutely infected mice (Fig. 4B–E), indicating that reduced viral fitness had not been responsible for impaired T cell expansion. To reconcile similar HSV loads in the TG of pretreated and untreated mice with the previously seen MPA-mediated reduction in HSV-specific CD8⁺ T cell numbers, we also completed in vivo studies in which TG-resident CD8⁺ T cells were depleted from HSV-infected mice using 3 distinct anti-CD8 mAb concentrations. Interestingly, a higher TG HSV copy numbers was seen only when TG-infiltrating CD8⁺ T cells were completely eliminated by anti-CD8 mAb treatment; even 90% reduction in CD8⁺ T cell numbers was associated with viral burdens identical to controls (Fig. 5). Such results were therefore entirely congruent with the observation that a 50% reduction in the number of TG-resident HSV-specific CD8⁺ T cells among pretreated mice does not alter HSV load in the TG during acute infection.

Fig. 4.

MPA does not affect HSV-1 replication. (A) The ability of MPA to alter in vitro HSV-1 replication was evaluated using multistep viral replication kinetics with Vero cell monolayers treated with MPA (10 μM) or vehicle (Ctrl) 24 h prior to infection (n = 9). Lower MPA concentrations were also tested, and none altered in vitro HSV-1 replication (data not shown). (B) Untreated or pretreated female B6 mice were infected with 10⁵ PFU HSV-1 per eye, and eye swabs collected at indicated dpi to compare viral titers by standard plaque assay (n = 12 per group). In (A) and (B), viral titers are shown as mean ± SD, two-way ANOVA was used to compare groups. In other experiments, pretreated or untreated intact B6 mice or untreated B6 mice administered i.v. ACV 1 dpi were infected with 10⁵ PFU HSV-1 per eye. Eye swabs were collected 2 dpi, and mice directly euthanized for corneal harvest (n = 18 per group). In (C), data points denote HSV-1 titers from corneal swabs of individual mice as determined by plaque assay (horizontal bars designate mean values). In (D), data bars denote mRNA expression in whole corneas of the immediate-early viral genes ICP0 and ICP4 and the leaky-late viral gene gH relative to expression of the housekeeping gene PCX as measured by quantitative real-time PCR (bars indicate mean ± SD). (In (C) and (D), *** p < 0.001 compared to all groups by one-way ANOVA and Tukey’s multiple comparison post test). In other experiments, pretreated and untreated B6 mice were infected, and sacrificed 8 dpi to excise TG. HSV-1 genome copy number per TG was determined by quantitative real-time PCR (n = 22 per group). Each data point represents the viral genome copy number from single TG. Comparison was performed using unpaired one-tailed Student t test, and horizontal bars indicate mean values. In all Fig. 4 experiments, mice were pretreated with 4 mg DMPA (all data shown are pooled results from 3 independent experiments).

Fig. 5.

HSV burden in the acutely infected TG is increased only when TG-infiltrating CD8⁺ T cells are fully eliminated. Female B6 mice were corneally infected with 10⁵ PFU HSV-1, and at 3 dpi were administered the indicated amounts of anti-CD8 mAb. Mice were euthanized 8 dpi, and TG excised to enumerate virus-specific T cells by flow cytometry and determine viral genome copy number per TG by quantitative real-time PCR (n = 5 per group). Each data point denotes the absolute number of CD8⁺ T cells or the HSV-1 genome copy number per TG (* p < 0.05; ** p < 0.01; *** p < 0.001 by one-way ANOVA and Tukey’s multiple comparison post test; horizontal bars indicate the mean values). Data shown are results from 1 of 2 independent experiments.

MPA decreased T cell proliferation in the lymph nodes draining acute infection

As pretreatment did not impair HSV-1 replication, we next determined if increased cell death or decreased cell proliferation contributed to MPA-mediated inhibition of T cell expansion. Levels of apoptosis in CD4⁺ and CD8⁺ T cells in DLN of pretreated B6 and BALB/cJ mice and untreated controls revealed no significant between-group differences (Suppl. Fig. 1A–B), but as assessed by BrdU incorporation, T cell proliferation was greatly inhibited in DLN of pretreated mice (Fig. 6). At 5 dpi, a time point of peak T cell priming [32], reduced proliferation was seen in CD4⁺, CD8⁺, and gB_498-505-specific CD8⁺ T cells of pretreated B6 mice (Fig. 6), and T cell proliferation was similarly suppressed in pretreated BALB/cJ and ovx B6 mice (Suppl. Fig. 2A–B). No defects in proliferation, however, were seen in T cells in the DLN or TG of pretreated B6 mice 8 dpi (data not shown), indicating that MPA preferentially suppressed T cell expansion at earlier time points after infection.

Fig. 6.

Pretreatment reduces proliferation of virus-specific T cells. Pretreated (4 mg DMPA) or untreated intact female B6 mice were corneally infected with 10⁵ PFU HSV-1. At 5 dpi, mice received i.v. BrdU, and 4 h later mice were euthanized. DLN were then processed into single-cell suspensions to determine BrdU incorporation by flow cytometry. (A) Figures show percentages of DLN CD4⁺, CD8⁺, and gB_498-505-specific CD8⁺ T cells that incorporated BrdU (n = 12 per group). Data shown were pooled from 2 independent experiments, and comparisons were made using a one-tailed Mann-Whitney U test (except for the between-group comparisons of BrdU⁺CD4⁺ T cells, which were made using an unpaired one-tailed Student t test) (horizontal bars accordingly indicate mean and median values). (B) Contour plots illustrate BrdU incorporation by each examined T cell subset. Data shown are representative, and numbers indicate percentages of BrdU⁺ cells.

Pretreatment decreased DC expression of CD40, CD70, and CD80 in lymph nodes draining acute mucosal tissue infection

Given the well-established role of DC in T cell priming [33, 34], we next explored the effects of pretreatment on DC activation. Among conventional (CD11c^hi) lymph node-resident DC subsets, CD11c^hiCD8α⁺CD11b⁻CD45R⁻ (CD8⁺) DC prime virus-specific CD8⁺ T cells [35] and CD11c^hiCD8α⁻CD11b⁺CD45R⁻ (CD8⁻) DC prime virus-specific CD4⁺ T cells [36], while among the more recently identified tissue-derived migratory DC subsets, CD11c^intMHC-II^hi tissue DC (tDC) and CD11c^intMHC-II^int inflammatory DC (iDC) prime virus-specific CD4⁺ and CD8⁺ T cells (Fig. 7B) [33, 37]. As earlier results revealed pervasive MPA-mediated suppression of T cell expansion (Fig. 1), we used flow cytometry to measure the expression of MHC molecules and the costimulatory molecules CD40, CD70, CD80, CD86 and TNFSF4 (OX40L) in all 4 of these DC subsets in the DLN of pretreated and untreated mice 2 dpi (Fig. 7A). Although similar expression of MHC-I, MHC-II, CD86, and TNFSF4 was observed, CD40, CD70, and CD80 levels were greatly diminished in pretreated mice among all DC subsets examined (Fig. 7A). Of note, CD40 expression was the activation molecule most dramatically decreased by pretreatment, while the highest levels of CD70 expression were seen in tDC.

Fig. 7.

Pretreatment impairs DC activation and T cell priming. (A) Pretreated (4 mg DMPA) and untreated female B6 mice were corneally infected with 10⁵ PFU HSV-1. Mice were euthanized 2 dpi. DLN were excised and rendered into single-cell suspensions, to determine expression levels for MHC and several costimulatory molecules in DC and B cells by flow cytometry. CD70, CD80, CD86, TNFSF4 (OX40L), MHC-I, MHC-II, and CD40 iMFI are shown as a relative measure of the amount of each protein expressed by the indicated DC subsets (n = 10 per group, data pooled from 2 independent experiments) (comparisons made with unpaired one-tailed Student t-tests; bars indicate mean ± one SD). Bottom right of panel shows representative contour plots for CD40 expression by various DC subsets (numbers indicate percentages of CD40⁺ cells). (B) Representative contour plots illustrate gating strategies that were used to define 4 main DLN DC subsets. (C) 2.5 × 10⁴ CD11c⁺ cells that were isolated from the DLN of pretreated or untreated B6 female mice 2 dpi were co-cultured 72 h with 5 × 10⁴ CD8⁺ splenic cells from pretreated or untreated naive gBT-I.1 mice labeled with a cell-tracing reagent, and the proliferation of CD8α⁺CD90.1⁺Vα2⁺ cells was determined by flow cytometry. Histograms show percentages of proliferating cells for the indicated culture conditions (data shown are representative results from 1 of 3 independent experiments).

Pretreatment decreased ability of DC to elicit T cell proliferation

Although pretreatment decreased DC expression of multiple costimulatory molecules, it remained to resolve if T cell expansion was suppressed by MPA-mediated effects on DC activation or a more direct effect on T cell proliferation. To distinguish between these 2 possibilities, ex vivo T cell proliferation assays were performed with DC and CD8⁺ T cells from pretreated and untreated mice. Since priming of unstimulated HSV-specific T cells begins 6 h after infection and peaks 2 dpi [32], CD11c⁺ cells from the DLN of pretreated and untreated B6 mice were isolated 2 dpi. Of note, no between-group differences in the frequencies of CD11c^hi cells or any DC subset were detected (data not shown). gB_498-505-specific splenic CD8⁺ T cells were next isolated from pretreated or untreated naïve HSV-1 gB_498-505-specific TCR transgenic (gBT-I.1) mice, labeled with a cell tracing reagent, and co-cultured directly ex vivo for 72 h with the CD11c⁺ cells from pretreated or untreated infected mice. Compared to untreated controls, cultures that contained CD11c⁺ cells from pretreated mice showed a 4-fold reduction in the proliferation of gB_498-505-specific CD8⁺ T cells (Fig. 7C). CD40, CD80, or CD86 blockade with mAb reduced T cell proliferation only 2-fold less than controls, results that suggested pretreatment suppressed proliferation via more than 1 costimulatory pathway. Conversely, CD8⁺ T cells from gBT-I.1 mice treated with MPA 7 days prior to selection and co-cultured with DC from untreated controls exhibited T cell proliferation similar to controls (Fig. 7C). Taken together, these results established that MPA-mediated inhibition of DC activation was responsible for the impaired T cell expansion seen among pretreated mice.

Reduced CD40 expression among pretreated mice inhibited T cell expansion

As CD40 was the activation molecule most affected by pretreatment, we hypothesized that in vivo blockade of CD40-CD154 interaction with anti-CD154 mAb (clone MR1) would recapitulate MPA-mediated inhibition of T cell expansion. While this mAb treatment did not affect T cell activation markers expression (Fig. 8C), T cell expansion was similarly suppressed among pretreated mice or mice administered anti-CD154 mAb (Fig. 8A). In addition, CD154^−/− mice on a BALB/c background showed inhibition of T cell expansion similar to that seen among pretreated BALB/cJ mice (Fig. 8D). However, neither CD154 antibody blockade nor absence of CD154 produced the decrease in CD8⁺ T cell GzmB expression seen among pretreated mice (Fig. 8B and 8E), suggesting that MPA affects on T cell function were not solely a result of diminished DC CD40 expression. Interestingly, pretreated mice administered rsCD154 concomitant with infection showed levels of virus-specific CD8⁺ T cell expansion and GzmB expression comparable to that seen among untreated controls (Fig. 8A–B). Combined, these results confirm and extend earlier studies that found CD40-CD154 interactions regulate the CD8⁺ T cell expansion produced by viral infection of mucosal tissue [38, 39].

MPA-mediated reduction of T cell expansion is site dependent

To determine if the ability of MPA to dampen anti-viral immunity was affected by route of infection, we assessed the proliferation and expansion of gB_498-505-specific CD8⁺ T cells among pretreated and untreated mice systemically (i.p. or i.v.) infected with HSV-1. No between-group differences in HSV-specific CD8⁺ T cell numbers or BrdU incorporation were seen (Fig. 9), indicating that compared to primary viral infection of mucosal tissue, systemic viral infection may be less susceptible to MPA-mediated inhibition of DC activation and T cell expansion.

Fig. 9.

MPA-mediated reduction of virus-specific T cell expansion is not observed with systemic HSV-1 infection. Untreated or pretreated (4 mg DMPA) intact female B6 mice were i.p. or i.v. infected with 10⁶ PFU HSV-1. At 6 dpi, mice were administered 1 mg BrdU (i.v.). 4 h later mice were euthanized, spleens processed into single-cell suspensions, and (A) percentages of BrdU⁺ cells and (B) numbers of gB_498-505-specific CD8⁺ T cells were determined by flow cytometric analysis. Comparisons were made using unpaired one-tailed Student t-tests; horizontal bars indicate means (n = 5 per group). Data shown are representative results from 1 of 2 independent experiments.

Discussion

Adaptive immunity evolved to provide appropriate host responses to commensal and pathogenic microorganisms. Activation of naïve T cells requires antigen recognition and costimulatory signaling [40], thus nonspecific activation is inhibited and tolerance to self-antigen promoted in uninfected tissue, where resting APC express very few costimulatory molecules [41]. With active infection, antigen binding and increases in IFN-γ stimulate APC to upregulate CD80 and CD86 [42], while T cells activated by antigen recognition and CD28 signaling upregulate CD154 expression [43]. Engagement between activated APC, that also upregulate CD40 expression, and the activated T cells, potentiates APC expression of CD70, CD80, CD86, and TNFSF4, and increases APC secretion of multiple T cell-activating cytokines (e.g., IL-12 and TNF) [44–46]. As APC-mediated activation of T cells authorizes additional APC to participate in the on-going response to infection, the reciprocal activation of the B7 and CD40 pathways has been termed “APC licensing.”

DLN DC from pretreated mice in our study had reduced expression of CD40, CD70, and CD80 after HSV-1 infection, while singular addition of CD40, CD80, or CD86 mAb to co-cultures that contained DC from untreated mice reduced ex vivo T cell proliferation less than what was seen in co-cultures containing DC from pretreated mice. These results imply pretreatment affected multiple costimulatory pathways and that impaired CD40 upregulation disrupted APC licensing, T cell expansion, and memory cell development, a conclusion consistent with earlier studies that showed anti-CD154 antibody inhibits mucosal tissue accumulation of CD8⁺ T cells [39], and CD40^−/− mice have reduced pools of memory CD8⁺ T cells after mucosal vesicular stomatitis virus (VSV) infection [38]. In our study, pretreated mice administered rsCD154 concomitant with HSV infection exhibited virus-specific T cell numbers similar to untreated controls, further suggesting that MPA profoundly impairs APC licensing, as even low levels of CD40-CD154 interaction promote costimulatory molecule expression, migration of DC to regional lymph nodes, and virus-specific T cell expansion (47, 48).

Our conclusions are supported by in vitro studies that show DC stimulated with CD154-transfected cells exhibit enhanced survival, increased IL-12 production, and higher CD80 expression [45, 49], and in vivo studies that show CD40^−/− mice display impaired early expansion of Mycobacterium tuberculosis-specific T cells and impaired generation of memory virus-specific CD8⁺ T cells [50, 51]. While prior investigations reported that progestin-containing compounds do not affect DC expression of CD80 or CD40 [52, 53], the DC in these earlier studies were matured in vitro from blood precursors while DC in our investigation were activated in vivo by viral infection of mucosal tissue. In addition to impaired T cell expansion, the inhibition of DC activation among pretreated mice in our study was associated with reduced numbers of other leukocyte subpopulations in acutely infected TG, results predicted by reports that DC control NK cell infiltration into infected tissues [54, 55], and that migration and accumulation of inflammatory cells to infected tissue is T cell-dependent [56, 57]. As CD154 plays a role in the development of bone marrow precursor cells [58], it remains a possibility that reduced expression of CD40 in pretreated mice may have contributed to the observed decrease in the numbers of cell lineage precursors. However, we found no differences in frequencies of any DC subset or any lymphoid or myeloid cell lineage in lymphoid organs or peripheral blood of uninfected mice administered DMPA (data not shown), results that suggest it is unlikely that a direct effect of DMPA on immune cell development was responsible for the reduced expansion of virus-specific lymphocytes in our study. In further support of this conclusion, earlier investigations have shown that progesterone treatment does not impair DC differentiation, but instead acts to promote DC differentiation and tissue accumulation [59, 60].

Effector T cell function (e.g., GzmB expression) was also impaired among pretreated mice, and reduced DC expression of CD40 may have contributed to this finding as optimal CTL function requires CD4⁺ T cell help responses regulated by CD40 signaling [61]. In support of this conclusion: CD154^−/− mice developed impaired CTL responses against VSV and adenovirus [62, 63]; several murine models showed that CD40-CD154 interactions were needed to acquire protective immune responses against intracellular pathogens [64–67]; while individuals with X-linked hyper-IgM syndrome, a disease linked to CD154 gene mutation, showed enhanced susceptibility to numerous microbial pathogens [68]. In the current study, T cell expansion among pretreated mice was unaffected if HSV infection was initiated through a non-mucosal route, a finding in agreement with earlier work that showed mucosal infection is more dependent than systemic infection on CD40-CD154 interaction for expansion (but not differentiation) of the CTL response [38, 39]. Taken together, these data highlight the central role of CD40-CD154 interaction during acute infection of mucosal tissue, while also indicating that MPA-mediated suppression of CD40 expression contributed to the impaired effector T cell function observed among pretreated mice in our investigation.

In conclusion, our study newly reveals that MPA impairs development of immunological memory. Although prior in vitro studies demonstrated that progesterone or MPA inhibited DC activation, this effect was shown at progesterone concentrations much higher than those achieved by pregnancy or Depo-Provera® injection for prevention of undesired pregnancy [69, 70]. As clinical studies indicate that steady state serum concentrations of MPA range between 0.7–10 ng/ml [26, 27, 71–73], further study is needed to learn if DC activation is impaired at these lower concentrations. In addition to the effects of MPA on virus-specific immunological memory, our study also provides novel elucidation of a mechanism by which progesterone may simultaneously promote tolerance and suppress nonspecific T cell activation in mucosal tissue. As female sex steroids help maintain tolerance to commensal organisms and allogeneic sperm and modulate defense against microbial pathogens, our results highlight the complexities of a system in which estrogen and progesterone are involved in the regulation of these seemingly divergent activities. Our results also emphasize the need for studies that are able to reveal the extent to which menstrual cycle changes, pregnancy, and exogenous sex steroids hormonal alter host susceptibility to infection by perturbing the balance between tolerance and immunity. Although MPA binds to the glucocorticoid receptor (GR) with much greater affinity than progesterone [74], there is little direct evidence to support that pharmacologically relevant doses of MPA modulate host immunity via GR binding. Therefore, it is also necessary to delineate the molecular mechanisms responsible for MPA’s immunomodulatory properties in order to facilitate both development and more confident selection of progestin-containing contraceptives that can prevent pregnancy without inhibiting host defense mechanisms or enhancing susceptibility to mucosal tissue infection.