Estradiol Enhances Antiviral CD4⁺ Tissue-Resident Memory T Cell Responses following Mucosal Herpes Simplex Virus 2 Vaccination through an IL-17-Mediated Pathway

Herpes simplex virus 2 (HSV-2) is a highly prevalent sexually transmitted infection for which there is currently no vaccine available. Interestingly, the female sex hormone estradiol has been shown to be protective against HSV-2. However, the underlying mechanisms by which this occurs remains relatively unknown. Our study demonstrates that under the influence of estradiol treatment, intranasal immunization with an attenuated strain of HSV-2 leads to enhanced establishment of antiviral memory T cell responses in the upper respiratory tract and female reproductive tract. In these sites, estradiol treatment leads to greater T_h17 memory cells, which precede enhanced T_h1 memory responses. Consequently, the T cell responses mounted by tissue-resident memory cells in the female reproductive tract of estradiol-treated mice are sufficient to protect mice against vaginal HSV-2 challenge. This study offers important insights regarding the regulation of mucosal immunity by hormones and on potential strategies for generating optimal immunity during vaccination.

ABSTRACT

Estradiol (E2) is a sex hormone which has been shown to be protective against sexually transmitted infections such as herpes simplex virus 2 (HSV-2). However, few studies have examined the underlying mechanisms by which this occurs. Here, we investigated the effect of E2 on the establishment of memory T cells post-intranasal immunization with HSV-2. CD4⁺ T cell responses first appeared in the upper respiratory tract (URT) within 3 days postimmunization before being detected in the female reproductive tract (FRT) at 7 days. E2 treatment resulted in greater and earlier T_h17 responses, which preceded augmented T_h1 responses at these sites. The CD4⁺ T cells persisted in the URT for up to 28 days, and E2 treatment resulted in higher frequencies of memory T cells. Intranasal immunization also led to the establishment of CD4⁺ tissue-resident memory T cells (T_RM cells) in the FRT, and E2 treatment resulted in increased T_h1 and T_h17 T_RM cells. When the migration of circulating T cells into the FRT was blocked by FTY720, immunized E2-treated mice remained completely protected against subsequent genital HSV-2 challenge compared to non-E2 controls, confirming that T_RM cells alone are adequate for protection in these mice. Finally, the enhanced vaginal T_h1 T_RM cells present in E2-treated mice were found to be modulated through an interleukin 17 (IL-17)-mediated pathway, as E2-treated IL-17A-deficient mice had impaired establishment of T_h1 T_RM cells. This study describes a novel role for E2 in enhancing CD4⁺ memory T cells and provides insight on potential strategies for generating optimal immunity during vaccination.

IMPORTANCE Herpes simplex virus 2 (HSV-2) is a highly prevalent sexually transmitted infection for which there is currently no vaccine available. Interestingly, the female sex hormone estradiol has been shown to be protective against HSV-2. However, the underlying mechanisms by which this occurs remains relatively unknown. Our study demonstrates that under the influence of estradiol treatment, intranasal immunization with an attenuated strain of HSV-2 leads to enhanced establishment of antiviral memory T cell responses in the upper respiratory tract and female reproductive tract. In these sites, estradiol treatment leads to greater T_h17 memory cells, which precede enhanced T_h1 memory responses. Consequently, the T cell responses mounted by tissue-resident memory cells in the female reproductive tract of estradiol-treated mice are sufficient to protect mice against vaginal HSV-2 challenge. This study offers important insights regarding the regulation of mucosal immunity by hormones and on potential strategies for generating optimal immunity during vaccination.

INTRODUCTION

Sex hormones play a major role in the development of the reproductive system by exerting profound effects on cell growth, development, differentiation, and homeostasis (1). Endogenous hormones, such as estrogen (E2) and progesterone (P4), as well exogenous administration of hormonal contraceptives, have been shown to regulate susceptibility to sexually transmitted viral infections (STIs), including herpes simplex virus 2 (HSV-2). Hormones can directly influence immune responses in the female reproductive tract (FRT), the primary site of heterosexual transmission of most STIs in women, as both innate and adaptive immune cells express receptors for E2 and P4 (2–4). Interestingly, we and others have shown that while P4 and P4-based hormonal contraceptives are associated with increased susceptibility and transmission of sexually transmitted viruses, E2 exerts an overall protective response (5–12). Previously, we showed that mice immunized under the influence of E2 were completely protected from subsequent genital HSV-2 challenge, with limited viral shedding and genital pathology (10, 11). Others have also reported a protective role for E2 in vivo when examining susceptibility to HSV-2 and other viral infections (13–17). However, the underlying mechanisms by which E2 mediates protection remain unclear. Better understanding of these mechanisms can provide greater insight into prophylactic protection strategies against HSV-2, which is critical because there is currently no cure or viable vaccine available for HSV-2.

HSV-2, the virus which causes genital herpes, is one of the most common STIs in the world. Rates of HSV-2 infection are disproportionately higher in women than in men; globally, approximately 14.8% of women are infected compared to 8.0% of men (18). While previous attempts at HSV-2 vaccine development have primarily focused on eliciting antibody responses for protection through systemic routes of immunization, limited success has been observed in clinical trials using these strategies (19, 20). Instead, recent efforts have shifted toward developing vaccine candidates capable of inducing both cellular and humoral immunity. Both clinical and experimental studies have shown that mucosal T cells play an important role in controlling HSV-2 infection. Studies examining genital lesions in infected women have reported that HSV-2 clearance is heavily dependent on CD8⁺ T cells, which participate in active immune surveillance at sites of HSV-2 reactivation (21–24). Additionally, recent studies examining T cell responses to HSV-2 in the FRT demonstrated that >90% of HSV-2-specific CD3⁺ T cells found in the cervixes of infected women were CD4⁺ T cells (25). These CD4⁺ T cells also demonstrated greater breadth of antigenic reactivity than the significantly minor (<5%) population of CD8⁺ T cells present. Similarly, animal studies have shown that compared to antibody responses, T helper 1 (T_h1) immunity and the production of interferon gamma (IFN-γ) are critical for protection against HSV-2 and that compared to systemic immunization, mucosal vaccination generates more balanced immunity at mucosal sites (26–32).

Studies conducted in mouse models of HSV-2 have shown that intranasal immunization provides better protection against subsequent challenge in the FRT than systemic immunization, and similar protection as observed following intravaginal immunization (29–32). While local immunization induces the strongest immune responses, intranasal immunization generates balanced protection composed of both antibodies and cellular immunity in the FRT (33). Additionally, intranasal immunization is considered a more practical method of vaccination, as it more feasible and less invasive than other routes (34). Limited studies examining the kinetics of T cell priming and dissemination following intranasal immunization have shown that initial priming and activation of T cells occurs in the upper respiratory tract (URT), which includes the nasal-associated lymphoid tissue (NALT) and cervical lymph nodes (cLNs) (35–40), and then the resulting antigen-specific T cells migrate to the FRT and the iliac LNs (iLNs) which drain the FRT. However, the precise mechanism by which intranasal immunization results in protection in the FRT is not yet fully understood.

We recently showed that E2 treatment induced vaginal dendritic cells (DCs) to prime robust T_h17 responses following intranasal immunization with HSV-2, and this coincided with greater accumulation of IFN-γ⁺ CD4⁺ effector T cells postchallenge (41). Consequently, E2-treated mice were completely protected against HSV-2 challenge. This study was the first to show that T_h17 cells are also involved in protective anti-HSV-2 immunity and that E2 can enhance T_h1 immunity in the FRT. In follow-up studies examining the antiviral role of interleukin 17 (IL-17), we found that IL-17 is critical for the induction of optimal T_h1 cell responses and overall protection against genital HSV-2 challenge (42). Following HSV-2 challenge, IL-17A-deficient mice (IL-17A^−/− mice) had impaired T_h1 cell responses, and this related to significantly greater mortality and disease severity than that in wild-type (WT) mice.

While our previous work examined the effect of E2 on CD4⁺ T cell responses generated post-intravaginal HSV-2 challenge, here, we were interested in understanding the effect of E2 during immunization. The purpose of the current study was to (i) better understand where T cell responses are initiated following intranasal immunization, and (ii) examine the effect of E2 during intranasal immunization. Furthermore, we wanted to examine if E2-mediated protection against HSV-2 could be attributed to greater establishment of CD4⁺ memory T cell responses postimmunization. To begin with, we examined the kinetics of the CD4⁺ T cell response in E2-treated and placebo-treated (mock) ovariectomized (OVX) female mice following intranasal vaccination with an attenuated strain of HSV-2. At early time points immediately following immunization (day 3), E2-treated mice first demonstrated greater T_h17 responses in the URT, which preceded enhanced T_h1 responses observed shortly thereafter (day 7). Similar results were observed in the FRT, where E2 treatment appeared to first enhance T_h17 responses (day 7), followed by greater T_h1 responses observed later (day 28). We next examined the establishment of memory T cells in these tissues and found that E2 enhanced CD44⁺ and CD103⁺ CD4⁺ T cells in both the NALT and cLNs. CD8⁺ memory T cells were also enhanced in the cLNs of E2-treated mice. Intranasal immunization also resulted in the establishment of T_h1 and T_h17 tissue-resident memory T cells (T_RM cells) in the FRT, which were enhanced in the presence of E2. Interestingly, the enhanced IFN-γ⁺ CD4⁺ T_RM cells appeared to be dependent on IL-17, as E2-treated IL-17A^−/− mice had impaired T_h1 T_RM cells in the FRT. Finally, we tested the antiviral efficacy of the CD4⁺ T_RM cells by treating immunized mice with the drug fingolimod (FTY720), which prevented the migration of circulating effector T cells into the FRT prior to intravaginal challenge with WT HSV-2. E2-treated mice which were administered FTY720 were still completely protected against HSV-2 challenge, thus demonstrating that CD4⁺ T_RM cells in the FRT were adequate for protection. Overall, our data demonstrate that E2 enhances CD4⁺ memory T cell responses, and our findings showcase a novel immunological mechanism by which E2 mediates greater protective outcomes during vaccination.

RESULTS

E2 treatment leads to greater induction of T_h17 cells following intranasal immunization, which precedes enhanced T_h1 responses.

Other studies have shown that post-intranasal immunization, T cell priming occurs in the URT (NALT and cLNs) and that this is followed by trafficking of primed T cells to the genital tract (35–40). Here, we wanted to determine the effect of E2 on CD4⁺ T cell responses following intranasal immunization with attenuated HSV-2 and the kinetics of that T cell response; therefore, we examined CD4⁺ T cells in the URT (NALT and cLNs) and the FRT at various time points following intranasal immunization.

E2- and mock-treated mice were immunized intranasally with thymidine kinase-deficient (TK⁻) HSV-2 (10⁴ PFU), and 3, 7, and 28 days later, CD4⁺ T cell responses at the site of immunization (NALT and cLNs), as well as distal sites related to HSV-2 challenge (FRT and iLNs), were examined using flow cytometric analysis. Cells were first gated on the total lymphocyte population, followed by single cells, and finally gated on the live population (Fig. 1A). Next, the expression of CD3 and CD4 was examined to identify the CD4⁺ T cells present, and functional differences were compared based on intracellular IFN-γ and IL-17 expression (Fig. 1A). As expected, the CD4⁺ T cell responses observed at the early time point (day 3) were quite limited and mainly restricted to the lymph nodes, with little to no response observed in the NALT and FRT (Fig. 1B). By day 7, T cell responses were increased in the NALT and FRT (Fig. 1C), and these T cell responses were sustained at 28 days postimmunization in the cLNs, NALT, and FRT (Fig. 1D). Interestingly, the time point analysis from days 3 to 28 demonstrated that in E2-treated mice, there was first a significant increase of T_h17 cells (IL-17⁺ CD4⁺), which preceded augmented T_h1 responses (IFN-γ⁺ CD4⁺). This was initially seen in the cLNs, where E2 treatment resulted in significantly greater proportions of T_h17 cells at day 3 postimmunization (Fig. 1B), which preceded the enhancement of T_h1 cells observed at day 7 (Fig. 1C). Likewise, this was also observed in the FRT and NALT, where there were first a greater proportion of T_h17 cells in E2-treated mice at day 7 (Fig. 1C) prior to the increased T_h1 cells seen at day 28 (Fig. 1D).

FIG 1.

Enhanced IL-17 levels precede greater IFN-γ responses in E2-treated mice following intranasal immunization. OVX WT (C57BL/6) mice (n = 5 to 10/group) were implanted with E2 or placebo (mock) pellets and immunized intranasally 2 weeks later with TK⁻ HSV-2 (10⁴ PFU/mouse). Three, 7, and 28 days postimmunization, cervical lymph nodes (cLNs), nasal-associated lymphoid tissue (NALT), iliac lymph nodes (iLNs), and vaginal tissue (FRT) were collected and processed. NALT or FRT tissues were pooled within groups. Cells were stimulated in vitro with a cell stimulation cocktail (CSC) for 15 h and then stained with a panel of antibodies (CD3, CD4, IL-17, and IFN-γ) and examined by flow cytometry. (A) Cells were gated based on the lymphocyte population, followed by single cells, and then CD4⁺ T cells were gated among total live CD3⁺ T cells. IFN-γ and IL-17 expression was examined using intracellular staining. The isotype control for IFN-γ and IL-17 staining is included (far right). The percentages of CD3⁺ CD4⁺ cells that were IFN-γ⁺ and IL-17⁺ were compared between E2-treated and mock-treated mice at day 3 (B), day 7 (C), and day 28 (D). Data shown represent means ± SEMs from 3 independent experiments. Data were analyzed using the two-way ANOVA with Sidak’s multiple-comparison test. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

Overall, these findings demonstrate a kinetic of the CD4⁺ T cell response following intranasal immunization, starting from the draining lymph nodes at the site of immunization (cLNs) to the distal sites related to HSV-2 infection (FRT). Furthermore, these results show that under the influence of E2, there is greater enhancement of T_h17 cells in cLNs, NALT, and FRT, which precedes an increase in T_h1 cells observed at these sites at a later time.

E2 enhances CD4⁺ but not CD8⁺ memory T cells in the NALT following intranasal immunization.

Having established that following intranasal immunization, CD4⁺ T cell responses were initiated within the URT and enhanced in the presence of E2, we next wanted to determine if the T cells induced at this site had a memory phenotype and if E2 influenced these T cell populations. To characterize the T cells present, we examined the expression of known memory markers, CD44 and CD103. Additionally, since others have shown a critical antiviral role for CD8⁺ T cells in the URT (43), we characterized both CD4⁺ and CD8⁺ T cells within the URTs of E2- and mock-treated mice 28 days post-intranasal immunization.

While E2 treatment did not affect the overall percentages of either CD4⁺ or CD8⁺ T cells in the NALT (Fig. 2A), there were significant differences in the phenotype of the CD4⁺ T cells present. IFN-γ and IL-17 production by CD4⁺ and CD8⁺ T cells was examined (Fig. 2B), and we found that E2-treated mice had significantly greater percentages and total numbers of both T_h1 (Fig. 2C) and T_h17 cells (Fig. 2E), while both of these populations were almost completely absent in mock-treated mice. Conversely, there were no significant differences in IFN-γ⁺ CD8⁺ T cells (Fig. 2D) in the NALT of E2-treated mice compared to that in mock controls, and there was very little to no production of IL-17 by CD8⁺ T cells (Fig. 2F).

FIG 2.

E2 enhances IFN-γ⁺ and IL-17⁺ CD4⁺ T cells in the NALT following intranasal immunization. OVX WT (C57BL/6) mice (n = 5 to 10/group) were implanted with E2 or placebo (mock) pellets and immunized intranasally 2 weeks later with TK⁻ HSV-2 (10⁴ PFU/mouse). Twenty-eight days postimmunization, nasal-associated lymphoid tissue (NALT) was collected, pooled, and processed. Cells were stimulated in vitro with a cell stimulation cocktail (CSC) for 15 h and then stained with a panel of antibodies (CD3, CD4, CD8, IL-17, and IFN-γ) and examined by flow cytometry. Cells were gated based on the lymphocyte population, followed by single cells, and then CD4⁺ and CD8⁺ T cells were gated among total live CD3⁺ T cells. (A) The percentages of CD3⁺ cells that were CD4⁺ and CD8⁺ were compared between E2-treated and mock-treated mice. Data shown represent means ± SEMs from 3 independent experiments. Data were analyzed using the two-way ANOVA with Tukey’s multiple-comparison test. (B) IL-17 and IFN-γ expression by T cells was examined. Representative plot shown from CD4⁺ T cells. The percentages and total numbers of IFN-γ-producing CD4⁺ (C) and CD8⁺ (D) T cells were compared between E2-treated and mock-treated mice. The percentages and total numbers of IL-17-producing CD4⁺ (E) and CD8⁺ (F) T cells were compared between E2-treated and mock-treated mice. Data shown represent means ± SEMs from 3 independent experiments. Data were analyzed using the unpaired, two-tailed t test with 95% confidence interval, with the ROUT method used to identify outliers. *, P < 0.05; **, P < 0.01; ns, no significance.

Next, we looked at the expression of CD44 (Fig. 3A) and CD103 (Fig. 3B) in order to examine memory T cell populations. We found that while E2 treatment significantly enhanced the proportions and total numbers of both CD44⁺ (Fig. 3C) and CD103⁺ (Fig. 3E) CD4⁺ T cells in the NALT, E2 did not influence CD8⁺ memory T cell populations (Fig. 3D and F). Taken together, these findings demonstrate that E2 enhances the establishment of CD4⁺ memory T cell populations in the NALT 28 days following intranasal immunization.

FIG 3.

E2 enhances CD4⁺ memory T cells (CD44⁺ and CD103⁺) in the NALT following intranasal immunization. OVX WT (C57BL/6) mice (n = 5 to 10/group) were implanted with E2 or placebo (mock) pellets and immunized intranasally 2 weeks later with TK⁻ HSV-2 (10⁴ PFU/mouse). Twenty-eight days postimmunization, nasal-associated lymphoid tissue (NALT) was collected, pooled, and processed. Cells were stimulated in vitro with a cell stimulation cocktail (CSC) for 15 h and then stained with a panel of antibodies (CD3, CD4, CD8, CD44, and CD103) and examined by flow cytometry. Cells were gated based on the lymphocyte population, followed by single cells, and then CD4⁺ and CD8⁺ T cells were gated among total live CD3⁺ T cells. CD44 (A) and CD103 (B) expression by T cells was examined. Representative plots shown from CD4⁺ T cells. Fluorescence minus one (FMO) controls for CD44 and CD103 are included. The percentages and total numbers of CD44⁺ CD4⁺ (C) and CD8⁺ (D) T cells were compared between E2-treated and mock-treated mice. The percentages and total numbers of CD103⁺ CD4⁺ (E) and CD8⁺ (F) T cells were compared between E2-treated and mock-treated mice. Data shown represent means ± SEMs from 3 independent experiments. Data were analyzed using the unpaired, two-tailed t test with 95% confidence interval, with the ROUT method used to identify outliers. *, P < 0.05; **, P < 0.01; ns, no significance.

E2 enhances CD4⁺ and CD8⁺ memory T cells in cLNs following intranasal immunization.

We repeated a similar analysis of T cells isolated from the cLNs which drain the NALT and found that E2-treated mice had greater proportions of CD8⁺ T cells in cLNs than mock-treated mice (Fig. 4A). When examining the expression of IFN-γ and IL-17 (Fig. 4B), we found that E2 treatment significantly enhanced both the percentages and total numbers of IFN-γ-producing CD4⁺ (Fig. 4C) and CD8⁺ T cells (Fig. 4D). E2-treated mice also had a greater number of T_h17 cells than mock-treated mice (Fig. 4E), while there were very few IL-17⁺ CD8⁺ T cells found within cLNs (Fig. 4F). Next, we looked at CD44⁺ (Fig. 5A) and CD103⁺ (Fig. 5B) memory T cell populations and found that in cLNs, E2 treatment significantly enhanced both CD44⁺ CD4⁺ (Fig. 5C) and CD44⁺ CD8⁺ memory T cells (Fig. 5D), as well as CD103⁺ CD4⁺ (Fig. 5E) and CD103⁺ CD8⁺ memory T cells (Fig. 5F). Overall, these results indicate that E2 treatment enhances the establishment of both CD4⁺ and CD8⁺ memory T cell populations in cLNs following intranasal immunization.

FIG 4.

E2 enhances IFN-γ⁺ and IL-17⁺ CD4⁺ T cells and IFN-γ⁺ CD8⁺ T cells in the cLNs following intranasal immunization. OVX WT (C57BL/6) mice (n = 5 to 10/group) were implanted with E2 or placebo (mock) pellets and immunized intranasally 2 weeks later with TK⁻ HSV-2 (10⁴ PFU/mouse). Twenty-eight days postimmunization, cervical lymph nodes (cLNs) were collected and processed. Cells were stimulated in vitro with a cell stimulation cocktail (CSC) for 15 h and then stained with a panel of antibodies (CD3, CD4, CD8, IL-17, and IFN-γ) and examined by flow cytometry. Cells were gated based on the lymphocyte population, followed by single cells, and then CD4⁺ and CD8⁺ T cells were gated among total live CD3⁺ T cells. (A) The percentages of CD3⁺ cells that were CD4⁺ and CD8⁺ were compared between E2-treated and mock-treated mice. Data shown represent means ± SEMs from 3 independent experiments. Data were analyzed using the two-way ANOVA with Tukey’s multiple-comparison test. (B) IL-17 and IFN-γ expression by T cells was examined. Representative plot shown from CD4⁺ T cells. The percentages and total numbers of IFN-γ-producing CD4⁺ (C) and CD8⁺ (D) T cells were compared between E2-treated and mock-treated mice. The percentages and total numbers of IL-17-producing CD4⁺ (E) and CD8⁺ (F) T cells were compared between E2-treated and mock-treated mice. Data shown represent means ± SEMs from 3 independent experiments. Data were analyzed using the unpaired, two-tailed t test with 95% confidence interval, with the ROUT method used to identify outliers. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, no significance.

FIG 5.

E2 enhances CD4⁺ and CD8⁺ memory T cells (CD44⁺ and CD103⁺) in the cLNs following intranasal immunization. OVX WT (C57BL/6) mice (n = 5 to 10/group) were implanted with E2 or placebo (mock) pellets and immunized intranasally 2 weeks later with TK⁻ HSV-2 (10⁴ PFU/mouse). Twenty-eight days postimmunization, cervical lymph nodes (cLNs) were collected and processed. Cells were stimulated in vitro with a cell stimulation cocktail (CSC) for 15 h and then stained with a panel of antibodies (CD3, CD4, CD8, CD44, and CD103) and examined by flow cytometry. Cells were gated based on the lymphocyte population, followed by single cells, and then CD4⁺ and CD8⁺ T cells were gated among total live CD3⁺ T cells. CD44 (A) and CD103 (B) expression by T cells was examined. Representative plots shown from CD4⁺ T cells. The percentages and total numbers of CD44⁺ CD4⁺ (C) and CD8⁺ (D) T cells were compared between E2-treated and mock-treated mice. The percentages and total numbers of CD103⁺ CD4⁺ (E) and CD8⁺ (F) T cells were compared between E2-treated and mock-treated mice. Data shown represent means ± SEMs from 3 independent experiments. Data were analyzed using the unpaired, two-tailed t test with 95% confidence interval, with the ROUT method used to identify outliers. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, no significance.

E2 enhances the establishment of CD4⁺ T_RM cells in the FRT following intranasal immunization.

After demonstrating that memory T_h17 and T_h1 cells are found in the URT following intranasal immunization and are enhanced in the presence of E2, we next wanted to determine if intranasal immunization with TK⁻ HSV-2 would also lead to the establishment of long-lasting CD4⁺ memory T cells in the FRT, the target site of protection from HSV-2. Specifically, we wanted to examine the establishment of CD4⁺ T_RM cells, which have been shown to be critical for mediating protection against HSV-2 (31, 44). Interestingly, previous studies have reported conflicting results regarding the generation of vaginal CD4⁺ T_RM cells following intranasal immunization (31, 44). Concurrently, we wanted to understand if E2 treatment would enhance the establishment of CD4⁺ T_RM cells, providing an explanation for a potential mechanism by which E2 leads to protection against HSV-2 in the FRT.

We first looked at the overall CD3⁺ T cell population within the FRTs of E2- and mock-treated mice 28 days post-intranasal immunization. Previous studies have shown that majority of the CD3⁺ T cells present in the FRTs of mice are CD4⁺ T cells; however, since most literature surrounding T_RM cells in the FRT has focused on the importance of CD8⁺ T cells, we also examined the population of CD8⁺ T cells present (45–47). We found that in mock-treated mice, CD4⁺ T cells constituted an approximately 4-fold higher frequency of the vaginal CD3⁺ T cell population than CD8⁺ T cells, and in E2-treated mice, there was an 8-fold higher frequency of CD4⁺ T cells than CD8⁺ T cells (Fig. 6A). Additionally, the percentage of vaginal CD4⁺ T cells in E2-treated mice was significantly higher than that in mock-treated mice, while there was no effect of E2 treatment on the small population of CD8⁺ T cells (Fig. 6A).

FIG 6.

E2 enhances the establishment of CD4⁺ T_RM cells in the FRT following intranasal immunization. OVX WT (C57BL/6) mice (n = 5 to 10/group) were implanted with E2 or placebo (mock) pellets and immunized intranasally 2 weeks later with TK⁻ HSV-2 (10⁴ PFU/mouse). Twenty-eight days postimmunization, vaginal tissue, spleen, and iliac lymph nodes (iLNs) were collected, pooled (vaginal tissue), and processed. Cells were stimulated in vitro with a cell stimulation cocktail (CSC) for 15 h and then stained with a panel of antibodies (CD3, CD4, CD8, IL-17, IFN-γ, CD44, CD103, CD69L, and CD69) and examined by flow cytometry. Cells were gated based on the lymphocyte population, followed by single cells, and then CD4⁺ and CD8⁺ T cells were gated among total live CD3⁺ T cells. (A) The percentages of CD3⁺ cells in the female reproductive tract (FRT) that were CD4⁺ and CD8⁺ were compared between E2-treated and mock-treated mice. Data shown represent means ± SEMs from 3 independent experiments. Data were analyzed using the two-way ANOVA with Tukey’s multiple-comparison test. (B) CD4⁺ T cells were gated among total live CD3⁺ T cells in the FRT, and CD4⁺ T_RM cells were defined as CD4⁺ CD44⁺ CD103⁻ CD69⁺ CD62L⁻. Fluorescence minus one (FMO) controls for CD62L and CD69 are included. (C) The percentages and total numbers of CD4⁺ T_RM cells in the FRT were compared between E2-treated and mock-treated mice. (D) The percentages of CD3⁺ CD4⁺ cells that were CD103⁺ in the FRT were compared between E2-treated and mock-treated mice. The percentages of CD3⁺ CD4⁺ T cells in the spleen (E) and iLNs (F) were compared between E2-treated and mock-treated mice. Data shown represent means ± SEMs from 3 to 5 independent experiments. Data were analyzed using the unpaired, two-tailed t test with 95% confidence interval. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001; ns, no significance.

Having established that CD4⁺ T cells were the predominant population of T cells present in the FRT post-intranasal immunization, we then further characterized these cells using an extensive panel of memory-related markers and a gating strategy used by previous studies, including our own, to specifically examine CD4⁺ T_RM cells (CD44⁺ CD103⁻ CD69⁺ CD62L⁻) in the FRT (Fig. 6B) (42, 44). We found that there were greater proportions and total numbers of CD4⁺ T_RM cells in the FRTs of E2-treated mice than in mock controls (Fig. 6C). Our primary gating strategy for CD4⁺ T_RM cells excludes CD103⁺ cells, as the expression of CD103 as a marker for CD4⁺ T cell residency in several tissues, including the FRT, is highly debated. Some studies consider CD103 a T_RM cell marker, while other studies have shown that vaginal CD4⁺ T_RM cells do not express CD103 (48). As such, we looked separately at CD103 expression by CD4⁺ T cells and found that E2 treatment also enhanced the CD103⁺ CD4⁺ T cell population in the FRT (Fig. 6D). Finally, for comparison, we examined CD4⁺ T cell responses at the 28-day time point in the spleen (Fig. 6E) and iLNs which drain the FRT (Fig. 6F) and found that E2 did not significantly impact the frequency of CD4⁺ T cells at these sites, suggesting that the effect of E2 on CD4⁺ memory T cells is site specific. Overall, these results suggest that intranasal immunization in the presence of E2 leads to the establishment of CD4⁺ T_RM cells in the FRT, further validating the use of intranasal vaccination as an effective mucosal route of immunization against HSV-2.

E2 increases the frequency of HSV-2-specific T_h1 and T_h17 T_RM cells in the FRT following intranasal immunization.

We next wanted to determine if the vaginal CD4⁺ T_RM cells established post-intranasal immunization represented antigen-specific populations and if these cells secreted cytokines known to generate protection against HSV-2 (IFN-γ and IL-17). Therefore, to further characterize the CD4⁺ T_RM cells present in the FRT, we examined the HSV-2 antigen specificity and phenotype of the FRT CD4⁺ T cells responding to HSV-2 antigen.

FRT cells were isolated from E2- and mock-treated mice 28 days following intranasal immunization and stimulated in vitro to induce cytokine production, as memory cells typically do not constitutively produce cytokines in the absence of active infection. To look at antigen-specific responses, we first conducted in vitro stimulation of the FRT cells using heat-inactivated HSV-2. Although the frequency of CD4⁺ T cells producing IFN-γ or IL-17 increased somewhat after stimulation with heat-inactivated HSV-2 and was enhanced in E2-treated mice, the numbers of cytokine-producing CD4⁺ T cells were not high enough to further characterize these memory cells based on the expression of T_RM markers (Fig. 7A). We therefore used a second model involving intranasal immunization of gDT-II transgenic mice to examine antigen-specific CD4⁺ T cell responses. These mice have been genetically modified such that a significant proportion of their CD4⁺ T cells express transgenes encoding a T cell receptor specific for the HSV gD-derived peptide epitope (gD_315–327) (49–51). Twenty-eight days post-intranasal immunization, FRT cells from gDT-II transgenic mice were stimulated in vitro with the corresponding gD peptide. Again, there was an antigen-specific response of IFN-γ- or IL-17-producing CD4⁺ T cells that was enhanced in E2-treated mice (Fig. 7B). However, we were unable to do any further phenotypic analysis with markers related to T_RM cells due to the small numbers of antigen-specific T cells. Finally, we used a cell stimulation cocktail (CSC) consisting of phorbol myristate acetate (PMA) and ionomycin to stimulate FRT cells isolated from WT mice, and this resulted in significant production of the target cytokines (Fig. 7C). We have previously used this method to examine T cell responses in other published work (41, 42, 52). To validate that the CSC-stimulated T cell responses resulted from the immunization with HSV-2, we also stimulated FRT cells from nonimmunized (naive) mice with CSC to measure any nonspecific responses. The comparison of the cytokine response by CD4⁺ T cells from the FRTs of immunized and nonimmunized mice indicated much higher frequencies (2- to 5-fold) of IL-17⁺ and IFN-γ⁺ CD4⁺ T cells from immunized mice (Fig. 7C), suggesting that CSC-stimulated cells in immunized mice mostly represent an antigen-specific response. We then used the CSC in vitro stimulation strategy to determine what frequency of FRT CD4⁺ T_RM cells (CD4⁺ CD44⁺ CD103⁻ CD69⁺ CD62L⁻) that expressed IL-17 or IFN-γ were present in immunized mice, comparing E2-treated and mock-treated mice. We found that E2-treated mice had greater proportions and total numbers of both IFN-γ⁺ (Fig. 7D and E) and IL-17⁺ CD4⁺ T_RM cells (Fig. 7F and G) in the FRT than mock-treated mice. Together, these findings indicate that E2 enhances antigen-specific T_h1 and T_h17 T_RM cells in the FRT following intranasal immunization.

FIG 7.

E2 enhances HSV-2 specific T_h1 and T_h17 T_RM cells in the FRT following intranasal immunization. OVX WT (C57BL/6) or gDT-II mice (n = 5 to 10/group) were implanted with E2 or placebo (mock) pellets and immunized intranasally 2 weeks later with TK⁻ HSV-2 (10⁴ PFU/mouse) or PBS (nonimmunized). Twenty-eight days postimmunization, vaginal tissue was collected, pooled, and processed. Cells from the female reproductive tract (FRT) were either left unstimulated or stimulated in vitro for 12 h with heat-inactivated HSV-2 (A), gD peptide (B), or cell stimulation cocktail (CSC) (C). Cells were stained with a panel of antibodies (CD3, CD4, IL-17, and IFN-γ) and examined by flow cytometry. Cells were gated based on the lymphocyte population, followed by single cells, and then CD4⁺ T cells were gated among total live CD3⁺ T cells. The percentages of IFN-γ- and IL-17-producing CD4⁺ T cells responding to heat-inactivated HSV-2, gD peptide, or CSC were compared between E2- and mock-treated mice. The isotype control for IFN-γ and IL-17 staining is included (A), along with CSC-stimulated cells from nonimmunized mice (C) for comparison. (D to G) CD4⁺ T cells were gated among total live CD3⁺ T cells, and CD4⁺ T_RM cells were defined as CD4⁺ CD44⁺ CD103⁻ CD69⁺ CD62L⁻. CD4⁺ T_RM cells were further gated based on IFN-γ and IL-17 expression. The percentages and total numbers of IFN-γ⁺ (D and E) and IL-17⁺ (F and G) CD4⁺ T_RM cells in the FRT were compared between E2-treated and mock-treated mice. Data shown represent means ± SEMs from 5 independent experiments. Data were analyzed using the unpaired, two-tailed t test with 95% confidence interval, with the ROUT method used to identify outliers. *, P < 0.05; ***, P < 0.001.

E2 augments T_h1 T_RM cells through an IL-17-mediated manner.

Given the observations that E2 treatment resulted in enhanced CD4⁺ T_RM cell responses in the FRT (Fig. 7) and that a greater induction of T_h17 cells preceded enhanced T_h1 immunity (Fig. 1), we wanted to understand the importance of IL-17 in augmenting IFN-γ responses. We have previously shown that E2-mediated protection coincides with greater T_h17 responses in the FRT and that T_h1 responses in the FRT are significantly reduced in the absence of IL-17 (41, 42). Therefore, to understand the importance of IL-17 in the establishment of T_h1 T_RM cells, we next examined the CD4⁺ T cell responses in IL-17A^−/− mice to see if the absence of IL-17 would impact the establishment of IFN-γ⁺ T_RM cells.

First, to distinguish if the vaginal IL-17-producing CD4⁺ T cells present in E2-treated mice consisted primarily of T_RM cells, we examined the total population of IL-17⁺ CD4⁺ T cells in the FRTs of E2-treated WT mice 28 days following intranasal immunization (Fig. 8A). IL-17⁺ CD4⁺ T cells were then gated based on the primary T_RM cell gating strategy, as shown in Fig. 7. We found that approximately 64% of all IL-17-producing CD4⁺ T cells were CD44⁺ CD103⁻ memory cells (Fig. 8A), suggesting that the remaining 36% of the IL-17⁺ CD4⁺ T cells present were not part of the typical population of T_RM cells. However, when we gated for the entire CD44⁺ T cell population, which also included CD103⁺ cells, >80% of the cells expressed both CD44⁺ and CD103⁺ (Fig. 8B). Overall, it appears that the majority of the IL-17-producing CD4⁺ T cells found in the FRTs of E2-treated mice following intranasal immunization are memory cells, as only a small proportion of these cells (<20%) did not express typical memory markers.

FIG 8.

E2-mediated enhancement of T_h1 T_RM cells is significantly diminished in the absence of IL-17. OVX WT (C57BL/6) and IL-17A^−/− mice (n = 5 to 10/group) were implanted with E2 or placebo (mock) pellets and immunized intranasally 2 weeks later with TK⁻ HSV-2 (10⁴ PFU/mouse). Twenty-eight days postimmunization, vaginal tissue was collected, pooled, and processed. Cells were stimulated in vitro for 15 h with a cell stimulation cocktail (CSC) and then stained with a panel of antibodies (CD3, CD4, IL-17, IFN-γ, CD44, CD103, CD62L, and CD69) and examined by flow cytometry. Cells were gated based on the lymphocyte population, followed by single cells, and then CD4⁺ T cells were gated among total live CD3⁺ T cells. The total population of IL-17-secreting CD4⁺ T cells in the female reproductive tracts (FRTs) of E2-treated WT mice was examined for CD44⁺ (A) and CD44⁺ CD103⁺ (B) expression. (C) Cells were gated based on the lymphocyte population, followed by single cells, and then CD4⁺ T cells were gated among total live CD3⁺ T cells. CD4⁺ T cells were further gated based on IFN-γ and IL-17 expression. The differences in IFN-γ⁺ and IL-17⁺ CD4⁺ T cells were compared between E2-treated IL-17A^−/− and WT mice. (D) CD4⁺ T cells were gated among total live CD3⁺ T cells in the FRT, and CD4⁺ T_RM cells were defined as CD4⁺ CD44⁺ CD103⁻ CD69⁺ CD62L⁻. The percentages of CD4⁺ T_RM cells in the FRT were compared between E2-treated IL-17A^−/− and WT mice. (E) CD4⁺ T_RM cells were further gated based on IFN-γ expression. The percentages of IFN-γ⁺ CD4⁺ T_RM cells in the FRT were compared between E2-treated IL-17A^−/− and WT mice. Data shown represent means ± SEMs from 4 independent experiments. Data were analyzed using the unpaired, two-tailed t test with 95% confidence interval. *, P < 0.05; ns, no significance.

To determine the importance of these IL-17⁺ CD4⁺ T cells in promoting greater T_h1 T_RM responses, we then examined IFN-γ⁺ CD4⁺ T cells in the FRTs of immunized IL-17A^−/− mice treated with E2. We found that the overall T_h1 cell population was greatly reduced in E2-treated IL-17A^−/− mice compared to that in E2-treated WT mice (Fig. 8C). When we looked specifically at CD4⁺ T_RM cells (CD4⁺ CD44⁺ CD103⁻ CD69⁺ CD62L⁻) (Fig. 8D) and IFN-γ-secreting CD4⁺ T_RM cells (Fig. 8E), we found that while there were no significant differences between the overall proportion of CD4⁺ T_RM cells in E2-treated IL-17A^−/− and WT mice, the frequency of T_h1 T_RM cells was significantly decreased in E2-treated IL-17A^−/− mice compared to that in control mice (Fig. 8E). Overall, these findings suggest that IL-17 is important for E2-mediated enhancement of T_h1 T_RM cells in the FRT.

E2-enhanced CD4⁺ T_RM cells are sufficient for protection against intravaginal HSV-2 challenge.

Finally, we wanted to test the antiviral efficacy of the CD4⁺ T_RM cells generated in the FRT following intranasal immunization. The goal was to determine the antiviral efficacy of the vaginal T_RM cells, independent of any help from circulating effector T cells which might migrate to the tissue upon HSV-2 challenge. To accomplish this, we used the drug fingolimod (FTY720), an antagonist of sphingosine-1-phosphate receptor 1 (S1PR1), which prevents lymphocyte egress from lymph nodes (53). FTY720 binds to S1PRs present on lymphocytes, causing the receptors to internalize and degrade, which results in an accumulation of lymphocytes in secondary lymphoid organs and leads to depletion of lymphocytes from circulation. Thus, FTY720 treatment prevents the circulation of T cells into and out of the FRT and allows us to study the ability of T_RM cells present in the FRT to respond to HSV-2 challenge independently of circulating effector T cells.

Three weeks following intranasal immunization, E2- and mock-treated (no hormone) mice were administered FTY720 or phosphate-buffered saline (PBS) daily via drinking water for 10 days prior to intravaginal challenge with WT HSV-2 (5 × 10³ PFU). To confirm the efficacy of FTY720, we checked if circulating T cells were absent in treated mice. Peripheral blood was collected on day 9 of FTY720 treatment, 1 day prior to challenge, and the number of CD4⁺ T cells present in the blood was determined. Mice administered FTY720 had significant depletion of CD4⁺ T cells in circulation compared to mice which received PBS (Fig. 9A). Both the frequencies and total numbers of CD4⁺ T cells were significantly lower in FTY720-treated animals than in PBS-treated controls (Fig. 9B). Once we confirmed the effectiveness of the drug, mice were challenged intravaginally with WT HSV-2 and daily FTY720 treatment continued, while survival, viral shedding, and genital pathology were monitored. We found that there was no difference in survival and pathology of mice regardless of whether there were circulating T cells present. Similar to E2-PBS control mice, E2-FTY720 mice, which must rely primarily on T_RM cells for protection, were completely protected against HSV-2 challenge (100% survival) (Fig. 9C). E2-treated mice administered FTY720 or PBS also had limited genital pathology (Fig. 9D) and significantly lower viral shedding (∼2-fold lower) (Fig. 9E and F) than immunized mice with no hormones (mock controls). Both pathology and viral shedding were comparable in both E2-treated groups. Likewise, both immunized mock-FTY720 and mock-PBS groups had similar disease outcomes as well; both groups succumbed to HSV-2 challenge by day 12 (Fig. 9C), all mice developed genital pathology (Fig. 9D), and both groups had similar levels of viral shedding (Fig. 9E and F). Overall, both E2-treated groups were better protected against HSV-2 challenge than immunized mice with no hormones. These results demonstrate that in the presence of E2, even without the help of any of circulating T cells (E2-FTY720), CD4⁺ T_RM cells generated in the FRT following intranasal immunization are sufficient for protection against subsequent intravaginal HSV-2 challenge. This suggests that the enhanced establishment of vaginal CD4⁺ T_RM cells observed in E2-treated mice may be responsible for E2-mediated protection against HSV-2. Moreover, these results show that intranasal immunization in the absence of E2 results in immune responses that are insufficient for protection against subsequent intravaginal HSV-2 challenge, thus further confirming that E2 plays a critical role in establishing protective memory responses during intranasal immunization.

FIG 9.

E2-enhanced CD4⁺ T_RM cells provide protection against intravaginal HSV-2 challenge. OVX WT (C57BL/6) mice (n = 5 to 10/group) were implanted with E2 or placebo (mock) pellets and immunized intranasally 2 weeks later with TK⁻ HSV-2 (10⁴ PFU/mouse). Three weeks postimmunization, mice were given drinking water containing 4 μg/ml of FTY720 or PBS for the control groups for 10 days prior to intravaginal challenge with WT HSV-2 (5 × 10³ PFU/mouse). Mice continued to receive treatment throughout the duration of the experiment. (A) On day 9 of FTY720 or PBS treatment and prior to intravaginal HSV-2 challenge, blood was collected, processed, stained with a panel of antibodies (CD45, CD3, and CD4), and examined by flow cytometry to confirm depletion of CD4⁺ T cells in circulation. Cells were gated based on the lymphocyte population, followed by single cells, and then CD3⁺ CD4⁺ T cells were gated among total live CD45⁺ T cells. (B) The percentages and total numbers of CD4⁺ T cells in the blood were compared between FTY720-treated and PBS-treated mice. Data represent means ± SEMs from 2 independent experiments. Data were analyzed using the unpaired, two-tailed t test with 95% confidence interval. Post-intravaginal challenge, survival was monitored (C) and pathology scores were recorded on a scale of 0 to 5 for up to 12 days (D). Significance in difference in survival (C) was calculated using the log rank (Mantel-Cox) test (***, P < 0.001). Data points superimposed on the x axes in panel D indicate mice without genital pathology. (E and F) Vaginal washes were collected daily for 6 days postchallenge, and HSV-2 shedding was calculated using a Vero cell-based assay. Each symbol represents a single animal. The dotted lines in panel E indicate the lower detection limit of the assay, and data points on this line indicate undetectable viral shedding. Data shown in panel F represent the average viral load (means ± SEMs) over 6 days. Data were analyzed using a one-way ANOVA with Tukey’s multiple-comparison test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

DISCUSSION

It is well known that hormones modulate immunity, thereby affecting susceptibility to pathogens and impacting vaccine efficacy. In the context of HSV-2, intranasal immunization under the influence of E2 has been associated with protection against intravaginal HSV-2 challenge in mice (11, 41). Interestingly, while intranasal immunization has often been used to generate protection in the FRT, the characterization of the T cell response generated following immunization and the mechanism by which intranasal immunization results in protection in the FRT has not been well described. We have previously examined potential mechanisms by which intranasal immunization under the influence of E2 results in protection against HSV-2. Our most recent studies showed that E2 mediates protection by enhancing T_h17 and T_h1 responses following HSV-2 challenge in the FRT (41). Similarly, T_h17 induction was also higher in mice which were naturally in the estrus stage of the cycle (E2 dominant) than in mice in diestrus (P4 dominant) or OVX mice, suggesting that high estrogen levels induce greater CD4⁺ T cell responses in the FRT (41). However, thus far, we had not characterized the memory responses generated following intranasal immunization and the effect of E2 on these responses. In the present study, we examined the initiation and dissemination of the CD4⁺ T cell memory responses following intranasal immunization and examined how E2 influenced this process.

We found that following intranasal immunization, CD4⁺ T cell responses were primarily initiated in the URT (cLNs) and were later detected at distal sites such as the FRT. Interestingly, E2 treatment enhanced CD4⁺ T cell responses in both the URT and FRT. At early time points following immunization (day 3), E2-treated mice had greater T_h17 cell responses in the cLNs, which preceded enhanced T_h1 responses observed later (day 7). Similar results were observed in the FRT and NALT, where E2-treated mice initially had greater T_h17 responses earlier (day 7), followed by enhanced T_h1 responses observed later (day 28). We also characterized T cell responses 28 days postimmunization to determine where long-lasting memory T cells are established. E2 enhanced CD4⁺ CD44⁺ and CD103⁺ memory T cells in both the NALT and cLNs, while CD8⁺ memory T cells were only enhanced in the cLNs. E2-treated mice also had greater T_h1 and T_h17 responses in the FRT, from T cells characterized as T_RM cells. Interestingly, IFN-γ⁺ CD4⁺ T_RM cells observed in E2-treated mice were enhanced through an IL-17-mediated pathway, as E2-treated IL-17A^−/− mice had impaired establishment of T_h1 T_RM cells. Furthermore, the CD4⁺ T_RM cells in E2-treated mice were able to protect against subsequent intravaginal HSV-2 challenge without help from any circulating effector T cells, thus demonstrating that the establishment of CD4⁺ T_RM cells in the FRT post-intranasal immunization is adequate for protection against HSV-2. To our knowledge, this is the first study to demonstrate that intranasal immunization under the influence of E2 enhances memory T cell populations in both the URT and FRT.

Unlike other memory subsets, such as effector and central memory T cells, T_RM cells respond more efficiently and rapidly to infection, primarily due to their anatomical location within nonlymphoid tissues (48). As such, vaccine strategies which can establish T_RM cells are considered more favorable for eliciting superior protection. In this work, we used an intranasal immunization model for vaccination against a sexually transmitted virus, HSV-2, which is a more practical route of mucosal vaccination than intravaginal immunization. Previous studies have reported that intranasal immunization fails to establish substantial, long-term CD4⁺ T cell immunity in the FRT (44). However, Sato et al. demonstrated that IFN-γ-producing CD4⁺ T cells could be found in the FRT for up to 3 weeks following intranasal HSV-2 immunization, although they did not characterize the subtypes of CD4⁺ memory T cells in depth or investigate the protective efficacy of the memory T cell population present (31). In the present study, we show that intranasal immunization under the influence of E2 results in the establishment of a substantial population of antigen-specific CD4⁺ T_RM cells in the FRT, thus providing additional support for the use of intranasal immunization as a viable vaccination route for generating protective immunity in the FRT. Furthermore, these CD4⁺ T_RM cells were shown to be adequate for mediating antiviral protection against subsequent intravaginal HSV-2 challenge. This may also be beneficial against other FRT pathogens where CD4⁺ T cell immunity is known to be protective.

Although most T_RM studies have focused on CD8⁺ T_RM cells, recent studies have also started to identify subsets of CD4⁺ T_RM cells, including T_h17 T_RM cells, which have been reported to be present in many tissues, including the nose, skin, and lungs (54–59). An elegant study by Vesely et al. used IL-17A tracking fate mouse models to show that majority of airway CD4⁺ T_RM cells found following infection with Klebsiella pneumonia are derived from T_h17 cells and that these cells are critical for protection against bacterial infection (55). In the context of the URT, Allen et al. found that intranasal immunization with a novel vaccine against Bordetella pertussis led to the establishment of T_h17 T_RM cells in the nose, which conferred long-term protection against nasal colonization and lung infection in mice (57). However, most studies examining T_RM cells in the URT have focused on bacterial and fungal pathogens. To the best of our knowledge, only one study has identified antiviral T_RM cells in the URT, and that was in the context of CD8⁺ T_RM cells (43). In this work, Pizzolla et al. demonstrated that influenza-specific CD8⁺ T_RM cells are established in the URT following intranasal immunization and were able to block the transmission of the virus into the lungs (43). Here, we show for the first time that E2 treatment leads to greater long-term memory populations of CD4⁺ T cells in the NALT and cLNs following intranasal immunization, suggesting that a similar phenomenon may also occur in other viral disease models. As such, it would be beneficial to determine if enhanced CD4⁺ memory T cells generated in the presence of E2 can help prevent dissemination of viral pathogens such as influenza into the lungs and, thereby, enhance protection.

Very few studies have reported the influence of hormones on memory T cells, and even fewer have examined specific subsets such as T_RM cells. A study by Rodriguez-Garcia et al. examined the effect of aging and changes in hormone levels on CD8⁺ memory T cells isolated from the FRT and found that with aging, there was a loss of CD103⁺ CD8⁺ T_RM cells in the cervix, while these populations were increased in the endometrium (60). This suggests that even within the same anatomical location (FRT), the effects of hormones on T_RM cells is site specific. Swaims-Kohlmeier et al. examined CCR7^hi CD4⁺ memory T cells in vaginal lavage samples collected from healthy women and found that proportions of these memory cells positively correlated with P4 levels, thus suggesting that P4 enhances CD4⁺ memory T cell populations (61). Conversely, Hall et al. reported that P4 treatment in mice resulted in significantly reduced numbers and activity of influenza-specific CD8⁺ T_RM cells in the lungs (62). Interestingly, the differential effects of P4 in the vaginal tract versus in the lungs again suggests that the induction of T_RM cells and the effects of hormones are most likely disease and tissue specific. Our findings add to this body of information by demonstrating that E2 increases CD4⁺ memory T cells in the NALT and cLNs, CD8⁺ memory T cells in the cLNs, and CD4⁺ T_RM cells in the FRT following intranasal immunization. Further work is required to understand the influence of hormones on molecular mechanisms involved in generating memory T cell responses in order to exploit this information for better vaccination strategies.

Our present study suggests that higher frequencies of T_h17 cells induced by E2 treatment play an important role in enhancing IFN-γ responses in the URT and FRT. Khader et al. similarly showed that in a Mycobacterium tuberculosis vaccination model, T_h17 cells accumulated in the lungs at earlier time points than T_h1 cells (63). Moreover, the absence of IL-17 resulted in significantly reduced IFN-γ-producing CD4⁺ T cells in the lungs (63). Additional studies have also reported important connections between T_h17 and T_h1 cells in both the lungs and FRT (64–67). For instance, we have previously shown in vitro that vaginal DCs isolated from IL-17A^−/− mice were significantly impaired at inducing IFN-γ production by CD4⁺ T cells compared to those from WT mice, thus suggesting there is an intrinsic impairment in the priming of T_h1 cell responses by vaginal DCs in the absence of IL-17 (41). This was confirmed in vivo, where we also showed in a separate study that there was a significant decrease in IFN-γ production by vaginal CD4⁺ T cells in IL-17A^−/− mice post-HSV-2 challenge (42). Likewise, Bai et al. demonstrated that mice treated with anti-IL-17 antibodies had significantly delayed clearance of Chlamydia muridarum in the lungs, and this corresponded with significantly reduced T_h1 responses (64). They also showed that DCs isolated from IL-17-neutralized mice induced lower levels of IFN-γ production by CD4⁺ T cells, and this was related to downregulation of DC activity in the absence of IL-17 (64). Our findings here suggest that a similar phenomenon may be occurring in the URT following intranasal immunization, where E2 may be differentially priming T cells and inducing greater T_h17 responses, which then leads to enhanced T_h1 cells. However, additional experiments examining T cell priming by nasal DCs isolated from E2-treated mice are required to determine the effect of E2 on T cell responses in the URT. Likewise, the mechanism by which IL-17 influences T_h1 memory responses is still unclear, and further work is needed to elucidate this mechanism.

In summary, this study shows for the first time that following intranasal immunization, E2 first enhances T_h17 responses in the URT and FRT, which precedes enhanced T_h1 immunity. Consequently, intranasal immunization under the influence of E2 results in greater establishment of CD4⁺ memory T cells at these sites. These findings also demonstrate a novel mechanism by which E2 protects against HSV-2, where the presence of E2 during intranasal immunization leads to enhanced T_h17 and T_h1 T_RM cells in the FRT. As such, when mice are then challenged with HSV-2, there is a rapid and robust CD4⁺ memory T cell response generated within the FRT, which protects against infection. This work has important implications in terms of vaccine development, as an effective vaccination strategy should aim to generate rapid long-lasting immunity. HSV-2 vaccine strategies which incorporate mucosal immunization, and also take into consideration the stage of the menstrual cycle during immunization, may promote effective T_h1 memory responses and generate better overall vaccine efficacy. Overall, the findings of this study emphasize the importance of hormones in influencing immune responses and provide insights for strategies which can help enhance memory responses in mucosal tissue following immunization.

MATERIALS AND METHODS

Mice.

C57BL/6 female mice were obtained from Charles River Laboratories Inc. (Saint-Constant, QC, Canada). IL-17A^−/− mice (C57BL/6 background) were generated by Yoichiro Iwakura (University of Tokyo, Tokyo, Japan). IL-17A^−/− and gDT-II transgenic mice were bred in the Central Animal Facility (McMaster University, Hamilton, Canada). The gDT-II mice express transgenes encoding a T cell antigen receptor (Vα3.2/Vβ2) that specifically recognizes the HSV gD-derived epitope, gD_315–327 (49–51). All mice were maintained under specific-pathogen-free and standard temperature-controlled conditions that followed a 12-h light/dark cycle. To ensure that mice remained specific pathogen free, routine quality assurance was conducted by serology and PCR, and this included testing dirty bedding, sentinels, direct resident animals, and exhaust air duct samples of racks. All animal studies were approved by, and in compliance with, the Animal Research Ethics Board at McMaster University in accordance with the Canadian Council of Animal Care guidelines.

Ovariectomy surgeries.

Endogenous hormones in mice were depleted by OVX, according to previously published protocols (11). Briefly, mice were administered an injectable anesthetic preparation of ketamine and xylazine intraperitoneally. Ovaries were removed by making two bilateral incisions, followed by small incisions through the peritoneal wall, and excising them through the incisions. Incisions were closed using surgical clips, and mice recovered for 10 to 14 days before the start of experiments.

Hormone treatment.

To examine the effect of E2, mice were treated with 17β-estradiol hormone pellets (0.010 mg/pellet) produced by Innovative Research of America (Sarasota, FL, USA), according to previously published protocols (11). These hormone pellets are designed to release 476 ng/day/mouse for 21 days. Briefly, 2 weeks following OVX surgery, mice were anesthetized with injectable anesthetic (ketamine and xylazine) and implanted subcutaneously with 17β-estradiol or placebo (mock) pellets. The level of serum E2 resulting from the pellets has previously been shown to correspond to that measured during the estrous cycle (68).

Intranasal immunization.

OVX mice were intranasally immunized with an attenuated strain of HSV: thymidine kinase-deficient (TK⁻) HSV-2. Mice were lightly anesthetized using isoflurane and then inoculated with 5 μl of TK⁻ HSV-2 into each nare with a micropipette, for a total of 10 μl (10⁴ PFU/mouse).

FTY720 treatment.

FTY720 treatment (Sigma-Aldrich, St. Louis, MO) was administered via drinking water to prevent T cell circulation into and out of the FRT. Briefly, 3 weeks following intranasal immunization with TK⁻ HSV-2, mice were given drinking water containing 4 μg/ml of FTY720 or PBS daily for 10 days prior to intravaginal challenge with WT HSV-2. FTY720 treatment was then continued postchallenge for the duration of the experiment.

Intravaginal challenge.

In FTY720 experiments, immunized mice were challenged intravaginally with WT HSV-2. Briefly, 4 weeks after intranasal immunization and following initiation of FTY720 treatment, mice were anaesthetized intraperitoneally and infected with 10 μl of WT HSV-2 strain 333 (5 × 10³ PFU/mouse) intravaginally. After inoculation, mice were placed on their backs for approximately 30 to 45 min to allow for the inoculum to infect the vaginal tract.

Collection of vaginal washes.

Vaginal washes were collected for up to 6 consecutive days postchallenge by pipetting 30 μl of phosphate-buffered saline (PBS) into and out of the vagina 5 to 6 times. This was repeated twice to collect a total volume of 60 μl/mouse that was stored at −80°C until required.

Genital pathology scoring.

Genital pathology was monitored daily postchallenge based on a five-point scale, as described previously (42): no infection (0), slight redness of external vagina (1), swelling and redness of vagina (2), severe swelling and redness of vagina and surrounding tissues (3), genital ulceration with severe redness and hair loss (4), and severe ulceration extending to surrounding tissues, ruffled hair, and lethargy (5). Animals were sacrificed before they reached stage 5.

Viral titration.

Viral shedding in vaginal washes was determined by conducting viral plaque assays on Vero cell (ATCC, Manassas, VA) monolayers, as described previously (42). Vero cells were grown in α-MEM (Gibco Laboratories, Burlington, ON, Canada) supplemented with 5% fetal bovine serum (FBS; Gibco Laboratories), 1% penicillin-streptomycin (Invitrogen, Burlington, ON, Canada), l-glutamate (BioShop Canada Inc., Burlington, ON, Canada), and 1% HEPES (Invitrogen). Cells were grown to confluence in 12-well plates, and washes were diluted in α-MEM and then added to monolayers. Infected monolayers were incubated at 37°C for 2 h and then overlaid with α-MEM, after which, infection was allowed to occur for 48 h at 37°C. Next, cells were fixed and stained with crystal violet, and viral plaques were enumerated under a microscope. The number of PFU per milliliter was calculated by taking a plaque count for every sample and accounting for the dilution factor.

Whole-blood analysis.

For peripheral blood analysis, whole-blood samples were collected, processed using ammonium-chloride-potassium lysing buffer (Sigma-Aldrich), and stained for cell surface markers using the following antibodies at concentrations based on the manufacturer’s specification sheets: CD4 BV421 (BioLegend, San Diego, CA, USA), CD3 phycoerythrin (PE)-cf594 (BD Biosciences, Mississauga, ON, Canada), and CD45 BV786 (BD Biosciences). Cells were incubated with these antibodies for 30 min before being analyzed by flow cytometry.

Single-cell preparation and cultures.

For iLN and cLN analyses, samples were collected, and a single-cell suspension was prepared by mechanically disrupting the tissue. For NALT samples, tissues were collected and pooled per group, and cell suspensions were prepared by gently teasing the tissue between glass slides followed by filtration through a 40-μm filter (Small Parts, Miami Lakes, FL, USA) (69, 70). The supernatants containing single cells were collected and centrifuged for 5 min (1,500 rpm) at 4°C. For vaginal tissue analysis, vaginal tracts were removed, pooled, cut lengthwise, washed to remove mucous, and minced into 2- to 4-mm pieces. The vaginal tissue pieces were enzymatically digested in 15 ml of RPMI 1640 containing 0.00157 g/ml collagenase A (Roche Life Science, USA) at 37°C on a stir plate for 2 h and filtered through a 40-μm filter to obtain a total tissue cell suspension (46). Vaginal cell samples were then centrifuged for 10 min (1,200 rpm) at 4°C.

Single-cell suspensions from different tissues were resuspended in 1 to 5 ml of RPMI 1640 medium supplemented with 10% FBS, 100 IU/ml penicillin, 100 μg/ml streptomycin, 1% l-glutamine, 0.1% 2-mercaptoethanol, 1× nonessential amino acids, and 1× sodium pyruvate (Gibco Life Technologies, Burlington, ON, Canada). Finally, mononuclear cells were counted, and cell preparations were seeded in a 24-well plate at 3 × 10⁶ cells/well for spleen and LN samples and 5 × 10⁵ to 1 × 10⁶ cells/well for vaginal and NALT samples; the total volume in the well was topped up to 1 ml with previously described supplemented RPMI 1640 medium. Cells were either left unstimulated (brefeldin A and monensin) at 37°C or underwent in vitro stimulation to determine the function of the T cells present. In vitro stimulation conditions included (i) 2 μl/ml of cell stimulation cocktail (CSC) plus protein transport inhibitors (500×) (cocktail of PMA, ionomycin, brefeldin A, and monensin [eBioscience, San Diego, CA, USA]) for 15 h, (ii) heat-inactivated HSV-2 (5 × 10⁴ PFU) for 12 h, or (iii) 25 μg/well of gD peptide (315 to 327) (Biomer Technology, Pleasanton, CA, USA) with sequence IPPNWHIPSIQDA for 12 h.

Flow cytometry.

Following 12 to 15 h of incubation at 37°C, cells were collected and stained with allophycocyanin (APC)-ef780 viability dye (eBioscience) for 30 min. Cells were washed and incubated for 5 to 10 min with 2 μl of Fc block (anti-mouse CD16/32; eBioscience) to reduce nonspecific Fc receptor staining. Cells were then stained for cell surface markers using the following antibodies at concentrations based on manufacturer’s specification sheets: CD4 BV421, CD3 PE-cf594, CD8 PE-Cy7 (BD Biosciences), CD103 BV510 (BD Biosciences), CD44 AF700 (BioLegend), CD62L BB515 (BD Biosciences), and CD69 PE (BD Biosciences). Cells were incubated with these antibodies for 30 min and then permeabilized and fixed using the Transcription Factor buffer set (BD Biosciences) according to the manufacturer’s protocol. Cells were then stained for intracellular markers using the following antibodies: IFN-γ fluorescein isothiocyanate (FITC) or FITC-IgG1 isotype control, and IL-17A APC or APC-IgG2 isotype control (eBioscience). The validity of staining was verified by fluorescence minus one (FMO) controls and/or appropriate isotype controls. Data were collected by flow cytometric analysis using a BD LSRII flow cytometer system (BD Bioscience Pharmingen), and results were analyzed using FlowJo software.

Statistical analysis.

Statistical analysis and graphical representation were performed using GraphPad Prism 6.0d (GraphPad Software, San Diego, CA). The Mantel-Cox log rank test was used to calculate significant differences in survival. Data are expressed as means ± standard errors of means (SEMs), typically derived from a minimum of 3 to 5 independent experiments, with n = 5 to 7 mice/group. Significance was calculated by comparing means using one-way or two-way analyses of variance (ANOVAs) or t tests, with appropriate additional tests, as indicated in the figure legends. Statistical significance was defined at a P value of <0.05.