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As women age, susceptibility to systemic and genital infections increases. Tissue-resident memory T cells (TRMs) are CD103 + CD8 + long-lived lymphocytes that provide critical mucosal immune protection. Mucosal dendritic cells (DCs) are known to induce CD103 expression on CD8 + T cells. While CD103 + CD8 + T cells are found throughout the female reproductive tract (FRT), the extent to which aging impacts their presence and induction by DCs remains unknown. Using hysterectomy tissues, we found that endometrial CD103 + CD8 + T cells were increased in postmenopausal compared to premenopausal women. Endometrial DCs from postmenopausal women were significantly more effective at inducing CD103 expression on allogeneic naïve CD8 + T cells than DCs from premenopausal women; CD103 upregulation was mediated through membrane-bound TGFβ signaling. In contrast, cervical CD103 + T cells and DC numbers declined in postmenopausal women with age. Decreases in DCs correlated with decreased CD103 + T cells in endocervix, but not ectocervix. Our findings demonstrate a previously unrecognized compartmentalization of TRMs in the FRT of postmenopausal women, with loss of TRMs and DCs in the cervix with aging, and increased TRMs and DC induction capacity in the endometrium. These findings are relevant to understanding immune protection in the FRT and to the design of vaccines for women of all ages., (© 2018 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Women acquire HIV through sexual transmission, with increasing incidence in women >50 years old. Identifying protective mechanisms in the female genital tract (FGT) is important to prevent HIV-acquisition in women as they age. Human genital and blood neutrophils inactivate HIV by releasing neutrophil extracellular traps (NETs), an innate protective mechanism against HIV-infection. However, how NET formation is triggered by HIV in different tissues and whether this mechanism is affected by aging remain unknown. We demonstrate that the mechanisms that trigger NET release in response to HIV are different in blood and genital tissues, and that NET release decreases with aging. In blood neutrophils, HIV stimulation independently activated calcium pathways and endosomal TLR8, but aging reduced calcium responses, resulting in delayed NET release. In contrast, calcium responses were absent in genital neutrophils and NET release was triggered preferentially through TLR8 activation, but aging impaired this pathway. HIV induced NET formation through non-lytic pathways in blood and FGT neutrophils, except for a small subset of NETs that incorporated annexin V and lactoferrin predominantly in blood, suggesting proinflammatory and lytic NET release. Our findings demonstrate that blood neutrophils cannot model genital neutrophil responses which has important implications to understanding protection against HIV acquisition. [ABSTRACT FROM AUTHOR]

Background: Immune function in the genital mucosa balances reproduction with protection against pathogens. As women age, genital infections, and gynecological cancer risk increase, however, the mechanisms that regulate cell-mediated immune protection in the female genital tract and how they change with aging remain poorly understood. Unconventional double negative (DN) T cells (TCRαβ + CD4-CD8-) are thought to play important roles in reproduction in mice but have yet to be characterized in the human female genital tract. Using genital tissues from women (27–77 years old), here we investigated the impact of aging on the induction, distribution, and function of DN T cells throughout the female genital tract. Results: We discovered a novel site-specific regulation of dendritic cells (DCs) and unconventional DN T cells in the genital tract that changes with age. Human genital DCs, particularly CD1a + DCs, induced proliferation of DN T cells in a TFGβ dependent manner. Importantly, induction of DN T cell proliferation, as well as specific changes in cytokine production, was enhanced in DCs from older women, indicating subset-specific regulation of DC function with increasing age. In human genital tissues, DN T cells represented a discrete T cell subset with distinct phenotypical and transcriptional profiles compared to CD4 + and CD8 + T cells. Single-cell RNA and oligo-tag antibody sequencing studies revealed that DN T cells represented a heterogeneous population with unique homeostatic, regulatory, cytotoxic, and antiviral functions. DN T cells showed relative to CD4 + and CD8 + T cells, enhanced expression of inhibitory checkpoint molecules and genes related to immune regulatory as well as innate-like anti-viral pathways. Flow cytometry analysis demonstrated that DN T cells express tissue residency markers and intracellular content of cytotoxic molecules. Interestingly, we demonstrate age-dependent and site-dependent redistribution and functional changes of genital DN T cells, with increased cytotoxic potential of endometrial DN T cells, but decreased cytotoxicity in the ectocervix as women age, with implications for reproductive failure and enhanced susceptibility to infections respectively. Conclusions: Our deep characterization of DN T cell induction and function in the female genital tract provides novel mechanistic avenues to improve reproductive outcomes, protection against infections and gynecological cancers as women age. [ABSTRACT FROM AUTHOR]

Mucosal tissues in the human female reproductive tract (FRT) are primary sites for both gynecological cancers and infections by a spectrum of sexually transmitted pathogens, including human immunodeficiency virus (HIV), that compromise women's health. While the regulation of innate and adaptive immune protection in the FRT by hormonal cyclic changes across the menstrual cycle and pregnancy are being intensely studied, little to nothing is known about the alterations in mucosal immune protection that occur throughout the FRT as women age following menopause. The immune system in the FRT has two key functions: defense against pathogens and reproduction. After menopause, natural reproductive function ends, and therefore, two overlapping processes contribute to alterations in immune protection in aging women: menopause and immunosenescence. The goal of this review is to summarize the multiple immune changes that occur in the FRT with aging, including the impact on the function of epithelial cells, immune cells, and stromal fibroblasts. These studies indicate that major aspects of innate and adaptive immunity in the FRT are compromised in a site‐specific manner in the FRT as women age. Further, at some FRT sites, immunological compensation occurs. Overall, alterations in mucosal immune protection contribute to the increased risk of sexually transmitted infections (STI), urogenital infections, and gynecological cancers. Further studies are essential to provide a foundation for the development of novel therapeutic interventions to restore immune protection and reverse conditions that threaten women's lives as they age. [ABSTRACT FROM AUTHOR]

The functional characterization and regulation of tissue resident and non-resident CD8+ T cells in the human female reproductive tract (FRT) as women age remains a gap in our knowledge. Here we characterized the cytotoxic activity and granular contents of CD8+ T cells from the FRT in pre- and postmenopausal women. We found that under steady-state conditions, CD8+ T cells from endometrium (EM), endocervix and ectocervix displayed direct cytotoxic activity, and that cytotoxicity increased in the EM after menopause. Cytotoxic activity was sensitive to suppression by TGFβ exclusively in the EM, and sensitivity to TGFβ was reduced after menopause. Under steady-state conditions, cytotoxic activity (measured as direct killing activity), cytotoxic potential (measured as content of cytotoxic molecules) and proliferation are enhanced in non-resident CD8+ (CD103−) T cells compared to tissue resident (CD103+) T cells. Upon activation, CD103+ T cells displayed greater degranulation compared to CD103− T cells, however the granular content of perforin, granzyme A (GZA) or granzyme B (GZB) was significantly lower. After menopause, degranulation significantly increased, and granular release switched from predominantly GZB in premenopausal to GZA in postmenopausal women. Postmenopausal changes affected both CD103+ and CD103− subpopulations. Finally, CD103+ T cells displayed reduced proliferation compared to CD103− T cells, but after proliferation, cytotoxic molecules were similar in each population. Our results highlight the complexity of regulation of cytotoxic function in the FRT before and after menopause, and are relevant to the development of protective strategies against genital infections and gynecological cancers as women age. [ABSTRACT FROM AUTHOR]

Half of the people living with HIV are women, and infection rates are increasing in women >50 years old. Women acquire HIV through sexual contact, but HIV transmission rates per sexual act are low. Yet, the critical immune responses that protect the female reproductive tract (FRT) against HIV infection and how these responses change with aging remain undefined. Here we investigate genital neutrophils and their ability to protect against HIV infection. Human hysterectomy tissues were used to determine neutrophil phenotype, function and tissue location using spatial transcriptomics. Formation of neutrophil extracellular traps (NETs) was analyzed with time-lapse imaging after stimulation with GFP-labeled viral-like particles (HIV-VLPs). NET-associated proteins were determined with mass spectrometry. Neutrophils are present in the FRT representing 5-30% of immune cells. Within FRT tissues, some neutrophils were located closely beneath the epithelium, while others were scattered throughout the stroma; intra-tissue location was associated with differential and unique transcriptional profiles. In response to HIV, neutrophils released NETs within minutes, and inactivated the virus preventing infection of target cells. NET release was mediated by HIV recognition through TLR8. As women aged, NET release progressively declined and TLR8 signaling was impaired. Proteomic analysis of NETs revealed incorporation of multiple proteins with anti-HIV activity and immunomodulatory properties. A better understanding of how HIV is inactivated in the FRT is essential to develop HIV prevention strategies for women of all ages. We propose that genital neutrophils represent a previously unrecognized form of mucosal protection against HIV. [ABSTRACT FROM AUTHOR]

Summary: As women age, susceptibility to systemic and genital infections increases. Tissue‐resident memory T cells (TRMs) are CD103+CD8+ long‐lived lymphocytes that provide critical mucosal immune protection. Mucosal dendritic cells (DCs) are known to induce CD103 expression on CD8+ T cells. While CD103+CD8+ T cells are found throughout the female reproductive tract (FRT), the extent to which aging impacts their presence and induction by DCs remains unknown. Using hysterectomy tissues, we found that endometrial CD103+CD8+ T cells were increased in postmenopausal compared to premenopausal women. Endometrial DCs from postmenopausal women were significantly more effective at inducing CD103 expression on allogeneic naïve CD8+ T cells than DCs from premenopausal women; CD103 upregulation was mediated through membrane‐bound TGFβ signaling. In contrast, cervical CD103+ T cells and DC numbers declined in postmenopausal women with age. Decreases in DCs correlated with decreased CD103+ T cells in endocervix, but not ectocervix. Our findings demonstrate a previously unrecognized compartmentalization of TRMs in the FRT of postmenopausal women, with loss of TRMs and DCs in the cervix with aging, and increased TRMs and DC induction capacity in the endometrium. These findings are relevant to understanding immune protection in the FRT and to the design of vaccines for women of all ages. [ABSTRACT FROM AUTHOR]

Problem The programmed death 1 ( PD-1)/ PD-L1 pathway regulates peripheral tolerance, immune responses, and is up-regulated in chronic viral infections, including HIV infection. However, expression of PD-1/ PD-L1 on immune cells from the human female reproductive tract ( FRT) and possible regulation by menopause and sex hormones are poorly understood. Method of study PD-1/ PD-L1 expression was analyzed on CD4+ and CD8+ T cells, CD163+ macrophages, and CD11c+ dendritic cells ( DC) from endometrium ( EM), endocervix ( CX) and ectocervix ( ECX). Expression after hormone treatment in culture was also evaluated. Results PD-1 and PD-L1 were constitutively expressed on CD4+ and CD8+ T cells from the FRT. PD-L1+ CD4+ T cells were increased in CX compared to EM and ECX, while no differences were found for PD-1 or between CD8+ T cells from different sites. Macrophages and DCs constitutively expressed PD-L1, but not PD-1, with no differences observed between FRT sites. Pre-menopausal FRT tissues showed increased PD-L1 expression on CD8+ T cells, but decreased expression on DCs when compared to post-menopausal women. In vitro estradiol treatment up-regulated PD-L1 expression specifically on CD8+T cells from CX, but had no effect on PD-1/ PD-L1 expression on the other cell types. Conclusion Our results suggest that PD-L1 may be involved in the differential regulation of FRT immune responses between pre-menopausal and post-menopausal women. [ABSTRACT FROM AUTHOR]

The conflicting results of recent pre-exposure prophylaxis (PrEP) trials utilizing tenofovir (TFV) to prevent HIV infection in women led us to evaluate the accumulation of intracellular TFV-diphosphate (TFV-DP) in cells from the female reproductive tract (FRT) and whether sex hormones influence the presence of TFV-DP in these cells. Following incubation with TFV, isolated epithelial cells, fibroblasts, CD4+ T cells and CD14+ cells from the FRT as well as blood CD4+ T cells and monocyte-derived macrophages convert TFV to TFV-DP. Unexpectedly, we found that TFV-DP concentrations (fmol/million cells) vary significantly with the cell type analyzed and the site within the FRT. Epithelial cells had 5-fold higher TFV-DP concentrations than fibroblasts; endometrial epithelial cells had higher TFV-DP concentrations than cells from the ectocervix. Epithelial cells had 125-fold higher TFV-DP concentrations than FRT CD4+ T cells, which were comparable to that measured in peripheral blood CD4+ T cells. These findings suggest the existence of a TFV-DP gradient in the FRT where epithelial cells > fibroblasts > CD4+ T cells and macrophages. In other studies, estradiol increased TFV-DP concentrations in endometrial and endocervical/ectocervical epithelial cells, but had no effect on fibroblasts or CD4+ T cells from FRT tissues. In contrast, progesterone alone and in combination with estradiol decreased TFV-DP concentrations in FRT CD4+ T cells. Our results suggest that epithelial cells and fibroblasts are a repository of TFV-DP that is under hormonal control. These cells might act either as a sink to decrease TFV availability to CD4+ T cells and macrophages in the FRT, or upon conversion of TFV-DP to TFV increase TFV availability to HIV-target cells. In summary, these results indicate that intracellular TFV-DP varies with cell type and location in the FRT and demonstrate that estradiol and/or progesterone regulate the intracellular concentrations of TFV-DP in FRT epithelial cells and CD4+ T cells. [ABSTRACT FROM AUTHOR]

Studies on HIV acquisition and transmission in women exposed to sexual trauma throughout their life cycle are lacking, but some findings suggest that rates of HIV acquisition through coercive sex are significantly higher than that seen in consensual sex. Sexual trauma can also occur as a result of female genital mutilation, which makes sex extremely painful and can cause increased abrasions, lacerations, and inflammation, which enhances the risk of HIV acquisition. This review presents an overview of the immune system in the human female reproductive tract ( FRT) from adolescence, through puberty to pregnancy and menopause. What is clear is that the foundation of information on immune protection in the FRT throughout the life cycle of women is extremely limited and at some stages such as adolescence and menopause are grossly lacking. Against this backdrop, forced or coercive sexual intercourse as well as genital mutilation further complicates our understanding of the biological risk factors that can result in transmission of HIV and other sexually transmitted infections. [ABSTRACT FROM AUTHOR]