Using both in situ tissue-based and ex vivo cytobrush-sampling protocols we confirm here that CD8 T cells comprise a significant component of the endocervical T cell infiltrate during C. trachomatis infection. Moreover, we also report that endocervical CD8 T cells are primarily of the TEM subtype, but, unlike their peripheral counterparts that express high levels of perforin, these cells have low levels of this protein. Since low perforin content is observed in CD8 TEM cells from both normal and C. trachomatis-infected tissues we hypothesize that the endocervical milieu drives this phenotype. That the endocervical CD8 TEM subpopulation is low in perforin may reflect an immunological response pattern that supports feto-maternal tolerance and facilitates a fine balance between protection of tissue integrity and defence against sexually transmitted pathogens.
Human studies and animal models of C. trachomatis infection have previously demonstrated recruitment of immune cells to the local site of infection. [18, 22–29]. Compelling evidence for the ability of Chlamydia-specific CD8 T cells to migrate to infected murine FGT has been presented by Roan and Starnbach who generated retrogenic mice expressing a T cell receptor specific for CrpA, a Chlamydia specific T cell antigen . They observed that adoptively-transferred Chlamydia- specific retrogenic CD8 T cells proliferated in genital tract-draining lymph nodes of naïve recipients in response to genital C. trachomatis challenge. Further, they also demonstrated that the adoptively transferred Chlamydia-specific CD8 T cells successfully migrated to the genital mucosa and acquired the ability to produce IFN gamma [ibid]. In our earlier study, in which we observed elevated endocervical T cell numbers during culture positive endocervical C. trachomatis infections, we also noted that T cells numbers significantly decline in these women post successful antibiotic treatment . We interpret these observations to suggest that active C. trachomatis infection is most likely responsible for the recruitment of T cells at the site of infection. We also observed that CD8 T cells constitute a significant proportion of the T cell infiltrates found in the endocervix [ibid]. Thus, our data from human study, and reports based on animal studies mutually support the finding that CD8 T cells migrate into the FGT in response to C. trachomatis infection.
Intriguingly, although animal and human studies suggest that CD8 T cells are recruited to the site of C. trachomatis infection, the role of their cytolytic activity in protective immunity remains uncertain. Some studies suggest that CD8 T cells can play a role in clearance of C. trachomatis infection [26, 31], while other studies implicate these cells to be responsible for immune-mediated pathology. As an example, Voorhis et al. found an association between perforin positive CD8 T cells and salpingeal tissue scarring in non-human primate model of C. trachomatis infection . Further, in a study by Murthy et al., CD8 T cells were suggested to mediate oviduct pathology (hydrosalpinx) following C. muridarum infection in mice, but this was via their TNFα activity. This finding was demonstrated by the restoration of hydrosalpinx upon repletion of mice genetically deficient in CD8 T cells with perforin-deficient, but not with TNFα-deficient, CD8 T cells . Therefore, currently, there is no direct evidence that perforin plays a role in immune-mediated pathology by CD8 T cells during C. trachomatis infection. Furthermore, evidence based on lung and genital infection studies in perforin-knockout mice suggests that perforin is not required for clearance of murine chlamydial infection . Based on these animal models, we could surmise that the perforin-dependent CD8 T cell cytolytic response may not play a critical role in controlling C. trachomatis burden in the human endocervix.
While studies by others and us suggest a limited role for perforin-dependent CD8 T cell cytolytic activity against C. trachomatis, we do not discount the possibility that CD8 T cells may be involved in perforin-independent immune mechanisms. Particularly, we believe that during C. trachomatis infection in the human endocervix, CD8 T cells may significantly contribute to the accumulation of IFN gamma at this site as demonstrated in murine studies by Starnbach’s group [18, 31]. This finding is supported by a report based on a human study that IFN gamma levels are elevated in the endocervix of C. trachomatis-infected women compared to uninfected controls .
Two of the possible mechanisms that could induce low perforin content in CD8 TEM in Chlamydia-infected endocervix are discussed here. These are: 1) CD8 TEM cells that have migrated to the endocervix in response to C. trachomatis infection exhibit active and continuous degranulation resulting in the loss of their perforin content, and; 2) The physiologic pressure in the female genital tract drives perforin downmodulation in CD8 TEM cells. To our knowledge, the differences in degranulation sensitivity and the rates of granule protein recovery between CD8 T cells and NK cells are not yet well defined. However, we note here that unlike the CD8 TEM cells, we observed that endocervical NK cells have a high perforin content (representative data shown in Figure 4), suggesting that our observations are not due to spontaneous and continuous degranulation of all cytolytic immune cells. Further, although not the focus of this study, we believe that our data suggests that NK cells could maintain a high cytolytic potential in the endocervix, and are likely to be a key immune cell population that mediates host cytolytic immune response to C. trachomatis infection. Consistent to this notion is our previous finding that the NK cell ligand expression in C. trachomatis infected endocervical epithelial cells renders these cells more susceptible to NK cell-mediated cytolysis .
To address the first possibility, a study on degranulation status of CD8 T cell infiltrates in the endocervix would need to be undertaken. One experimental approach to test this possibility would be the concurrent measurement of the surface expression of CD107a, a marker of degranulation , and the intracellular perforin content of CD8 T cells derived from the endocervix. In principle, the low perforin content of CD8 T cells in the endocervix of women could result from their degranulation in response to stimulation of resident Chlamydia-specific, and other pathogen – specific CD8 T cells at this site. As we have not tested for degranulation in this study, we do not discount that this is a plausible mechanism that could underlie the low perforin content of CD8 T cells in the endocervix. Therefore, further study needs to be undertaken to test this possibility.
The second possibility, that physiological pressure in the endocervix drives the low perforin content of CD8 T cells, was explored in this study. Specifically, we have provided a proof-of-principle that the IFN gamma exposure of cervical epithelial cells could potentially downmodulate perforin expression of CD8 TEM cells in the same microenvironment. IFN gamma has been well established to induce indoleamine-2,3-dioxygenase (IDO) expression in epithelial cells. We also confirmed this in the HeLa 229 cells that were utilized in our in vitro study by immunoblotting HeLa 229 lysates with an IDO1-specific antibody (data not shown). IDO has been reported to impair the cytotoxic function of CD8 T cells by reducing perforin expression in these cells . Therefore, IFN gamma secretion by infiltrating CD8 T cells during C. trachomatis infection may lead to an increase in the levels of IDO in the endocervix that could induce the decrease in perforin content of the local CD8 TEM cells. While we have not yet been able to test this hypothesis in vivo, we believe that further investigation into IDO and perforin regulation in the endocervix during C. trachomatis infection is warranted to determine the mechanisms and functional significance of the low perforin content of CD8 TEM cells in this unique anatomical region.
Interestingly, there are indications that our hypothesis, proposing that IFN gamma and IDO could mediate the downmodulation perforin expression of endocervical CD8 T cells during C. trachomatis infection, could be generally operant in the FGT even in the absence of chlamydial infection. The first series of studies that support our hypothesis includes the observation that endometrial tissues have high levels of IFN gamma at the secretory stage of the menstrual cycle but not during the proliferative phase . Consistent with these findings are immunohistochemical and mRNA-based analyses demonstrating that IDO expression is low during the proliferative stage, but is elevated during the secretory phase [37, 38]. Significantly, an elegant study by Wira’s group demonstrated that T cells isolated from tissues during proliferative phase of menstrual cycle demonstrate cytolytic capacity but those during the secretory phase do not . Therefore, the presence of IFN gamma and elevated levels of IDO expression during secretory phase, which can downmodulate perforin expression in CD8 T cells, coincides with the dampening of cytolytic T cell activity.
The second series of studies that support our hypothesis is based on the analyses of IDO expression during pregnancy. Elevated IDO expression has been found in the epithelium of cervical glands, Fallopian tubes and endometrial stromal cells during decidualization in both animal and human studies [37, 38, 40, 41]. Interestingly, CD8 T cells comprise the largest fraction of T cells at the fetal-maternal interface . However, analysis of perforin expression of CD8 T cells during pregnancy revealed that while peripheral CD8 T cells express perforin, this cytolytic molecule is deficient in decidual CD8 T cells [43, 44]. These finding is consistent with the studies by Mellor et al. suggesting that IDO-mediated T cell dysfunction plays a significant role in the induction of feto-maternal tolerance [45–48]. Therefore, it seems apparent that the IFN gamma-IDO-perforin axis is an important component of the regulation of CD8 T cell cytolytic activity in the female genital tract.
If our hypothesis that IFN gamma-mediated induction of IDO is the primary mechanism involved in lowering perforin content of CD8 TEM cells in the endocervix, studies on the CD8 T cell immune repertoire of C. trachomatis infected women stratified based on their stage of menstrual cycle at the time of sample collection would be desirable, as this would clarify whether elevation of IFN gamma levels during C. trachomatis infection could overcome the low IDO expression during the proliferative stage. This would shed light on whether regardless of the stage of the menstrual cycle, the CD8 TEM cells in the endocervix are consistently low in perforin content during C. trachomatis infection.