BACKGROUND
cd <dig> t cells are key cellular components in the control of many intracellular microbial infections via their cytolytic function.
chlamydia trachomatis serovars d-k are intracellular bacteria that infect the columnar epithelial cells of the genital tract.
epithelial cells can present antigens in the context of mhc class i and activate a cd <dig> t cell immune response.
thus, investigation of the cd <dig> t cell cytolytic response to c. trachomatis infection is important as it could reveal a mechanism by which the bacterium is deprived of its intracellular niche.
the major cd <dig> t cell cytolytic pathway involves the perforin and granzyme mediated induction of apoptosis  <cit> .
perforin mediates the delivery of granzymes to target cells by homopolymerization in the plasma membrane in a ca2+ dependent manner producing pores that acts as a channel for granzyme entry  <cit> .
perforin is suggested to be necessary in cd <dig> t cell cytolytic activity, as perforin deficient mice have reduced efficiency in controlling viral infection  <cit> .
during the course of infection, cd <dig> t cells differentiate and this is accompanied by changes in the expression of surface markers and functional capacity  <cit> .
naïve t cells are activated when they encounter their specific peptide-mhc complexes on professional antigen presenting cells  <cit> .
a memory t cell differentiation pathway has been established by the group of sallusto and lanzavecchia, and others, whereby subsequent to antigen encounter, t cells proliferate and undergo phenotypic changes that modify their tissue homing properties  <cit> .
antigen-specific cells possessing a naïve-like phenotype  are recruited into a pre-memory subset before reaching the central memory  and effector memory  stages that are characterized as cd45ra-ccr7+ and cd45ra-ccr7- respectively.
eventually, these memory t cells reach a terminally differentiated effector stage  characterized as cd45ra+ ccr7-.
progression of a t cell through these subsets is associated with the acquisition of effector function and loss of proliferative potential .
effector cd <dig> t cells acquire the capacity to migrate to extra lymphoid sites to sites of infection, and deliver perforin and granzyme at the immunological synapse to kill infected target cells  <cit> .
cd <dig> t cell differentiation in response to infection is thus characterized by the acquisition of immunological properties that allow them to successfully clear intracellular pathogens.
evidence that specific tissue microenvironments significantly influence cd <dig> t cell phenotype and function is accumulating.
for example, masopust et al., illustrated that the gastrointestinal microenvironment promotes differentiation of a unique memory cd <dig> t cell population, and that cd <dig> t cells can switch phenotypes with changes in anatomic location  <cit> .
the influence of the anatomic micromilieu was also illustrated by a study by shacklett et al., who demonstrated that unlike those in peripheral blood, cd <dig> t cells resident in the gastrointestinal tract of normal macaques were low in perforin.
the absence of perforin in normal gi tissue was interpreted to be a mechanism by which the anatomical integrity of this mucosal site is protected, though it provides advantages for pathogens  <cit> .
the female genital tract , like the gi tract, is also frequently exposed to foreign antigens, including commensal and pathogenic microorganisms.
furthermore, as a reproductive site, the fgt must tolerate foreign antigens to support conception.
we thus hypothesized that cd <dig> t cells in the endocervix may also have limited cytolytic potential compared to their peripheral counterparts.
therefore, to investigate the phenotype of cd <dig> t cells in the fgt in the absence and presence of c. trachomatis infection, we sampled endocervical cd <dig> t cells from women to characterize the immune cell population, and assess the cytolytic potential of cd <dig> t cells at this site.
in addition, using an in vitro approach, we further tested whether the presence of ifn gamma in a microenvironment could influence the perforin expression of cd <dig> tem cells.
RESULTS
cd <dig> t cells infiltrate the human endocervix during c. trachomatis infection
consistent with our previous findings with cytobrush-retrieved endocervical t cells, immunohistological staining for cd <dig> and cd <dig> t cell infiltrates in six c. trachomatis-negative and four c. trachomatis-positive banked endocervical tissues demonstrated that the number of cd3+ and cd8+ t cells is significantly higher in c. trachomatis-positive than in c. trachomatis-negative endocervical tissues  .
interestingly, when we stained serial sections of endocervical tissues for cd <dig> and perforin, we observed that the majority of cd <dig> positive cells were perforin negative .
quantitative analysis of perforin-expressing cd <dig> t cells, however, was limited by the immunohistological single staining technique’s inability to differentiate these cells from other immune cells that also express perforin, such as eosinophils, basophils, mast cells and nk cells.
thus, while our immunohistochemical data provided significant insights on the make-up of t cell infiltrates and suggested that cd <dig> t cells expressed limited amounts of perforin, we recognized that a multiparameter flow cytometric analysis would be needed to support and extend these observations.
the cd <dig> t cell repertoire in the human endocervix is distinct from the periphery
to further analyze the endocervical immune cell repertoire, we performed multiparameter flow cytometric analyses to determine the mononuclear leukocyte types in isolated peripheral blood mononuclear cells  and cytobrush-retrieved endocervical cells from  <dig> c. trachomatis-infected and  <dig> uninfected young women attending the delgado clinic.
the gating strategy utilized in this study is shown in a representative analysis flow chart shown in figure  <dig>  utilizing this strategy, we identified the lymphocyte population based on forward  and side-scatter  profile, from which the nk cell, cd <dig> t cell, and cd <dig> t cell subpopulations were delineated.
we then assessed the perforin and granzyme expression in each of these cell types from paired samples obtained from the blood and endocervix.
nk cells and cd <dig> t cells were utilized as cellular controls, while granzyme b was utilized as an intracellular staining control.
perforin and granzyme b expression of the different immune cells from the blood and endocervix of a representative c. trachomatis-negative and c. trachomatis-positive women is shown in figure  <dig>  consistent with previous reports, we observed that perforin expression is relatively low in cd <dig> t cells, and high in nk cells  <cit> .
we then further delineated the cd <dig> t cell population into memory subsets based on the expression of cd45ra and ccr <dig> surface markers, as described by the groups of sallusto and lanzavecchia .
representative distributions of cd <dig> t cells in blood and endocervix of c. trachomatis-negative and c. trachomatis-positive women are shown in figure 5b.
analyses of blood and endocervical cd <dig> t cell subtype percent- distribution from  <dig> women using repeated anova indicated a significant tissue site interaction .
further, a paired comparison of mean percentage cd <dig> t cell subtypes in blood and endocervix, using t- test with confirmatory analysis using the wilcoxon signed rank test, indicated a significantly higher percentage of tem cells in the endocervix , with a significantly lower percentage of naïve cells .
taken together these data indicate that, while the different cd <dig> t cell subtypes are relatively distributed in the blood, the endocervix is primarily populated by effector memory  cd <dig> t cells .
effector memory cd <dig> t cells  in the human endocervix have limited perforin expression
based on the observation that the endocervix is primarily populated by cd <dig> tem cells, we focused our subsequent analyses on this subpopulation.
here, the cytolytic potential of cd <dig> tem cells was assessed by analyzing flow cytometry data for the percentage of perforin expressing cd <dig> tem cells, using difference scores  and non-parametric analysis.
based on the wilcoxon rank sum test, a significant difference in the percentage of perforin-expressing cd <dig> tem cells from blood and endocervix were observed in both c. trachomatis-negative  and in c. trachomatis-positive  women as shown in figures 6a and 6c respectively.
furthermore, pair-wise comparison of the percentage of perforin+ tem in the blood and endocervix of individual samples demonstrated that in both c. trachomatis-negative  and c. trachomatis-positive patients  there was a general trend for the endocervical tem to have lower percentage of perforin positive cells compared to tem derived from the blood.
however, further analyses, with repeated measures anova, using c. trachomatis infection status as a grouping factor, showed that although there were differences in blood vs. endocervix, no interaction with c. trachomatis-infection status was observed.
these data show that despite the significant increase in cd <dig> t cells infiltrating the endocervix during c. trachomatis-infection, the relative low perforin level of the cd <dig> t tem cell population is still observed.
ifn gamma exposure of epithelial cells could potentially mediate the downmodulation of perforin expression of tem cd <dig> t cells in the cervical microenvironment
our ex vivo data suggested that endocervical cd <dig> t tem cells have a low perforin content.
therefore, we investigated one of the factors that could influence this phenotype.
we hypothesized that ifn gamma could be one of the mediating factors that drives the decrease in perforin content of endocervical cd <dig> tem cells based on the following observations from previous studies: 1) ifn gamma levels in the female genital tract are elevated during the secretory stage of menstruation  <cit>  and during decidualization in successful pregnancies  <cit> ; 2) animal models of c. trachomatis infections have demonstrated that t cells with the capacity to secrete ifn gamma migrate to the site of chlamydia infection  <cit> ; 3) high levels of ifn gamma are found in the fgt of c. trachomatis-infected patients  <cit> ; and 4) ifn gamma can induce the expression of indoleamine- <dig> -dioxygenase  in epithelial cells  <cit> ; and ido, a tryptophan catabolic enzyme, could downmodulate perforin expression in cd <dig> t cells  <cit> .
therefore, based on these previous findings, we developed a pbl-hela  <dig> cervical epithelial cell co-culture model to investigate whether the exposure of epithelial cells to ifn gamma has an effect on perforin expression by cd <dig> tem cells.
to do this, pbls were cultured alone or with a monolayer of hela  <dig> cells and with or without  <dig> u/ml of ifn gamma .
after  <dig> hours in culture, the pbls were then stained to identify the different cd <dig> t cell subpopulations and intracellular perforin and granzyme expression.
the gating strategy to delineate the different cd <dig> t cell subpopulation is shown in figure 7b.
this gating allowed us to identify the cd <dig> tem cell subpopulation from the pbls, and to assess the perforin expression of these cells in the absence or presence of ifn gamma.
we found that the level of perforin expression by the control cd <dig> tem cells from pbls cultured in the absence of hela  <dig> cells did not shift in the presence of ifn gamma .
however, when we co-cultured pbls with hela cells, the addition of ifn gamma in the culture medium resulted in the negative shift in the fluorescence intensity of perforin, suggesting a downmodulation of the expression of this protein .
therefore, our in vitro data suggest that ifn gamma could be potential mediator of the downmodulation of perforin expression in cd <dig> tem cells.
discussion
using both in situ tissue-based and ex vivo cytobrush-sampling protocols we confirm here that cd <dig> t cells comprise a significant component of the endocervical t cell infiltrate during c. trachomatis infection.
moreover, we also report that endocervical cd <dig> 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 cd <dig> tem cells from both normal and c. trachomatis-infected tissues we hypothesize that the endocervical milieu drives this phenotype.
that the endocervical cd <dig> 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.
<cit> .
compelling evidence for the ability of chlamydia-specific cd <dig> 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  <cit> .
they observed that adoptively-transferred chlamydia-specific retrogenic cd <dig> 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 cd <dig> t cells successfully migrated to the genital mucosa and acquired the ability to produce ifn gamma .
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  <cit> .
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 cd <dig> t cells constitute a significant proportion of the t cell infiltrates found in the endocervix .
thus, our data from human study, and reports based on animal studies mutually support the finding that cd <dig> t cells migrate into the fgt in response to c. trachomatis infection.
intriguingly, although animal and human studies suggest that cd <dig> 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 cd <dig> t cells can play a role in clearance of c. trachomatis infection  <cit> , 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 cd <dig> t cells and salpingeal tissue scarring in non-human primate model of c. trachomatis infection  <cit> .
further, in a study by murthy et al., cd <dig> t cells were suggested to mediate oviduct pathology  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 cd <dig> t cells with perforin-deficient, but not with tnfα-deficient, cd <dig> t cells  <cit> .
therefore, currently, there is no direct evidence that perforin plays a role in immune-mediated pathology by cd <dig> 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  <cit> .
based on these animal models, we could surmise that the perforin-dependent cd <dig> 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 cd <dig> t cell cytolytic activity against c. trachomatis, we do not discount the possibility that cd <dig> t cells may be involved in perforin-independent immune mechanisms.
particularly, we believe that during c. trachomatis infection in the human endocervix, cd <dig> t cells may significantly contribute to the accumulation of ifn gamma at this site as demonstrated in murine studies by starnbach’s group  <cit> .
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  <cit> .
two of the possible mechanisms that could induce low perforin content in cd <dig> tem in chlamydia-infected endocervix are discussed here.
these are: 1) cd <dig> 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 cd <dig> tem cells.
to our knowledge, the differences in degranulation sensitivity and the rates of granule protein recovery between cd <dig> t cells and nk cells are not yet well defined.
however, we note here that unlike the cd <dig> tem cells, we observed that endocervical nk cells have a high perforin content , 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  <cit> .
to address the first possibility, a study on degranulation status of cd <dig> 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  <cit> , and the intracellular perforin content of cd <dig> t cells derived from the endocervix.
in principle, the low perforin content of cd <dig> t cells in the endocervix of women could result from their degranulation in response to stimulation of resident chlamydia-specific, and other pathogen – specific cd <dig> 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 cd <dig> 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 cd <dig> 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 cd <dig> tem cells in the same microenvironment.
ifn gamma has been well established to induce indoleamine- <dig> -dioxygenase  expression in epithelial cells.
we also confirmed this in the hela  <dig> cells that were utilized in our in vitro study by immunoblotting hela  <dig> lysates with an ido1-specific antibody .
ido has been reported to impair the cytotoxic function of cd <dig> t cells by reducing perforin expression in these cells  <cit> .
therefore, ifn gamma secretion by infiltrating cd <dig> 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 cd <dig> 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 cd <dig> 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 cd <dig> 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  <cit> .
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  <cit> .
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  <cit> .
therefore, the presence of ifn gamma and elevated levels of ido expression during secretory phase, which can downmodulate perforin expression in cd <dig> 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  <cit> .
interestingly, cd <dig> t cells comprise the largest fraction of t cells at the fetal-maternal interface  <cit> .
however, analysis of perforin expression of cd <dig> t cells during pregnancy revealed that while peripheral cd <dig> t cells express perforin, this cytolytic molecule is deficient in decidual cd <dig> t cells  <cit> .
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  <cit> .
therefore, it seems apparent that the ifn gamma-ido-perforin axis is an important component of the regulation of cd <dig> 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 cd <dig> tem cells in the endocervix, studies on the cd <dig> 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 cd <dig> tem cells in the endocervix are consistently low in perforin content during c. trachomatis infection.
CONCLUSIONS
the findings in this study suggest that perforin-dependent cd <dig> t cell-mediated cytoxicity may play a limited role in immune control of human endocervical c. trachomatis infection.
however, further studies are needed to clearly establish the mechanism that drives the decrease in perforin levels in normal and c. trachomatis-infected endocervix.
nevertheless, despite the challenging nature of human studies in the female genital tract, we have generated ex vivo data that provides important insights into the endocervical immunological repertoire that may influence the outcome of c. trachomatis infection at this site.

 cd t cells are key cellular components in the control of many intracellular microbial infections via their cytolytic function. chlamydia trachomatis serovars d-k are intracellular bacteria that infect the columnar epithelial cells of the genital tract. epithelial cells can present antigens in the context of mhc class i and activate a cd t cell immune response. thus, investigation of the cd t cell cytolytic response to c. trachomatis infection is important as it could reveal a mechanism by which the bacterium is deprived of its intracellular niche. the major cd t cell cytolytic pathway involves the perforin and granzyme mediated induction of apoptosis. perforin mediates the delivery of granzymes to target cells by homopolymerization in the plasma membrane in a ca2+ dependent manner producing pores that acts as a channel for granzyme entry. perforin is suggested to be necessary in cd t cell cytolytic activity, as perforin deficient mice have reduced efficiency in controlling viral infection. during the course of infection, cd t cells differentiate and this is accompanied by changes in the expression of surface markers and functional capacity. naïve t cells are activated when they encounter their specific peptide-mhc complexes on professional antigen presenting cells. a memory t cell differentiation pathway has been established by the group of sallusto and lanzavecchia, and others, whereby subsequent to antigen encounter, t cells proliferate and undergo phenotypic changes that modify their tissue homing properties. antigen-specific cells possessing a naïve-like phenotype are recruited into a pre-memory subset before reaching the central memory and effector memory stages that are characterized as cd45ra-ccr7+ and cd45ra-ccr7- respectively. eventually, these memory t cells reach a terminally differentiated effector stage characterized as cd45ra+ ccr7-. progression of a t cell through these subsets is associated with the acquisition of effector function and loss of proliferative potential. effector cd t cells acquire the capacity to migrate to extra lymphoid sites to sites of infection, and deliver perforin and granzyme at the immunological synapse to kill infected target cells. cd t cell differentiation in response to infection is thus characterized by the acquisition of immunological properties that allow them to successfully clear intracellular pathogens. evidence that specific tissue microenvironments significantly influence cd t cell phenotype and function is accumulating. for example, masopust et al., illustrated that the gastrointestinal microenvironment promotes differentiation of a unique memory cd t cell population, and that cd t cells can switch phenotypes with changes in anatomic location. the influence of the anatomic micromilieu was also illustrated by a study by shacklett et al., who demonstrated that unlike those in peripheral blood, cd t cells resident in the gastrointestinal tract of normal macaques were low in perforin. the absence of perforin in normal gi tissue was interpreted to be a mechanism by which the anatomical integrity of this mucosal site is protected, though it provides advantages for pathogens. the female genital tract, like the gi tract, is also frequently exposed to foreign antigens, including commensal and pathogenic microorganisms. furthermore, as a reproductive site, the fgt must tolerate foreign antigens to support conception. we thus hypothesized that cd t cells in the endocervix may also have limited cytolytic potential compared to their peripheral counterparts. therefore, to investigate the phenotype of cd t cells in the fgt in the absence and presence of c. trachomatis infection, we sampled endocervical cd t cells from women to characterize the immune cell population, and assess the cytolytic potential of cd t cells at this site. in addition, using an in vitro approach, we further tested whether the presence of ifn gamma in a microenvironment could influence the perforin expression of cd tem cells.  cd t cells infiltrate the human endocervix during c. trachomatis infection consistent with our previous findings with cytobrush-retrieved endocervical t cells, immunohistological staining for cd and cd t cell infiltrates in six c. trachomatis-negative and four c. trachomatis-positive banked endocervical tissues demonstrated that the number of cd3+ and cd8+ t cells is significantly higher in c. trachomatis-positive than in c. trachomatis-negative endocervical tissues. interestingly, when we stained serial sections of endocervical tissues for cd and perforin, we observed that the majority of cd positive cells were perforin negative. quantitative analysis of perforin-expressing cd t cells, however, was limited by the immunohistological single staining technique’s inability to differentiate these cells from other immune cells that also express perforin, such as eosinophils, basophils, mast cells and nk cells. thus, while our immunohistochemical data provided significant insights on the make-up of t cell infiltrates and suggested that cd t cells expressed limited amounts of perforin, we recognized that a multiparameter flow cytometric analysis would be needed to support and extend these observations. the cd t cell repertoire in the human endocervix is distinct from the periphery to further analyze the endocervical immune cell repertoire, we performed multiparameter flow cytometric analyses to determine the mononuclear leukocyte types in isolated peripheral blood mononuclear cells and cytobrush-retrieved endocervical cells from c. trachomatis-infected and uninfected young women attending the delgado clinic. the gating strategy utilized in this study is shown in a representative analysis flow chart shown in figure  utilizing this strategy, we identified the lymphocyte population based on forward and side-scatter profile, from which the nk cell, cd t cell, and cd t cell subpopulations were delineated. we then assessed the perforin and granzyme expression in each of these cell types from paired samples obtained from the blood and endocervix. nk cells and cd t cells were utilized as cellular controls, while granzyme b was utilized as an intracellular staining control. perforin and granzyme b expression of the different immune cells from the blood and endocervix of a representative c. trachomatis-negative and c. trachomatis-positive women is shown in figure  consistent with previous reports, we observed that perforin expression is relatively low in cd t cells, and high in nk cells. we then further delineated the cd t cell population into memory subsets based on the expression of cd45ra and ccr surface markers, as described by the groups of sallusto and lanzavecchia. representative distributions of cd t cells in blood and endocervix of c. trachomatis-negative and c. trachomatis-positive women are shown in figure 5b. analyses of blood and endocervical cd t cell subtype percent- distribution from women using repeated anova indicated a significant tissue site interaction. further, a paired comparison of mean percentage cd t cell subtypes in blood and endocervix, using t- test with confirmatory analysis using the wilcoxon signed rank test, indicated a significantly higher percentage of tem cells in the endocervix, with a significantly lower percentage of naïve cells. taken together these data indicate that, while the different cd t cell subtypes are relatively distributed in the blood, the endocervix is primarily populated by effector memory cd t cells. effector memory cd t cells in the human endocervix have limited perforin expression based on the observation that the endocervix is primarily populated by cd tem cells, we focused our subsequent analyses on this subpopulation. here, the cytolytic potential of cd tem cells was assessed by analyzing flow cytometry data for the percentage of perforin expressing cd tem cells, using difference scores and non-parametric analysis. based on the wilcoxon rank sum test, a significant difference in the percentage of perforin-expressing cd tem cells from blood and endocervix were observed in both c. trachomatis-negative and in c. trachomatis-positive women as shown in figures 6a and 6c respectively. furthermore, pair-wise comparison of the percentage of perforin+ tem in the blood and endocervix of individual samples demonstrated that in both c. trachomatis-negative and c. trachomatis-positive patients there was a general trend for the endocervical tem to have lower percentage of perforin positive cells compared to tem derived from the blood. however, further analyses, with repeated measures anova, using c. trachomatis infection status as a grouping factor, showed that although there were differences in blood vs. endocervix, no interaction with c. trachomatis-infection status was observed. these data show that despite the significant increase in cd t cells infiltrating the endocervix during c. trachomatis-infection, the relative low perforin level of the cd t tem cell population is still observed. ifn gamma exposure of epithelial cells could potentially mediate the downmodulation of perforin expression of tem cd t cells in the cervical microenvironment our ex vivo data suggested that endocervical cd t tem cells have a low perforin content. therefore, we investigated one of the factors that could influence this phenotype. we hypothesized that ifn gamma could be one of the mediating factors that drives the decrease in perforin content of endocervical cd tem cells based on the following observations from previous studies: 1) ifn gamma levels in the female genital tract are elevated during the secretory stage of menstruation and during decidualization in successful pregnancies; 2) animal models of c. trachomatis infections have demonstrated that t cells with the capacity to secrete ifn gamma migrate to the site of chlamydia infection; 3) high levels of ifn gamma are found in the fgt of c. trachomatis-infected patients; and 4) ifn gamma can induce the expression of indoleamine- -dioxygenase in epithelial cells; and ido, a tryptophan catabolic enzyme, could downmodulate perforin expression in cd t cells. therefore, based on these previous findings, we developed a pbl-hela cervical epithelial cell co-culture model to investigate whether the exposure of epithelial cells to ifn gamma has an effect on perforin expression by cd tem cells. to do this, pbls were cultured alone or with a monolayer of hela cells and with or without u/ml of ifn gamma. after hours in culture, the pbls were then stained to identify the different cd t cell subpopulations and intracellular perforin and granzyme expression. the gating strategy to delineate the different cd t cell subpopulation is shown in figure 7b. this gating allowed us to identify the cd tem cell subpopulation from the pbls, and to assess the perforin expression of these cells in the absence or presence of ifn gamma. we found that the level of perforin expression by the control cd tem cells from pbls cultured in the absence of hela cells did not shift in the presence of ifn gamma. however, when we co-cultured pbls with hela cells, the addition of ifn gamma in the culture medium resulted in the negative shift in the fluorescence intensity of perforin, suggesting a downmodulation of the expression of this protein. therefore, our in vitro data suggest that ifn gamma could be potential mediator of the downmodulation of perforin expression in cd tem cells. discussion using both in situ tissue-based and ex vivo cytobrush-sampling protocols we confirm here that cd t cells comprise a significant component of the endocervical t cell infiltrate during c. trachomatis infection. moreover, we also report that endocervical cd 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 cd tem cells from both normal and c. trachomatis-infected tissues we hypothesize that the endocervical milieu drives this phenotype. that the endocervical cd 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.. compelling evidence for the ability of chlamydia-specific cd 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 cd 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 cd t cells successfully migrated to the genital mucosa and acquired the ability to produce ifn gamma. 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 cd t cells constitute a significant proportion of the t cell infiltrates found in the endocervix. thus, our data from human study, and reports based on animal studies mutually support the finding that cd t cells migrate into the fgt in response to c. trachomatis infection. intriguingly, although animal and human studies suggest that cd 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 cd t cells can play a role in clearance of c. trachomatis infection, 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 cd t cells and salpingeal tissue scarring in non-human primate model of c. trachomatis infection. further, in a study by murthy et al., cd t cells were suggested to mediate oviduct pathology 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 cd t cells with perforin-deficient, but not with tnfα-deficient, cd t cells. therefore, currently, there is no direct evidence that perforin plays a role in immune-mediated pathology by cd 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 cd 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 cd t cell cytolytic activity against c. trachomatis, we do not discount the possibility that cd t cells may be involved in perforin-independent immune mechanisms. particularly, we believe that during c. trachomatis infection in the human endocervix, cd t cells may significantly contribute to the accumulation of ifn gamma at this site as demonstrated in murine studies by starnbach’s group. 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 cd tem in chlamydia-infected endocervix are discussed here. these are: 1) cd 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 cd tem cells. to our knowledge, the differences in degranulation sensitivity and the rates of granule protein recovery between cd t cells and nk cells are not yet well defined. however, we note here that unlike the cd tem cells, we observed that endocervical nk cells have a high perforin content, 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 cd 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 cd t cells derived from the endocervix. in principle, the low perforin content of cd t cells in the endocervix of women could result from their degranulation in response to stimulation of resident chlamydia-specific, and other pathogen – specific cd 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 cd 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 cd 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 cd tem cells in the same microenvironment. ifn gamma has been well established to induce indoleamine- -dioxygenase expression in epithelial cells. we also confirmed this in the hela cells that were utilized in our in vitro study by immunoblotting hela lysates with an ido1-specific antibody. ido has been reported to impair the cytotoxic function of cd t cells by reducing perforin expression in these cells. therefore, ifn gamma secretion by infiltrating cd 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 cd 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 cd 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 cd 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. 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 cd 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. interestingly, cd t cells comprise the largest fraction of t cells at the fetal-maternal interface. however, analysis of perforin expression of cd t cells during pregnancy revealed that while peripheral cd t cells express perforin, this cytolytic molecule is deficient in decidual cd t cells. 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. therefore, it seems apparent that the ifn gamma-ido-perforin axis is an important component of the regulation of cd 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 cd tem cells in the endocervix, studies on the cd 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 cd tem cells in the endocervix are consistently low in perforin content during c. trachomatis infection.  the findings in this study suggest that perforin-dependent cd t cell-mediated cytoxicity may play a limited role in immune control of human endocervical c. trachomatis infection. however, further studies are needed to clearly establish the mechanism that drives the decrease in perforin levels in normal and c. trachomatis-infected endocervix. nevertheless, despite the challenging nature of human studies in the female genital tract, we have generated ex vivo data that provides important insights into the endocervical immunological repertoire that may influence the outcome of c. trachomatis infection at this site. 
the cytolytic function of cd t cells is important in the protective immunity against many intracellular pathogens, and requires the cytolytic granule perforin to facilitate the entry of other molecules that mediate the lysis of target cells. interestingly, our in vitro co-culture study suggests that the exposure of hela cervical epithelial cells to ifn gamma could potentially induce a decrease in perforin content in cd tem cells in the same microenvironment. the low perforin content of cd tem cells in the endocervix, the local site of c. trachomatis infection in women, may reflect the unique immunological environment that balances immune protection against sexually transmitted infections and immune- tolerance to support conception. a subsequent flow cytometric analysis of paired blood and endocervix-derived cells revealed that while all the cd t cell subsets: naïve, effector memory, central memory and terminally differentiated effector memory can be found in the blood, the endocervix is populated mainly by the tem cd t cell subset. the local tissue microenvironment plays an important role in the induction, homing, maintenance and development of effector functions of t cells. our previous flow-cytometry-based study of cervical-cytobrush retrieved cells indicated that cd t cells are significantly increased in the c. trachomatis-infected human endocervix. our histological data revealed that c. trachomatis-infected tissues have significantly higher numbers of cd and cd t cells compared to non-infected tissues, and that the majority of cd8+ cells do not express perforin in situ. determination of perforin expression of the cd t cell population in the endocervix would therefore provide insights on the granule-mediated cytolytic potential of these cells at this site. our data also showed that perforin expression in the tem population is significantly lower in the endocervix than in the blood of c. trachomatis positive women, as well as in c. trachomatis-negative individuals.
 the local tissue microenvironment plays an important role in the induction, homing, maintenance and development of effector functions of t cells. thus, site-specific differences in phenotypes of mucosal and systemic t cell populations have been observed. chlamydia trachomatis most commonly infects the endocervix in women, yet little is known about chlamydia-specific effector t cell immunity at this unique mucosal site. our previous flow-cytometry-based study of cervical-cytobrush retrieved cells indicated that cd t cells are significantly increased in the c. trachomatis-infected human endocervix. the cytolytic function of cd t cells is important in the protective immunity against many intracellular pathogens, and requires the cytolytic granule perforin to facilitate the entry of other molecules that mediate the lysis of target cells. determination of perforin expression of the cd t cell population in the endocervix would therefore provide insights on the granule-mediated cytolytic potential of these cells at this site.  our histological data revealed that c. trachomatis-infected tissues have significantly higher numbers of cd and cd t cells compared to non-infected tissues, and that the majority of cd8+ cells do not express perforin in situ. a subsequent flow cytometric analysis of paired blood and endocervix-derived cells revealed that while all the cd t cell subsets: naïve, effector memory, central memory and terminally differentiated effector memory can be found in the blood, the endocervix is populated mainly by the tem cd t cell subset. our data also showed that perforin expression in the tem population is significantly lower in the endocervix than in the blood of c. trachomatis positive women, as well as in c. trachomatis-negative individuals. interestingly, our in vitro co-culture study suggests that the exposure of hela cervical epithelial cells to ifn gamma could potentially induce a decrease in perforin content in cd tem cells in the same microenvironment.  the low perforin content of cd tem cells in the endocervix, the local site of c. trachomatis infection in women, may reflect the unique immunological environment that balances immune protection against sexually transmitted infections and immune- tolerance to support conception. 