The contribution of Chlamydia-specific CD8⁺ T cells to upper genital tract pathology

Abstract

Genital chlamydial infections lead to severe upper reproductive tract pathology in a subset of untreated women. We demonstrated previously that tumor necrosis factor (TNF)-α-producing CD8(+) T cells contribute significantly to chlamydial upper genital tract pathology in female mice. In addition, we observed that minimal chlamydial oviduct pathology develops in OT-1 transgenic (OT-1) mice, wherein the CD8(+) T-cell repertoire is restricted to recognition of the ovalbumin peptide Ova(257-264), suggesting that non-Chlamydia-specific CD8(+) T cells may not be responsible for chlamydial pathogenesis. In the current study, we evaluated whether antigen-specific CD8(+) T cells mediate chlamydial pathology. Groups of wild-type (WT) C57BL/6J, OT-1 mice, and OT-1 mice replete with WT CD8(+) T cells (1 × 10(6) cells per mouse intravenously) were infected intravaginally with C. muridarum (5 × 10(4) IFU/mouse). Serum total anti-Chlamydia antibody and total splenic anti-Chlamydia interferon (IFN)-γ and TNF-α responses were comparable among the three groups of animals. However, Chlamydia-specific IFN-γ and TNF-α production from purified splenic CD8(+) T cells of OT-1 mice was minimal, whereas responses in OT-1 mice replete with WT CD8(+) T cells were comparable to those in WT animals. Vaginal chlamydial clearance was comparable between the three groups of mice. Importantly, the incidence and severity of oviduct and uterine horn pathology was significantly reduced in OT-1 mice but reverted to WT levels in OT-1 mice replete with WT CD8(+) T cells. Collectively, these results demonstrate that Chlamydia-specific CD8(+) T cells contribute significantly to upper genital tract pathology.

Figure 1. Immune responses in OT-1 mice replete with WT CD8⁺ T cells

(A) A group (n = 3) of OT-1 mice were replete with CFSE labeled CD8⁺ T cells on day 0 and euthanized on day 4 to evaluate the frequency of CD3⁺ CD8⁺ CFSE ⁺ cells in the spleen. A representative histogram is shown with a mouse in this group to an OT-1 mouse control. Results are representative of two independent experiments. (B) Groups (n = 10–12) of mice (WT mice (n=12), OT-1 mice (n=12), and OT-1 mice replete with CD8⁺ T cells (n=10)) were pre-treated (5 days prior to infection) with Depo-provera ®, and challenged (on day 0) with 5×10⁴ IFU of C. muridarum. Mice were bled on day 40 and serum anti-chlamydial total antibody levels determined. Mean ± SEM of the reciprocal antibody titer corresponding to 50% maximal binding is shown. Results pooled from two independent experiments is shown. (C) Groups (n = 4) of mice (WT mice, OT-1 mice, and OT-1 mice replete with CD8⁺ T cells) were pre-treated (5 days prior to infection) with Depo-provera ®, and challenged (on day 0) with 5×10⁴ IFU of C. muridarum. On day 14, total splenocyte IFN-γ and TNF-α production in response to in vitro stimulation with UV-inactivated C. muridarum was evaluated. Mean ± SEM of the cytokine levels in each group is shown. Results are representative of two independent experiments.

Figure 2. Splenic CD8⁺ T cell cytokine response against chlamydial infection

Groups (n = 4) of mice (WT mice, OT-1 mice, and OT-1 mice replete with CD8⁺ T cells) were pre-treated (5 days prior to infection) with Depo-provera ®, and challenged (on day 0) with 5×10⁴ IFU of C. muridarum. On day 14, Splenic CD8⁺ T cell IFN-γ and TNF-α production in response to in vitro stimulation with live C. muridarum infected antigen-presenting cells. Mean ± SEM of the cytokine levels in each group is shown. * Significant (p ≤ 0.05; One-way ANOVA with Holm-Sidak method for multiple group comparisons) difference between OT-1 mice and wild type mice, and between OT-1 mice and OT-1 mice replete with WT CD8⁺ T cells when stimulated in vitro with chlamydial antigens. Results are representative of two independent experiments.

Figure 3. Chlamydial shedding after genital C. muridarum challenge

Groups (n = 10–12) of mice (WT mice (n=12), OT-1 mice (n=12), and OT-1 mice replete with CD8⁺ T cells (n=10)) were pre-treated (5 days prior to infection) with Depo-provera ®, and challenged (on day 0) with 5×10⁴ IFU of C. muridarum. Chlamydial shedding was enumerated at the indicated timepoints for a period of 33 days. The mean ± SEM of vaginal chlamydial shedding per group at each time point is shown. Results pooled from two independent experiments is shown.

Figure 4. Upper genital tract pathology after genital C. muridarum challenge

Groups (n = 10–12) of mice (WT mice (n=12), OT-1 mice (n=12), and OT-1 mice replete with CD8⁺ T cells (n=10)) were pre-treated (5 days prior to infection) with Depo-provera ®, and challenged (on day 0) with 5×10⁴ IFU of C. muridarum. (A–D) On day 80, oviduct and uterine horn pathology was evaluated. The percentage and the number of pathological and normal tissues in the oviduct (A) and uterine horns (B) is shown. *, significant (p ≤ 0.05, Fisher’s exact test) difference in the incidence of dilatation within the group of OT-1 mice compared to wild type mice or OT-1 mice replete with WT CD8⁺ T cells. The macroscopic oviduct diameter (C) and uterine horn diameter (D) were measured. Each individual marker represents one oviduct or uterine horn, and the mean ± SEM of oviduct/uterine horn diameter is also shown. The horizontal line at 0.5 mm and 1 mm depicts the distinction between normal and dilated oviducts and uterine horns, respectively. *, significant (p ≤ 0.05, One-way ANOVA with Dunn’s test for multiple group compairsons) difference in severity of dilatation between OT-1 mice and wild type mice, and between OT-1 mice and OT-1 mice replete with WT CD8⁺ T cells. Results pooled from two independent experiments are shown.