An Adaptive Chlamydia trachomatis-Specific IFN-γ-Producing CD4+ T Cell Response Is Associated With Protection Against Chlamydia Reinfection in Women

Abstract

Background: Adaptive immune responses that mediate protection against Chlamydia trachomatis (CT) remain poorly defined in humans. Animal chlamydia models have demonstrated that CD4⁺ Th1 cytokine responses mediate protective immunity against reinfection. To better understand protective immunity to CT in humans, we investigated whether select CT-specific CD4⁺ Th1 and CD8⁺ T cell cytokine responses were associated with protection against CT reinfection in women. Methods: Peripheral blood mononuclear cells were collected from 135 CT-infected women at treatment and follow-up visits and stimulated with CT antigens. CD4⁺ and CD8⁺ T-cells expressing IFN-γ, TNF-α, and/or IL-2 were assessed using intracellular cytokine staining and cytokine responses were compared between visits and between women with vs. without CT reinfection at follow-up. Results: A CD4⁺TNF-α response was detected in the majority (77%) of study participants at the treatment visit, but a lower proportion had this response at follow-up (62%). CD4⁺ IFN-γ and CD4⁺ IL-2 responses occurred less frequently at the treatment visit (32 and 18%, respectively), but increased at follow-up (51 and 41%, respectively). CD8⁺ IFN-γ and CD8⁺ TNF-α responses were detected more often at follow-up (59% for both responses) compared to the treatment visit (30% for both responses). At follow-up, a CD4⁺IFN-γ response was detected more often in women without vs. with reinfection (60 vs. 33%, P = 0.005). Conclusions: Our findings suggest that a CT-specific CD4⁺ IFN-γ response is associated with protective immunity against CT reinfection and is thus an important component of adaptive immunity to CT in women.

Keywords: CD4+IFN-γ responses; Chlamydia trachomatis; T cell responses; protection; reinfection.

Figure 1

Gating strategy: Gating strategy used to analyze the quantitative and qualitative T cell cytokine responses at both treatment and follow-up visits after a 7-h PBMC stimulation with Chlamydia trachomatis (CT) antigens (Pgp3, MOMP and EB) by intracellular cytokine staining with flow cytometry. Expanded lymphocytes were first selected based upon forward (FSC) and side scatters (SSC) before gating for a singlet population of live cells. Then they were either selected for CD4⁺ T cells (CD3⁺CD4⁺) or CD8⁺ T cells (CD3⁺CD8⁺) before analyzing cytokine production for IFN-γ, TNF-α and IL-2 using a similar gating strategy.

Figure 2

Th1 cytokine hierarchy changed from TNF-α-producing to IFN-γ-producing CD4⁺ T cells in chlamydia-infected women after therapy: Bar graph representing the change in the percentage of subjects with positive CD4⁺ (A) and CD8⁺ (B) cytokine-positive T cell responses from the Tx (black bars) to FU visits (white bars) among a cumulative 135 women and further stratified in 93 women at a 3-month visit and 42 women at a 6-month visit. Standard error of mean (SEM) is shown on the bars. The numbers on top of the bars represent the fold change in the percent positive CD4⁺/CD8⁺ cytokine positive responses from Tx to FU visit for the respective cytokines after PBMC stimulation. An extension of McNemar's chi-square test was used to determine statistical significance. P < 0.05 was considered statistically significant. ***P < 0.001, **P < 0.005, and *P < 0.05.

Figure 3

T cell responses against individual Chlamydia trachomatis antigens: Bar graph representing the change in the percentage of subjects with positive CD4⁺ (Left) and CD8⁺ (Right) cytokine-positive T cell responses from the Tx (black bars) to FU visits (white bars) among 135 women (cumulative). Standard error of mean (SEM) is shown on the bars. An extension of McNemar's chi-square test was used to determine statistical significance. P < 0.05 was considered statistically significant. ***P < 0.001, **P < 0.005, and *P < 0.05.

Figure 4

Increased magnitude of Chlamydia trachomatis (CT)-specific T cell cytokine responses at follow-up: Box and whisker plot with 5–95% interquartile range showing the frequency of CT-specific positive CD4⁺ T cell (A) and CD8⁺ T cell (B) responses as measured by cytokine-producing T cells after 7 h of PBMC stimulation with either recombinant Pgp3 protein (left panels), MOMP peptides (middle panels), or EB (right panels) and subtraction of the background responses at treatment (Tx, gray boxes) or follow up (FU, open boxes) visits in 135 women with CT infection at the Tx visit. Compared to the Tx visit, women had a stronger magnitude of CD4⁺ and CD8⁺ TNF-α and IFN-γ T cell responses detected at FU (P < 0.001). Line within the box represents the median and the symbols above and below the whiskers represent the outliers that are either greater than 95th or less than 5th percentile. A log transformed mixed model was used to compare Tx vs. FU. P < 0.05 was considered statistically significant. ***P < 0.001.

Figure 5

Women with a CD4⁺IFN-γ response detected at follow-up had a lower frequency of Chlamydia trachomatis (CT) reinfection: Bar graph illustrating the percentage of women with positive CD4⁺ T cell (A) and CD8⁺ T cell (B) responses detected after their PBMCs were stimulated with any of the three CT antigens (Pgp3, MOMP, EB), with women stratified based on their CT status at follow-up (R = reinfection, NR = no reinfection). A significantly higher proportion of women from the NR group (white bars) had a CD4⁺ IFN-γ response detected than women from the R group (black bar; P = 0.005; OR 3.00 [95% CI 1.40, 6.42]) for cumulative follow-up. Standard error of mean (SEM) is shown on the bars. An extension of McNemar's chi-square test was used to compare R vs. NR matched groups. P < 0.05 was considered statistically significant. **P < 0.005, and *P < 0.05.

Figure 6

Differential and temporal CD4⁺IFN-γ T cell response with different Chlamydia trachomatis (CT) antigens. Bar graph depicting the percentage of women with a CD4⁺IFN-γ T cell response detected after their PBMCs were stimulated with any of the three CT antigens (Pgp3, MOMP, EB), with women stratified based on their CT status at follow-up (R = reinfection, NR = no reinfection). Stimulation with different CT antigens led to differential CD4⁺IFN-γ responses, with MOMP stimulation resulting in significantly higher CD4⁺IFN-γ T cell response from women in the NR group compared to women in the R group at 3-month and 6-month follow-up visit respectively. Standard error of mean (SEM) is shown on the bars. An extension of McNemar's chi-square test was used to compare R vs. NR matched groups. P < 0.05 was considered statistically significant. *P < 0.05.

Figure 7

No difference in the magnitude of the CD4⁺ IFN-γ response detected in women with vs. without reinfection at follow-up: Box and whisker plot with 5–95% interquartile range showing the percentage of Chlamydia trachomatis (CT)-specific IFN-γ-producing CD4⁺ T cells at follow-up when stimulated with CT antigens: Pgp3, MOMP and EB. No significant difference in the magnitude of the CD4⁺ IFN-γ response in the reinfection (R) group (n = 45; white box) vs. the no reinfection (NR) groups (n = 90; gray box). Line within the box represents the median and the symbols above and below the whiskers represent the outliers that are either greater than 95th or less than 5th percentile. A log transformed mixed model was used to compare R vs. NR matched groups. P < 0.05 was considered statistically significant.

Figure 8

Women presenting for follow-up after chlamydia treatment predominantly had a dual cytokine-producing CD4⁺ T cell response detected: Percentage of women with CD4⁺ dual TNF-α IFN-γ responses and IFN-γ IL-2 responses detected after their PBMCs were stimulated with any of the three Chlamydia trachomatis (CT) antigens among those with a positive CD4⁺ IFN-γ response. (A) Bar graph showing the difference in the percentage of women with CT infection at enrollment who had a CD4⁺ dual cytokine response detected between the two visits: treatment (Tx) visit (white bars) and follow-up (FU) visit (black bars). A significantly higher proportion of women at the FU visit had a CD4⁺ dual cytokine response detected compared to those at the Tx visit (P < 0.001). (B) Bar graph depicting the difference in the percentage of women with CD4⁺ dual TNF-α +FN-γ responses and IFN-γ+IL-2 responses detected (determined using dual gating) from the reinfection (R) (white bars) vs. the no reinfection (NR) (black bars) groups, after their PBMCs were stimulated with any of the three CT antigens. There was no significant difference in the frequency of these dual CD4⁺ cytokine responses between women from the R and NR groups. Standard error of mean (SEM) is shown on the bars. An extension of McNemar's chi-square test was used to compare R vs. NR matched groups. P < 0.05 was considered statistically significant. ***P < 0.001, **P < 0.005, and *P < 0.05.