Cytotoxic CD4+ T Cells Are Induced during Infection with Chlamydia trachomatis

Abstract

Chlamydia trachomatis is the most common cause of bacterial sexually transmitted infection in both men and women. Immunity to C. trachomatis involves many cell types, but CD4+ T cells play a key role in protecting the host during natural infection. Specifically, IFN-γ production by CD4+ T cells is the main effector responsible for bacterial clearance, yet the exact mechanism by which IFN-γ confers protection is poorly defined. In our efforts to define the specific mechanisms for bacterial clearance, we now show that IFN-γ upregulates expression of MHC class II (MHCII) on nonhematopoietic cells during C. trachomatis infection in vivo. We also find that MHCII expression on epithelial cells of the upper genital tract contributes to the efficient clearance of bacteria mediated by pathogen-specific CD4+ Th1 cells. As we further cataloged the protective mechanisms of C. trachomatis-specific CD4+ T cells, we found that the T cells also express granzyme B (GzmB) when coincubated with infected cells. In addition, during C. trachomatis infection of mice, primed activated-naive CD4+ Th1 cells displayed elevated granzyme transcripts (GzmA, GzmB, GzmM, GzmK, GzmC) compared with memory CD4+ T cells in vivo. Finally, using intracellular cytokine staining and a GzmB-/- mouse strain, we show that C. trachomatis-specific CD4+ Th1 cells express GzmB upon Ag stimulation, and that this correlates with Chlamydia clearance in vivo. Together these results have led us to conclude that Chlamydia-specific CD4+ Th1 cells develop cytotoxic capacity through engagement with nonhematopoietic MHCII, and this correlates to C. trachomatis clearance.

Figure 1.. IFNγ sensing by the non-hematopoietic compartment is required for bacterial clearance.

A) Schematic representation for the generation of the various IFNγ-sensing bone marrow chimeric mice. B) C. trachomatis-specific CD4⁺ T cells were skewed towards the Th1 phenotype and transferred into the groups of mice. Mice were then challenged with 5 × 10⁶ IFU of C. trachomatis. Five days post-infection, the upper genital tract was harvested and bacterial burden was assessed by qPCR. C) Percentages and total numbers of uterine epithelial cells (CD326⁺) were analyzed for the expression of MHCII by flow cytometry. Data shown in panel B and C, are representative of 3 independent experiments performed with at least 5 mice per group and analyzed using Kruskal-Wallis test with Dunn’s multiple comparisons test (Panel B) and ordinary one-way analysis of variance (ANOVA) with Dunnett’s multiple-comparison test (Panel C). Error bars = SEM, ns= non-significant, *, P< 0.05, **, P<0.01.

Figure 2.. MHCII expression in non-hematopoietic cells is required for NR1 Th1 CD4⁺ T cell-mediated protection against C. trachomatis

A) Schematic representation of the MHCII-expression deficient chimeric mice. B) NR1 CD4⁺ T cells were skewed to the Th1 phenotype and transferred into the different groups of mice. Mice were then challenged with 5 × 10⁶ IFU of C. trachomatis. Five days post-infection, the upper genital tract was harvested and bacterial burden was assessed by qPCR. Data in panel B are pooled from 3 independent experiments performed with at least 5 mice per group and analyzed using Kruskal-Wallis test with Dunn’s multiple comparisons test). Error bars = SEM, ns= non-significant, * P< 0.05.

Figure 3.. Activated-Naïve CD4⁺ T cells, but not memory CD4⁺ T cells, upregulate GzmB following secondary infection.

RNA sequencing data from Helble et al [27] were reanalyzed to assess the expression of different granzymes. Briefly, CD90.2^+/+ mice received 10⁶ CD90.1^+/− NR1 T cells one day prior to transcervical infection with 5×10⁶ IFU of C. trachomatis. Four weeks later, mice received 10⁶ CD90.1^+/+ NR1 T cells and were subsequently reinfected with 5×10⁶ IFU of C. trachomatis. Five days of post-secondary infection, draining iliac lymph nodes were harvested, and equivalent numbers of memory (CD90.1^+/−) and naive (CD90.1^+/+) NR1 T cells were sorted, and the transcriptomes of the two populations were analyzed using the R package DESeq2. A) In the heat map of differentially expressed granzyme transcripts, blue denotes downregulation and red denotes upregulation and was generated using the pheatmap R package. B) Volcano plot of transcriptomes from activated-naive vs. memory NR1 T cells was generated using the ggplot2 R package. Genes upregulated in Activated-naive T cells are on the left, and genes upregulated in memory T cells are on the right. The horizontal dotted line is located at -log10(0.05), while the vertical dotted lines are at −0.6 and 0.6.

Figure 4.. NR1 Th1 CD4⁺ T cells acquire cytotoxic characteristics when exposed to C. trachomatis-infected cells.

A) Mouse Embryonic Fibroblast (MEF) were stimulated with various concentrations of IFNγ (0–2000 U/ml) for 24 h, and expression of MHCII was analyzed by flow cytometry. The bar graph shows the MFI of MHCII⁺ MEFs cells with the mixes of cells shown. Data are a representative of 3 independent experiments. B) MEF cells were infected with C. trachomatis at a Multiplicity Of Infection (MOI) of 1 for 18 h or 24 h, and expression of MHCII was then analyzed by flow cytometry. The bar graph shows the MFI of MHCII⁺ MEFs cells with the mixes of cells shown. Data are representative of 3 independent experiments. C) Histogram plots of Chlamydia specific Th1 CD4⁺ T cells co-incubated with non-infected or infected (MOI= 1) MEF cells. Cells were harvested 3 days after infection and GzmB expression was determined by flow cytometry. Two control groups, a positive control where epithelial cells were treated with IFNγ 4 h prior to incubation and an isotype control, were included in the experiment. The bar graph shows the MFI of GzmB⁺ T cells with the mixes of cells shown. D) MFI plots of Chlamydia specific Th1 CD4⁺ T cells co-incubated with non-infected or infected (MOI= 1) MEF cells. Cells were harvested 3 days after infection and GzmB expression was determined by flow cytometry. Two control groups, a positive control where epithelial cells were treated with IFNγ 4 h prior to incubation and an isotype control, were included in the experiment. The bar graph shows the MFI of GzmB⁺ T cells with the mixes of cells shown. E) Uninfected or infected (MOI of 1) WT primary epithelial cells were co-incubated with Chlamydia-specific Th1 CD4⁺ T cells for 72 h. LDH in the co-culture supernatants was then measured using a commercial assay. Uninfected epithelial cells alone, infected epithelial cells alone, and infected CD4⁺ T cells alone were included as controls. The mean value of LDH-release from an additional control group (Lysis buffer) was considered as the positive control and set to 100%. Data shown in A and B are representative of 3 independent experiments with two technical replicates for each condition. Data shown in panels C and D were pooled from the results of three independent experiments performed with two technical replicates for each condition. Data from all panels were analyzed using ordinary one-way analysis of variance (ANOVA) with Dunnett’s multiple-comparison test to epithelial cells alone control. Error bars = SEM, ns= non-significant, * P< 0.05, ** P<0.01, *** P< 0.001,**** P< 0.0001

Figure 5.. Chlamydia-specific CD4 Th1 cells express Granzyme B upon antigen recognition in vivo, correlating with C. trachomatis clearance.

A) The NR1 CD4⁺ T cells gating strategy. B) Representative flow-cytometry plots of CPD eFluor 450 and granzyme B (GzmB) staining, gated on CD4⁺TCRα⁺CD90.1⁺ T cells from non-infected (naïve) or infected B6 or MCHII^−/− mice. C) Absolute numbers and percentages of GzmB⁺ cells among CD4⁺TCRα⁺CD90.1⁺ T cells from non-infected (naïve) or infected B6 and MCHII^−/− mice. D) Total numbers and percentage of total CD4⁺ T cells gated on live cells. E) Total numbers and percentage of total TCRα⁺CD90.1⁺ T cells gated on CD4⁺ T cells. F) Bacterial burden in the spleen was analyzed by qPCR 4 days post-infection. Data were pooled from 4 independent experiments performed with at least 3 mice each and were analyzed by the Kruskal-Wallis test with Dunn’s multiple comparisons test. Error bars = SEM, ns= non-significant, * P< 0.05, ** P<0.01, *** P< 0.001,**** P< 0.0001

Figure 6.. Granzyme B produced by Chlamydia-specific CD4⁺ Th1 cell can directly contribute to C. trachomatis clearance in mice.

A) Representative flow-cytometry plots of CPD eFluor 450 and granzyme B (GzmB) staining, gated on CD4⁺TCRα⁺CD90.1⁺ T cells for GzmB-competent NR1 cells or CD4⁺TCRα⁺CD45.2⁺ for GzmB deficient NR1 cells from non-infected (naïve) or infected B6, PepBoy, or MCHII^−/− mice. B) Bacterial burden in the spleen was analyzed by qPCR 4 days post-infection. Data were pooled from 3 independent experiments performed with at least 3 mice each and were analyzed by the Kruskal-Wallis test with Dunn’s multiple comparisons test. Error bars = SEM, ns= non-significant, * P< 0.05, ** P<0.01, *** P< 0.001,**** P< 0.0001