Differential CD28 and inducible costimulatory molecule signaling requirements for protective CD4+ T-cell-mediated immunity against genital tract Chlamydia trachomatis infection

Abstract

Th1 cells and gamma interferon (IFN-gamma) production play critical roles in protective immunity against genital tract infections by Chlamydia trachomatis. Here we show that inducible costimulatory molecule (ICOS)(-/-) mice develop greatly augmented host resistance against chlamydial infection. Protection following a primary infection was characterized by strong Th1 immunity with enhanced CD4(+) T-cell-mediated IFN-gamma production in the genital tract and high expression of T-bet in the draining para-aortic lymph node. This Th1 dominance was associated with low expression of interleukin 10 (IL-10) mRNA in the uteruses of protected ICOS(-/-) mice. By contrast, CD28(-/-) mice were severely impaired in their adaptive immune response, demonstrating a lack of CD4(+) T cells and IFN-gamma in the genital tract, with a substantial delay in bacterial elimination compared to that seen in wild-type (WT) mice. Upon reinfection, WT mice exhibited a transient local infection with evidence of regulatory T-cell (Treg)/Foxp3 mRNA and a more balanced Th1 and Th2 response in the genital tract than ICOS(-/-) mice, whereas 90% of the latter mice developed sterile immunity, poor expression of local Treg/Foxp3 mRNA, and macroscopic signs of enhanced local immunopathology. Therefore, different requirements for CD28 signaling and ICOS signaling clearly apply to host protection against a genital tract infection by C. trachomatis. Whereas, CD28 signaling is critical, ICOS appears to be dispensable and can have a dampening effect on Th1 development by driving Th2 immunity and anti-inflammation through IL-10 production and promotion of the Foxp3(+) Treg populations in the genital tract. Both the CD28-deficient and the ICOS-deficient mice demonstrated poor specific antibody production, supporting the fact that antibodies are not needed for protection against genital tract chlamydial infections.

FIG. 1.

ICOS^−/− mice, but not CD28^−/− mice, develop protective immunity in the genital tract to C. trachomatis infection. Bacterial shedding was determined at given intervals after a primary infection (A) or after reinfection (B) with 1 × 10⁶ IFU of C. trachomatis EBs given intravaginally to WT, CD28^−/−, and ICOS^−/− mice. (C) The percentages of infected mice per group at given time intervals after the primary infection or postreinfection (D) were calculated. Results represent mean absorbances ± standard errors of the mean (A and B), and the percentages (C and D) of mice positive for chlamydial antigen shedding, as determined by Chlamydia Mikrotrak EIA. Values given are the combined means of three independent experiments, each including at least 10 mice per group. * (P < 0.05) and ** (P < 0.01) denote statistically significant differences between the WT and either CD28^−/− or ICOS^−/− groups, while # (P < 0.05) and ## (P < 0.01) are statistically significant differences between the CD28^−/− and ICOS^−/− groups.

FIG. 2.

Greatly impaired systemic and local Chlamydia-specific antibody production in ICOS^−/− and CD28^−/− mice. Serum IgG (A) and vaginal IgG (B) from WT, CD28^−/−, and ICOS^−/− mice were measured by ELISA after the clearance of a primary infection. Responses are given as individual log₁₀ titers, with the combined mean of the group indicated by a line. Results represent two independent experiments. * (P < 0.05) and ** (P < 0.01) denote statistically significant differences between the WT and either CD28^−/− or ICOS^−/− groups, while # (P < 0.05) and ## (P < 0.01) denote statistically significant differences between CD28^−/− and ICOS^−/− groups.

FIG. 3.

Augmented CD4⁺ T-cell responses in ICOS^−/− mice but impaired responses in CD28^−/− mice to genital tract infection with C. trachomatis. (A) Cell proliferation of PALN cells isolated 10 days after clearance of infection and cultured with or without heat-inactivated C. trachomatis EBs. Results are given as the mean cpm ± standard error of the mean above background in cultures with medium alone and represent pooled pairs from three independent experiments with 6 to 10 mice per group. (B) CD4⁺ T-cell infiltrates in the genital tract mucosa of naïve, WT, CD28^−/−, and ICOS^−/− mice during a secondary challenge infection were determined. Cross sections of the uterus were peroxidase labeled with biotin-conjugated anti-CD4 monoclonal antibodies, followed by a commercial ABC/AEC kit. Mean CD4⁺ T-cell counts of three fields (each 1,250 μm² at a magnification of ×20), from the left and right uterine horns of at least three mice per group, per experiment were performed. Data represent the means + standard errors of the means of three independent experiments. Naïve represents the maximum CD4⁺ T-cell count in all groups. Representative sections of CD4⁺ T-cell infiltrates in naïve WT mice (C), naïve CD28^−/− mice (D), and naïve ICOS^−/− mice (E) are shown. Right panels (F to H) represent reinfected WT (F) mice, CD28^−/− (G) mice, and ICOS^−/− (H) mice on day 2 of the reinfection. Large photographs at a magnification of ×10, with a magnification of ×40 for the insets. * (P < 0.05) and ** (P < 0.01) denote statistically significant differences between the WT and either CD28^−/− or ICOS^−/− groups, while # (P < 0.05) and ## (P < 0.01) denote statistically significant differences between CD28^−/− and ICOS^−/− groups.

FIG. 4.

Early and enhanced IFN-γ production in ICOS^−/− mice in response to reinfection with C. trachomatis. T cells containing IFN-γ were enumerated in frozen cross sections of the uteruses of infected mice. Results are given as average percentages ± standard errors of the means of total CD4⁺ T cells that double stained for IFN-γ⁺ at the indicated time points postreinfection. Cells were counted in at least three fields (1,250 μm² each at ×20 magnification), from left and right uterine horns from three independent experiments with at least three mice per group. (A) Frozen sections were colabeled with anti-IFN-γ (red, Texas Red) and anti-CD4⁺(green, FITC) and double-labeled of IFN-γ⁺ CD4⁺ T cells (yellow) were counted (B). Negative controls include the uteruses from Chlamydia-infected IFN-γ^−/− mice (C) or isotype controls (not shown) labeled and scored identically to tissues from the WT, ICOS^−/−, or CD28^−/− mice. Photographs shown at a magnification of ×40. * (P < 0.05) and ** (P < 0.01) denote statistically significant differences between the WT and either CD28^−/− or ICOS^−/− groups, while # (P < 0.05) and ## (P < 0.01) denote statistically significant differences between CD28^−/− and ICOS^−/− groups.

FIG. 5.

Striking Th1 skewing of protective immunity in ICOS^−/− mice. Uterine tissues were collected from WT, CD28^−/−, and ICOS^−/− mice at different time points after reinfection for mRNA extraction. Quantitative analyses using real-time RT-PCR were undertaken to determine the relative levels of (A) IFN-γ or (B) IL-10 mRNA in the effector tissues of reinfected mice at peak expression, on day 2 postreinfection. Values represent the means (WT, CD28^−/−, or ICOS^−/− mice) or means naive (WT, CD28^−/−, or ICOS^−/− mice) from three independent experiments with two to three mice per group in each experiment. Values were normalized against mRNA expression levels for the HRPT gene, the housekeeping gene.

FIG. 6.

Detection of Th1 and Th2 transcription factors provide compelling evidence for Th1 dominance in protective immunity in ICOS^−/− mice. PALN and uterine tissues from WT, CD28^−/−, and ICOS^−/− mice were collected on day 8 of the primary infection (1°; A and C) and at peak expression on day 2 of the reinfection (2°; B and D). Real-time RT-PCR was used to determine the T-bet (A and B) and GATA-3 (C and D) mRNA transcription factor expression levels. Values are calculated as mean postinfection (WT, CD28^−/−, or ICOS^−/− mice) minus mean naive (WT, CD28^−/− or ICOS^−/− mice, respectively) from one representative experiment of three with two to three mice per group. Values were normalized against mRNA expression levels for CD3-γ. * (P < 0.05) and ** (P < 0.01) denote statistically significant differences between WT and either CD28^−/− or ICOS^−/− groups.

FIG. 7.

ICOS^−/− mice exhibit a reduced ability to generate Treg cells in response to a genital tract chlamydial infection. Using quantitative real-time RT-PCR, we determined the Foxp3 mRNA expression levels in uterine and PALN tissues after a primary and a secondary challenge infection with Chlamydia. (A) A macroscopic picture of the degree of tissue inflammation is given as comparisons between the uninfected tissue and tissue at day 7 after clearance of a primary infection, from a representative experiment with WT, CD28^−/−, and ICOS^−/− mice. Representative H&E staining (magnification ×20) reflects the degree of inflammatory infiltrates in the oviducts of WT mice (B) and ICOS^−/− mice (C) after clearance of a primary infection. (D) Oviduct area reflects the degree of inflammation. The area was measured from left and right uterine horns from three independent experiments with at least three mice per group on day 8 of the primary infection. PALN and uterine tissues from WT, CD28^−/−, and ICOS^−/− mice were collected on day 8 of the primary infection (E) or on day 2 of the reinfection (F), and real time RT-PCR values for Foxp3 mRNA levels were calculated for uterus and PALN in relation to the total CD3-γ mRNA content in each sample. Values are calculated as mean postinfection (WT, CD28^−/−, or ICOS^−/− mice), mean naive (WT, CD28^−/−, or ICOS^−/− mice, respectively). * (P < 0.05) and ** (P < 0.01) denote statistically significant differences between WT and either CD28^−/− or ICOS^−/− groups.