Immunity to murine Chlamydia trachomatis genital tract reinfection involves B cells and CD4(+) T cells but not CD8(+) T cells

Abstract

CD4(+) T-helper type 1 (Th1) responses are essential for the resolution of a primary Chlamydia trachomatis genital tract infection; however, elements of the immune response that function in resistance to reinfection are poorly understood. Defining the mechanisms of immune resistance to reinfection is important because the elements of protective adaptive immunity are distinguished by immunological memory and high-affinity antigen recognition, both of which are crucial to the development of efficacious vaccines. Using in vivo antibody depletion of CD4(+) and CD8(+) T cells prior to secondary intravaginal challenge, we identified lymphocyte populations that functioned in resistance to secondary chlamydial infection of the genital tract. Depletion of either CD4(+) or CD8(+) T cells in immune wild-type C57BL/6 mice had a limited effect on resistance to reinfection. However, depletion of CD4(+) T cells, but not CD8(+) T cells, in immune B-cell-deficient mice profoundly altered the course of secondary infection. CD4-depleted B-cell-deficient mice were unable to resolve a secondary infection, shed high levels of infectious chlamydiae, and did not resolve the infection until 3 to 4 weeks following the discontinuation of anti-CD4 treatment. These findings substantiated a predominant role for CD4(+) T cells in host resistance to chlamydial reinfection of the female genital tract and demonstrated that CD8(+) T cells are unnecessary for adaptive immune resistance. More importantly, however, this study establishes a previously unrecognized but very significant role for B cells in resistance to chlamydial reinfection and suggests that B cells and CD4(+) T cells may function synergistically in providing immunity in this model of chlamydial infection. Whether CD4(+) T cells and B cells function independently or dependently is unknown, but definition of those mechanisms is fundamental to understanding optimum protective immunity and to the development of highly efficacious immunotherapies against chlamydial urogenital infections.

FIG. 1

Effect of anti-CD4 or anti-CD8 treatment on the resolution of secondary C. trachomatis genital tract infection of wild-type B6 mice. Mice were infected vaginally with 100 ID₅₀ of C. trachomatis, and the course of the primary infection was monitored. Following resolution of the primary infection, immune mice were divided into four groups of five mice each and treated with either anti-CD4, anti-CD8, rat Ig, or PBS as described in Materials and Methods. Treated mice were challenged vaginally with 100 ID₅₀ of C. trachomatis on day 56 following primary infection, and the infection was monitored by swabbing the vaginal vault and enumerating IFU on HeLa cell monolayers. Data are presented as log₁₀ IFU and represent the mean ± the standard error of the mean of triplicate determinations of five mice. P < 0.05 for anti-CD4-, anti-CD8-, rat Ig-, and PBS-treated mice compared to primary infection of mice at days 3, 7, 10, and 14 post infectious challenge.

FIG. 2

Effect of anti-CD4 or anti-CD8 treatment on the resolution of a secondary C. trachomatis genital tract infection of B-cell-deficient mice. Mice were infected vaginally with 100 ID₅₀ of C. trachomatis, and the course of the primary infection was monitored. Following resolution of the primary infection, immune mice were divided into four groups of five mice each and treated with either anti-CD4, anti-CD8, rat Ig, or PBS as described in Materials and Methods. Treated mice were challenged vaginally with 100 ID₅₀ of C. trachomatis on day 56, and the infection was monitored by swabbing the vaginal vault and enumerating IFU on HeLa cell monolayers. Data are presented as log₁₀ IFU and represent the mean ± the standard error of the mean of triplicate determinations of five mice. P < 0.05 for anti-CD8-, rat Ig-, and PBS-treated mice compared to primary infection of mice at days 3, 7, 10, 14, and 21 days post infectious challenge. P < 0.05 for anti-CD4-treated mice compared to primary infection of mice at days 3, 14, 21, 28, 35, and 42 post infectious challenge.

FIG. 3

Tissue depletion of CD4⁺ and CD8⁺ T cells following secondary infectious challenge of antibody-treated wild-type B6 mice. Seven days following a secondary C. trachomatis infection, mice were sacrificed and their genital tracts were removed and processed for immunohistochemical analysis. In vivo treatment: PBS (A to C), anti-CD4 (D to F), and anti-CD8 (G to I). In vitro immunohistochemical staining: anti-CD4 (A, D, and G), anti-CD8 (B, E, and H), and control rat Ig (C, F, and I). Representative tissues from two or three mice are shown. Tissues harvested at 3 days post secondary infectious challenge showed a similar staining pattern. Original magnification, ×300.

FIG. 4

Tissue depletion of CD4⁺ and CD8⁺ T cells following secondary infectious challenge of antibody-treated, B-cell-deficient mice. Seven days following secondary C. trachomatis infection, mice were sacrificed and their genital tracts were removed and processed for immunohistochemical analysis. In vivo treatment: PBS (A to C), anti-CD4 (D to F), and anti-CD8 (G to I). In vitro immunohistochemical staining: anti-CD4 (A, D, and G), anti-CD8 (B, E, and H), and control rat Ig (C, F, and I). Representative tissues from two or three mice are shown. Tissues harvested at 3 days post secondary infectious challenge showed a similar staining pattern. Original magnification, ×300.

FIG. 5

In vitro production of IFN-γ (A), IL-4 (B), and IL-10 (C) by spleen cells from wild-type B6 and B-cell-deficient mice following in vivo treatment with either PBS, rat Ig, anti-CD4, or anti-CD8. Spleens were harvested from two or three mice per group 7 days after secondary infection with C. trachomatis and restimulated in vitro with heat-killed chlamydiae. The values shown are the concentrations of cytokines (mean ± standard deviation of triplicate determinations) present in the supernatants after 72 h of culture. At 24 and 72 h, supernatants were also analyzed for IL-12 but IL-12 was never detectable.