CD8+ T cells recognize an inclusion membrane-associated protein from the vacuolar pathogen Chlamydia trachomatis

Abstract

During infection with Chlamydia trachomatis, CD8(+) T cells are primed, even though the bacteria remain confined to a host cell vacuole throughout their developmental cycle. Because CD8(+) T cells recognize antigens processed from cytosolic proteins, the Chlamydia antigens recognized by these CD8(+) T cells very likely have access to the host cell cytoplasm during infection. The identity of these C. trachomatis proteins has remained elusive, even though their localization suggests they may play important roles in the biology of the organism. Here we use a retroviral expression system to identify Cap1, a 31-kDa protein from C. trachomatis recognized by protective CD8(+) T cells. Cap1 contains no strong homology to any known protein. Immunofluorescence microscopy by using Cap1-specific antibody demonstrates that this protein is localized to the vacuolar membrane. Cap1 is virtually identical among the human C. trachomatis serovars, suggesting that a vaccine incorporating Cap1 might enable the vaccine to protect against all C. trachomatis serovars. The identification of proteins such as Cap1 that associate with the inclusion membrane will be required to fully understand the interaction of C. trachomatis with its host cell.

Figure 1

Design of the pBIB-KS retroviral expression vectors used for eukaryotic expression of prokaryotic chromosomal DNA.

Figure 2

IFN-γ ELISPOT analysis to identify transduced cell pools recognized by Chlamydia-specific T cells. Twelve wells are shown, including positive pools 2C7 and 2E10. Positive wells contained confluent layers of ELISPOTs, precluding numerical quantitation.

Figure 3

Assays to identify peptides able to target P815 cells for lysis by Chlamydia-specific T cells. In A, ⁵¹Cr-labeled P815 cells were pulsed with the indicated peptides, washed, and incubated with the Chlamydia-specific T-cell line 69 at an effector to target ratio (E/T) of 10:1. Targeting was evaluated by measuring the amount of ⁵¹Cr release. In B, Cap1_139–147 was chemically synthesized and tested at the dilutions shown for its ability to target P815 cells for lysis by the C. trachomatis-specific T cells. The E/T ratio used in the assay was 8:1.

Figure 4

Cap1_139–147 peptide was tested for the ability to target cells for lysis by Cap1_139–147-specific T cells in a ⁵¹Cr release assay. In A and B, the cells were tested with (solid bars) and without (open bars) Cap1_139–147 peptide. The target cells shown in A are L-929 (H-2^k) cells individually transfected with the genes encoding the D^d, K^d, or L^d molecules. L-929 cells alone are shown as a negative control. In B, the target cells are RMA-S cells (H-2^b) and RMA-S cells transfected with the gene encoding K^d. The E/T ratio used in the assay was 10:1. In C, the target cells were C. trachomatis-infected () or uninfected () Balb/3T3 cells.

Figure 5

Chromium release assays showing recognition of Cap1_139–147 by T cells primed during murine infection with C. trachomatis. Mice were immunized i.p. with 10⁸ IFU of C. trachomatis serovar L2. Two weeks after infection, spleen cells from an immunized mouse (A) or a control mouse injected with PBS (B) were cultured for 5 days with Cap1_139–147-coated syngeneic spleen cells. Target cells were Cap1_139–147-coated () or untreated () P815 cells. Graphs are representative of three experimental and three control mice.

Figure 6

Fluorescence immunocytochemistry of McCoy cells infected with C. trachomatis (A–C) or C. psittaci (D), all fixed 24 h postinfection. In A and D, cells were stained with rabbit anti-Cap1 antiserum. In B, cells were stained with preimmune serum. In C, cells were labeled with an antiserum directed against C. trachomatis elementary bodies.