Identification of Chlamydia pneumoniae-derived mouse CD8 epitopes

Abstract

Chlamydia pneumoniae is a common intracellular human pathogen that has been associated with several severe pathological conditions, including coronary heart disease and atherosclerosis. There is no vaccine against C. pneumoniae infection, but CD8(+) T cells have been shown to be crucial for protection during experimental infection. However, the effector functions and epitope specificity of the protective CD8(+) T cell remain unknown. The aim of this study was to identify C. pneumoniae-derived mouse CD8 epitopes by using a recent epitope prediction method. Of four C. pneumoniae proteins (the major outer membrane protein, outer membrane protein 2, polymorphic outer membrane protein 5, and heat shock protein 60), 53 potential CD8(+) T-cell epitopes were predicted by H-2 class I binding algorithms. Nineteen of the 53 peptides were identified as CD8 epitopes by testing for induction of a cytotoxic response after immunization. To test whether the predicted epitopes are naturally processed and presented by C. pneumoniae-infected cells, we generated a panel of seven peptide-specific cytotoxic T lymphocyte lines that were subsequently tested for recognition of C. pneumoniae-infected target cells. By using this strategy, we were able to identify three C. pneumoniae CD8 epitopes that were, indeed, processed and presented on infected cells. Identification of these natural CD8 epitopes provides tools for characterization of CD8(+) T-cell function in vivo and generation of epitope-specific prevention strategies.

FIG. 1.

Peptide specificity of the seven long-term CTL lines. CTL lines were enriched from splenocytes of BALB/c (H-2^d) and C57BL/6 (H-2^b) mice that had been subcutaneously immunized with a mixture of TiterMax adjuvant and C. pneumoniae-derived peptide. The splenocytes were restimulated in vitro weekly with the corresponding peptide and irradiated autologous splenocytes. The cytotoxic activity of the CTL lines was tested with a 5-h ⁵¹Cr release assay on target cells alone (○), target cells coated with corresponding peptide (•), or target cells coated with another peptide specific for the same H-2 allele from the same protein (▴). P815 (for H-2^d effectors) and RMA (for H-2^b effectors) cells were used as target cells. The E/T cell ratio describes the number of CTLs (effectors) in comparison with the number of target cells in each well. Percent specific lysis was calculated as follows: % specific lysis = 100 × [(experimental release − spontaneous release)/(maximum release − spontaneous release)].

FIG. 2.

(A) Expression of CD4 and CD8 molecules on a CTL line specific for peptide 8511. After enrichment by Ficoll-Paque gradient centrifugation, the viable cells were stained with either FITC-conjugated anti-mouse CD8 and phycoerythrin-conjugated anti-mouse CD4 antibodies or with irrelevant anti-mouse antibodies (rat IgG2a and rat IgG2b). The labeled cells were fixed with 1% paraformaldehyde, and 10,000 events were subjected to flow cytometric analysis. Similar results (>95% of the cells were CD8⁺) were also obtained with the CTL lines specific for the other peptides. (B) Neutralization of CD8⁺ cells reduced the cytotoxic activity of CTL line 8511 compared to that of an untreated culture. CTL effector cells were preincubated with 20 μg of anti-CD4 or -CD8 monoclonal antibodies (Ab) for 45 min before the addition of ⁵¹Cr-labeled peptide-pulsed target cells. The E/T cell ratio was 40. A similar reduction of cytotoxic activity after neutralization of CD8⁺ cells was also detected with other CTL lines, inhibition varying from 10 to 60%.

FIG. 3.

(A) Lysis of C. pneumoniae-infected target cells by CTL line 8482 in a 10-h ⁵¹Cr release assay. The CTL line was stimulated with splenocytes of a mouse immunized with Omp2-derived peptide 8482. BALB/3T3 target cells were infected in vitro with C. pneumoniae and labeled with ⁵¹Cr. Uninfected control cells were treated similarly without addition of C. pneumoniae. An E/T cell ratio of 10 was used. (B) The TNF-α production of CTL line 8482 in the presence of infected BALB/3T3 cells was significantly higher than the background level or the response to uninfected target cells. We stimulated 3 × 10⁵ CTLs with the same number of infected target cells (E/T cell ratio of 1) on 24-well plate for 48 h, after which the supernatants were collected and analyzed by ELISA. BD, below detection limit.