CD8+-T-cell responses of Mycobacterium-infected mice to a newly identified major histocompatibility complex class I-restricted epitope shared by proteins of the ESAT-6 family

Abstract

Here we describe the identification of a new CD8(+)-T-cell epitope, the GYAGTLQSL nonamer, shared by the TB10.3 and TB10.4 proteins of the Mycobacterium tuberculosis ESAT-6 family. Cytotoxic T cells from mycobacterium-infected mice efficiently recognized this epitope. GYAGTLQSL-specific T-cell hybridomas, which were able to recognize Mycobacterium bovis BCG-infected macrophages, were generated and now allow investigation of mycobacterial-antigen processing through the major histocompatibility complex class I pathway.

FIG.1.

CTL responses to mycobacterial nonamers predicted to bind to H-2K^d. CTL activity in the lymph nodes of BALB/c mice (n = 3) immunized s.c. with individual peptides emulsified in incomplete Freund adjuvant (A), in the splenocytes of mice (n = 4) injected s.c. with 10⁷ CFU of BCG Pasteur 1173P2 (B), or in the splenocytes of mice infected s.c. with 10⁶ CFU of M. tuberculosis (H37Rv) (C) is shown. Eight days after the last injection of H37Rv-derived peptides or 4 weeks after infection with BCG or H37Rv, CTL activity against unloaded or peptide-loaded P815 targets was measured in a standard 5-h ⁵¹Cr release assay run in duplicate. The percentage of specific lysis was calculated with the formula 100 × (experimental release − spontaneous release)/(maximum release − spontaneous release). Maximum release was obtained by the addition of 1% Triton X-405 to labeled target cells. Results are representative of at least two independent experiments. Standard deviations were always <5%.

FIG. 2.

Alignment of the protein sequences of Rv0288 (TB10.4) and Rv3019c (TB10.3). The sequence of the TB10.3/4:20-28 epitope is underlined.

FIG. 3.

Requirement of immunization with live BCG for induction of TB10.3/4:20-28-specific CD8⁺ CTLs. (A) TB10.3/4:20-28-specific CTL responses. BALB/c mice were immunized by a single s.c. injection of 10⁷ CFU of live BCG or by two injections at a 15-day interval of 10⁷ CFU of heat-killed BCG. Splenocytes were harvested at day 21 for CTL assay. (B) TB10.3/4:20-28-specific CTL activity of total CD4⁺- or CD8⁺-T-cell-depleted splenocytes of BCG-immunized BALB/c mice. T-subset depletion was assessed by negative selection with biotinylated anti-CD4 (GK1.5) or anti-CD8 (H35-17-2) monoclonal antibodies and streptavidin-coupled magnetic beads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany).

FIG. 4.

Generation of H-2K^d-restricted TB10.3/4:20-28-specific T-cell hybridomas from BCG-immunized BALB/c mice. (A) Specific IL-2 production by four TB10.3/4:20-28-specific T-cell hybridomas (XH3, YB8, WC7, and KC6) (10⁵ cells/well) subsequent to overnight stimulation with various concentrations of TB10.3/4:20-28 peptide in the presence of syngeneic bone marrow-derived dendritic cells (10⁵ cells/well) used as an antigen-presenting cell. (B) Specific IL-2 production (monitored by CTLL-2 bioassay) by XH3, YB8, WC7, and KC6 T-cell hybridomas in response to stimulation by Raw 264.7 macrophages incubated with 1 μg of TB10.3/4:20-28 peptide per ml or previously infected with BCG at a final concentration of 5 × 10⁵ CFU/ml under antibiotic-free conditions for 4 days.