HLA-B*35-restricted CD8(+)-T-cell epitope in Mycobacterium tuberculosis Rv2903c

Abstract

Few human CD8(+) T-cell epitopes in mycobacterial antigens have been described to date. Here we have identified a novel HLA-B*35-restricted CD8(+) T-cell epitope in Mycobacterium tuberculosis Rv2903c based on a reverse immunogenetics approach. Peptide-specific CD8 T cells were able to kill M. tuberculosis-infected macrophages and produce gamma interferon and tumor necrosis factor alpha.

FIG. 1.

Screening of Rv2903c-derived peptides for binding to HLA-B*3501. RMA-S B*3501 cells were pulsed with 50 μM unlabeled competitor peptide and 1 μM FL-labeled reference peptide. Results are expressed as percent inhibition of the maximum signal of the FL-labeled reference peptide. The vertical dotted line identifies peptides that inhibited the maximum signal by >75%. Positive controls were peptides HIV-1_SF2 Nef_72–80 (aa 72 to 80) (4T6R), FPVTPRVPL, and EBV TEGU (aa 1974 to 1982), HPLTNNLPL. Peptide Flu-A NP (aa 265 to 273), ILRGSVAHK, was used as a negative control.

FIG. 2.

HLA-B*3501-restricted cytokine production by activated CD8 T cells in response to mycobacterial peptides. Short-term cultures of PBMC stimulated with peptide Rv2903c (aa 201 to 209) were cocultivated for 6 h with RMA-S B*3501 cells pulsed with a control peptide, HIV-1_SF2 Nef_72–80 (4T6R) (panels a and c), or the specific peptide (panels b and d). Cytokine secretion was blocked by brefeldin A. Surface markers CD8 and CD69 and intracellular IFN-γ (lower panels) and TNF-α (upper panels) were stained following fixation and permeabilization of cells. Dot plots show log fluorescence intensity. CD8⁺ bright T cells were gated in the fluorescence-3 channel versus side scatter light plots. Quadrant markers for CD69, IFN-γ, and TNF-α were set using the proper isotype control reagents. Frequencies of positive cells are given as a percentage of the gated CD8⁺ T cells. Abbreviations: PE, phycoerythrin; FITC, fluorescein isothiocyanate.