In vitro expansion of T-cell-receptor V alpha 2.3(+) CD4(+) T lymphocytes in HLA-DR17(3), DQ2(+) individuals upon stimulation with Mycobacterium tuberculosis

Abstract

The T-cell receptor (TCR) V alpha/beta gene product expression upon in vitro stimulation with mycobacteria was investigated to assess whether T-cell proliferation was associated with any specific TCR V gene usage. T-cell-enriched populations from peripheral blood of Mycobacterium bovis BCG-vaccinated healthy blood donors were stimulated in vitro with live or killed M. tuberculosis or with a soluble extract thereof. TCR V alpha/beta repertoire analysis of reactive CD4(+) and CD8(+) T cells revealed a selective HLA-DR17(3), DQ2-restricted expansion of V alpha 2.3(+) CD4(+) T cells upon stimulation with live M. tuberculosis or its soluble extract. Third-complementarity-determining-region (CDR3) length analysis of the expanded V alpha 2.3(+) T cells indicated an oligoclonal pattern with short CDR3 lengths in six of seven HLA-DR17(3), DQ2(+) individuals tested. In addition, V alpha/V beta repertoire analysis of T lymphocytes from a DR17(3), DQ2(+) donor before and after BCG vaccination revealed that positivity of skin test reactivity was associated with expansion of V alpha 2.3(+) CD4(+) T lymphocytes with preferential use of a short CDR3 peak length after in vitro stimulation. Separation of M. tuberculosis soluble extract by fast protein liquid chromatography (FPLC) purification indicated that fractions corresponding to molecular masses of 60 to 70 and 15 to 25 kDa were particularly effective in eliciting V alpha 2.3(+) CD4(+) T-cell expansion.