Mycobacterium tuberculosis-reactive CD8⁺ T lymphocytes: The relative contribution of classical versus nonclassical HLA restriction

Previous studies in mice and humans models have suggested an important role for CD8⁺ T cells in host defense to Mycobacterium tuberculosis (Mtb). In humans, CD8⁺ Mtb-reactive T cells have been described that are HLA-A2-, B52-, as well as CD1-restricted. Recently, we have described Mtb-specific CD8⁺ T cells that are neither HLA-A-, B-, or C- nor group 1 CD1-restricted. At present, little is known about the relative contribution of each of these restriction specificities to the overall CD8⁺ response to Mtb. An IFN-γ enzyme-linked immunospot assay was used to determine the frequency of Mtb- reactive CD8⁺ T cells directly from PBMC. The effector cell frequency among five healthy purified protein derivative-positive subjects was 1/7,600 ± 4,300 compared with 1/16,000 ± 7,000 in six purified protein derivative- negative controls. To determine the frequencies of classically, CD1-, and nonclassically restricted cells, a limiting dilution analysis was performed. In one purified protein derivative-positive subject, 192 clones were generated using Mtb-infected dendritic cells (DC). Clones were assessed for reactivity against control autologous DC, Mtb-infected autologous DC, and HLA-mismatched CD1⁺ targets (DC), as well as HLA-mismatched CD1⁺ targets (macrophages). Of the 96 Mtb-reactive CD8⁺ T cell clones, four (4%) were classically restricted and 92 (96%) were nonclassically restricted. CD1- restricted cells were not detected. Of the classically restricted cells, two were HLA-B44 restricted and one was HLA-B14 restricted. These results suggest that while classically restricted CD8⁺ lymphocytes can be detected, they comprise a relatively small component of the overall CD8⁺ T cell response to Mtb. Further definition of the nonclassical response may aid development of an effective vaccine against tuberculosis.