Enumeration of T cells reactive with Mycobacterium tuberculosis organisms and specific for the recombinant mycobacterial 64‐kDa protein

Abstract

The major goal of the present study was to develop a limiting dilution system for the enumeration of T cells which respond to mycobacterial antigens. Purified T cells from M. tuberculosis-immune mice were restimulated with mycobacterial antigens and accessory cells, and after 4 days expanded with antigen, accessory cells and T cell growth factor. After another 3 days, proliferative responses were determined. Similar cultures performed without antigen served as controls. Limiting dilution analysis revealed that approximately 1/2000 to 1/3000 T cells from M. tuberculosis-immune mice responded to whole M. tuberculosis organisms while T cells from normal mice did not respond. Similar T cell numbers reacted with several mycobacterial strains indicating expression of shared T cell antigens. Using a semi-purified recombinant 64-kDa protein from M. bovis the frequency of T cells generated after immunization with M. tuberculosis which reacted with a single mycobacterial protein could be estimated. We found that approximately 1/5 of the M. tuberculosis-reactive T cells recognized this particular antigen. Immunization with the recombinant 64-kDa protein in an adjuvant containing trehalose dimycolate, monophosphoryl lipid A and mycobacterial cell wall skeleton stimulated an equally high number of M. tuberculosis-reactive T cells (1/2000). These findings demonstrate that a high proportion of tuberculosis-responsive T cells are directed against the 64-kDa protein and that immunization with this antigen in an appropriate adjuvant system is capable of stimulating high numbers of M. tuberculosis-reactive T cells. Limiting dilution analysis with a panel of mycobacterial proteins or peptides may allow their ranking from immunodominant to immunosilent and facilitate identification of antigens or epitopes relevant to protection.

Treatment of murine collagen‐induced arthritis by the stress protein BiP via interleukin‐4–producing regulatory T cells: A novel function for an ancient protein

“…An interesting property of heatshock proteins (HSPs) is their highly immunogenic nature (24). To investigate the nature of the response stimulated by BiP, DBA/1 mice were immunized subcutaneously with 200 g BiP either solubilized in PBS or emulsified in IFA.…”

Phase I trial of DNA-hsp65 immunotherapy for advanced squamous cell carcinoma of the head and neck

“…This approach was considered to be particularly promising for tumors in which dominant antigens are poorly characterized as is the case of HNSCC. Second, considering that Hsp65 is an immunodominant antigen in mycobacteria, 36 the expression of this foreign protein by tumor cells in the context of HLA class I molecules could lead to breakdown of the tolerogenic environment found in cancer patients. As with any new type of therapy, there are important safety concerns.…”

Yersinia enterocolitica immunodominant 60 kDa antigen, common to a broad range of bacteria, is a heat-shock protein

“…In addition, it stimulates the proliferation of T cells in immune mice, suggesting that it is likely to be involved in the development of cellmediated immunity (Kaufmann et al, 1987). In contrast, Willem et al (1985) suggested that arthritis was induced by a T-cell clone which recognized an epitope on the mycobacterial 65 kDa antigen.…”