Cell-mediated immune responses to the 18 kilodalton protein of Mycobacterium leprae

Abstract

The cell-mediated immune system plays an important role in protective immunity against Mycobacterium leprae. The aim of work presented in this thesis was to study various T cell responses in mice to one protein of M. leprae, designated the 18 kDa protein. Mice were immunised with recombinant 18 kDa protein and lymph node cells tested later in proliferative T cell assays. The in vitro response was specific for the 18 kDa protein and proliferation dependent on the immunising dose as well as in vitro antigen concentration. The proliferative response required CD4+ T cells, which recognised the antigen in the context of major histocompatibility class II molecules. The T cell response was tested in various recombinant and congenic pairs of mouse strains. BALB/cJ (H-2d), BALB.B (H-2b), B10,BR (H-2k), and B10.M (H-2f) mice were all high responder strains, while the C57BL/10J (H-2b) mouse strain was a low responder. By comparing the genetic characteristics of these high and low responding strains, it was deduced that both H-2 and non-H-2 gene(s) contributed to the magnitude of responsiveness.F1 progeny mice from high and low responder parents responded in a way that showed that high responsiveness was inherited in a dominant manner. Limiting dilution assays indicated that the frequency of cells proliferating in response to the 18 kDa protein correlated with high and low responsiveness of cells from secondary lymphoid tissue. The low frequency of C57BL10/J responding lymphocytes to the 18 kDa protein probably accounted for the reduced in vitro IL-2 production observed in this mouse strain. The T cell response of mice immunised with either the 18 kDa or M. leprae antigens revealed the following. The 18 kDa protein-primed lymph node cells responded to both M. leprae and. M. tuberculosis, suggesting a shared 18 kDa protein T cell epitope by these pathogens. The M. leprae-primed lymph node cells showed no significant response to the 18 kDa protein, but responded similarly to M. leprae and M. tuberculosis antigens. The results were obtained using both bulk cultures and limiting dilution experiments. This indicated that the l8 kDa protein was probably not a dominant antigen in primary murine immune responses against M. leprae. Proliferative T cell epitopes on the 18 kDa protein were examined in several mouse strains. Each mouse strain responded to one dominant region after immunising with 18 kDa protein. The dominant T cell epitope was not identical in all strains. The peptide consisting of residues 106-l25 of the 18 kDa protein was dominant in BALB/cJ and BALB.B mice, whereas the B10.BR mice responded to peptide 31-50 and C57BL/10J mice to peptide 16-35. Lymph node cells primed with individual synthetic peptides and challenged with 18 kDa protein in vitro revealed additional T cell epitopes which were not recognised following immunisation with 18 kDa protein. Cytotoxic T cell epitopes on the l8 kDa molecule were investigated, using an in vitro-priming system. Only one region of the 18 kDa protein elicited cytotoxic T cell responses. This T cell epitope was restricted to mouse strains of H-2b haplotype. The cytotoxic response was a feature of CD8+ T cells and the lysis of target cells depended upon peptide concentration. Cells infected with a recombinant retrovirus carrying the 18 kDa gene were used to prime mice for the induction of cytotoxic T cells in vivo. Cytotoxic T cell responses were detected but none were specific for the 18 kDa protein. Inflammatory T cell responses to the 18 kDa protein were investigated in a number of mouse strains, using a delayed-type hypersensitivity footpad assay. The C57BL/10J mouse strain elicited a similar or even greater response than other strains tested, in contrast to proliferative T cell responses where C57BL/10J was a low responder strain. The number of immunizations influenced the type of immune response elicited in mice. Multiple immunisations with the 18 kDa protein resulted in a reduced delayed-type hypersensitivity response concomitant with the appearance of antigen-specific IgG antibodies. Several 18 kDa-specific T cell lines derived from BALB/cJ, B10.BR, and C57BL/10J mouse strains were generated. T cell lines from the BALB/cJ strain showed characteristics of TH1 and TH0 phenotypes and one line responded to peptide 46-65 of the 18 kDa protein. The B10.BR- and C57BL/10J-derived T cell lines responded to peptide 31-50 and 106-125 respectively and secreted IL-3 upon stimulation, but neither IL-2 nor IL-4 were detected. Various subsets of T cells might play different roles in the immune response against pathogens. The T cell subsets involved in protection might not be identical to those eliciting a strong immunological reaction. T cell responses against intracellular parasites are discussed in relation to factors that govern protective immunity.