Reversal of T cell unresponsiveness by augmentation of antigen presenting cell function.

We have previously described epitopes of the 18 kDa protein of Mycobacterium leprae which stimulate T and B cell responses in different strains of mice. A series of overlapping 20-mer peptides that span the 18 kDa protein were used as immunogens to examine T and B cell recognition of different epitopes. Strain-specific variation in the epitopes which induce the strongest responses was affected by genes linked to the H-2 complex and the T cell responses revealed by re-challenge with antigen were at least partially controlled by factors other than T cell specificity. We have examined the responses to one such antigen, peptide 1-20, which contains strongly immunogenic epitopes for T and B cells. T cells from draining lymph nodes of peptide 1-20 immunized B10.BR, but not BALB/c mice, proliferated in vitro in response to rechallenge with peptide 1-20 or whole protein. Immunization with the same peptide also induced specific antibody only in B10.BR mice. However, immunization of BALB/c mice results in 'silent' priming of T cells since these can be induced to respond in vitro to this antigen when cultured with activated macrophages as antigen presenting cells (APC). The failure of APC from mBALB/c mice primed with peptide 1-20 to stimulate CD4+ proliferation when re-challenged in vitro and the failure to elicit antibody responses to peptide 1-20 are presumably due to the same defect in antigen-presenting cell function, since presentation of peptide 1-20 by activated macrophages is sufficient to restore both responses.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of T cell stimulatory epitopes from the 18 kDa protein of Mycobacterium leprae.

We have used different mouse strains to examine in vivo and in vitro responses to the 18 kDa protein of Mycobacterium leprae, which appears to be strongly immunogenic in both mice and humans. B and T cell stimulatory epitopes recognised by different strains of mice have been mapped using overlapping peptides that span the entire 18 kDa protein. Previous work established that immunization of mice with the 18 kDa protein results in specific antibody production to common B cell epitopes and immunization of mice with peptides containing these B cell epitopes resulted in the induction of specific IgG to only a limited subset of epitopes in each strain. Now we report that T cells purified from mice immunized with peptides that stimulate antibody production, proliferate in vitro when rechallenged. The proliferating T cells produce levels of IL-2 and IFN-gamma, that indicate antigen-specific T helper type 1 cells are present in significant numbers. Thus, a comparison of in vivo and in vitro data suggests that T cells bearing the phenotype associated with potentially protective cell-mediated responses can be primed in vivo by epitopes on small peptides. Since T cells from both strains of mice are capable of responding to the immunogenic synthetic peptides in vitro, but give different responses to the same peptides in vivo, factors other than epitope structure appear to influence T cell subset activation. This may have important implications for diseases such as leprosy where a polarized T cell response appears to develop and for the development of synthetic subunit vaccines.

Immune responses to the 18-kDa protein of Mycobacterium leprae. Similar B cell epitopes but different T cell epitopes seen by inbred strains of mice.

Antibody responses to the 18-kDa protein of Mycobacterium leprae have been analyzed in different strains of mice. High, intermediate, and low responder strains have been identified and these response patterns show clear linkage to genes encoded in the H-2 complex. Three peptides, residues 1-50, 51-100, and 101-148 have been synthesized, as well as a series of 20-mer peptides, which span the entire 18-kDa protein. Repeated immunization of different strains of mice with the 18-kDa protein resulted in IgG responses to epitopes found on all three synthetic peptides. Immunization of BALB/cJ and B10.BR mice, two high responder strains, with 18-kDa protein resulted in high levels of IgG antibody to epitopes found on peptides 1-20, 16-35, 31-50, 46-65, and 76-95. B10.BR mice also contained IgG that bound peptide 61-80 and BALB/cJ mice produced IgG that bound peptide 91-110. Although B10.BR mice produced IgG that bound the 50-mer peptide 101-148, this IgG was not detected by binding to peptides 91-110, 106-125, 121-140, and 131-148. Immunization of B10.BR mice with individual overlapping 20-mer peptides as Ag revealed that peptides 1-20, 16-35, 31-50, and 76-95 elicited high titers of IgG that bound both the immunizing peptide as well as 18-kDa protein. As these peptides induce antibody synthesis they must contain both B cell and T cell epitopes. By contrast, immunization of BALB/cJ mice with the same 20-mer peptides, all of which contain B cell epitopes for this strain, failed to elicit IgG responses with one exception. Peptide 91-110 induced IgG that bound peptide 91-110, but not the intact 18-kDa protein. We conclude that peptides 1-20, 16-35, 31-50, and 76-95 either lack T cell epitopes for BALB/cJ mice, or activate different T cell subpopulations in the two strains. We suggest that the induction of IgG responses to small peptide Ag is an in vivo assay of the activity of Th2 cell subpopulations.

The mapping of epitopes of the 18-kDa protein of Mycobacterium leprae recognized by murine T cells in a proliferation assay.

The 18-kDa protein of Mycobacterium leprae was purified from recombinant plasmids pUL108 and pML-3 grown in Saccharomyces cerevisiae and Escherichia coli, respectively. Significant lymphoproliferative responses were observed when T cells from immunized mice were challenged in culture with purified 18-kDa protein. Synthetic peptides have been prepared that span most of the 148 amino acid residues that constitute the sequence of the 18-kDa protein and used to map epitopes recognized by T cells. When mice were immunized with 18-kDa protein and lymph node cells subsequently prepared and challenged in microculture proliferative assays by using synthetic peptides, only one region of the intact protein appeared stimulatory. This T cell epitope was located between residues 116 and 121, adjacent to an epitope between residues 110 and 115 which we have previously shown to bind the L5 mAb. Immunization of mice with peptides, and subsequent challenge of lymph node cells in assays by using the 18-kDa protein as Ag revealed that residues 111-125 were the most effective in priming responses. Furthermore, the ability of 18-kDa primed lymph node cells to recognize determinants on both M. leprae and Mycobacterium tuberculosis indicates that in addition to possessing an M. leprae-specific B cell determinant, the 18-kDa protein contains a cross-reactive T cell epitope(s).

Characterization of an antibody-binding epitope from the 18-kDa protein on Mycobacterium leprae.

A murine mAb, designated L5, appears to be specific for an epitope on a protein from Mycobacterium leprae of restricted distribution within the mycobacteria. This protein, of Mr 18,000 (18 kDa) is of interest because monoclonal antibodies raised against it do not appear to cross-react with other mycobacterial pathogens. The L5 antibody-binding epitope has been mapped by two complementary methods; expression of gene fragments and synthesis of short peptides. This L5-binding region of the 18-kDa protein (amino acids 109 to 115) shows some homology to a region of the GroEL heat shock family of proteins. Characterization of this antibody-binding epitope may lead to a reagent of use in early diagnosis of infection.