The peptide-binding properties of the nonclassical major histocompatibility complex (MHC) class 1b molecule Qa-1 were investigated using a transfected hybrid molecule composed of the alpha 1 and alpha 2 domains of Qa-1b and the alpha 3 domain of H-2Db. This allowed the use of a monoclonal antibody directed against H-2Db whilst retaining the peptide-binding groove of Qa-1b. By comparison with classical MHC class I molecules, intracellular maturation of the chimeric molecule was inefficient with weak intracellular association with beta 2-microglobulin. However, at the cell surface the hybrid molecules were stably associated with beta 2-microglobulin and were recognized by cytotoxic T lymphocyte (CTL) clones specific for the Qa-1b-presented peptide Qdm (AMAPRTLLL). A whole-cell binding assay was used to determine which residues of Qdm were important for binding to Qa-1b and CTL clones served to identify residues important for T cell recognition. Substitutions at position 1 and 5 did not reduce the efficiency of binding and had little effect on CTL recognition. In contrast, substitutions at position 9 resulted in loss of MHC class I binding. Mass spectrometric analysis of peptides eluted from immunopurified Qa-1b/Db molecules indicated that Qdm was the dominant peptide. The closely related peptide, AMVPRTLLL, which is derived from the signal sequence of H-2Dk, was also present, although it was considerably less abundant. The mass profile suggested the presence of additional peptides the majority of which consisted of eight to ten amino acid residues. Finally, the finding that a peptide derived from Klebsiella pneumoniae can bind raises the possibility that this non-classical MHC class I molecule may play a role in the presentation of peptides of microorganisms.

Immunization of mice with a recombinant vaccinia virus expressing the human papillomavirus type 16 (HPV-16) E6 gene elicits specific antibody, proliferative and cytotoxic T lymphocyte responses. T and B cell epitopes were mapped by using synthetic peptides. This study provides the background to future investigation aimed at developing prophylactic and therapeutic vaccines against HPV-16 infection and cervical cancer.

The transforming protein E7 of human papilloma virus type 16 can stimulate cytotoxic T lymphocytes (CTL) which can protect experimental animals against growth of E7 expressing tumour cells. In this study we compared CTL responses in mice immunized with either E7 protein in MF59 adjuvant or with recombinant vaccinia virus expressing E7 (Vac-E7). We have chosen H-2d mice because no E7-specific CTL responses have been described in this MHC haplotype. Immunization of these mice with Vac-E7 generated CTL which lysed target cells infected with Vac-E7 or transfected with the E7 gene. CTL from mice immunized with E7 protein in MF59 adjuvant showed specificity for the same target cells. Antibody blocking experiments revealed that both immunization with Vac-E7 and E7 protein stimulated CD8+ effector CTL. The find specificity of CTL induced by the two immunization protocols was similar. A major CTL epitope was mapped to the carboxyl terminal amino acids 48-98 of the E7 protein. Peptide isolation from E7 expressing cells followed by HPLC separation indicated that CTL induced by immunization with protein and Vac-E7 recognized the same HPLC purified peptide fractions. Together, the study suggests that vaccines based on protein can activate CTL with similar fine specificity to CTL induced by vaccines based on recombinant vaccinia virus.

CD8+ T cells have been implicated in protective anti-tuberculous immune responses, but little is known about the identity of mycobacterial antigens recognized by CD8+ T cells. In this study we identified the Mycobacterium tuberculosis 38 kDa protein as a target for murine CD8+ cytotoxic T lymphocytes (CTL) which were induced by vaccination of C57BL/6 mice with DNA delivered with a plasmid, with transfected tumour cells or by infection with tubercle bacilli. Using overlapping synthetic peptides covering the whole protein sequence, peptides predicted to contain H-2Kb or H-2Db motifs, as well as naturally processed peptides, we were able to identify CTL epitopes. Differences were demonstrated in peptide specificity between CTL from immunized or M. tuberculosis-infected mice. The identified CTL epitopes could be important for future analysis of the involvement of CD8+ T cells in M. tuberculosis infections and for vaccine development.

CD68, a haematopoietic differentiation marker of the monocyte-macrophage lineage, is expressed in various human malignancies including chronic and acute myeloid leukaemia (AML). While the majority of normal CD34 + cells are negative for CD68 expression, CD34 + cells from AML patients produce elevated amounts of this protein. The purpose of this study was to identify CTL epitopes in the human CD68 protein. Mouse CD68 was also analysed to search for epitopes that could be used in murine tumor model. Peptides binding to murine H2 b class I molecules were identified and used to stimulate CTL responses from allogeneic donor mice to avoid immunological tolerance. High avidity CTL clones specific for three different peptide epitopes did not kill CD68-expressing murine target cells, indicating that endogenous antigen processing failed to produce sufficient amounts of these peptides. In contrast, allorestricted human CTL specific for an HLA-A2-binding peptide of CD68 recognised not only picomolar concentrations of peptide, but also displayed low levels of killing against HLA-A2-positive K562 and THP-1 leukemia cell lines and blast cells from AML patients. These data suggest that human leukaemia cells express limited amounts of CD68-derived peptides, and that high avidity CTL capable of recognising sub-picomolar concentrations of peptides are required for efficient killing of leukaemia cells. Leukemia (2002) IntroductionThe success of tumour immunotherapy is largely dependent on the choice of target antigen. Tumour-associated antigens, that are targeted by various immunotherapy strategies fall into three categories: viral antigens, products of mutated genes and normal cellular proteins. The latter category, which comprises differentiation antigens, has been extensively exploited for the treatment of solid tumours, such as melanoma and prostate cancer. 1 Self-tolerance to differentiation antigens expressed in non-haematopoietic cells is probably not absolute, providing an explanation for the detection of CTL against these antigens in tumour patients. The use of haemopoietic differentiation antigens, however attractive it may appear for the purposes of targeting cellular immune responses against leukaemic blast cells, is limited by potent tolerogenic effects of haemopoietic cells. Hence, it is likely that autologous T cells specific for haemopoietic differentiation antigens have been deleted in the thymus or rendered unresponsive by peripheral tolerance mechanisms.In the past few years, we have developed a strategy to circumvent tolerance to self proteins. 2 The allo-restricted strategy is based on the observation that T cell tolerance is self-MHC restricted. Hence, T cells from A2-negative donor individuals can mount CTL responses against epitopes from self-proteins presented by A2 class I molecules. Using this strategy, we have previously identified CTL epitopes in the self-proteins mdm2 and cyclin D1, and shown that the CTL selectively killed tumour cells expressing large quantities of these proteins but not normal ce...