Identification of naturally processed HLA-A2--restricted proinsulin epitopes by reverse immunology.

Type 1 diabetes is thought to result from the destruction of beta-cells by autoantigen-specific T-cells. Observations in the NOD mouse model suggest that CD8+ cytotoxic T-cells play an essential role in both the initial triggering of insulitis and its destructive phase. However, little is known about the epitopes derived from human beta-cell autoantigens and presented by HLA class I molecules. We used a novel reverse immunology approach to identify HLA-A2-restricted, naturally processed epitopes derived from proinsulin, an autoantigen likely to play an important role in the pathogenesis of type 1 diabetes. Recombinant human proinsulin was digested with purified proteasome complexes to establish an inventory of potential COOH-terminals of HLA class I-presented epitopes. Cleavage data were then combined with epitope predictions based on the SYFPEITHI and BIMAS algorithms to select 10 candidate epitopes; 7 of these, including 3 with a sequence identical to murine proinsulin, were immunogenic in HLA-A2 transgenic mice. Moreover, six of six tested peptides were processed and presented by proinsulin-expressing cells. These results demonstrate the power of reverse immunology approaches. Moreover, the novel epitopes may be of significant interest in monitoring autoreactive T-cells in type 1 diabetes.

Peptides derived from the onconeural HuD protein can elicit cytotoxic responses in HHD mouse and human.

Anti-Hu syndrome is a paraneoplastic neurologic disease seemingly associated with an efficient antitumoral immune response against HuD protein expressed by both small cell lung cancer (SCLC) and neurons. Since anti-Hu antibodies are not pathogenic, and oligoclonal CD8(+) T cells infiltrate neoplastic and nervous tissues, we examined MHC class I-restricted immunogenicity of human HuD. Among 14 HuD-derived peptides potentially immunogenic in HLA-A*0201 restriction, 10 had actual in vitro binding capacity to the HLA molecule, 8 elicited specific cytotoxic T lymphocytes (CTLs) in a humanized murine model after peptidic vaccination, 2 also elicited specific CTLs in healthy humans, and 1 was naturally processed and presented to the immune system.

Role of T cell help and endoplasmic reticulum targeting in protective CTL response against influenza virus.

We report on the induction of primary and long-term memory cytotoxic T lymphocyte (CTL) responses against the nucleoprotein of the influenza virus A/PR8/34 in mice immunized with plasmid DNA targeted to B lymphocytes in the spleen. We found that the magnitude of the CTL response and the size of the pool of memory CTL was greater when the CTL response was induced in presence of T cell help. Interestingly, immunization with a signal sequence-competent transgene was markedly superior to immunization with a transgene lacking the endoplasmic reticulum (ER) targeting sequence, in inducing CTL. We also found a correlation between in vivo protection from lethal virus challenge and (1) the availability of T cell help and (2) ER targeting. Immunization of dendritic cell-deficient mice suggests that B lymphocytes function as antigen-presenting cells in this model of immunization. Collectively, the results suggest that somatic transgene immunization is a conceptually new approach to induce effective anti-viral CTL responses and to assess the parameters critical for long-lasting and protective CTL responses in vivo.

Identification of a human telomerase reverse transcriptase peptide of low affinity for HLA A2.1 that induces cytotoxic T lymphocytes and mediates lysis of tumor cells.

Telomerase reverse transcriptase (TRT) is a tumor-associated antigen expressed in the vast majority of human tumors and is presently one of the most promising target candidates for a therapeutic cancer vaccine. TRT is also expressed at low level in selected tissues and should be considered a self antigen. In the present study we sought to develop cytotoxic T lymphocytes (CTL) responses directed against human (h)TRT peptides with low relative affinity for which the available repertoire is to be preferentially spared from tolerance. This was accomplished by using analogue peptides of hTRT whose relative affinity for the MHC was increased by a targeted (-->Tyr) substitution in position one. By immunizing HLA A2.1 transgenic mice with these analogue peptides, we identified one such low relative affinity peptide (p572) that is endogenously processed and presented by HLA A2.1 in tumor cells, and is recognized by specific CTL. We used the highly immunogenic analogue peptide to successfully induce TRT-specific CTL in cancer patients and normal donors. CTL against p572-lysed human and mouse tumor cells but not activated autologous B cells. This peptide represents, therefore, an important candidate component of a cancer vaccine based on a TRT substrate and validates the strategy of targeting peptides with low affinity for the MHC for cancer immunotherapy.

Comparative analysis of the CD8(+) T cell repertoires of H-2 class I wild-type/HLA-A2.1 and H-2 class I knockout/HLA-A2.1 transgenic mice.

HHD transgenic mice which express HLA-A2.1 monochain molecules in a H-2 class I(-) context have an improved capacity to develop HLA-A2.1-restricted cytotoxic T lymphocyte (CTL) responses as compared with classical A2.1/K(b) transgenic mice, which express heterodimeric HLA-A2.1 molecules in a H-2 class I wild-type context. A detailed TCR analysis of HLA-A2.1-restricted CD8(+) T cells educated and mobilized in both strains of mice was undertaken. Focusing on TCR beta chains, comparative PCR analysis of naive and immune CD8(+) T cell repertoires were performed. In spite of lower cell surface expression of HLA class I molecules and lower overall number of CD8(+) T cells, HHD mice educate a qualitatively normally diversified CD8(+) T cell repertoire and mobilize a larger variety of CD8(+) T cells in response to HLA-A2.1-restricted antigens compared with A2.1/K(b) mice. These observations provide the molecular bases accounting for the fact that HHD mice represent the most versatile animal model currently available for preclinical studies of HLA-A2.1-restricted CTL responses.

Use of a lentiviral flap vector for induction of CTL immunity against melanoma. Perspectives for immunotherapy.

BACKGROUND: A central triple-stranded DNA structure created during HIV-1 reverse transcription, the central flap, acts as a cis-active nuclear import determinant of the HIV-1 DNA genome. Insertion of the sequences responsible for formation of the central DNA flap into an HIV-1-derived vector strongly enhances its transduction efficiency. METHODS: HIV-1 vectors with or without inclusion of the DNA flap and encoding the same melanoma polyepitope were constructed to transduce dendritic cells (DCs) and to evaluate their capacity for induction of melanoma-specific cytotoxic T-lymphocyte (CTL) responses ex vivo and in vivo. RESULTS: HIV-1 vectors including the DNA flap transduced up to 100% of immature mouse and human DCs. Inoculation of HLA-A2.1 transgenic mice with this flap vector elicited vigorous and multi-specific long-term anti-melanoma CTL responses, whereas the parental vector lacking the flap sequence was less efficient. Furthermore, human DCs transduced ex vivo with the recombinant DNA flap vector displayed efficient multi-specific primary human CTL responses against melanoma. CONCLUSION: Lentiviral vectors including the DNA flap should be powerful tools both for active immunization and for the ex vivo priming of CTL for tumor immunotherapy.