Development of a Humanized HLA-A2.1/DP4 Transgenic Mouse Model and the Use of This Model to Map HLA-DP4-Restricted Epitopes of HBV Envelope Protein

A new homozygous humanized transgenic mouse strain, HLA-A2.1+/+HLA-DP4+/+ hCD4+/+mCD4−/−IAβ−/−β2m−/− (HLA-A2/DP4), was obtained by crossing the previously characterized HLA-A2+/+β2m−/− (A2) mouse and our previously created HLA-DP4+/+ hCD4+/+mCD4−/−IAβ−/− (DP4) mouse. We confirmed that the transgenes (HLA-A2, HLA-DP4, hCD4) inherited from the parental A2 and DP4 mice are functional in the HLA-A2/DP4 mice. After immunizing HLA-A2/DP4 mice with a hepatitis B DNA vaccine, hepatitis B virus-specific antibodies, HLA-A2-restricted and HLA-DP4-restricted responses were observed to be similar to those in naturally infected humans. Therefore, the present study demonstrated that HLA-A2/DP4 transgenic mice can faithfully mimic human cellular responses. Furthermore, we reported four new HLA-DP4-restricted epitopes derived from HBsAg that were identified in both vaccinated HLA-A2/DP4 mice and HLA-DP4-positive human individuals. The HLA-A2/DP4 mouse model is a promising preclinical animal model carrying alleles present to more than a quarter of the human population. This model should facilitate the identification of novel HLA-A2- and HLA-DP4-restricted epitopes and vaccine development as well as the characterization of HLA-DP4-restricted responses against infection in humans

Induction of viral and tumour specific CTL responses using antibody targeted HLA class I peptide complexes

The production of cytotoxic T cells with specificity for cancer cells is a rapidly evolving branch of cancer therapeutics. A variety of approaches aim to amplify anti-tumour cytotoxic T cell responses using purified peptides, tumour cell lysates or recombinant HLA/peptide complexes in differing antigen presenting systems. Using a two-step biotin-streptavidin antibody targeting system, recombinant HLA-class I/peptide complexes were attached to the surface of B cells via the anti-CD20 B9E9-scFvSA antibody-streptavidin fusion protein. Flow cytometry with a conformation dependant monoclonal antibody to HLA class I indicated that targeted HLA-class I/peptide complexes remain on the surface of B cells in culture for periods in excess of 72 h. PBMCs were stimulated in vitro for 8–14 days using the autologous B cells as antigen presenting cells. Following a single cycle of stimulation specific cytotoxic T cell responses to targeted HLA-A2 complexes containing the M1, BMLF1 and Melan A peptides could be demonstrated by tetramer staining and Cr release assays. With the HLA-A2/BMLF1 complex up to 2.99% of CD8+ve cells were tetramer positive producing 20% lysis (E : T 10 : 1) of CIR-A2 target cells in an in vitro cytotoxicity assay compared to baseline levels of 0.09% tetramer +ve and 2% lysis in the unstimulated population. PBMCs from a healthy donor treated with two cycles of stimulations with targeted HLA-A2/Melan A complexes, demonstrated expansion of the melanA tetramer +ve population from 0.03% to 1.4% producing 15% lysis of Melan A pulsed target cells. With further consideration to the key variables of HLA/peptide complex density, the ratio of stimulator to effector cells and optimum cytokine support, this system should offer an easy and effective method for the in vitro amplification of specific cytotoxic T cell responses and warrants development for the in vivo induction of cytotoxic T cell responses in cancer therapy

Epidermal growth factor receptor (<it>EGFR</it>) mutations and expression in squamous cell carcinoma of the esophagus in central Asia

<p>Abstract</p> <p>Background</p> <p>Esophageal squamous cell carcinoma (ESCC) shows geographic variations in incidence, with high incidences (>50/10⁵ person-years) in central Asia, including North Eastern Iran (Golestan) and Northern India (Kashmir). In contrast to Western countries, smoking does not appear to be a significant risk factor for ESCC in central Asia. In lung adenocarcinoma, activating mutations in the gene encoding epidermal growth factor receptor (<it>EGFR</it>) are frequent in tumors of never smokers of Asian origin, predicting therapeutic sensitivity to <it>Egfr</it>-targeting drugs.</p> <p>Methods</p> <p>In this study 152 cases of histologically confirmed ESCC from Iran (Tehran and Golestan Province) and North India (Kashmir Valley) have been analyzed for <it>EGFR</it> mutation by direct sequencing of exons 18–21. <it>Egfr</it> protein expression was evaluated by immunohistochemistry in 34 samples from Tehran and <it>HER2</it> mutations were analyzed in 54 cases from Kashmir.</p> <p>Results</p> <p>A total of 14 (9.2%) <it>EGFR</it> variations were detected, including seven variations in exons. Among those, four (2.6%) were already documented in lung cancers, two were reported as polymorphisms and one was a potentially new activating mutation. All but one variation in introns were previously identified as polymorphisms. Over-expression of <it>Egfr</it> was detected in 22/34 (65%) of tested cases whereas no <it>HER2</it> mutation was found in 54 cases from Kashmir.</p> <p>Conclusion</p> <p>Overall, <it>EGFR</it> mutations appear to be a rare event in ESCC in high incidence areas of central Asia, although a very small proportion of cases may harbor mutations predicting sensitivity to anti-<it>Egfr</it> drugs.</p