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Abstract: Mice were immunized twice with a pool of five peptides selected among twenty 8–9-mer peptides for their ability to form stable complexes at 37°C with recombinant H-2Kb (half-lives 10–15h). Vaccine-induced immunity of splenic CD8+ T cells was studied in a 24h IFNγ Elispot assay. Surprisingly, IFNγ spot-formation was observed without addition of peptide to the assay culture at 3 weeks and 3 months after immunization. To clarify if IFNγ spot formation in the absence of peptide exposure ex vivo is caused by the peptide-pool per se, mice were immunized with single peptides. Three of the five peptides induced normal peptide immunity i.e. the specific T cell reactivity in the Elispot culture was strictly dependent on exposure to the immunizing peptide ex vivo. However, immunization with two of the peptides, a VSV- and a Mycobacterium-derived peptide, resulted in IFNγ spot formation without peptide in the Elispot culture. Immunization with a mixture of the VSV-peptide and a “normal” peptide also resulted in IFNγ spot formation without addition of peptide to the assay culture. Peptide-tetramer staining of CD8+ T cells from mice immunized with a mixture of VSV-peptide and “normal” peptide showed peptide specific binding by CD8+ T cells for both of the peptides. Thus, although immunization with certain peptides alone or in a mixture of peptides may result in IFNγ spot formation without peptide in the assay culture, specific immunity against the individual immunizing peptide in the mixture remains intact. Our data suggest that certain peptides exhibit sustained immunogenicity in vivo for prolonged periods of time. [Copyright &y& Elsevier]

In fully HLA-matched allogeneic hematopoietic cell transplantation (HCT), the main mechanism of the beneficial graft-versus-tumor (GVT) effect and of detrimental graft-versus-host disease (GVHD) is believed to be caused by donor cytotoxic T cells directed against disparate recipient minor histocompatibility antigens (miHAs). The most common origin of disparate miHAs is nonsynonymous single nucleotide polymorphism (nsSNP) differences between donors and patients. To date, only some 30 miHAs have been identified and registered, but considering the many different HLA types in the human population, as well as all the possible nsSNP differences between any 2 individuals, it is likely that many miHAs have yet to be discovered. The objective of the current study was to predict novel HLA-A– and HLA-B–restricted miHAs in a cohort of patients treated with nonmyeloablative conditioning allogeneic HCT (matched related donor, n = 70; matched unrelated donor, n = 56) for a hematologic malignancy. Initially, the cohort was genotyped for 53 nsSNPs in 11 known miHA source proteins. Twenty-three nsSNPs within 6 miHA source proteins showed variation in the graft-versus-host (GVH) direction. No correlation between the number of disparate nsSNPs and clinical outcome was seen. Next, miHAs in the GVH direction were predicted for each patient–donor pair. Using the NetMHCpan predictor, we identified peptides encompassing an nsSNP variant uniquely expressed by the patient and with predicted binding to any of the HLA-A or -B molecules expressed by the patient and donor. Patients with more than the median of 3 predicted miHAs had a significantly lower 5-year overall survival (42% vs 70%, P = .0060; adjusted hazard ratio [HR], 2.6, P = .0047) and significantly higher treatment-related mortality (39% vs 10%, P = .0094; adjusted HR, 4.6, P = .0038). No association between the number of predicted miHAs and any other clinical outcome parameters was observed. Collectively, our data suggest that the clinical outcome of HCT is affected not by disparate nsSNPs per se, but rather by the HLA-restricted presentation and recognition of peptides encompassing these. Our data also suggest that 6 of the 11 proteins included in the current study could contain more miHAs yet to be identified, and that the presence of multiple miHAs confers a higher risk of mortality after nonmyeloablative conditioning HCT. Furthermore, our data suggest a possible role for in silico based miHA predictions in donor selection as well as in selecting candidate miHAs for further evaluation in in vitro and in vivo experiments. [Copyright &y& Elsevier]

Abstract: Development of methods for efficient in vitro stimulation and expansion of peptide specific CD8+ T cells is compelling not only with respect to adoptive T cell therapy but also regarding analysis of T cell responses and search for new immunogenic peptides. In the present study, a new approach to in vitro T cell stimulation was investigated. By use of an antigenic peptide derived from the cytomegalovirus (CMVp) we tested the stimulatory efficacy of recombinant plate bound MHC molecules (PB-MHC), being immobilized in culture plates. A single stimulation of non-adherent peripheral blood mononuclear cells (NA-PBMCs) with PB-MHC/CMVp resulted in significant expansion of CMVp specific CD8+ T cells, which was comparable to that achieved by CMVp pulsed mature dendritic cells (DCs). By repeated exposure of NA-PBMCs to PB-MHC/CMVp more than 60% CMVp specific CD8+ T cells, representing a 240-fold expansion, were reached after only two stimulations. Although stimulation with PB-MHC/CMVp clearly demonstrated efficient peptide specific expansion of CD8+ T cells, there was a tendency to proliferative exhaustion of the cells after 3–4 stimulations. Thus, it will be of interest to examine the effect of new stimulatory cocktails, e.g. cytokines and co-stimulatory molecules, by use of the present rapid and easy-to-use method of expanding peptide specific T cells. [Copyright &y& Elsevier]

Abstract: The purpose of the present study is to perform a global screening for new immunogenic HLA class I (HLA-I) restricted cytotoxic T cell (CTL) epitopes of potential utility as candidates of influenza A-virus diagnostics and vaccines. We used predictions of antigen processing and presentation, the latter encompassing 12 different HLA class I supertypes with >99% population coverage, and searched for conserved epitopes from available influenza A viral protein sequences. Peptides corresponding to 167 predicted peptide–HLA-I interactions were synthesized, tested for peptide–HLA-I interactions in a biochemical assay and for influenza-specific, HLA-I-restricted CTL responses in an IFN-γ ELISPOT assay. Eighty-nine peptides could be confirmed as HLA-I binders, and 13 could be confirmed as CTL targets. The 13 epitopes, are highly conserved among human influenza A pathogens, and all of these epitopes are present in the emerging bird flu isolates. Our study demonstrates that present technology enables a fast global screening for T cell immune epitopes of potential diagnostics and vaccine interest. This technology includes immuno-bioinformatics predictors with the capacity to perform fast genome-, pathogen-, and HLA-wide searches for immune targets. To exploit this new potential, a coordinated international effort to analyze the precious source of information represented by rare patients, such as the current victims of bird flu, would be essential. [Copyright &y& Elsevier]