Soluble recombinant CMVpp65 spanning multiple HLA alleles for reconstitution of antiviral CD4+ and CD8+ T-cell responses after allogeneic stem cell transplantation.
ABSTRACT The reactivation of the human cytomegalovirus (CMV) can be prevented or controlled by the adoptive transfer of ex vivo expanded donor-derived CMV-specific T lymphocytes. Several methods for expansion and adoptive transfer of CMV-specific T cells have been developed using either defined CMV peptides or peptide pools for antigen-specific T-cell stimulation. The majority of studies have focused on the lower matrix protein (pp65) and the immediate-early protein-1 (IE-1) of CMV as immunodominant targets. We investigated the behavior of secretory CMVpp65 (sCMVpp65) with respect to its capacity to stimulate pp65-specific T cells independently of human lymphocyte antigen (HLA) type and even in donors unresponsive to the immunodominant HLA-A*0201-restricted CMVpp65495-503 peptide. To facilitate the eukaryotic expression and isolation procedures, we constructed an HLA-A*0201/CMVpp65 fusion protein that is secreted into the supernatant of human embryonic kidney 293 (HEK293) cells. CMV-specific CD4 and CD8 T cells generated by culturing unfractionated peripheral blood mononuclear cells in the presence of recombinant sCMVpp65 did not differ in function with regard to cytotoxicity and interferon-gamma (IFN-gamma) production compared with cytotoxic T cells induced using the well-studied HLA-A*0201-restricted CMVpp65495-503 peptide. We demonstrated that polyclonal CMV-specific T cells could be generated from CMV-seropositive individuals expressing HLA alleles for which no immunogenic epitopes have been identified so far. The production of recombinant sCMVpp65 can easily be adapted to good manufacturing practice conditions and can be used to generate large numbers of immunogenic pathogen-derived proteins for therapeutic applications.
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ABSTRACT: Matching of human leukocyte antigen (HLA) alleles between donors and recipients plays a major role in hematopoietic stem cell transplantation (HSCT). Null or questionably expressed HLA allelic variants are a major issue in HLA matching, because the aberrant expression of such alleles can have a major impact on the outcome of HSCT and/or its complications such as graft-versus-host disease. The goal of this study was to investigate the potential of a recently developed cytokine-induced secretion assay to differentiate the expression levels of HLA-A*32:11Q (questionable) into a null (N) or low (L) expression variant. An amino acid mutation at position 164 of HLA-A*32:11Q disrupts the disulfide bridge in the α2 domain. HLA-A*32:11Q is not detectable by standard microlymphocytotoxicity assay. To this end, we cloned soluble HLA-A*32:11Q and a reference allele (HLA-A*32:01) into expression vectors and transfected/transduced HEK293 and K562 cells. Allele-expressing K562 cells were simultaneously transfected/transduced with a β2-microglobulin (B2M)-encoding vector to ensure the intact HLA structure with B2M. After treatment with proinflammatory cytokines, secreted soluble HLA molecules were determined by enzyme-linked immunosorbent assay in the supernatant and intracellular accumulation of the recombinant proteins by flow cytometry. HLA-A*32:11Q was nearly undetectable in untreated transfectants. Cytokine treatment increased the secretion of HLA-A*32:11Q to detectable levels and resulted in intracellular accumulation of the allele. There was no difference in mRNA transcription between the A*32 alleles. On the basis of these results, we recommend reclassification of HLA-A*32:11Q as a low expression (L) variant.Tissue Antigens 05/2012; 79(5):340-50. · 2.35 Impact Factor
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ABSTRACT: The glycoprotein CD86 is an important costimulatory molecule that has been shown to be predominantly expressed on APCs, such as dendritic cells, macrophages, and B cells. More recently, CD86 was also detected on T cells in specific pathological conditions. The mechanisms of how CD86 might be induced and its functional role in T cells are not well understood. In the present study, we showed that treatment with IL-2 markedly upregulated CD86, but not CD80, in human CD4(+) and CD8(+) T cells. This upregulation occurred in the absence of bystander cells, and isolated naive CD4(+) or CD8(+) T cells exhibited different time-dependent CD86-expression patterns in response to IL-2. Upregulation of CD86 on activated T cells was reduced by Abs that block IL-2 and IL-2Rα (CD25), indicating a receptor-mediated mechanism. IL-2-dependent CD86 upregulation was blocked by pharmacological inhibitors of the NFAT and mammalian target of rapamycin pathways and was largely reduced by simultaneous exposure to IFN-α. Importantly, a marked increase in CD86 on T cells was also observed in vivo in IL-2-treated patients. In conclusion, IL-2 upregulates CD86 expression on human CD4(+) and CD8(+) T cells via a receptor-dependent mechanism that involves the NFAT and mammalian target of rapamycin pathways.The Journal of Immunology 02/2012; 188(4):1620-9. · 5.36 Impact Factor
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ABSTRACT: Adoptive immunotherapy with donor-derived antiviral T cells can prevent viral complications such as with cytomegalovirus (CMV) and Epstein-Barr virus (EBV) . In this context accurate monitoring of cellular immunity is essential and requires suitable quantitative and qualitative assays for high-throughput screening. We comparatively analyzed 57 HLA-typed healthy donors for memory T-cell responses to CMV- and EBV-derived proteins, peptide pools and single HLA-restricted peptides by five commonly used immunoassays in parallel: enzyme-linked immunospot (ELISPOT), cytokine secretion assay (CSA), intracellular cytokine staining (ICS), enzyme-linked immunosorbent assay (ELISA) and pMHC multimer staining. T-cell responses varied greatly between the different target antigens in the investigated assays. IFN-γ ELISPOT consistently detected the highest T-cell response levels against CMV and EBV. CMV-specific T cells were detected in 100% of CMV-seropositive donors tested using CMVpp65 and/or overlapping CMVpp65 peptide pool. CMV-specific T cells in HLA-A*02:01-positive /CMV-seropositive donors were identified directly by HLA-A02/CMVpp65 (A02pp65) multimer staining and, after short in vitro stimulation with HLA-A*02:01-restricted pp65 peptide, by ELISPOT, ELISA, ICS and CSA. A peptide-specific T-cell response was detected in only 4 HLA-A*02:01-positive donors (50%). Despite A02pp65 peptide negativity, T-cell responses to CMVpp65 protein and/or overlapping peptide pool were detected. Comparing the specific immune response against EBV antigens in healthy donors overall, BZLF1-specific T cells (<92.9% peptides, <56.3% peptide pool) were more frequent than EBNA-specifi8c T cells (<64.3% peptides, <46.9% peptide pool) with higher percentage of positive findings for single HLA-restricted EBV peptides. T-cell response against HLA-B*08 peptide epitopes was predominant (multimer staining: EBNA3A: 9/14 and BZLF1: 7/14, IFN-γ ELISPOT: EBNA3A: 13/14 and BZLF1: 11/14). The fact that responses to EBV-specific antigens were not detected in every single EBV-seropositive donor as well as that the T-cell frequencies in response to the investigated EBV antigens differed strongly in the donor cohort indicates that these epitopes are less immunodominant than CMVpp65. Taken together, precise monitoring of T-cell immunity against infectious agents in potential T-cell donors and post-transplant recipients requires individual selection of antigens and immunoassays for the efficient detection and generation of clinically relevant T cells. Due to its lower detection limit and direct visualization of each IFN-γ-secreting cell we identified ELISPOT analysis to be preferable for high-throughput pre-screening. CSA was found to be advantageous for a more detailed analysis of antigen-specific T-cell subsets.Journal of Immunological Methods 05/2014; · 2.01 Impact Factor