The construction of a novel A2/pp65 MHC tetramer enabled the design of a rapid and precise flow cytometric method allowing quantitative and qualitative analysis of CMV-specific T-cells. The number of A2/pp65 tetramer binding CTLs in blood may prove to be clinically relevant in assessing the immune response to CMV.
SUMMARYThe availability of tetrameric complexes of HLA class I molecules folded with immunodominant peptides makes it possible to utilize flow cytometry for rapid and highly specific visualization of virus specific CD8 + T cells. An alternate technique is to incubate whole blood with specific antigens and to subsequently detect and characterize responding T cells (e.g. by performing intracellular staining of interferon-gamma). By using an HLA-A2 tetramer construct folded with the same immunodominant CMV-peptide as that used for peptide pulsing, we monitored both the presence and functional capacity of CMV-specific CD8 + T cells. In addition T cell activation was assayed by determination of CD38 and CD69 expression. Twelve organ transplant patients and 31 healthy blood donors with latent CMV infection were investigated using CMV pp65 tetramer staining and intracellular staining of interferongamma after CMV pp65 peptide pulsing or CMV lysate pulsing. CMV-specific T cells were detected in similar absolute numbers as well as frequencies of T cells in the two groups investigated. However, the CMV-specific CD8 + T cells in immunosuppressed individuals showed a decreased functional response to the CMV-peptide, as evidenced by reduced interferon-gamma production when compared to healthy blood donors (19%; 42%, P < 0·005). In addition, CD38 expression was markedly higher in immunosuppressed patients compared to healthy blood donors (24%; 6%, P < 0·005). In a case report we demonstrate that reactivation of CMV can occur in an immunosuppressed patient with high number of CMV-specific T cells, but without functional capacity. Hence, these findings reflect impaired activation of cytotoxic T cells controlling latent CMV infection in immunosuppressed patients.
Ex vivo propagation of graft infiltrating lymphocytes has become a useful method for the examination of the cellular response after allogeneic transplantation. The aim of the present study was to investigate if this method can be used also for isolation of xenograft infiltrating cells, and, if so, to further characterize these cells. The concordant mouse-to-rat heart transplantation model was used for the experiments. Recipient rats were treated either with 15-deoxyspergualin (DSG) or with a combination of DSG and cyclosporine A (CyA) or left untreated. Transplants were removed beating on day 2 (untreated) or day 8 (DSG and CyA + DSG) and biopsies were incubated in culture medium for 48 h, resulting in propagation of cells from the biopsies into culture medium. The propagated cells were counted and phenotyped using flow cytometry. In parallel, the transplants were examined morphologically and immunohistochemically. Infiltrating cells could be isolated from all grafts in all groups. The number of propagated T lymphocytes during cellular rejection (DSG-treatment) was about 3.5 times higher than during 'non-rejection' (CyA + DSG) and six times higher than during acute vascular rejection (untreated). These findings were supported both by the morphological and the immunohistochemical findings. In conclusion, we have shown that graft infiltrating lymphocytes can be isolated from xenogeneic heart transplants by incubation of biopsies for 48 h, resulting in spontaneous propagation of immune cells into culture medium. Propagated cells could be further characterized by flow cytometry. Thus, the technique presented here can be used as a tool for studies of xenogeneic cellular rejection.
AP-PCR is a useful technique for distinguishing the identity of bacterial isolates from patients and blood components. A patient with bacteremia can contaminate a PC in conjunction with a platelet transfusion. With AP-PCR, the PC could be ruled out as the cause of the posttransfusion sepsis.
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