Cellular responses to cytomegalovirus in immunosuppressed patients: circulating CD8+ T cells recognizing CMVpp65 are present but display functional impairment.

The 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 interferon-gamma 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.

Characterization of CMVpp65-specific CD8+ T lymphocytes using MHC tetramers in kidney transplant patients and healthy participants.

BACKGROUND: Cytomegalovirus (CMV) is a ubiquitous herpesvirus that infects 50-90% of individuals in different populations. After primary infection, the virus persists latently in myeloid cells under the control of specific T-cells. Reactivation of CMV infection may cause lethal organ dysfunction and is frequently seen in immunosuppressed individuals. CD8+ cytotoxic T-cells (CTL) have a primary role in suppressing CMV reactivation, and the dominating CTL response is directed against pp65. METHODS: MHC tetramers, that is, complexes between HLA class I (or class II) molecules and antigenic peptides conjugated to fluorochromes allow the direct visualization of antigen-specific receptor-carrying T-cells using flow cytometry. We constructed a novel MHC tetramer for identification of CMVpp65-specific CD8+ T-cells using HLA-A2 molecules folded with the immunodominant NLVPMVATV peptide. RESULTS: The A2/pp65 tetramer specifically stained CMV-directed T-cell lines, and sorted cells showed CMV-specific cytotoxicity. High proportions (0.1-9%) of the CD8+ T-cells were A2/pp65 tetramer+ in healthy HLA-A2+ CMV carriers and in immunosuppressed kidney transplant patients with latent infection. Patients with reactivated CMV infection exhibited up to 15% A2/pp65 tetramer+ cells, which seemed to correlate with CMV load over time. A2/pp65 tetramer+ cells expressed T-cell activation markers. CONCLUSIONS: 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.

Ex vivo propagation and characterization of lymphocytes from rejecting rat-kidney allografts.

Today, most clinically used methods for analysis of alloreactivity in organ transplantation are based on humoral immunity. In order to study the cellular alloresponse, a rat kidney transplantation model with culturing of graft infiltrating lymphocytes was developed. Kidney transplantations between inbred rat strains were performed with the animals initially immunosuppressed with cyclosporine. In order to initiate acute cellular rejection, immunosuppression was withdrawn after 10 days. Infiltrating lymphocytes were analysed using an in vitro culture system, allowing cells to propagate from the biopsies to culture medium. The propagated cells were counted and analysed for subtype activation markers and donor-specificity using flow cytometry and a proliferation assay. Syngeneically transplanted animals and animals given constant immunosuppression upon transplantation were used as controls. During rejection, significantly more T lymphocytes were propagating from the biopsies compared to controls. A higher percentage of the propagated T lymphocytes in the rejection group expressed activation markers [CD25 and major histocompatibility complex (MHC) class II antigen] compared to spleen- and peripheral blood T lymphocytes from the same individuals. Propagated mononuclear cells from biopsies in the rejection group were proliferating and showed donor-specific reactivity whereas mononuclear spleen cells from animals in the same group did not show this donor specificity. In conclusion, we have presented a rat kidney allotransplantation model with in vitro propagation of graft-infiltrating, activated and donor-specific T lymphocytes. This technique offers a possibility to study cellular reactivity in allotransplantation.

Retrograde transmission of Proteus mirabilis during platelet transfusion and the use of arbitrarily primed polymerase chain reaction for bacteria typing in suspected cases of transfusion transmission of infection.

BACKGROUND: When bacteria are found, after a platelet transfusion, in the recipient's blood as well as in the platelet concentrate (PC), a causal relationship is normally suspected, with the PC as the causative agent. The other alternative, that the patient has bacteremia and contaminated the PC, is less well documented in the literature. CASE REPORT: Arbitrarily primed polymerase chain reaction (AP-PCR) was used for testing strains of Proteus mirabilis isolated from a patient's blood before and after a platelet transfusion and from the PC. Because of a febrile reaction after a platelet transfusion, bacterial culture was performed on the PC used, showing growth of P. mirabilis. The same species was found in the patient's blood after the transfusion. Posttransfusion sepsis caused by a contaminated PC was suspected, and anti-sepsis treatment was given to the recipient. Later, it became apparent that the patient had had bacteremia before the transfusion and that P. mirabilis was one of the species in the isolate. With AP-PCR, the identity of the three P. mirabilis isolates could be distinguished. CONCLUSION: 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.