sq-23377 and Epstein-Barr-Virus-Infections

SLAM-associated protein (SAP) is an adaptor molecule that facilitates critical effector functions in immune cells, and its deficiency causes X-linked lymphoproliferative disease type 1 in which effector responses directed against EBV are severely compromised. The primary objective of this study was to phenotypically and functionally characterize a rare, CD8 T cell-restricted bimodal SAP expression pattern observed in healthy, human donors with the widely used 1C9-SAP mAb clone. We initially observed this pattern during the clinical validation of our flow cytometry-based assay to diagnose X-linked lymphoproliferative disease type 1 in our laboratory. For this validation study, we used multiparameter flow cytometry to identify cytosolic SAP expression in lymphocyte subsets, and CD8 T cells from the donors displaying the rare SAP expression pattern mentioned above were separately further evaluated by intracellular cytokine and CD107a staining to examine polyfunctionality following PMA/ionomycin and HLA class I allele-restricted EBV peptide epitope-induced T cell activation. Our data revealed that SAP 1C9-hi CD8 T cells clearly displayed higher polyfunctional responses versus SAP 1C9-lo CD8 T cells following PMA/ionomycin stimulation. Furthermore, polyfunctional EBV-specific CD8 T cell responses segregated with the SAP 1C9-hi CD8 T cells and not the SAP 1C9-lo CD8 T cells. Additionally, and rather intriguingly, short- and long-term T cell stimulation selectively diminished the signal for the 1C9-hi subset. Overall, our data suggest that although rare, this unique SAP expression pattern merits further evaluation as it has the potential to provide some insight into fundamental processes as they might relate to host-pathogen dynamics.

Topics: Adult; Antibodies, Monoclonal, Murine-Derived; Blood Donors; CD8-Positive T-Lymphocytes; Cells, Cultured; Epitopes, T-Lymphocyte; Epstein-Barr Virus Infections; Female; Flow Cytometry; Herpesvirus 4, Human; Host-Pathogen Interactions; Humans; Immunoglobulin G; Ionomycin; Lymphocyte Activation; Male; Phenotype; Signal Transduction; Signaling Lymphocytic Activation Molecule Associated Protein; Tetradecanoylphorbol Acetate

In vitro stimulation of peripheral blood lymphocytes (PBL) from healthy Epstein-Barr Virus (EBV) seropositive individuals with autologous lymphoblastoid cell lines (LCL) gives rise to CD4+ and CD8+ T cells both of which are cytotoxic for autologous lymphoblastoid cells. Activated EBV-specific CD4+ T cells are cytotoxic towards autologous LCL but, paradoxically, CD4+ T cells have also been shown to enhance tumour formation in SCID/Hu mice. Here, we show that despite being cytotoxic, CD4+ T-cell lines from different donors show considerable variation in their ability to inhibit the long-term growth of autologous LCLs in vitro. Following re-stimulation in vitro with PMA and ionomycin, CD4+ T cells produced IFNgamma, TNFalpha, TNFbeta, IL-2, IL-4, IL-10 and IL-13. TNFalpha, TNFbeta and IL-10 production were also detected in LCL. IL-6 was only detected in trace amounts in either cell type. The ratio of IFNgamma to IL-4 production varied between the CD4+ T-cell lines, indicating differences in the Th1/Th2 balance of the response. When CD4+ T cells were re-stimulated using autologous LCL as antigen-presenting cells, they produced more IL-4 and less IFNgamma or IL-13 when compared with cells re-stimulated by phorbol myristate acetate (PMA) and ionomycin. Using two colour cytokine staining, we showed that many individual CD4+ T cells produced IFNgamma along with either IL-4 or IL-13. Purified CD4+ T cells completely inhibited the outgrowth of autologous LCL in five out of nine cases, and partially inhibited outgrowth in the remaining four. There was no correlation between the pattern of CD4+ T-cell cytokine production and the capacity to inhibit outgrowth of autologous LCL. The killing of LCLs was contact-dependant and not mediated by soluble factors. We conclude that the ability of CD4+ T cells to inhibit autologous LCL growth is not directly related to T-helper cell cytokine production, but may depend on cytoxicity through surface ligands such as CD95L (FasL) and TNFalpha-related apoptosis-inducing ligand (TRAIL).

Topics: Antibodies, Viral; CD4-Positive T-Lymphocytes; Cell Division; Cell Line; Cell Line, Transformed; Cytokines; Epstein-Barr Virus Infections; Herpesvirus 4, Human; Humans; Ionomycin; T-Lymphocytes, Cytotoxic; Tetradecanoylphorbol Acetate; Tissue Donors