Early studies showed that preincubation with anti-CD8 antibodies can block conjugate formation between effector and target cells (16) and inhibit CD8+T-cell activation in response to cognate pMHCI presented on the target cell surface (17-20)

Early studies showed that preincubation with anti-CD8 antibodies can block conjugate formation between effector and target cells (16) and inhibit CD8+T-cell activation in response to cognate pMHCI presented on the target cell surface (17-20). D-Luciferin sodium salt heterogeneity in the ability of anti-CD8 antibodies to trigger T-cell effector function provides an explanation for the apparent incongruity observed in previous studies and should be taken into consideration when interpreting results generated with these reagents. Furthermore, the ability of antibody-mediated CD8-engagement to deliver an activation signal underscores the importance of CD8 in CD8+T-cell signalling. Keywords:anti-CD8 antibody, CD8+T-cell activation, pMHCI tetramer, T-cells, surface plasmon resonance == INTRODUCTION == CD8+T-cells MAT1 are essential for the control of viral infection and the natural eradication of cancer. CD8+T-cells recognize short peptides, 8-13 amino acids in length, presented at the target cell surface bound to major histocompatibility complex class I (MHCI) molecules. T-cell antigen recognition is unique in nature as it involves the binding of a single ligand (peptide-MHC) by two receptors (TCR and coreceptor) (1,2). The CD8 glycoprotein, which serves as the coreceptor on MHCI-restricted T-cells, acts to enhance the antigen sensitivity of CD8+T-cells by binding to a largely invariant region of MHCI at a site distinct from the TCR docking platform. CD8 has multiple enhancing effects on early T-cell activation events, including: (i) promotion and stabilization of TCR/pMHCI binding at the cell surface (3-5); (ii) recruitment of essential signalling molecules to the intracellular side of the TCR/CD3/ complex (6-11); and, (iii) localization of TCR/pMHCI complexes within specialized membrane micro-domains that act as potentially privileged sites for initiation of the TCR-mediated signalling cascade (12,13). CD8 binding also controls the level of T-cell crossreactivity (14) and can differentially affect the deployment of CD8+T-cell effector functions (15). Anti-CD8 antibodies have been used widely to investigate the role of CD8 in CD8+T-cell activation. Early studies showed that preincubation with anti-CD8 antibodies can block conjugate formation between effector and target cells (16) and inhibit CD8+T-cell activation in response to cognate pMHCI presented on the target cell surface (17-20). These findings provided key evidence that CD8 was important in the process of CD8+T-cell activation. However, considerable heterogeneity between different CD8+T-cells was apparent in terms of their ability to activate in the presence of anti-CD8 antibodies and, as a result, these reagents were used D-Luciferin sodium salt as tools to classify CD8+T-cells as either CD8-dependent or CD8-independent (21,22). Antibody-mediated ligation of T-cell surface molecules, such as CD2, CD3 and CD28 (23,24), can D-Luciferin sodium salt result in effector function. In contrast, studies of antibody-mediated CD8 ligation in the absence of TCR engagement have yielded conflicting results. D-Luciferin sodium salt Early studies demonstrated that induction of CD8 crosslinking at the cell surface can result in p56lckphosphorylation similar to that seen with anti-CD3 antibodies (25) and elicit downstream effector functions, such as chemokine release (26) and potent cytotoxicity (27). However, in conflict with these data, more recent studies suggest that CD8 ligation alone may actually deliver a negative signal (28,29). To date, a cohesive explanation for these widely disparate findings with anti-CD8 antibodies has remained elusive. Furthermore, there has been no systematic study of the effects of multiple different anti-human CD8 antibodies on CD8+T-cells with different specificities. Here, we report on the ability of a panel of seven monoclonal anti-human CD8 antibodies to induce chemokine/cytokine release and cytotoxicity by six different human CD8+T-cell clones specific for a total of five different pMHCI antigens. The data, supported by parallel observations in a mouse system, reveal that considerable heterogeneity exists in D-Luciferin sodium salt the ability of anti-CD8 antibodies to activate CD8+T-cells. These results elucidate the apparent incongruity that has been observed in previous studies and mandate that the disparate effects of anti-CD8 antibodies are considered in the interpretation of results generated with these reagents. ==.