(536f) Function-Based Screening of HIV-Specific CD8 T Cells Using Arrays of Subnanoliter Wells

Virus-specific Cytotoxic T Lymphocytes (CTLs) play an important role in restricting the replication of persistent viruses like the Human Immunodeficiency Virus (HIV). The restriction is, however, only transient for most individuals and fails to afford long-term, durable protection. The reasons for the failure remain poorly understood. We present a high-throughput approach to monitor effector-mediated lysis of peptide-loaded targets by pairing both target cells and effector CTLs in arrays of subnanoliter wells. By employing this assay, we were able to screen clinical samples of HIV patients for cytolytic CD8 T cells and isolate those cells exclusively based on their lytic functionality. Functional studies on clonal lines established from these cells indicate that the lytic functionality is preserved and that these cells are able to inhibit viral replication in vitro. Additionally, we have used the assay to probe the correlation between CD8 T cells from HIV patients cells primed ex vivo for immediate cytotoxicity and short-term interferon gamma secretion?a common surrogate marker used for evaluating the efficacy of vaccine candidates. Lastly, the screening and isolation of HIV-specific CD8 T cells from tissue biopsies (colon, duodenum) based on their profiles of cytokine secretion will be discussed.