In vitro T-cell proliferative response to the flavivirus, west Nile

West Nile virus (WNV)-specific murine T-cell proliferation in vitro was investigated in terms of conditions that optimize antigen-specific responses and reduce background proliferation. The responder populations consisted of splenocytes from WNV-primed mice enriched for L3T4+ T cells. Ia+ antigen-presenting cells (APC) were derived from splenocytes of WNV-primed or naive mice. Antigen was a lysate prepared from WNV-infected Vero cells at 12 h postinfection. Strong virus-specific proliferative responses were observed when antigen-pulsed APC were cocultured with responders at a 1:1 ratio. Substantial nonspecific proliferation occurred when culture medium supplemented with 5% fetal bovine serum (FBS) was used, whereas with 1% normal mouse serum a higher degree of antigen specificity was evident, although the magnitude of the responses was lower. The best separation between antigen-specific and background proliferation was obtained by using an exogenous source of T-cell growth factors to amplify for 2 days the proliferation of L3T4+ cells triggered by an initial 3 days of culture with antigen-pulsed APC. This investigation has defined optimal conditions for investigating the stimulation of WNV-primed L3T4+ T-cell proliferation in response to the presentation of viral gene products by Ia+ APC. This assay should permit detailed analysis of the efficiency of various APC populations and identification of viral antigens that stimulate the proliferation of Class II MHC-restricted T cells.