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.
Kulkarni
A B
AB
Division of Cell Biology, John Curtin School of Medical Research, Australian National University, Canberra.
Mullbacher
A
A
Blanden
R V
RV
D000818:Animals; D000938:Antigen-Presenting Cells; D000956:Antigens, Viral / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology / Q000528:radiation effects; D000939:Epitopes / Q000276:immunology; D007156:Immunologic Memory; D008213:Lymphocyte Activation; D051379:Mice; D008808:Mice, Inbred CBA; D013047:Specific Pathogen-Free Organisms; D013154:Spleen / Q000166:cytology; D016176:T-Lymphocyte Subsets / Q000276:immunology / Q000528:radiation effects; D006377:T-Lymphocytes, Helper-Inducer / Q000276:immunology*; D014901:West Nile Fever / Q000276:immunology*; D014902:West Nile virus / Q000276:immunology*
D000956:Antigens, Viral; D000939:Epitopes