DETECTION OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE 1-SPECIFIC MEMORY CYTOTOXIC T-LYMPHOCYTES IN FRESHLY DONATED AND FROZEN-THAWED PERIPHERAL-BLOOD MONONUCLEAR-CELLS

Loss of anti-human immunodeficiency virus type 1 (HIV-1) memory cytotoxic T-lymphocyte (CTLm) responses is associated with disease progression in HIV-1 infection. In this study, nonspecific stimulation of peripheral blood mononuclear cells (PBMC) from HIV-1-infected homosexual men with anti-CIM monoclonal antibody (MAb) was compared with antigen-specific stimulation with inactivated, autologous B lymphoblastoid cells (B-LCL) infected with a vaccinia virus vector encoding HIV-1 IIIb Gag, Pol, and Env (VV-GPE) for activation of HIV-1-specific CTLm responses in a bulk lysis assay and by precursor frequency analysis. The results show that VV-GPE-infected B-LCL stimulated on average 10-fold greater anti-HIV-1 CTLm activity, as detected in the bulk lysis assay, and 55-fold-greater CTLm precursor frequencies specific for the three HIV-1 structural proteins than did stimulation with anti-CD3 MAb. This effect was noted with both freshly donated and frozen-thawed PBMC. The lysis was mediated by CD8(+) T cells and was restricted by the major histocompatibility class I complex, These data indicate that antigen-specific stimulation with VV-GPE-infected B-LCL is a highly efficient method for detection of anti-HIV-1 CTLm responses that is applicable to noncurrent prospective studies with frozen PBMC.