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 cytoto xic T-lymphocyte (CTLm) responses is associated with disease progressi on in HIV-1 infection. In this study, nonspecific stimulation of perip heral blood mononuclear cells (PBMC) from HIV-1-infected homosexual me n with anti-CIM monoclonal antibody (MAb) was compared with antigen-sp ecific 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 response s in a bulk lysis assay and by precursor frequency analysis. The resul ts show that VV-GPE-infected B-LCL stimulated on average 10-fold great er 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 eff ect was noted with both freshly donated and frozen-thawed PBMC. The ly sis was mediated by CD8(+) T cells and was restricted by the major his tocompatibility class I complex, These data indicate that antigen-spec ific stimulation with VV-GPE-infected B-LCL is a highly efficient meth od for detection of anti-HIV-1 CTLm responses that is applicable to no ncurrent prospective studies with frozen PBMC.