CTL are held to be an important host defense mechanism in persistent herpes-virus infections. We have therefore studied the nature and specificity of human cytomegalovirus (HCMV)-specific CTL in normal persistently infected individuals. This was achieved by using vaccinia recombinants encoding viral genes expressed at different stages of the virus replicative cycle, a structural glycoprotein gB (vac.gB) and the major 72-kD immediate early nonstructural protein (vac.IE) of HCMV, combined with limiting dilution analysis of the CTL response. In two subjects, 43 and 58% of HCMV CTL precursors (CTLp) lysed vac.IE-infected cells, in contrast to less than 6% lysing gB-infected cells. HCMV-specific CTL could also be generated by secondary in vitro stimulation with vac.gB- but not vac.IE-infected autologous fibroblasts. The high frequency of 72-kD IE protein-specific CTL suggests that this is at least a major recognition element for the HCMV-specific CTL response in asymptomatic persistently infected individuals, and CTL with this specificity may be important in maintaining the normal virus/host equilibrium.

Human virus-specific cytotoxic T (Tc) cells may be important in maintaining the virus/host equilibrium during persistent herpes virus infections such as that with human cytomegalovirus (HCMV). We have previously shown that HCMV-specific Tc cells are present in peripheral blood in normal asymptomatic seropositive individuals (L. K. Borysiewicz et al., Eur. J. Immunol. 1983. 13: 804). In this study we have used limiting dilution analysis to estimate the precursor frequency of these Tc cells and to further delineate their specificity for viral proteins expressed at different stages of the virus replicative cycle. HCMV-specific Tc precursor cells were present in peripheral blood lymphocytes (PBL) at a frequency of 1/5000 to 20,000 E+ PBL. This frequency was higher than that observed for varicella-zoster virus (VZV)-specific Tc cells (1/30,000 to greater than 500,000) in asymptomatic individuals and was similar to the VZV Tc precursor cell frequencies observed following clinical reactivation (1/30,000). When the stage specificity of clonally derived HCMV-specific Tc cells was analyzed, using target cells treated with phosphonoformate to allow expression of only the nonstructural viral proteins, the majority (60%) of Tc cells lysed these cells. A number of Tc cells lysed only cells which expressed the structural or late HCMV proteins. These results suggest a high precursor frequency of HCMV-specific Tc cells in PBL, and that there are subpopulations of such Tc cells specific for HCMV antigens expressed at different stages of the virus replicative cycle. However, the relative frequencies of these subpopulations suggest that the immunodominant HCMV antigens with respect to the Tc response are expressed at immediate early and/or early times.

We have previously identified two subsets of CD8+, CD57+ lymphocytes in normal peripheral blood: i) T cells expressing high levels [CD8high(CD57+)] and ii) natural killer cells expressing low levels of surface CD8[CD8low(CD57+)]. We investigated the cytotoxic and suppressive function of CD8high(CD57+) T lymphocytes from normal, healthy individuals using standard chromium-release assays and limiting dilution analysis. In normal, healthy subjects, this cell subset suppressed the generation of cytotoxic T lymphocytes (CTL) to autologous, Epstein-Barr virus (EBV)-transformed B cell lines (BCL). Depletion of CD8high(CD57+) T lymphocytes from peripheral blood mononuclear cells (PBMC) resulted in a three- to sevenfold rise in CTL precursor frequency to autologous EBV-transformed BCL, but not allogeneic PBMC or BCL by LDA. Replacement of CD8high(CD57+) T lymphocytes in limiting dilution cultures led to the dose-dependent suppression of EBV-specific, but not allogeneic, CTL generation. Supernatant from CD8high(CD57+) T lymphocytes cultured with autologous BCL did not exhibit suppression, suggesting that soluble factors were not responsible. As CD8high(CD57+) T lymphocytes did not, themselves, exhibit cytotoxicity against autologous BCL, removal of BCL stimulator cells in co-culture was not the mechanism of suppression. Furthermore, while the CD8high(CD57+) T lymphocytes from healthy subjects suppressed the generation of CTL to autologous BCL, they did not suppress the cytotoxic activity of established mixed lymphocyte reactions or peptide-specific CTL clones, as has been reported in bone marrow transplant recipients and human immunodeficiency virus patients. This suggests that CD8high(CD57+) T lymphocytes from healthy subjects suppress the generation of, rather than killing by, CTL in a contact-dependent manner. To our knowledge, this is the first identification of a phenotypically distinct subset of human CD8+ T cells that can suppress generation of antigen-specific major histocompatibility complex class I-restricted CTL.

Little is known about the nature and specificity of T-cell-mediated responses to picornaviruses in humans. In this study, the nature of the T-cell response to seven picornaviruses, including polioviruses, coxsackieviruses B3 and B4, human rhinovirus 14, and encephalomyocarditis virus, was determined. Twenty-nine individuals responded to poliovirus type 3, coxsackievirus B3, and encephalomyocarditis virus by proliferation of T cells, and from such cultures, 130 virus-specific T-cell lines were established. T-cell lines generated in response to encephalomyocarditis virus were exclusively strain specific. However, the majority of T-cell lines established in response to viruses, other than encephalomyocarditis virus, were cross-reactive to each other. Their cross-reactivity was confirmed in 2 of the 30 picornavirus-specific clonally derived T-cell lines from two subjects, but the majority of these lines were serotype specific. T-cell epitopes adjacent to each of the B-cell antigenic sites in VP1 of poliovirus type 3 were identified. The response to the region adjacent to B-cell antigenic site 1 (residues 97 to 114) was dominant between individuals. The localization of this major CD4 T-cell epitope may permit the construction of chimeric viruses utilizing the natural picornavirus T-cell response to augment production of antibody specific for inserted sequences.