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Cytotoxic T lymphocytes (CTL) recognize and kill virus-infected cells and contribute to immunologic control of viral replication. For many herpesviruses (e.g., Epstein-Barr and cytomegalovirus), virus-specific CTL responses can be readily detected in infected persons, but CTL responses against Kaposi's sarcoma-associated herpesvirus (KSHV) appear to be weak and remain poorly characterized. Using a human leukocyte antigen (HLA) binding motif-based epitope prediction algorithm, we identified 37 HLA-A*0201 binding peptides from 8 KSHV open-reading frames (ORFs). After in vitro stimulation of peripheral blood mononuclear cells from KSHV-infected persons, CTL responses against 1 peptide in the KSHV kaposin protein (ORF K12) were detected in 2 HLA-A*0201-positive subjects. The optimal CTL epitope was identified by HLA restriction analysis and peptide titration assays. These data describe a latent phase viral gene product targeted by CTL that may be relevant for KSHV immunopathogenesis.

Ammonium-chloride-containing solutions (AC) are routinely used to lyse red blood cells during preparation of PBMC. Although exposure to AC has been described to affect the ultrastructural appearance of large granular lymphocytes and to temporarily inhibit cytolytic activity of PBMC preparations, the cellular basis of this phenomenon has not been studied. Here, the inhibitory effect of AC on human CTL and NK-mediated cytotoxicity has been analyzed in 4-h 51Cr-release assays. The results show that NK killing of K562 leukemia cells and xenogeneic endothelial cells is inhibited by AC exposure. The effect is dose-dependent and reversible, because recovery of cytotoxicity is observed within 15 h of re-culturing. AC does not reduce the viability of NK cells and the inhibitory effect is not mediated by the exhaustive release of granzymes upon AC treatment. In contrast, antigen-specific CTL killing of EBV-transformed B-lymphoblastoid cell lines and xenogeneic PHA lymphoblasts was less sensitive to AC and data are presented suggesting that FasL-induced apoptosis is not inhibited by AC. In conclusion, perforin-mediated NK killing is AC-sensitive whereas CTL killing and FasL-mediated killing appear to be AC-resistant. Therefore, AC represents a powerful tool to study different mechanisms of cell-mediated cytotoxicity and may be helpful in assessing antigen-specific CTL cytotoxicity without the influence of NK cell-mediated background killing.

Kaposi's sarcoma-associated herpes virus (KSHV) is a recently identified human gamma2-herpesvirus associated with Kaposi's sarcoma, primary effusion lymphoma, and Castleman's disease. We reasoned that CTL responses may provide host defense against this virus, and consequently, KSHV may have evolved strategies to evade the CTL-mediated immune surveillance. In this study six B cell lines latently infected with KSHV were found to express reduced levels of HLA class I surface molecules compared with B cell lines transformed by the related gamma-herpesvirus EBV. KSHV-infected cells also required higher concentrations of soluble peptides to induce efficient CTL-mediated lysis than control cell lines and were unable to process and/or present intracellularly expressed Ag. Incubation of the KSHV-infected cell lines with high concentrations of soluble HLA class I binding peptides did not restore the deficient HLA class I surface expression. To assess the underlying mechanisms of these phenomena, TAP-1 and TAP-2 gene expression was analyzed. While no attenuation in TAP-2 expression was observed, TAP-1 expression was significantly reduced in all KSHV cell lines compared with that in controls. These results indicate that KSHV can modulate HLA class I-restricted Ag presentation to CTL, which may allow latently infected cells to escape CTL recognition and persist in the infected host.

Immune escape from cytotoxic T-lymphocyte (CTL) responses has been shown to occur not only by changes within the targeted epitope but also by changes in the flanking sequences which interfere with the processing of the immunogenic peptide. However, the frequency of such an escape mechanism has not been determined. To investigate whether naturally occurring variations in the flanking sequences of an immunodominant human immunodeficiency virus type 1 (HIV-1) Gag CTL epitope prevent antigen processing, cells infected with HIV-1 or vaccinia virus constructs encoding different patient-derived Gag sequences were tested for recognition by HLA-A*0201-restricted, p17-specific CTL. We found that the immunodominant p17 epitope (SL9) and its variants were efficiently processed from minigene expressing vectors and from six HIV-1 Gag variants expressed by recombinant vaccinia virus constructs. Furthermore, SL9-specific CTL clones derived from multiple donors efficiently inhibited virus replication when added to HLA-A*0201-bearing cells infected with primary or laboratory-adapted strains of virus, despite the variability in the SL9 flanking sequences. These data suggest that escape from this immunodominant CTL response is not frequently accomplished by changes in the epitope flanking sequences.

Cellular immune responses are thought to be an important antiviral host defense, but the relationship between virus-specific T-helper and cytotoxic-T-lymphocyte (CTL) responses has not been defined. To investigate a potential link between these responses, we examined functional human immunodeficiency virus type 1 (HIV-1)-specific memory CTL precursor frequencies and p24-specific proliferative responses in a cohort of infected untreated persons with a wide range of viral loads and CD4 cell counts. Levels of p24-specific proliferative responses positively correlated with levels of Gag-specific CTL precursors and negatively correlated with levels of plasma HIV-1 RNA. These data linking the levels of HIV-specific CTL with virus-specific helper cell function during chronic viral infection provide cellular immunologic parameters to guide therapeutic and prophylactic vaccine development.

Therapeutic suppression of human immunodeficiency virus type 1 (HIV-1) replication may help elucidate interactions between the host cellular immune responses and HIV-1 infection. We performed a detailed longitudinal evaluation of two subjects before and after the start of highly active antiretroviral therapy (HAART). Both subjects had evidence of in vivo-activated and memory cytotoxic T-lymphocyte precursor (CTLp) activity against multiple HIV-1 gene products. After the start of therapy, both subjects had declines in the levels of in vivo-activated HIV-1-specific CTLs and had immediate increases in circulating HIV-1-specific CTL memory cells. With continued therapy, and continued suppression of viral load, levels of memory CTLps declined. HLA A*0201 peptide tetramer staining demonstrated that declining levels of in vivo-activated CTL activity were associated with a decrease in the expression of the CD38(+) activation marker. Transient increases in viral load during continued therapy were associated with increases in the levels of virus-specific CTLps in both individuals. The results were confirmed by measuring CTL responses to discrete optimal epitopes. These studies illustrate the dynamic equilibrium between the host immune response and levels of viral antigen burden and suggest that efforts to augment HIV-1-specific immune responses in subjects on HAART may decrease the incidence of virologic relapse.

To address the issue of clonal exhaustion in humans, we monitored HLA class I-restricted, epitope-specific CTL responses in an in utero HIV-1-infected infant from 3 mo through 5 years of age. Serial functional CTL precursor assays demonstrated persistent, vigorous, and broadly directed HIV-1 specific CTL activity with a dominant response against an epitope in HIV-1 Gag-p17 (SLYNTVATL, aa 77-85). A clonal CTL response directed against the immunodominant, HLA-A*0201-restricted epitope was found to persist over the entire observation period, as shown by TCR analysis of cDNA libraries generated from PBMC. The analysis of autologous viral sequences did not reveal any escape mutations within the targeted epitope, and viral load measurement indicated ongoing viral replication. Furthermore, inhibition of viral replication assays indicated that the epitope was properly processed from autologous viral protein. These data demonstrate that persistent exposure to high levels of viral Ag does not necessarily lead to clonal exhaustion and that epitope-specific clonal CTL responses induced within the first weeks of life can persist for years without inducing detectable viral escape variants.

Despite detailed analysis of the HIV-1-specific cytotoxic T lymphocyte response by various groups, its relation to viral load and viral sequence variation remains controversial. We analyzed HLA-A*0201 restricted cytotoxic T lymphocyte responses in 17 HIV-1-infected individuals with viral loads ranging from < 400 to 221,000 HIV RNA molecules per milliliter of plasma. In 13 out of 17 infected subjects, CTL responses against the SLYNTVATL epitope (p17 Gag; aa 77-85) were detectable, whereas two other HLA-A*0201 restricted epitopes (ILKEPVHGV, IV9; and VIYQYMDDL, VL9) were only recognized by six and five individuals out of 17 individuals tested, respectively. Naturally occurring variants of the SL9 epitope were tested for binding to HLA-A*0201 and for recognition by specific T cell clones generated from five individuals. Although these variants were widely recognized, they differed by up to 10,000-fold in terms of variant peptide concentrations required for lysis of target cells. A comparison of viral sequences derived from 10 HLA-A*0201-positive individuals to sequences obtained from 11 HLA-A*0201-negative individuals demonstrated only weak evidence for immune selective pressure and thus question the in vivo efficacy of immunodominant CTL responses present during chronic HIV-1 infection.