Virus-specific CD8 ϩ T cells are associated with declining viremia in acute human immunodeficiency virus (HIV)1 infection, but do not correlate with control of viremia in chronic infection, suggesting a progressive functional defect not measured by interferon ␥ assays presently used. Here, we demonstrate that HIV-1-specific CD8 ϩ T cells proliferate rapidly upon encounter with cognate antigen in acute infection, but lose this capacity with ongoing viral replication. This functional defect can be induced in vitro by depletion of CD4 ϩ T cells or addition of interleukin 2-neutralizing antibodies, and can be corrected in chronic infection in vitro by addition of autologous CD4 ϩ T cells isolated during acute infection and in vivo by vaccine-mediated induction of HIV-1-specific CD4 ϩ T helper cell responses. These data demonstrate a loss of HIV-1-specific CD8 ϩ T cell function that not only correlates with progressive infection, but also can be restored in chronic infection by augmentation of HIV-1-specific T helper cell function. This identification of a reversible defect in cell-mediated immunity in chronic HIV-1 infection has important implications for immunotherapeutic interventions.

Viral mutational escape can reduce or abrogate recognition by the T cell receptor (TCR) of virus-specific CD8+ T cells. However, very little is known about the impact of cytotoxic T lymphocyte (CTL) epitope mutations on interactions between peptide–major histocompatibility complex (MHC) class I complexes and MHC class I receptors expressed on other cell types. Here, we analyzed a variant of the immunodominant human leukocyte antigen (HLA)-B2705–restricted HIV-1 Gag KK10 epitope (KRWIILGLNK) with an L to M amino acid substitution at position 6 (L6M), which arises as a CTL escape variant after primary infection but is sufficiently immunogenic to elicit a secondary, de novo HIV-1–specific CD8+ T cell response with an alternative TCR repertoire in chronic infection. In addition to altering recognition by HIV-1–specific CD8+ T cells, the HLA-B2705–KK10 L6M complex also exhibits substantially increased binding to the immunoglobulin-like transcript (ILT) receptor 4, an inhibitory MHC class I–specific receptor expressed on myelomonocytic cells. Binding of the B2705–KK10 L6M complex to ILT4 leads to a tolerogenic phenotype of myelomonocytic cells with lower surface expression of dendritic cell (DC) maturation markers and co-stimulatory molecules. These data suggest a link between CTL-driven mutational escape, altered recognition by innate MHC class I receptors on myelomonocytic cells, and functional impairment of DCs, and thus provide important new insight into biological consequences of viral sequence diversification.

Human immunodeficiency virus type 1 (HIV-1) evades CD8؉ T-cell responses through mutations within targeted epitopes, but little is known regarding its ability to generate de novo CD8 ؉ T-cell responses to such mutants. Here we examined gamma interferon-positive, HIV-1-specific CD8 ؉ T-cell responses and autologous viral sequences in an HIV-1-infected individual for more than 6 years following acute infection. Fourteen optimal HIV-1 T-cell epitopes were targeted by CD8 ؉ T cells, four of which underwent mutation associated with dramatic loss of the original CD8 ؉ response. However, following the G 357 S escape in the HLA-A11-restricted Gag 349-359 epitope and the decline of wild-type-specific CD8 ؉ T-cell responses, a novel CD8 ؉ T-cell response equal in magnitude to the original response was generated against the variant epitope. CD8 ؉ T cells targeting the variant epitope did not exhibit cross-reactivity against the wild-type epitope but rather utilized a distinct T-cell receptor V␤ repertoire. Additional studies of chronically HIV-1-infected individuals expressing HLA-A11 demonstrated that the majority of the subjects targeted the G 357 S escape variant of the Gag 349-359 epitope, while the wild-type consensus sequence was significantly less frequently recognized. These data demonstrate that de novo responses against escape variants of CD8 ؉ T-cell epitopes can be generated in chronic HIV-1 infection and provide the rationale for developing vaccines to induce CD8 ؉ T-cell responses directed against both the wild-type and variant forms of CD8 epitopes to prevent the emergence of cytotoxic T-lymphocyte escape variants.

Human immunodeficiency virus type 1 (HIV-1)-specific CD8؉ T cells in early infection are associated with the dramatic decline of peak viremia, whereas their antiviral activity in chronic infection is less apparent. The functional properties accounting for the antiviral activity of HIV-1-specific CD8 ؉ T cells during early infection are unclear. Using cytokine secretion and tetramer decay assays, we demonstrated in intraindividual comparisons that the functional avidity of HIV-1-specific CD8 ؉ T cells was consistently higher in early infection than in chronic infection in the presence of high-level viral replication. This change of HIV-1-specific CD8 ؉ T-cell avidity between early and chronic infections was linked to a substantial switch in the clonotypic composition of epitope-specific CD8 ؉ T cells, resulting from the preferential loss of high-avidity CD8 ؉ T-cell clones. In contrast, the maintenance of the initially recruited clonotypic pattern of HIV-1-specific CD8 ؉ T cells was associated with low-level set point HIV-1 viremia. These data suggest that high-avidity HIV-1-specific CD8 ؉ T-cell clones are recruited during early infection but are subsequently lost in the presence of persistent high-level viral replication.

The relative contributions of HLA alleles and T-cell receptors (TCRs) to the prevention of mutational viral escape are unclear. Here, we examined human immunodeficiency virus type 1 (HIV-1)-specific CD8 ؉ T-cell responses restricted by two closely related HLA class I alleles, B*5701 and B*5703, that differ by two amino acids but are both associated with a dominant response to the same HIV-1 Gag epitope KF11 (KAFSPEVIP MF). When this epitope is presented by HLA-B*5701, it induces a TCR repertoire that is highly conserved among individuals, cross-recognizes viral epitope variants, and is rarely associated with mutational escape. In contrast, KF11 presented by HLA-B*5703 induces an entirely different, more heterogeneous TCR ␤-chain repertoire that fails to recognize specific KF11 escape variants which frequently arise in clade C-infected HLA-B*5703 ؉ individuals. These data show the influence of HLA allele subtypes on TCR selection and indicate that extensive TCR diversity is not a prerequisite to prevention of allowable viral mutations. Sequence mutations resulting in loss of recognition by CD8ϩ

CD8(+) T cells play a crucial role in the control of viral infections by direct elimination of infected cells and secretion of a number of soluble factors. Recent data suggest that HIV-1-specific CD8(+) T cell subsets may differ in their ability to exert these effector functions. Here, we directly compared the cytokine secretion patterns and cytotoxic capacity of HIV-1-specific CD8(+) T cells, using a flow-cytometric cytotoxicity assay based on caspase-3 activation in dying target cells. These experiments revealed considerable intraindividual and interindividual differences among epitope-specific T-cell effector functions: while the frequency of HIV-1-specific CD8(+) T cells secreting interferon-gamma but no tumor necrosis factor-alpha (TNF-alpha) following antigenic stimulation was only weakly correlated to their cytotoxic activity (R = 0.05, P =.57), a subset of CD8(+) T cells secreting both inter-feron-gamma and TNF-alpha was substantially more strongly associated with cytotoxicity (R = 0.67, P <.001). This subset of CD8(+) T cells also exhibited stronger intracellular perforin expression and more pronounced direct ex vivo HIV-1-specific cytoxicity than CD8(+) T cells secreting solely interferon-gamma following sorting of these subpopulations according to their cytokine profile. These results suggest that HIV-1-specific cytotoxicity of CD8(+) T cells is preferentially mediated by a subset of CD8(+) T cells secreting both interferon-gamma and TNF-alpha.

Studies in acute human immunodeficiency virus type 1 (HIV-1) infection indicate viral evolution under CD8T-cell immune selection pressure, but the effects of ongoing immune pressure on epitope evolution during chronic infection are not well described. In this study, we performed a detailed longitudinal analysis of viral sequence variation within persistently targeted cytotoxic T-lymphocyte (CTL) epitopes in two HIV-1-infected persons during 6 years of persistent viremia. Responses were quantitated using freshly isolated peripheral blood lymphocytes in direct lytic assays as well as by gamma interferon (IFN-␥) Elispot assays on cryopreserved cells. Seven targeted epitopes were identified in each person. In the majority of cases, the dominant epitope sequence did not change over time, even in the presence of responses of sufficient magnitude that they were detectable using fresh peripheral blood mononuclear cells in direct lytic assays. Only 4 of the 14 autologous epitopes tested represented potential CTL escape variants; however, in most cases strong responses to these epitopes persisted for the 6 years of study. Although persistent IFN-␥ responses were detected to all epitopes, direct lytic assays demonstrated declining responses to some epitopes despite the persistence of the targeted sequence in vivo. These data indicate limited viral evolution within persistently targeted CD8 T-cell epitopes during the chronic phase of infection and suggest that these regions of the virus are either refractory to sequence change or that persistently activated CD8 T-cell responses in chronic infection exert little functional selection pressure.

Viral epitopes within regions of low HIV-1 clade B diversity and high inter-clade homology can be recognized in the clade A, B and C variants and indicate a wide degree of cross-isolate and cross-clade recognition by HIV-1-specific T cells. These regions may therefore be of particular relevance for the design of HIV-1 vaccines.