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Despite progress toward understanding the correlates of protective T cell immunity in HIV infection, the optimal approach to Ag delivery by vaccination remains uncertain. We characterized two immunodominant CD8 T cell populations generated in response to immunization of BALB/c mice with a replication-deficient adenovirus serotype 5 vector expressing the HIV-derived Gag and Pol proteins at equivalent levels. The Gag-AI9/H-2K(d) epitope elicited high-avidity CD8 T cell populations with architecturally diverse clonotypic repertoires that displayed potent lytic activity in vivo. In contrast, the Pol-LI9/H-2D(d) epitope elicited motif-constrained CD8 T cell repertoires that displayed lower levels of physical avidity and lytic activity despite equivalent measures of overall clonality. Although low-dose vaccination enhanced the functional profiles of both epitope-specific CD8 T cell populations, greater polyfunctionality was apparent within the Pol-LI9/H-2D(d) specificity. Higher proportions of central memory-like cells were present after low-dose vaccination and at later time points. However, there were no noteworthy phenotypic differences between epitope-specific CD8 T cell populations across vaccine doses or time points. Collectively, these data indicate that the functional and phenotypic properties of vaccine-induced CD8 T cell populations are sensitive to dose manipulation, yet constrained by epitope specificity in a clonotype-dependent manner., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Persistent exposure to cognate Ag leads to the functional impairment and exhaustion of HIV-specific CD8 T cells. Ag withdrawal, attributable either to antiretroviral treatment or the emergence of epitope escape mutations, causes HIV-specific CD8 T cell responses to wane over time. However, this process does not continue to extinction, and residual CD8 T cells likely play an important role in the control of HIV replication. In this study, we conducted a longitudinal analysis of clonality, phenotype, and function to define the characteristics of HIV-specific CD8 T cell populations that persist under conditions of limited antigenic stimulation. Ag decay was associated with dynamic changes in the TCR repertoire, increased expression of CD45RA and CD127, decreased expression of programmed death-1, and the emergence of polyfunctional HIV-specific CD8 T cells. High-definition analysis of individual clonotypes revealed that the Ag loss-induced gain of function within HIV-specific CD8 T cell populations could be attributed to two nonexclusive mechanisms: 1) functional improvement of persisting clonotypes; and 2) recruitment of particular clonotypes endowed with superior functional capabilities.

Recent studies have revealed the critical role of programmed death-1 (PD-1) in exhaustion of HIV- and SIV-specific CD8(+) T cells. In this study, we show that high expression of PD-1 correlates with increased ex vivo spontaneous and CD95/Fas-induced apoptosis, particularly in the "effector-memory" CD8(+) T cell population from HIV(+) donors. High expression of PD-1 was linked to a proapoptotic phenotype characterized by low expression of Bcl-2 and IL7-R alpha, high expression of CD95/Fas and high mitochondrial mass. Expression of PD-1 and CD57 was differentially associated with the maturation status of CD8(+) T cells in HIV infection. CD57 was linked to higher apoptosis resistance, with cells expressing a PD-1(L)CD57(H) phenotype exhibiting lower levels of cell death. The majority of HIV-specific CD8(+) T cells were found to express a PD-1(H)CD57(L) or PD-1(H)CD57(H) phenotype. No correlation was found between PD-1 expression and ex vivo polyfunctionality of either HIV- or CMV-specific CD8(+) T cells. Contrary to CD57, high expression of PD-1 was characterized by translocation of PD-1 into the area of CD95/Fas-capping, an early necessary step of CD95/Fas-induced apoptosis. Thus, our data further support the role of PD-1 as a preapoptotic factor for CD8(+) T cells in HIV infection.

Antiviral CD8(+) T cells can elaborate at least two effector functions, cytokine production and cytotoxicity. Which effector function is elaborated can determine whether the CD8(+) T cell response is primarily inflammatory (cytokine producing) or antiviral (cytotoxic). In this study we demonstrate that cytotoxicity can be triggered at peptide concentrations 10- to 100-fold less than those required for cytokine production in primary HIV- and CMV-specific human CD8(+) T cells. Cytolytic granule exocytosis occurs at peptide concentrations insufficient to cause substantial TCR down-regulation, providing a mechanism by which a CD8(+) T cell could engage and lyse multiple target cells. TCR sequence analysis of virus-specific cells shows that individual T cell clones can degranulate or degranulate and produce cytokine depending on the Ag concentration, indicating that response heterogeneity exists within individual CD8(+) T cell clonotypes. Thus, antiviral CD8(+) T cell effector function is determined primarily by Ag concentration and is not an inherent characteristic of a virus-specific CD8(+) T cell clonotype or the virus to which the response is generated. The inherent ability of viruses to induce high or low Ag states may be the primary determinant of the cytokine vs cytolytic nature of the virus-specific CD8(+) T cell response.

The excessive immunological response triggered by microbial superantigens has been implicated in the etiology of a wide range of human diseases but has been most clearly defined for the staphylococcal and streptococcal toxic shock syndromes. Because MHC class II presentation of superantigens to T cells is not MHC-restricted, the possibility that HLA polymorphisms could influence superantigenicity, and thus clinical susceptibility to the toxicity of individual superantigens, has received little attention. In this study, we demonstrate that binding of streptococcal and staphylococcal superantigens to HLA class II is influenced by allelic differences in class II. For the superantigen streptococcal pyrogenic exotoxin A, class II binding is dependent on DQ alpha-chain polymorphisms such that HLA-DQA1*01 alpha-chains show greater binding than DQA1*03/05 alpha-chains. The functional implications of differential binding on T cell activation were investigated in various experimental systems using human T cells and murine Vbeta8.2 transgenic cells as responders. These studies showed quantitative and qualitative differences resulting from differential HLA-DQ binding. We observed changes in T cell proliferation and cytokine production, and in the Vbeta specific changes in T cell repertoire that have hitherto been regarded as a defining feature of an individual superantigen. Our observations reveal a mechanism for the different outcomes seen following infection by toxigenic bacteria.

Escape from the CD8(+) T cell response through epitope mutations can lead to loss of immune control of HIV replication. Theoretically, escape from CD8(+) T cell recognition is less likely when multiple TCRs target individual MHC/peptide complexes, thereby increasing the chance that amino acid changes in the epitope could be tolerated. We studied the CD8(+) T cell response to six immunodominant epitopes in five HIV-infected subjects using a novel approach combining peptide stimulation, cell surface cytokine capture, flow cytometric sorting, anchored RT-PCR, and real-time quantitative clonotypic TCR tracking. We found marked variability in the number of clonotypes targeting individual epitopes. One subject recognized a single epitope with six clonotypes, most of which were able to recognize and lyse cells expressing a major epitope variant that arose. Additionally, multiple clonotypes remained expanded during the course of infection, irrespective of epitope variant frequency. Thus, CD8(+) T cells comprising multiple TCR clonotypes may expand in vivo in response to individual epitopes, and may increase the ability of the response to recognize virus escape mutants.