Despite the pressing need for an AIDS vaccine, the determinants of protective immunity to HIV remain concealed within the complexity of adaptive immune responses. We dissected immunodominant virus-specific CD8⁺ T cell populations in Mamu-A*01⁺ rhesus macaques with primary SIV infection to elucidate the hallmarks of effective immunity at the level of individual constituent clonotypes, which were identified according to the expression of distinct T cell receptors (TCRs). The number of public clonotypes, defined as those that expressed identical TCR β-chain amino acid sequences and recurred in multiple individuals, contained within the acute phase CD8⁺ T cell population specific for the biologically constrained Gag CM9 (CTPYDINQM; residues 181–189) epitope correlated negatively with the virus load set point. This independent molecular signature of protection was confirmed in a prospective vaccine trial, in which clonotype engagement was governed by the nature of the antigen rather than the context of exposure and public clonotype usage was associated with enhanced recognition of epitope variants. Thus, the pattern of antigen-specific clonotype recruitment within a protective CD8⁺ T cell population is a prognostic indicator of vaccine efficacy and biological outcome in an AIDS virus infection.

Primary simian immunodeficiency virus (SIV) infections of rhesus macaques result in the dramatic depletion of CD4⁺ CCR5⁺ effector–memory T (T_EM) cells from extra-lymphoid effector sites, but in most infections, an increased rate of CD4⁺ memory T cell proliferation appears to prevent collapse of effector site CD4⁺ T_EM cell populations and acute-phase AIDS. Eventually, persistent SIV replication results in chronic-phase AIDS, but the responsible mechanisms remain controversial. Here, we demonstrate that in the chronic phase of progressive SIV infection, effector site CD4⁺ T_EM cell populations manifest a slow, continuous decline, and that the degree of this depletion remains a highly significant correlate of late-onset AIDS. We further show that due to persistent immune activation, effector site CD4⁺ T_EM cells are predominantly short-lived, and that their homeostasis is strikingly dependent on the production of new CD4⁺ T_EM cells from central–memory T (T_CM) cell precursors. The instability of effector site CD4⁺ T_EM cell populations over time was not explained by increasing destruction of these cells, but rather was attributable to progressive reduction in their production, secondary to decreasing numbers of CCR5⁻ CD4⁺ T_CM cells. These data suggest that although CD4⁺ T_EM cell depletion is a proximate mechanism of immunodeficiency, the tempo of this depletion and the timing of disease onset are largely determined by destruction, failing production, and gradual decline of CD4⁺ T_CM cells.

Vaccinia virus immunization provides lifelong protection against smallpox, but the mechanisms of this exquisite protection are unknown. We used polychromatic flow cytometry to characterize the functional and phenotypic profile of CD8⁺ T cells induced by vaccinia virus immunization in a comparative vaccine trial of modified vaccinia virus Ankara (MVA) versus Dryvax immunization in which protection was assessed against subsequent Dryvax challenge. Vaccinia virus–specific CD8⁺ T cells induced by both MVA and Dryvax were highly polyfunctional; they degranulated and produced interferon γ, interleukin 2, macrophage inflammatory protein 1β, and tumor necrosis factor α after antigenic stimulation. Responding CD8⁺ T cells exhibited an unusual phenotype (CD45RO⁻CD27^intermediate). The unique phenotype and high degree of polyfunctionality induced by vaccinia virus also extended to inserted HIV gene products of recombinant NYVAC. This quality of the CD8⁺ T cell response may be at least partially responsible for the profound efficacy of these vaccines in protection against smallpox and serves as a benchmark against which other vaccines can be evaluated.

The role of CD4⁺ T cells in the control of persistent viral infections beyond the provision of cognate help remains unclear. We used polychromatic flow cytometry to evaluate the production of the cytokines interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and interleukin (IL)-2, the chemokine macrophage inflammatory protein (MIP)-1β, and surface mobilization of the degranulation marker CD107a by CD4⁺ T cells in response to stimulation with cytomegalovirus (CMV)-specific major histocompatibility complex class II peptide epitopes. Surface expression of CD45RO, CD27, and CD57 on responding cells was used to classify CD4⁺ T cell maturation. The functional profile of virus-specific CD4⁺ T cells in chronic CMV infection was unique compared with that observed in other viral infections. Salient features of this profile were: (a) the simultaneous production of MIP-1β, TNF-α, and IFN-γ in the absence of IL-2; and (b) direct cytolytic activity associated with surface mobilization of CD107a and intracellular expression of perforin and granzymes. This polyfunctional profile was associated with a terminally differentiated phenotype that was not characterized by a distinct clonotypic composition. Thus, mature CMV-specific CD4⁺ T cells exhibit distinct functional properties reminiscent of antiviral CD8⁺ T lymphocytes.

Here, we report on the expression of programmed death (PD)-1 on human virus-specific CD8⁺ T cells and the effect of manipulating signaling through PD-1 on the survival, proliferation, and cytokine function of these cells. PD-1 expression was found to be low on naive CD8⁺ T cells and increased on memory CD8⁺ T cells according to antigen specificity. Memory CD8⁺ T cells specific for poorly controlled chronic persistent virus (HIV) more frequently expressed PD-1 than memory CD8⁺ T cells specific for well-controlled persistent virus (cytomegalovirus) or acute (vaccinia) viruses. PD-1 expression was independent of maturational markers on memory CD8⁺ T cells and was not directly associated with an inability to produce cytokines. Importantly, the level of PD-1 surface expression was the primary determinant of apoptosis sensitivity of virus-specific CD8⁺ T cells. Manipulation of PD-1 led to changes in the ability of the cells to survive and expand, which, over several days, affected the number of cells expressing cytokines. Therefore, PD-1 is a major regulator of apoptosis that can impact the frequency of antiviral T cells in chronic infections such as HIV, and could be manipulated to improve HIV-specific CD8⁺ T cell numbers, but possibly not all functions in vivo.

Acute simian immunodeficiency virus (SIV)/human immunodeficiency virus infection is accompanied by a massive destruction of CD4 memory T cells across all the tissue compartments. These early events set the course toward disease progression and immunodeficiency. Here, we demonstrate that prior vaccination reduces this destruction during acute SIV Mac₂₅₁ infection, leading to better survival and long-term outcome. Systemic vaccination with a DNA-prime recombinant adenovirus boost regimen preserved memory CD4 T cells throughout the body. The vaccine regimen induced broad CD4 and CD8 T cell responses in all tissues examined and, importantly, induced antibodies that neutralized the primary isolate of SIV used for challenge. Finally, we demonstrate that the extent of preservation of the CD4 memory compartment during the acute phase provides a strong predictor for subsequent progression to death. Our data provide a mechanism to explain clinical observations that acute-phase viral loads predict long-term disease progression and underscore the need for interventions that protect against early destruction of CD4 memory T cells during acute infection.

The forces that govern clonal selection during the genesis and maintenance of specific T cell responses are complex, but amenable to decryption by interrogation of constituent clonotypes within the antigen-experienced T cell pools. Here, we used point-mutated peptide–major histocompatibility complex class I (pMHCI) antigens, unbiased TCRB gene usage analysis, and polychromatic flow cytometry to probe directly ex vivo the clonal architecture of antigen-specific CD8 ⁺ T cell populations under conditions of persistent exposure to structurally stable virus-derived epitopes. During chronic infection with cytomegalovirus and Epstein-Barr virus, CD8 ⁺ T cell responses to immunodominant viral antigens were oligoclonal, highly skewed, and exhibited diverse clonotypic configurations; TCRB CDR3 sequence analysis indicated positive selection at the protein level. Dominant clonotypes demonstrated high intrinsic antigen avidity, defined strictly as a physical parameter, and were preferentially driven toward terminal differentiation in phenotypically heterogeneous populations. In contrast, subdominant clonotypes were characterized by lower intrinsic avidities and proportionately greater dependency on the pMHCI–CD8 interaction for antigen uptake and functional sensitivity. These findings provide evidence that interclonal competition for antigen operates in human T cell populations, while preferential CD8 coreceptor compensation mitigates this process to maintain clonotypic diversity. Vaccine strategies that reconstruct these biological processes could generate T cell populations that mediate optimal delivery of antiviral effector function.

Clinical trials have indicated that autologous hematopoietic stem cell transplantation (HSCT) can persistently suppress inflammatory disease activity in a subset of patients with severe multiple sclerosis (MS), but the mechanism has remained unclear. To understand whether the beneficial effects on the course of disease are mediated by lympho-depletive effects alone or are sustained by a regeneration of the immune repertoire, we examined the long-term immune reconstitution in patients with MS who received HSCT. After numeric recovery of leukocytes, at 2-yr follow-up there was on average a doubling of the frequency of naive CD4⁺ T cells at the expense of memory T cells. Phenotypic and T cell receptor excision circle (TREC) analysis confirmed a recent thymic origin of the expanded naive T cell subset. Analysis of the T cell receptor repertoire showed the reconstitution of an overall broader clonal diversity and an extensive renewal of clonal specificities compared with pretherapy. These data are the first to demonstrate that long-term suppression of inflammatory activity in MS patients who received HSCT does not depend on persisting lymphopenia and is associated with profound qualitative immunological changes that demonstrate a de novo regeneration of the T cell compartment.

All thymically selected T cells are inherently cross-reactive, yet many data indicate a fine specificity in antigen recognition, which enables virus escape from immune control by mutation in infections such as the human immunodeficiency virus (HIV). To address this paradox, we analyzed the fine specificity of T cells recognizing a human histocompatibility leukocyte antigen (HLA)-A2–restricted, strongly immunodominant, HIV gag epitope (SLFNTVATL). The majority of 171 variant peptides tested bound HLA-A2, but only one third were recognized. Surprisingly, one recognized variant (SLYNTVATL) showed marked differences in structure when bound to HLA-A2. T cell receptor (TCR) recognition of variants of these two peptides implied that they adopted the same conformation in the TCR–peptide–major histocompatibility complex (MHC) complex. However, the on-rate kinetics of TCR binding were identical, implying that conformational changes at the TCR–peptide–MHC binding interface occur after an initial permissive antigen contact. These findings have implications for the rational design of vaccines targeting viruses with unstable genomes.

The mechanisms underlying CD4⁺ T cell depletion in human immunodeficiency virus (HIV) infection are not well understood. Comparative studies of lymphoid tissues, where the vast majority of T cells reside, and peripheral blood can potentially illuminate the pathogenesis of HIV-associated disease. Here, we studied the effect of HIV infection on the activation and depletion of defined subsets of CD4⁺ and CD8⁺ T cells in the blood, gastrointestinal (GI) tract, and lymph node (LN). We also measured HIV-specific T cell frequencies in LNs and blood, and LN collagen deposition to define architectural changes associated with chronic inflammation. The major findings to emerge are the following: the GI tract has the most substantial CD4⁺ T cell depletion at all stages of HIV disease; this depletion occurs preferentially within CCR5⁺ CD4⁺ T cells; HIV-associated immune activation results in abnormal accumulation of effector-type T cells within LNs; HIV-specific T cells in LNs do not account for all effector T cells; and T cell activation in LNs is associated with abnormal collagen deposition. Taken together, these findings define the nature and extent of CD4⁺ T cell depletion in lymphoid tissue and point to mechanisms of profound depletion of specific T cell subsets related to elimination of CCR5⁺ CD4⁺ T cell targets and disruption of T cell homeostasis that accompanies chronic immune activation.