Monitoring HIV-specific CD8+ T cell responses by intracellular cytokine production

Human immunodeficiency virus (HIV)-specific CD8+ T cells play an important role in controlling HIV infection. Accurate monitoring of these cells is crucial in determining the effects of HIV therapy and vaccine efficacy. Using an intracellular cytokine staining based assay, we are able to directly quantify functional HIV-specific CD8+ T cells. This assay is highly reproducible, and can be performed using both fresh and cryopreserved peripheral blood cells. Importantly, this assay can be used to examine multiple HIV-peptide epitopes simultaneously, and is independent of patient HLA haplotype. Here, we examine the HIV-specific CD8+ T cell response to 95 optimized HIV-derived cytotoxic T lymphocyte (CTL) epitopes in 21 HIV-infected patients of varying HLA haplotype, using peptide mixes and matrices. We find that when using mixes of multiple HIV peptides, the CD8+ T cell response to the mixture is equivalent to the sum of the responses to the individual peptides contained therein. Detailed comparison of the responses in patients suggests that most patients generate a diverse CD8+ T cell response, recognizing multiple HIV epitopes derived from HIV Gag, Pol, Env, or Nef. Although some patients sharing HLA alleles occasionally recognize common peptides, rarely are responses to those peptides dominant within the same group of patients. These results confirm our previous findings that the responses to single HIV-peptides are rarely representative of the entire HIV response.     

Impaired memory CD8 T cell responses against an immunodominant retroviral cryptic epitope

The immunodominant cryptic epitope SYNTGRFPPL, encoded within open reading frame 2 of the LP-BM5 retroviral gag gene, is critical for protection against retroviral-induced pathogenesis. The goal of this study was to dissect the memory response against this unique immunodominant cryptic epitope. Unlike the protective acute effector population of SYNTGRFPPL-specific CD8 T cells, long-lived SYNTGRFPPL-specific CD8 T cells lacked the ability to protect susceptible mice infected with LP-BM5 retrovirus. Compared to memory CD8 T cells against a conventional epitope with similar MHC-I specificity, primed and restimulated using similar conditions, long-lived SYNTGRFPPL-specific CD8 T cells were impaired in their ability to recall against antigen, with reduced cytolytic capabilities and cytokine production. Since similar priming and restimulation regimes were utilized to generate each effector CD8 T cell population, this study has potentially broad implications with regard to the selection criteria of potent, highly conserved cryptic epitopes for use in epitope-based vaccines.     

HIV-specific CD8 T cells express low levels of IL-7Ralpha: implications for HIV-specific T cell memory

Chronic infections in mice can result in defects in memory CD8 T cell properties including low expression of the IL-7Ralpha (CD127). To determine whether defects in memory CD8 T cell formation exist during human chronic infections and to what extent these defects may be allele- or epitope-specific, we compared influenza (Flu), vaccinia (VV) and EBV-specific CD8 T cells to HIV-specific CD8 T cells, using a panel of 13 HIV tetramers. Compared to Flu, VV or EBV, HIV tetramer+ CD8 T cells expressed significantly lower levels of CD127, and this reduction was pervasive across all epitopes and alleles tested and over a wide range of viral loads and CD4 counts. These results indicate impaired HIV-specific memory CD8 T cell differentiation, regardless of level of control of viremia, epitopes targeted or restricting HLA alleles.     

Dynamic imaging of chemokine-dependent CD8+ T cell help for CD8+ T cell responses

Naive T lymphocytes move efficiently in lymphoid tissues while scanning dendritic cells in search of cognate complexes of peptide in major histocompatibility molecules. However, T cell migration ceases after recognition of cognate antigen. We show here that during the initiation of antigen-specific CD8(+) T cell responses, naive CD8(+) polyclonal T cells 'preferentially' interacted in an antigen-independent way with mature dendritic cells competent to present antigen to antigen-specific CD8(+) T cells. These antigen-independent interactions required expression of the chemokine receptor CCR5 on polyclonal T cells and increased the efficiency of the induction of naive, low-precursor-frequency CD8(+) T cell responses. Thus, antigen-specific CD8(+) T cells favor the priming of naive CD8(+) T cells by promoting the CCR5-dependent recruitment of polyclonal CD8(+) T cells to mature dendritic cells.     

Increased CD95/Fas-induced apoptosis of HIV-specific CD8(+) T cells.

Why HIV-specific CD8(+) T cells ultimately fail to clear or control HIV infection is not known. We show here that HIV-specific CD8(+) T cells exhibit increased sensitivity to CD95/Fas-induced apoptosis. This apoptosis is 3-fold higher compared to CMV-specific CD8(+) T cells from the same patients. HIV-specific CD8(+) T cells express the CD45RA(-)CD62L(-) but lack the CD45RA(+)CD62L(-) T cell effector memory (T(EM)) phenotype. This skewing is not found in CMV- and EBV-specific CD8(+) T cells in HIV-infected individuals. CD95/Fas-induced apoptosis is much higher in the CD45RA(-)CD62L(-) T(EM) cells. However, cytotoxicity and IFNgamma production by HIV-specific CD8(+) T cells is not impaired. Our data suggest that the survival and differentiation of HIV-specific CD8(+) T cells may be compromised by CD95/Fas apoptosis induced by FasL-expressing HIV-infected cells.     

Robust CD4+ and CD8+ T cell responses to SIV using mRNA-transfected DC expressing autologous viral Ag

A potentially powerful strategy for therapeutic HIV vaccination is the use of DC transfected with mRNA encoding autologous viral Ag, as epitopes presented by transfected DC would exactly reflect those expressed by infected cells in the individual. Using human and rhesus macaque monocyte-derived DC, we show that nucleofection is a superior method for mRNA transfection, resulting in high-level protein expression and DC maturation. DC transfected with SIV gag isolated from an infected monkey stimulated robust Ag-specific recall T cell responses of similar magnitude to those induced by peptide-pulsed PBMC that were predominantly CD8+ T cell mediated. Enhanced CD4+ T cell responses were stimulated when Gag was redirected into the lysosomal pathway via the targeting signal derived from lysosome-associated membrane protein-1 (LAMP-1). Rhesus DC transfected with lysosome-targeted gag encoding an escape mutation in an immunodominant CTL epitope stimulated CD4+ and CD8+ T cell responses of almost equivalent magnitude directed towards undefined epitopes outside of the mutated region. Finally, gag-transfected DC from SIV-infected monkeys stimulated significant Ag-specific recall T cell responses in an entirely autologous system. These findings demonstrate that mRNA-transfected DC expressing SIV Ag derived from infected monkeys stimulate broad and relevant T cell responses, supporting this approach for therapeutic HIV vaccine development.     

Mycobacterium-specific human CD8 T cell responses.

Tuberculosis (TB) remains a global health problem. There is an intense effort to identify correlates of protective immunity and to design new TB vaccines. CD8 T cells are thought to play a significant role in controlling Mycobacterium tuberculosis infection. Relatively little has been published about the antigens and epitopes targeted by mycobacteria-specific CD8 T cells. Here we present an update of our 1999 overview of human CD8 T cell epitopes in mycobacterial antigens and discuss related issues relevant to TB diagnosis and vaccine development.     

Tracking tumor-specific CD8+ T cell responses

In a recent article, Puig-Saus et al. computationally predict and experimentally validate neoantigen-specific T cell responses in patients with melanoma. They identify a restricted set of neoantigens recognized by polyclonal CD8⁺ T cells as a unique feature of anti-PD-1 responders and engineer autologous tumor-responsive T cells expressing neoantigen-specific TCRs, providing proof-of-concept for future cellular therapies.     

HIV-specific cytolytic CD4 T cell responses during acute HIV infection predict disease outcome

Early immunological events during acute HIV infection are thought to fundamentally influence long-term disease outcome. Whereas the contribution of HIV-specific CD8 T cell responses to early viral control is well established, the role of HIV-specific CD4 T cell responses in the control of viral replication after acute infection is unknown. A growing body of evidence suggests that CD4 T cells-besides their helper function-have the capacity to directly recognize and kill virally infected cells. In a longitudinal study of a cohort of individuals acutely infected with HIV, we observed that subjects able to spontaneously control HIV replication in the absence of antiretroviral therapy showed a significant expansion of HIV-specific CD4 T cell responses-but not CD8 T cell responses-compared to subjects who progressed to a high viral set point (P = 0.038). Markedly, this expansion occurred before differences in viral load or CD4 T cell count and was characterized by robust cytolytic activity and expression of a distinct profile of perforin and granzymes at the earliest time point. Kaplan-Meier analysis revealed that the emergence of granzyme A(+) HIV-specific CD4 T cell responses at baseline was highly predictive of slower disease progression and clinical outcome (average days to CD4 T cell count <350/μl was 575 versus 306, P = 0.001). These data demonstrate that HIV-specific CD4 T cell responses can be used during the earliest phase of HIV infection as an immunological predictor of subsequent viral set point and disease outcome. Moreover, these data suggest that expansion of granzyme A(+) HIV-specific cytolytic CD4 T cell responses early during acute HIV infection contributes substantially to the control of viral replication.     

Cytokines and the inception of CD8 T cell responses

The activation and differentiation of CD8 T cells is a necessary first step that endows these cells with the phenotypic and functional properties required for the control of intracellular pathogens. The induction of the CD8 T cell responses typically results in the development of a massive overall population of effector cells, comprising both highly functional but short-lived terminally differentiated cells, as well as a smaller subset of precursors that are predisposed to survive and transition into the memory T cell pool. In this review, we discuss how inflammatory cytokines and IL-2 bias the initial response towards short-lived effector generation, and also highlight the potential counterbalancing role of IL-21.     

Unravelling the mechanisms of help for CD8+ T cell responses

CD8+ T cells are critically important for immune defense against many viral and bacterial pathogens, and are also key components of cancer immunotherapy. Help from CD4+ T cells is usually essential for optimal CD8+ T cell responses, driving the primary response, the survival of memory cells, and the generation of protective and therapeutic immunity. Understanding the mechanisms of help is thus essential for vaccine design, and for restoring protective immunity in immunosuppressed individuals. Our laboratory has developed an immunization protocol using peptide-pulsed dendritic cells to stimulate help-dependent primary, memory, and secondary CD8+ T cell responses. We have used gene-targeted and T cell receptor transgenic mice to identify two distinct pathways that generate help-dependent and help-independent CD8+ T cell responses, respectively, and are now starting to define the molecular mechanisms underlying these two pathways.     

Frequency and function of HIV-specific CD8(+) T cells

The virus-specific CD8(+) T cell responses of 27 HIV-infected patients were studied, including a unique cohort of long term nonprogressors (LTNP) with normal CD4(+) T cell counts, low levels of plasma viral RNA, strong proliferative responses to HIV antigens and an over-representation of the HLA B*5701 class I allele. The frequencies of CD8(+) T cells specific to the majority of HIV gene products were measured by flow cytometric detection of intracellular interferon-gamma (IFN-gamma) in response to HIV-vaccinia recombinant infected autologous B cells. Very high frequencies (1.4-22%) of circulating CD8(+) T cells were found to be HIV-specific and were not only found in LTNP with reduced plasma virus. No correlation was evident between the frequency of HIV-specific CD8(+) T cells and levels of plasma viremia. In each case, the vast majority of cells (up to 17.2%) responded to Gag-Pol gene products. Although similar frequencies of Gag peptide-specific CD8(+) T cells were found in LTNP and progressors by either intracellular IFN-gamma or MHC class I tetramer staining, the breadth of these responses was greater in patients with progressive HIV infection compared with the LTNP group. The frequency of CD8(+) T cells specific for a single peptide was not representative of an individual patient's total HIV-specific CD8(+) T cell response. These data demonstrate that high numbers of HIV-specific CD8(+) T cells exist even in patients with high level viremia and progressive disease. Further, they suggest that other qualitative parameters of the CD8(+) T cell response may differentiate some patients with very low levels of plasma virus and nonprogressive infection.     

Memory CD8+ T cell responses to cancer

Long-lived memory CD8⁺ T cells play important roles in tumor immunity. Studies over the past two decades have identified four subsets of memory CD8⁺ T cells - central, effector, stem-like, and tissue resident memory - that either circulate through blood, lymphoid and peripheral organs, or reside in tissues where cancers develop. In this article, we will review studies from both pre-clinical mouse models and human patients to summarize the phenotype, distribution and unique features of each memory subset, and highlight specific roles of each subset in anti-tumor immunity. Moreover, we will discuss how stem-cell like and resident memory CD8⁺ T cell subsets relate to exhausted tumor-infiltrating lymphocytes (TIL) populations. These studies reveal how memory CD8⁺ T cell subsets together orchestrate durable immunity to cancer.     

Functions of tetramer-stained HIV-specific CD4(+) and CD8(+) T cells

Significant insight has been gained into constraints on the sensitivity and specificity of staining with class I tetramers. The function of the populations that are defined varies with the clinical situation. Insight has also been gained into the determinants of the CD8(+) T cell response during primary and chronic HIV infection. Human class II tetramers have been synthesised but their role in defining CD4(+) T cell function in HIV infection remains to be determined.     

IL-2 production correlates with effector cell differentiation in HIV-specific CD8+ T cells

Background  

Diminished IL-2 production and lack of effector differentiation have been reported for HIV-specific T cells. In this study, we examined the prevalence of these phenomena using 8-color cytokine flow cytometry, and tested the hypothesis that these two findings were causally related. We analyzed cytokine profiles and memory/effector phenotypes of HIV-specific and CMV-specific T cells using short-term in vitro stimulation with HIV or CMV peptide pools. Nineteen HIV-positive subjects with progressive disease and twenty healthy, HIV-negative subjects were examined.     

Vaccinia assay for the rapid detection of functional HIV-specific CD8+ cytotoxic T lymphocytes

Human immunodeficiency virus (HIV)-specific T cells play a critical role in anti-virus immunity. Therefore, during the clinical development of immune-based therapies, it is important to perform a diagnostic test that rapidly quantifies and characterizes cellular immune responses. For detection of functional HIV-specific CD8(+) cytotoxic T cells (CTL), we used a rapid vaccinia assay that employs recombinant vaccinia virus-infected peripheral blood mononuclear cells (PBMC). This assay was compared with the traditional 51Cr-release CTL assay and with the peptide pool-based enzyme-linked immunospot assay (ELISPOT). We demonstrated a close correlation between these assays by using identical antigens in parallel assays. However, the vaccinia assay was the least expensive and time- and labor-consuming test. Regarding sensitivity, the vaccinia assay was similar to both the peptide pool-based ELISPOT and CTL assays. We showed that human histocompatibility leukocyte antigen (HLA)-Dr(+) cells are responsible for presenting recombinant vaccinia antigens to T cells. The recombinant vaccinia-based assay is a rapid, sensitive and quantitative test and is thus suitable for the detection of functional antigen-specific CD8(+) CTL in large-scale clinical studies.     

Neutrophils sustain pathogenic CD8+ T cell responses in the heart

This study explores the influence of innate immunity on CD8(+) T-cell responses against heart tissue. Adoptive transfer of ovalbumin-specific CD8(+) effector T cells into CMy-mOva mice, which express ovalbumin in cardiac myocytes, results in a lethal acute myocarditis. The inflammatory infiltrate in the heart includes neutrophils as well as T cells. We used anti-Ly6G antibody to transiently deplete neutrophils at the time of onset of disease. By day 7 after receiving 5 x 10(5) CD8(+) effector T cells, 100% of control Ig-treated CMy-mOva mice had died, while 85% of anti-Ly6G-treated mice survived indefinitely. CD8(+) T-cell infiltration and tissue damage were present in both groups, but the disease was limited in the anti-Ly6G-treated mice, with a rapid disappearance of the adoptively transferred CD8(+) T cells within 11 days. Recovery occurred even though blood neutrophil counts began to rise 48 hours after the last anti-Ly6G treatment. Recovery was associated with a chronic CD4(+) cell infiltrate, and a rapid decline in expression of IFN-gamma and IP-10 mRNA in the myocardium. Neutrophil depletion did not effect survival of CMy-mOva mice that received 3 x 10(6) CD8(+) T cells. These data show that granulocytic inflammation sustains CD8(+) T-cell-mediated heart disease, which has important implications for the pathogenesis and treatment of acute myocarditis and allograft rejection.     

Detailed analysis of Epstein-Barr virus-specific CD4+ and CD8+ T cell responses during infectious mononucleosis

We studied simultaneously Epstein-Barr virus (EBV)-specific CD4(+) and CD8(+) T cell responses during and after infectious mononucleosis (IM), using a previously described 12-day stimulation protocol with EBNA1 or BZLF1 peptide pools. Effector function of EBV-specific T cells was determined after restimulation by measuring intracellular interferon-gamma production. During IM, BZLF1-specifc CD4(+) T cell responses were dominant compared with CD8(+) T cell responses. EBNA1-specific CD4(+) and CD8(+) T cell responses were low and remained similar for 6 months. However, 6 months after IM, BZLF1-specific CD4(+) T cell responses had declined, but CD8(+) T cell responses had increased. At diagnosis, EBV-specific CD8(+) T cells as studied by human leucocyte antigen class I tetramer staining comprised a tetramer(bright)CD8(bright) population consisting mainly of CD27(+) memory T cells and a tetramer(dim)CD8(dim) population consisting primarily of CD27(-) effector T cells. The remaining EBV-specific CD8(+) T cell population 6 months after the diagnosis of IM consisted mainly of tetramer(bright)CD8(bright) CD27(+) T cells, suggesting preferential preservation of memory T cells after contraction of the EBV-specific T cell pool.     

IL-15 ex vivo overcomes CD4+ T cell deficiency for the induction of human antigen-specific CD8+ T cell responses

CD4(+) Th cells are important for the induction and maintenance of antigen-specific CD8(+) T cell function, so their loss or dysfunction in HIV-infected or cancer patients could reduce the patients' ability to control viral infection. Previous work in murine systems indicated that IL-15 codelivered with vaccines could overcome CD4(+) Th cell deficiency for induction of functionally efficient CD8(+) T cells and maintenance of viral-specific CTLs, but its efficacy in helping primary human CD8(+) T cell responses is unknown. In the present study, a peptide-pulsed, DC-based human coculture ex vivo system was used to study the role of IL-15 in overcoming CD4(+) Th deficiency to elicit CD8(+) T cell responses in CD4-depleted PBMCs from healthy individuals and PBMCs from HIV-1-infected patients. We found that IL-15 could overcome CD4(+) Th deficiency to induce primary and recall memory CD8(+) T cell responses in healthy individuals. Moreover, in CD4-deficient, HIV-1-infected patients with diminished CD8(+) T cell responses, IL-15 greatly enhanced CD8(+) T cell responses to alloantigen. These results suggest that IL-15 may be useful in the development of therapeutic and preventive vaccines against cancers and viral infections in patients defective in CD4(+) Th cell.