Identification of a Human Cyclin D1-Derived Peptide that Induces Human Cytotoxic CD4 T Cells

Cyclin D1 is over-expressed in various human tumors and therefore can be a potential oncogenic target antigen. However, only a limited number of T cell epitopes has been characterized. We aimed at identifying human cyclin D1-derived peptides that include both CD4 and CD8 T cell epitopes and to test if such multi-epitope peptides could yield improved cytotoxic CD8 T cell responses as well as cytotoxic CD4 T cells. Five HLA-DR.B1-binding peptides containing multiple overlapping CD4 epitopes and HLA-A0201-restricted CD8 T cell epitopes were predicted by computer algorithms. Immunogenicity of the synthetic peptides was assessed by stimulating T cells from healthy donors in vitro and the epitope recognition was measured by IFN-γ ELISPOT and ⁵¹Chromium release assays. A HLA-DR.B1 peptide, designed “DR-1”, in which a HLA-A0201-binding epitopes (D1-1) was imbedded, induced CD3 T cell responses against both DR-1 and D1-1 peptides in IFN-γ ELISPOT assay. This suggested processing of the shorter D1-1 epitope from the DR-1 sequence. However, only DR-1-stimulated CD4 or CD3 T cells possessed cytotoxicity against peptide-pulsed autologous DCs and a cancer cell line, that expresses a high level of cyclin D1. Monoclonal antibody to HLA-DR abrogated the epitope-specific responses of both CD3 and CD4 T cells, demonstrating class II-mediated killing. Our studies suggest a possible role of CD4 T cells in anti-tumor immunity as cytotoxic effectors against HLA-DR expressing cancers and provide a rationale for designing peptide vaccines that include CD4 epitopes.     

Cross-Presentation of a Spread-Defective MCMV Is Sufficient to Prime the Majority of Virus-Specific CD8+ T Cells

CD8+ T cells can be primed by peptides derived from endogenous proteins (direct presentation), or exogenously acquired protein (cross-presentation). However, the relative ability of these two pathways to prime CD8+ T cells during a viral infection remains controversial. Cytomegaloviruses (CMVs) can infect professional antigen presenting cells (APCs), including dendritic cells, thus providing peptides for direct presentation. However, the viral immune evasion genes profoundly impair recognition of infected cells by CD8+ T cells. Nevertheless, CMV infection elicits a very strong CD8+ T cell response, prompting its recent use as a vaccine vector. We have shown previously that deleting the immune evasion genes from murine cytomegalovirus (MCMV) that target class I MHC presentation, has no impact on the size or breadth of the CD8+ T cell response elicited by infection, suggesting that the majority of MCMV-specific CD8+ T cells in vivo are not directly primed by infected professional APCs. Here we use a novel spread-defective mutant of MCMV, lacking the essential glycoprotein gL, to show that cross-presentation alone can account for the majority of MCMV-specific CD8+ T cell responses to the virus. Our data support the conclusion that cross-presentation is the primary mode of antigen presentation by which CD8+ T cells are primed during MCMV infection.     

Visualizing Early Splenic Memory CD8+ T Cells Reactivation against Intracellular Bacteria in the Mouse

Memory CD8⁺ T cells represent an important effector arm of the immune response in maintaining long-lived protective immunity against viruses and some intracellular bacteria such as Listeria monocytogenes (L.m). Memory CD8⁺ T cells are endowed with enhanced antimicrobial effector functions that perfectly tail them to rapidly eradicate invading pathogens. It is largely accepted that these functions are sufficient to explain how memory CD8⁺ T cells can mediate rapid protection. However, it is important to point out that such improved functional features would be useless if memory cells were unable to rapidly find the pathogen loaded/infected cells within the infected organ. Growing evidences suggest that the anatomy of secondary lymphoid organs (SLOs) fosters the cellular interactions required to initiate naive adaptive immune responses. However, very little is known on how the SLOs structures regulate memory immune responses. Using Listeria monocytogenes (L.m) as a murine infection model and imaging techniques, we have investigated if and how the architecture of the spleen plays a role in the reactivation of memory CD8⁺ T cells and the subsequent control of L.m growth. We observed that in the mouse, memory CD8⁺ T cells start to control L.m burden 6 hours after the challenge infection. At this very early time point, L.m-specific and non-specific memory CD8⁺ T cells localize in the splenic red pulp and form clusters around L.m infected cells while naïve CD8⁺ T cells remain in the white pulp. Within these clusters that only last few hours, memory CD8⁺ T produce inflammatory cytokines such as IFN-γ and CCL3 nearby infected myeloid cells known to be crucial for L.m killing. Altogether, we describe how memory CD8⁺ T cells trafficking properties and the splenic micro-anatomy conjugate to create a spatio-temporal window during which memory CD8⁺ T cells provide a local response by secreting effector molecules around infected cells.     

Role of human immunodeficiency virus (HIV)-specific T-cell immunity in control of dual HIV-1 and HIV-2 infection

Progressive immune dysfunction and AIDS develop in most cases of human immunodeficiency virus type 1 (HIV-1) infection but in only 25 to 30% of persons with HIV-2 infection. However, the natural history and immunologic responses of individuals with dual HIV-1 and HIV-2 infection are largely undefined. Based on our previous findings, we hypothesized that among patients with dual infection the control of HIV-1 is associated with the ability to respond to HIV-2 Gag epitopes and to maintain HIV-specific CD4⁺ T-cell responses. To test this, we compared the HIV-specific ex vivo IFN-γ enzyme-linked immunospot (ELISPOT) assay responses of 19 dually infected individuals to those of persons infected with HIV-1 or HIV-2 only. Further, we assessed the functional profile of HIV Gag-specific CD4⁺ and CD8⁺ T cells from nine HIV dually infected patients by using a multicolor intracellular cytokine staining assay. As determined by ELISPOT assay, the magnitude and frequency of IFN-γ-secreting T-cell responses to gene products of HIV-1 were higher than those to gene products of HIV-2 (2.64 versus 1.53 log₁₀ IFN-γ spot-forming cells/10⁶ cells [90% versus 63%, respectively].) Further, HIV-1 Env-, Gag-, and Nef- and HIV-2 Gag-specific responses were common; HIV-2 Nef-specific responses were rare. HIV-specific CD4⁺ T helper responses were detected in nine of nine dually infected subjects, with the majority of these T cells producing gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) and, to a lesser extent, interleukin-2. The HIV-1 plasma viral load was inversely correlated with HIV-2 Gag-specific IFN-γ-/TNF-α-secreting CD4⁺ and HIV-2 Gag-specific IFN-γ-secreting CD8⁺ T cells. In conclusion, the T-cell memory responses associated with containment of single HIV-1 and HIV-2 infection play a similar significant role in the immune control of dual HIV-1 and HIV-2 infection.     

Polyfunctional Type-1, -2, and -17 CD8+ T Cell Responses to Apoptotic Self-Antigens Correlate with the Chronic Evolution of Hepatitis C Virus Infection

Caspase-dependent cleavage of antigens associated with apoptotic cells plays a prominent role in the generation of CD8⁺ T cell responses in various infectious diseases. We found that the emergence of a large population of autoreactive CD8⁺ T effector cells specific for apoptotic T cell-associated self-epitopes exceeds the antiviral responses in patients with acute hepatitis C virus infection. Importantly, they endow mixed polyfunctional type-1, type-2 and type-17 responses and correlate with the chronic progression of infection. This evolution is related to the selection of autoreactive CD8⁺ T cells with higher T cell receptor avidity, whereas those with lower avidity undergo prompt contraction in patients who clear infection. These findings demonstrate a previously undescribed strict link between the emergence of high frequencies of mixed autoreactive CD8⁺ T cells producing a broad array of cytokines (IFN-γ, IL-17, IL-4, IL-2…) and the progression toward chronic disease in a human model of acute infection.     

High Frequencies of Virus-Specific CD8+ T-Cell Precursors

A productive CD8⁺ T-cell response to a viral infection requires rapid division and proliferation of virus-specific CD8⁺ T cells. Tetramer-based enrichment assays have recently given estimates of the numbers of peptide-major histocompatibility complex-specific CD8⁺ T cells in naïve mice, but precursor frequencies for entire viruses have been examined only by using in vitro limiting-dilution assays (LDAs). To examine CD8⁺ T-cell precursor frequencies for whole viruses, we developed an in vivo LDA and found frequencies of naïve CD8⁺ T-cell precursors of 1 in 1,444 for vaccinia virus (VV) (∼13,850 VV-specific CD8⁺ T cells per mouse) and 1 in 2,958 for lymphocytic choriomeningitis virus (LCMV) (∼6,761 LCMV-specific CD8⁺ T cells per mouse) in C57BL/6J mice. In mice immune to VV, the number of VV-specific precursors, not surprisingly, dramatically increased to 1 in 13 (∼1,538,462 VV-specific CD8⁺ T cells per mouse), consistent with estimates of VV-specific memory T cells. In contrast, precursor numbers for LCMV did not increase in VV-immune mice (1 in 4,562, with ∼4,384 LCMV-specific CD8⁺ T cells per VV-immune mouse). Using H-2D^b-restricted LCMV GP33-specific P14-transgenic T cells, we found that, after donor T-cell take was accounted for, approximately every T cell transferred underwent a full proliferative expansion in response to LCMV infection. This high efficiency was also seen with memory populations, suggesting that most antigen-specific T cells will proliferate extensively at a limiting dilution in response to infections. These results show that frequencies of naïve and memory CD8⁺ T cell precursors for whole viruses can be remarkably high.The immune response to a viral infection often involves the rapid proliferation of CD8⁺ effector T cells that recognize virus-infected targets expressing 8- to 11-amino-acid-long peptides on class I major histocompatibility complex (MHC) molecules. This recognition is mediated by membrane-bound T-cell receptors (TCRs) that are generated through largely random DNA recombination events of the many TCRα and -β genes, encoding polypeptide chains that heterodimerize to form the recognition structure of T cells. The recombination of the segments also involves addition or deletion of nucleotides during the joining process, causing even greater diversity, and these processes allow for a very broad range of T-cell specificities, with a calculated theoretical diversity of ∼10¹⁵ TCRs in the mouse (7). By use of PCR, CDR3 spectratyping, and sequencing techniques, it was estimated that there are approximately 2 × 10⁶ distinct TCR specificities in a mouse spleen (1, 5). This is far below the theoretical level of T-cell diversity, but considering estimates of T-cell degeneracy that propose that a single TCR can recognize up to 10⁶ peptide-MHC (pMHC) complexes (17, 36), it is likely that the functional diversity is much greater than the number of individual TCRs.It has been of interest to calculate the number of T cells that would either recognize or respond to a pathogen or to a specific pMHC complex. Early estimates of numbers of CD8⁺ T cells that are specific to a single virus, i.e., precursor frequencies, took advantage of an in vitro limiting-dilution assay (LDA) and calculated CD8⁺ T-cell virus-specific precursor frequencies to be on the order of 1 in 100,000 in naïve mice and predicted that these cells needed to undergo about 15 divisions to reach the higher precursor frequencies found at day 8 postinfection (29, 30). The efficiency of such assays, however, is relatively poor. Later studies estimated the number of pMHC-specific CD8⁺ T cells in a naïve mouse by CDR3 sequencing. H-2K^d-restricted T cells specific to HLA residues 170 to 179 (HLA 170-179) were sorted by tetramer from human tumor-immunized mice, and their Vβ CDR3 regions were sequenced. After a plateau suggesting that the majority of the different TCRs had been sequenced was reached, exhaustive sequencing was then used to identify the frequencies of these sequences in naïve mice. These studies found that there were about 600 CD8⁺ T cells specific for that pMHC complex in naïve mice (4). A second strategy used an in vivo competition assay with H-2D^b-restricted lymphocytic choriomeningitis virus (LCMV) GP33-specific P14-transgenic T cells to estimate the number of GP33-specific CD8 T cells in naïve mice and calculated the number to be between 100 to 200 cells per mouse (2).Others estimated numbers of pMHC-specific T cells by sequencing the CDR3β regions of antigen-specific T cells that had expanded during an acute infection. By calculating a measure of CDR3 diversity and then assuming a logarithmic distribution of diversity, they extrapolated the number of T-cell clones that responded to an acute infection. With this technique, 300 to 500 H-2D^b-restricted mouse hepatitis virus (MHV)-encoded S510 clonotypes were calculated to be in the central nervous systems of acutely infected mice, with ∼100 to 900 clonotypes calculated to be in chronically infected mice (24). Later studies used a gamma interferon (IFN-γ) capture assay instead of tetramer sorting and estimated 1,100 to 1,500 H-2D^b-restricted S510-specific clonotypes and 600 to 900 clonotypes of the subdominant H-2K^b-restricted MHV S598 peptide-specific T cells in the spleens of acutely infected mice (25). Those studies also estimated that there were 1,000 to 1,200 different H-2D^b-restricted GP33-specific clonotypes that could respond to an LCMV infection.More-recent studies have taken advantage of magnetic tetramer binding enrichment and double tetramer staining of cells from the spleen and lymph nodes of naïve mice to determine pMHC precursor frequencies, with the assumption that most CD8⁺ T cells in a naïve mouse reside in lymphoid organs and will react with tetramers. This technique was first described by Moon et al. for CD4⁺ T cells, and it detected ∼190 I-A^b 2W1S 52-68-specific T cells, ∼20 I-A^b Salmonella enterica serovar Typhimurium FLiC 427-441-specific T cells, and ∼16 I-A^b chicken ovalbumin (OVA) 323-339-specific T cells per mouse (19). This same technique was then used to determine numbers of pMHC-specific CD8⁺ T cells for epitopes derived from a variety of viruses and found 15 to 1,070 pMHC-specific CD8⁺ T cells per mouse, depending on the specificity of the pMHC tetramer (10, 15, 23). Determinations of CD8⁺ T-cell precursor frequencies in humans are currently not experimentally attainable, but exhaustive sequencing of an HLA-A2.1-restricted influenza A virus (IAV) M1 58-66-specific T-cell response has suggested that there are at least 141 different clonotypes that can grow out in response to an in vitro stimulation with peptide, providing a minimum number of T cells that can respond to this pMHC complex in humans (22).Most of the assays estimate the number of T cells specific to single peptides in individual mice. These assays, therefore, do not determine the numbers of CD8⁺ T cells that can proliferate in response to an entire virus, especially if the virus is known to have many epitopes or if epitopes for the virus have not been described. By examining the average number of pMHC-specific CD8⁺ T cells in a naïve mouse and comparing this to the number of pMHC-specific CD8⁺ T cells that are in a mouse at the peak of the T-cell response, it can be calculated that CD8⁺ T cells divide approximately 12 to 14 times after virus infection (23). Considering that the progeny of one precursor after only 12 divisions can result in just over 4,000 cells, and since recent experiments using H-2K^b-restricted chicken OVA 257-264-specific OT-1-transgenic T cells have confirmed that the progeny from a single cell can be detected in a mouse after infection (31), an in vivo LDA was set up to take advantage of the extensive division and proliferation of virus-specific CD8⁺ T cells in order to determine virus-specific CD8⁺ T-cell precursor frequencies.Here, we show that by transferring limiting amounts of carboxyfluorescein succinimidyl ester (CFSE)-labeled Thy1.1⁺ Ly5.2⁺ heterogeneous CD8⁺ T cells into Thy1.2⁺ Ly5.1⁺ hosts, we are able to calculate CD8⁺ T-cell precursor frequencies for whole viruses. Our calculations are based on finding the number of donor CD8⁺ T cells that results in low-level-CFSE (CFSElo) (i.e., proliferated) donor CD8 T cells in 50% of the hosts. Using probit or Reed and Muench 50% endpoint calculations (3, 26), we are able to calculate CD8⁺ T-cell precursor frequencies. We show here that frequencies of naïve CD8⁺ T-cell precursors for whole viruses are quite high and that our in vivo LDA calculates whole-virus precursor frequencies in line with determinations using other methods with naïve and immune mice.     

Antigen-Specific Expansion of Cytotoxic T Lymphocytes in Acute Measles Virus Infection

Skewing of the T-cell receptor repertoire of CD8⁺ T cells has been shown in some persistent infections with viruses, such as human immunodeficiency virus, simian immunodeficiency virus, and Epstein-Barr virus. We have demonstrated that similar distortions also occur in nonpersistent measles virus infection. In addition, two of four children immunized with live, attenuated measles virus showed larger and more persistent CD8⁺ T-cell expansions than their naturally infected counterparts. The expanded lymphocyte populations were monoclonal or oligoclonal and lysed target cells infected with recombinant vaccinia virus expressing measles virus protein. These results demonstrate that the expansions of CD8⁺ T lymphocytes are antigen driven.     

Protective Efficacy of Cross-Reactive CD8+ T Cells Recognising Mutant Viral Epitopes Depends on Peptide-MHC-I Structural Interactions and T Cell Activation Threshold

Emergence of a new influenza strain leads to a rapid global spread of the virus due to minimal antibody immunity. Pre-existing CD8⁺ T-cell immunity directed towards conserved internal viral regions can greatly ameliorate the disease. However, mutational escape within the T cell epitopes is a substantial issue for virus control and vaccine design. Although mutations can result in a loss of T cell recognition, some variants generate cross-reactive T cell responses. In this study, we used reverse genetics to modify the influenza NP_336–374 peptide at a partially-solvent exposed residue (N->A, NPN3A mutation) to assess the availability, effectiveness and mechanism underlying influenza-specific cross-reactive T cell responses. The engineered virus induced a diminished CD8⁺ T cell response and selected a narrowed T cell receptor (TCR) repertoire within two Vβ regions (Vβ8.3 and Vβ9). This can be partially explained by the H-2D^bNPN3A structure that showed a loss of several contacts between the NPN3A peptide and H-2D^b, including a contact with His155, a position known to play an important role in mediating TCR-pMHC-I interactions. Despite these differences, common cross-reactive TCRs were detected in both the naïve and immune NPN3A-specific TCR repertoires. However, while the NPN3A epitope primes memory T-cells that give an equivalent recall response to the mutant or wild-type (wt) virus, both are markedly lower than wt->wt challenge. Such decreased CD8⁺ responses elicited after heterologous challenge resulted in delayed viral clearance from the infected lung. Furthermore, mice first exposed to the wt virus give a poor, low avidity response following secondary infection with the mutant. Thus, the protective efficacy of cross-reactive CD8⁺ T cells recognising mutant viral epitopes depend on peptide-MHC-I structural interactions and functional avidity. Our study does not support vaccine strategies that include immunization against commonly selected cross-reactive variants with mutations at partially-solvent exposed residues that have characteristics comparable to NPN3A.     

Impaired hepatitis C virus (HCV)-specific effector CD8+ T cells undergo massive apoptosis in the peripheral blood during acute HCV infection and in the liver during the chronic phase of infection

A majority of patients infected with hepatitis C virus (HCV) do not sustain an effective T-cell response, and viremia persists. The mechanism leading to failure of the HCV-specific CD8⁺ T-cell response in patients developing chronic infection is unclear. We investigated apoptosis susceptibility of HCV-specific CD8⁺ T cells during the acute and chronic stages of infection. Although HCV-specific CD8⁺ T cells in the blood during the acute phase of infection and in the liver during the chronic phase were highly activated and expressed an effector phenotype, the majority was undergoing apoptosis. In contrast, peripheral blood HCV-specific CD8⁺ T cells during the chronic phase expressed a resting memory phenotype. Apoptosis susceptibility of HCV-specific CD8⁺ T cells was associated with very high levels of programmed death-1 (PD-1) and low CD127 expression and with significant functional T-cell deficits. Further evaluation of the “death phase” of HCV-specific CD8⁺ T cells during acute HCV infection showed that the majority of cells were dying by a process of cytokine withdrawal, mediated by activated caspase 9. Contraction during the acute phase occurred rapidly via this process despite the persistence of the virus. Remarkably, in the chronic phase of HCV infection, at the site of infection in the liver, a substantial frequency of caspase 9-mediated T-cell death was also present. This study highlights the importance of cytokine deprivation-mediated apoptosis with consequent down-modulation of the immune response to HCV during acute and chronic infections.     

Dynamic Interaction between STLV-1 Proviral Load and T-Cell Response during Chronic Infection and after Immunosuppression in Non-Human Primates

We used mandrills (Mandrillus sphinx) naturally infected with simian T-cell leukemia virus type 1 (STLV-1) as a model for evaluating the influence of natural STLV-1 infection on the dynamics and evolution of the immune system during chronic infection. Furthermore, in order to evaluate the role of the immune system in controlling the infection during latency, we induced immunosuppression in the infected monkeys. We first showed that the STLV-1 proviral load was higher in males than in females and increased significantly with the duration of infection: mandrills infected for 10–6 years had a significantly higher proviral load than those infected for 2–4 years. Curiously, this observation was associated with a clear reduction in CD4+ T-cell number with age. We also found that the percentage of CD4⁺ T cells co-expressing the activation marker HLA-DR and the mean percentage of CD25⁺ in CD4⁺ and CD8⁺ T cells were significantly higher in infected than in uninfected animals. Furthermore, the STLV-1 proviral load correlated positively with T-cell activation but not with the frequency of T cells secreting interferon γ in response to Tax peptides. Lastly, we showed that, during immunosuppression in infected monkeys, the percentages of CD8⁺ T cells expressing HLA-DR⁺ and of CD4⁺ T cells expressing the proliferation marker Ki67 decreased significantly, although the percentage of CD8⁺ T cells expressing HLA-DR⁺ and Ki67 increased significantly by the end of treatment. Interestingly, the proviral load increased significantly after immunosuppression in the monkey with the highest load. Our study demonstrates that mandrills naturally infected with STLV-1 could be a suitable model for studying the relations between host and virus. Further studies are needed to determine whether the different compartments of the immune response during infection induce the long latency by controlling viral replication over time. Such studies would provide important information for the development of immune-based therapeutic strategies.     

Human Mucosal Associated Invariant T Cells Detect Bacterially Infected Cells

Control of infection with Mycobacterium tuberculosis (Mtb) requires Th1-type immunity, of which CD8⁺ T cells play a unique role. High frequency Mtb-reactive CD8⁺ T cells are present in both Mtb-infected and uninfected humans. We show by limiting dilution analysis that nonclassically restricted CD8⁺ T cells are universally present, but predominate in Mtb-uninfected individuals. Interestingly, these Mtb-reactive cells expressed the Vα7.2 T-cell receptor (TCR), were restricted by the nonclassical MHC (HLA-Ib) molecule MR1, and were activated in a transporter associated with antigen processing and presentation (TAP) independent manner. These properties are all characteristics of mucosal associated invariant T cells (MAIT), an “innate” T-cell population of previously unknown function. These MAIT cells also detect cells infected with other bacteria. Direct ex vivo analysis demonstrates that Mtb-reactive MAIT cells are decreased in peripheral blood mononuclear cells (PBMCs) from individuals with active tuberculosis, are enriched in human lung, and respond to Mtb-infected MR1-expressing lung epithelial cells. Overall, these findings suggest a generalized role for MAIT cells in the detection of bacterially infected cells, and potentially in the control of bacterial infection.     

Random T-cell receptor recruitment in human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T cells from genetically identical twins infected with the same HIV-1 strain

Human immunodeficiency virus type 1 (HIV-1) cytotoxic T-lymphocyte escape mutations represent both a major reason for loss of HIV immune control and a considerable challenge for HIV-1 vaccine design. Previous data suggest that initial HIV-1-specific CD8⁺ T-cell responses are determined largely by viral and host genetics, but the mechanisms influencing the subsequent viral evolution are unclear. Here, we show a random recruitment of T-cell receptor (TCR) alpha and beta clonotypes of the initial HIV-1-specific CD8⁺ T cells during primary infection in two genetically identical twins infected simultaneously with the same virus, suggesting that stochastic TCR recruitment of HIV-1-specific CD8⁺ T cells contributes to the diverse and unpredictable HIV-1 sequence evolution.     

Distinct Mechanisms Trigger Apoptosis in Human Immunodeficiency Virus Type 1-Infected and in Uninfected Bystander T Lymphocytes

Apoptosis is a main feature of AIDS pathogenesis and is thought to play a role in the progressive decrease of CD4⁺ T lymphocytes in infected individuals. To determine whether apoptosis occurs in infected and/or in uninfected peripheral blood T lymphocytes, we have used a recombinant human immunodeficiency virus type 1 (HIV-1) infectious clone expressing the green fluorescent protein (GFP). Using flow cytometry, we have determined the incidence of apoptosis by either terminal transferase dUTP nick end labeling or annexin-V assays in different cell subpopulations, i.e., in CD4⁺ or CD8⁺ T cells that were GFP positive or negative. After HIV-1 infection of purified peripheral blood lymphocytes, we observed that apoptosis occurred mostly in infected CD4⁺ peripheral blood lymphocytes. Remarkably, the presence of monocyte-derived macrophages in the culture increased dramatically the apoptosis of uninfected bystander T lymphocytes, while apoptosis in HIV-infected T lymphocytes was not changed. We therefore demonstrate that HIV-induced apoptosis results from at least two distinct mechanisms: (i) direct apoptosis in HIV-infected CD4⁺ T lymphocytes and (ii) indirect apoptosis in uninfected T cells mediated by antigen-presenting cells.     

Genome-wide characterization of a viral cytotoxic T lymphocyte epitope repertoire

A genome-wide search using major histocompatibility complex (MHC) class I binding and proteosome cleavage site algorithms identified 101 influenza A PR8 virus-derived peptides as potential epitopes for CD8+ T cell recognition in the H-2b mouse. Cytokine-based flow cytometry, ELISPOT, and cytotoxic T lymphocyte assays reveal that 16 are recognized by CD8+ T cells recovered directly ex vivo from infected animals, accounting for greater than 70% of CD8+ T cells recruited to lung after primary infection. Only six of the 22 highest affinity MHC class I binding peptides comprise cytotoxic T lymphocyte epitopes. The remaining non-immunogenic peptides have equivalent MHC affinity and MHC-peptide complex half-lives, eliciting T cell responses when given in adjuvant and with T cell receptor-ligand avidity comparable with their immunogenic counterparts. As revealed by a novel high sensitivity nanospray tandem mass spectrometry methodology, failure to process those predicted epitopes may contribute significantly to the absent response. These results have important implications for rationale design of CD8+ T cell vaccines.     

Evidence of CD8+ T-cell-mediated selective pressure on human immunodeficiency virus type 1 nef in HLA-B*57+ elite suppressors

Elite suppressors (ES) are human immunodeficiency virus type 1 (HIV-1)-infected patients who maintain viral loads of <50 copies/ml without treatment. The observation that the HLA-B*57 allele is overrepresented in these patients implies that HIV-1-specific CD8⁺ T cells play a key role in suppressing viral replication. We have previously shown that while CD8⁺ T-cell escape mutations are rarely seen in proviral Gag sequences in resting CD4⁺ T cells from peripheral blood, they are present in every clone amplified from the low levels of free virus in the plasma of HLA-B*57⁺ ES. In this study, we compared the pattern of mutations in Nef sequences amplified from peripheral blood CD4⁺ T cells and from plasma virus. We show that Nef mutations are present in plasma virus but are rare in the cellular sequences and provide evidence that these plasma Nef variants represent novel escape mutants. The results provide further evidence of CD8⁺ T-cell-mediated selective pressure on plasma virus in ES and suggest that there must be ongoing HIV-1 replication in spite of the very low viral loads seen for these patients.     

Ligand-independent exhaustion of killer immunoglobulin-like receptor-positive CD8+ T cells in human immunodeficiency virus type 1 infection

Virus-specific CD8⁺ T cells play a central role in the control of viral infections, including human immunodeficiency virus type 1 (HIV-1) infection. However, despite the presence of strong and broad HIV-specific CD8⁺ T-cell responses in chronic HIV-1 infection, these cells progressively lose critical effector functions and fail to clear the infection. Mounting evidence suggests that the upregulation of several inhibitory regulatory receptors on the surface of CD8⁺ T cells during HIV-1 infection may contribute directly to the impairment of T-cell function. Here, we investigated the role of killer immunoglobulin receptors (KIR), which are expressed on NK cells and on CD8⁺ T cells, in regulating CD8⁺ T-cell function in HIV-1 infection. KIR expression was progressively upregulated on CD8⁺ T cells during HIV-1 infection and correlated with the level of viral replication. Expression of KIR was associated with a profound inhibition of cytokine secretion, degranulation, proliferation, and activation by CD8⁺ T cells following stimulation with T-cell receptor (TCR)-dependent stimuli. In contrast, KIR⁺ CD8⁺ T cells responded potently to TCR-independent stimulation, demonstrating that these cells are functionally competent. KIR-associated suppression of CD8⁺ T-cell function was independent of ligand engagement, suggesting that these regulatory receptors may constitutively repress TCR activation. This ligand-independent repression of TCR activation of KIR⁺ CD8⁺ T cells may represent a significant barrier to therapeutic interventions aimed at improving the quality of the HIV-specific CD8⁺ T-cell response in infected individuals.     

Transduction of Human T Cells with a Novel T-Cell Receptor Confers Anti-HCV Reactivity

Hepatitis C Virus (HCV) is a major public health concern, with no effective vaccines currently available and 3% of the world''s population being infected. Despite the existence of both B- and T-cell immunity in HCV-infected patients, chronic viral infection and HCV-related malignancies progress. Here we report the identification of a novel HCV TCR from an HLA-A2-restricted, HCV NS3:1073–1081-reactive CTL clone isolated from a patient with chronic HCV infection. We characterized this HCV TCR by expressing it in human T cells and analyzed the function of the resulting HCV TCR-transduced cells. Our results indicate that both the HCV TCR-transduced CD4⁺ and CD8⁺ T cells recognized the HCV NS3:1073–1081 peptide-loaded targets and HCV⁺ hepatocellular carcinoma cells (HCC) in a polyfunctional manner with cytokine (IFN-γ, IL-2, and TNF-α) production as well as cytotoxicity. Tumor cell recognition by HCV TCR transduced CD8⁻ Jurkat cells and CD4⁺ PBL-derived T cells indicated this TCR was CD8-independent, a property consistent with other high affinity TCRs. HCV TCR-transduced T cells may be promising for the treatment of patients with chronic HCV infections.     

Antiviral antibodies are necessary to prevent cytotoxic T-lymphocyte escape in mice infected with a coronavirus

Mutation within virus-derived CD8 T-cell epitopes can effectively abrogate cytotoxic T-lymphocyte (CTL) recognition and impede virus clearance in infected hosts. These so-called “CTL escape variant viruses” are commonly selected during persistent infections and are associated with rapid disease progression and increased disease severity. Herein, we tested whether antiviral antibody-mediated suppression of virus replication and subsequent virus clearance were necessary for preventing CTL escape in coronavirus-infected mice. We found that compared to wild-type mice, B-cell-deficient mice did not efficiently clear infectious virus, uniformly developed clinical disease, and harbored CTL escape variant viruses. These data directly demonstrate a critical role for antiviral antibody in protecting from the selective outgrowth of CTL escape variant viruses.