IL-15 promotes activation and expansion of CD8+ T cells in HIV-1 infection

In HIV-1–infected patients, increased numbers of circulating CD8⁺ T cells are linked to increased risk of morbidity and mortality. Here, we identified a bystander mechanism that promotes CD8 T cell activation and expansion in untreated HIV-1–infected patients. Compared with healthy controls, untreated HIV-1–infected patients have an increased population of proliferating, granzyme B⁺, CD8⁺ T cells in circulation. Vβ expression and deep sequencing of CDR3 revealed that in untreated HIV-1 infection, cycling memory CD8 T cells possess a broad T cell repertoire that reflects the repertoire of the resting population. This suggests that cycling is driven by bystander activation, rather than specific antigen exposure. Treatment of peripheral blood mononuclear cells with IL-15 induced a cycling, granzyme B⁺ phenotype in CD8⁺ T cells. Moreover, elevated IL-15 expression in the lymph nodes of untreated HIV-1–infected patients correlated with circulating CD8⁺ T cell counts and was normalized in these patients following antiretroviral therapy. Together, these results suggest that IL-15 drives bystander activation of CD8⁺ T cells, which predicts disease progression in untreated HIV-1–infected patients and suggests that elevated IL-15 may also drive CD8⁺ T cell expansion that is linked to increased morbidity and mortality in treated patients.     

Dual-Affinity Re-Targeting proteins direct T cell–mediated cytolysis of latently HIV-infected cells

Enhancement of HIV-specific immunity is likely required to eliminate latent HIV infection. Here, we have developed an immunotherapeutic modality aimed to improve T cell–mediated clearance of HIV-1–infected cells. Specifically, we employed Dual-Affinity Re-Targeting (DART) proteins, which are bispecific, antibody-based molecules that can bind 2 distinct cell-surface molecules simultaneously. We designed DARTs with a monovalent HIV-1 envelope-binding (Env-binding) arm that was derived from broadly binding, antibody-dependent cellular cytotoxicity–mediating antibodies known to bind to HIV-infected target cells coupled to a monovalent CD3 binding arm designed to engage cytolytic effector T cells (referred to as HIVxCD3 DARTs). Thus, these DARTs redirected polyclonal T cells to specifically engage with and kill Env-expressing cells, including CD4⁺ T cells infected with different HIV-1 subtypes, thereby obviating the requirement for HIV-specific immunity. Using lymphocytes from patients on suppressive antiretroviral therapy (ART), we demonstrated that DARTs mediate CD8⁺ T cell clearance of CD4⁺ T cells that are superinfected with the HIV-1 strain JR-CSF or infected with autologous reservoir viruses isolated from HIV-infected–patient resting CD4⁺ T cells. Moreover, DARTs mediated CD8⁺ T cell clearance of HIV from resting CD4⁺ T cell cultures following induction of latent virus expression. Combined with HIV latency reversing agents, HIVxCD3 DARTs have the potential to be effective immunotherapeutic agents to clear latent HIV-1 reservoirs in HIV-infected individuals.     

Potent inhibition of HIV-1 by TRIM5-cyclophilin fusion proteins engineered from human components

New World monkeys of the genus Aotus synthesize a fusion protein (AoT5Cyp) containing tripartite motif-containing 5 (TRIM5) and cyclophilin A (CypA) that potently blocks HIV-1 infection. We attempted to generate a human HIV-1 inhibitor modeled after AoT5Cyp, by fusing human CypA to human TRIM5 (hT5Cyp). Of 13 constructs, 3 showed substantial HIV-1–inhibitory activity when expressed in human cell lines. This activity required capsid binding by CypA and correlated with CypA linkage to the TRIM5a capsid-specificity determinant and the ability to form cytoplasmic bodies. CXCR4- and CCR5-tropic HIV-1 clones and primary isolates were inhibited from infecting multiple human macrophage and T cell lines and primary cells by hT5Cyp, as were HIV-2_ROD, SIV_AGMtan, FIV_PET, and a circulating HIV-1 isolate previously reported to be AoT5Cyp resistant. The anti–HIV-1 activity of hT5Cyp was surprisingly more effective than that of the well-characterized rhesus TRIM5α, especially in T cells. hT5Cyp also blocked HIV-1 infection of primary CD4⁺ T cells and macrophages and conferred a survival advantage to these cells without disrupting their function. Extensive attempts to elicit HIV-1 resistance to hT5Cyp were unsuccessful. Finally, Rag2^–/–γc^–/– mice were engrafted with human CD4⁺ T cells that had been transduced by optimized lentiviral vectors bearing hT5Cyp. Upon challenge with HIV-1, these mice showed decreased viremia and productive infection in lymphoid organs and preserved numbers of human CD4⁺ T cells. We conclude that hT5Cyp is an extraordinarily robust inhibitor of HIV-1 replication and a promising anti–HIV-1 gene therapy candidate.     

Tim-3 expression defines a novel population of dysfunctional T cells with highly elevated frequencies in progressive HIV-1 infection

Progressive loss of T cell functionality is a hallmark of chronic infection with human immunodeficiency virus 1 (HIV-1). We have identified a novel population of dysfunctional T cells marked by surface expression of the glycoprotein Tim-3. The frequency of this population was increased in HIV-1–infected individuals to a mean of 49.4 ± SD 12.9% of CD8⁺ T cells expressing Tim-3 in HIV-1–infected chronic progressors versus 28.5 ± 6.8% in HIV-1–uninfected individuals. Levels of Tim-3 expression on T cells from HIV-1–infected inviduals correlated positively with HIV-1 viral load and CD38 expression and inversely with CD4⁺ T cell count. In progressive HIV-1 infection, Tim-3 expression was up-regulated on HIV-1–specific CD8⁺ T cells. Tim-3–expressing T cells failed to produce cytokine or proliferate in response to antigen and exhibited impaired Stat5, Erk1/2, and p38 signaling. Blocking the Tim-3 signaling pathway restored proliferation and enhanced cytokine production in HIV-1–specific T cells. Thus, Tim-3 represents a novel target for the therapeutic reversal of HIV-1–associated T cell dysfunction.     

Relationship of SARS-CoV-2–specific CD4 response to COVID-19 severity and impact of HIV-1 and tuberculosis coinfection

T cells are involved in control of coronavirus disease 2019 (COVID-19), but limited knowledge is available on the relationship between antigen-specific T cell response and disease severity. Here, we used flow cytometry to assess the magnitude, function, and phenotype of SARS coronavirus 2–specific (SARS-CoV-2–specific) CD4⁺ T cells in 95 hospitalized COVID-19 patients, 38 of them being HIV-1 and/or tuberculosis (TB) coinfected, and 38 non–COVID-19 patients. We showed that SARS-CoV-2–specific CD4⁺ T cell attributes, rather than magnitude, were associated with disease severity, with severe disease being characterized by poor polyfunctional potential, reduced proliferation capacity, and enhanced HLA-DR expression. Moreover, HIV-1 and TB coinfection skewed the SARS-CoV-2 T cell response. HIV-1–mediated CD4⁺ T cell depletion associated with suboptimal T cell and humoral immune responses to SARS-CoV-2, and a decrease in the polyfunctional capacity of SARS-CoV-2–specific CD4⁺ T cells was observed in COVID-19 patients with active TB. Our results also revealed that COVID-19 patients displayed reduced frequency of Mycobacterium tuberculosis–specific CD4⁺ T cells, with possible implications for TB disease progression. These results corroborate the important role of SARS-CoV-2–specific T cells in COVID-19 pathogenesis and support the concept of altered T cell functions in patients with severe disease.     

Factors Secreted by Human T Lymphotropic Virus Type I (HTLV-I)–infected Cells Can Enhance or Inhibit Replication of HIV-1 in HTLV-I–uninfected Cells: Implications for In Vivo Coinfection with HTLV-I and HIV-1

It remains controversial whether human T lymphotropic virus type I (HTLV-I) coinfection leads to more rapid progression of human immunodeficiency virus (HIV) disease in dually infected individuals. To investigate whether HTLV-I infection of certain cells can modulate HIV-1 infection of surrounding cells, primary CD4⁺ T cells were treated with cell-free supernatants from HTLV-I–infected MT-2 cell cultures. The primary CD4⁺ T cells became resistant to macrophage (M)-tropic HIV-1 but highly susceptible to T cell (T)-tropic HIV-1. The CC chemokines RANTES (regulated on activation, normal T cell expressed and secreted), macrophage inflammatory protein (MIP)-1α, and MIP-1β in the MT-2 cell supernatants were identified as the major suppressive factors for M-tropic HIV-1 as well as the enhancers of T-tropic HIV-1 infection, whereas soluble Tax protein increased susceptibility to both M- and T-tropic HIV-1. The effect of Tax or CC chemokines on T-tropic HIV-1 was mediated, at least in part, by increasing HIV Env-mediated fusogenicity. Our data suggest that the net effect of HTLV-I coinfection in HIV-infected individuals favors the transition from M- to T-tropic HIV phenotype, which is generally indicative of progressive HIV disease.     

ESAT-6–dependent cytosolic pattern recognition drives noncognate tuberculosis control in vivo

IFN-γ is a critical mediator of host defense against Mycobacterium tuberculosis (Mtb) infection. Antigen-specific CD4⁺ T cells have long been regarded as the main producer of IFN-γ in tuberculosis (TB), and CD4⁺ T cell immunity is the main target of current TB vaccine candidates. However, given the recent failures of such a TB vaccine candidate in clinical trials, strategies to harness CD4-independent mechanisms of protection should be included in future vaccine design. Here, we have reported that noncognate IFN-γ production by Mtb antigen–independent memory CD8⁺ T cells and NK cells is protective during Mtb infection and evaluated the mechanistic regulation of IFN-γ production by these cells in vivo. Transfer of arenavirus- or protein-specific CD8⁺ T cells or NK cells reduced the mortality and morbidity rates of mice highly susceptible to TB in an IFN-γ–dependent manner. Secretion of IFN-γ by these cell populations required IL-18, sensing of mycobacterial viability, Mtb protein 6-kDa early secretory antigenic target–mediated (ESAT-6–mediated) cytosolic contact, and activation of NLR family pyrin domain–containing protein 3 (NLRP3) inflammasomes in CD11c⁺ cell subsets. Neutralization of IL-18 abrogated protection in susceptible recipient mice that had received noncognate cells. Moreover, improved Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine–induced protection was lost in the absence of ESAT-6–dependent cytosolic contact. Our findings provide a comprehensive mechanistic framework for antigen-independent IFN-γ secretion in response to Mtb with critical implications for future intervention strategies against TB.     

Integration features of intact latent HIV-1 in CD4+ T cell clones contribute to viral persistence

Latent intact HIV-1 proviruses persist in a small subset of long-lived CD4⁺ T cells that can undergo clonal expansion in vivo. Expanded clones of CD4⁺ T cells dominate latent reservoirs in individuals on long-term antiretroviral therapy (ART) and represent a major barrier to HIV-1 cure. To determine how integration landscape might contribute to latency, we analyzed integration sites of near full length HIV-1 genomes from individuals on long-term ART, focusing on individuals whose reservoirs are highly clonal. We find that intact proviruses in expanded CD4⁺ T cell clones are preferentially integrated within Krüppel-associated box (KRAB) domain–containing zinc finger (ZNF) genes. ZNF genes are associated with heterochromatin in memory CD4⁺ T cells; nevertheless, they are expressed in these cells under steady-state conditions. In contrast to genes carrying unique integrations, ZNF genes carrying clonal intact integrations are down-regulated upon cellular activation. Together, the data suggest selected genomic sites, including ZNF genes, can be especially permissive for maintaining HIV-1 latency during memory CD4⁺ T cell expansion.     

Productive Infection of Neonatal CD8+ T Lymphocytes by HIV-1

CD8⁺ T lymphocytes confer significant but ultimately insufficient protection against HIV infection. Here we report that activated neonatal CD8⁺ T cells can be productively infected in vitro by macrophage-tropic (M-tropic) HIV-1 isolates, which are responsible for disease transmission, whereas they are resistant to T cell–tropic (T-tropic) HIV strains. Physiological activation of CD8-α/β⁺ CD4⁻ T cell receptor–α/β⁺ neonatal T cells, including activation by allogeneic dendritic cells, induces the accumulation of CD4 messenger RNA and the expression of CD4 Ag on the cell surface. The large majority of anti-CD3/B7.1–activated cord blood CD8⁺ T cells coexpress CD4, the primary HIV receptor, as well as CCR5 and CXCR4, the coreceptors used by M- and T-tropic HIV-1 strains, respectively, to enter target cells. These findings are relevant to the rapid progression of neonatal HIV infection. Infection of primary HIV-specific CD8⁺ T cells may compromise their survival and thus significantly contribute to the failure of the immune system to control the infection. Furthermore, these results indicate a previously unsuspected level of plasticity in the neonatal immune system in the regulation of CD4 expression by costimulation.     

HLA-E–restricted HIV-1–specific CD8+ T cell responses in natural infection

CD8⁺ T cell responses restricted by MHC-E, a nonclassical MHC molecule, have been associated with protection in an SIV/rhesus macaque model. The biological relevance of HLA-E–restricted CD8⁺ T cell responses in HIV infection, however, remains unknown. In this study, CD8⁺ T cells responding to HIV-1 Gag peptides presented by HLA-E were analyzed. Using in vitro assays, we observed HLA-E–restricted T cell responses to what we believe to be a newly identified subdominant Gag-KL9 as well as a well-described immunodominant Gag-KF11 epitope in T cell lines derived from chronically HIV-infected patients and also primed from healthy donors. Blocking of the HLA-E/KF11 binding by the B7 signal peptide resulted in decreased CD8⁺ T cell responses. KF11 presented via HLA-E in HIV-infected cells was recognized by antigen-specific CD8⁺ T cells. Importantly, bulk CD8⁺ T cells obtained from HIV-infected individuals recognized infected cells via HLA-E presentation. Ex vivo analyses at the epitope level showed a higher responder frequency of HLA-E–restricted responses to KF11 compared with KL9. Taken together, our findings of HLA-E–restricted HIV-specific immune responses offer intriguing and possibly paradigm-shifting insights into factors that contribute to the immunodominance of CD8⁺ T cell responses in HIV infection.     

Engagement of Cytotoxic T Lymphocyte–associated Antigen 4 (CTLA-4) Induces Transforming Growth Factor β (TGF-β) Production by Murine CD4+ T Cells

Evidence indicates that cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) may negatively regulate T cell activation, but the basis for the inhibitory effect remains unknown. We report here that cross-linking of CTLA-4 induces transforming growth factor β (TGF-β) production by murine CD4⁺ T cells. CD4⁺ T helper type 1 (Th1), Th2, and Th0 clones all secrete TGF-β after antibody cross-linking of CTLA-4, indicating that induction of TGF-β by CTLA-4 signaling represents a ubiquitous feature of murine CD4⁺ T cells. Stimulation of the CD3–T cell antigen receptor complex does not independently induce TGF-β, but is required for optimal CTLA-4–mediated TGF-β production. The consequences of cross-linking of CTLA-4, together with CD3 and CD28, include inhibition of T cell proliferation and interleukin (IL)-2 secretion, as well as suppression of both interferon γ (Th1) and IL-4 (Th2). Moreover, addition of anti–TGF-β partially reverses this T cell suppression. When CTLA-4 was cross-linked in T cell populations from TGF-β1 gene–deleted (TGF-β1^−/−) mice, the T cell responses were only suppressed 38% compared with 95% in wild-type mice. Our data demonstrate that engagement of CTLA-4 leads to CD4⁺ T cell production of TGF-β, which, in part, contributes to the downregulation of T cell activation. CTLA-4, through TGF-β, may serve as a counterbalance for CD28 costimulation of IL-2 and CD4⁺ T cell activation.     

PD1-based DNA vaccine amplifies HIV-1 GAG-specific CD8+ T cells in mice

Viral vector–based vaccines that induce protective CD8⁺ T cell immunity can prevent or control pathogenic SIV infections, but issues of preexisting immunity and safety have impeded their implementation in HIV-1. Here, we report the development of what we believe to be a novel antigen-targeting DNA vaccine strategy that exploits the binding of programmed death-1 (PD1) to its ligands expressed on dendritic cells (DCs) by fusing soluble PD1 with HIV-1 GAG p24 antigen. As compared with non–DC-targeting vaccines, intramuscular immunization via electroporation (EP) of the fusion DNA in mice elicited consistently high frequencies of GAG-specific, broadly reactive, polyfunctional, long-lived, and cytotoxic CD8⁺ T cells and robust anti-GAG antibody titers. Vaccination conferred remarkable protection against mucosal challenge with vaccinia GAG viruses. Soluble PD1–based vaccination potentiated CD8⁺ T cell responses by enhancing antigen binding and uptake in DCs and activation in the draining lymph node. It also increased IL-12–producing DCs and engaged antigen cross-presentation when compared with anti-DEC205 antibody-mediated DC targeting. The high frequency of durable and protective GAG-specific CD8⁺ T cell immunity induced by soluble PD1–based vaccination suggests that PD1-based DNA vaccines could potentially be used against HIV-1 and other pathogens.     

Upregulation of Tim-3 and PD-1 expression is associated with tumor antigen–specific CD8+ T cell dysfunction in melanoma patients

The paradoxical coexistence of spontaneous tumor antigen–specific immune responses with progressive disease in cancer patients furthers the need to dissect the molecular pathways involved in tumor-induced T cell dysfunction. In patients with advanced melanoma, we have previously shown that the cancer-germline antigen NY-ESO-1 stimulates spontaneous NY-ESO-1–specific CD8⁺ T cells that up-regulate PD-1 expression. We also observed that PD-1 regulates NY-ESO-1–specific CD8⁺ T cell expansion upon chronic antigen stimulation. In the present study, we show that a fraction of PD-1⁺ NY-ESO-1–specific CD8⁺ T cells in patients with advanced melanoma up-regulates Tim-3 expression and that Tim-3⁺PD-1⁺ NY-ESO-1–specific CD8⁺ T cells are more dysfunctional than Tim-3⁻PD-1⁺ and Tim-3⁻PD-1⁻ NY-ESO-1–specific CD8⁺ T cells, producing less IFN-γ, TNF, and IL-2. Tim-3–Tim-3L blockade enhanced cytokine production by NY-ESO-1–specific CD8⁺ T cells upon short ex vivo stimulation with cognate peptide, thus enhancing their functional capacity. In addition, Tim-3–Tim-3L blockade enhanced cytokine production and proliferation of NY-ESO-1–specific CD8⁺ T cells upon prolonged antigen stimulation and acted in synergy with PD-1–PD-L1 blockade. Collectively, our findings support the use of Tim-3–Tim-3L blockade together with PD-1–PD-L1 blockade to reverse tumor-induced T cell exhaustion/dysfunction in patients with advanced melanoma.There is ample evidence that patients with melanoma can develop immune responses directed against antigens expressed by their own tumor (Boon et al., 2006). Among these antigens, cancer-germline antigens (CGAs) are expressed by tumors of many different histological types, including melanoma, but not by normal tissues, except testis. Because germ cells in testis do not express HLA molecules on their surface (Haas et al., 1988), CGAs represent strictly tumor-specific T cell targets (Boon et al., 2006). Among CGAs, NY-ESO-1 has been shown to stimulate spontaneous cellular and humoral responses that are detectable only in patients with advanced NY-ESO-1–expressing cancer (Stockert et al., 1998; Jäger et al., 2000; Mandic et al., 2005; Fourcade et al., 2008). Understanding the failure of spontaneous NY-ESO-1–specific T cell responses to promote regression of NY-ESO-1⁺ tumors is therefore critical for the design of novel therapeutic interventions aimed at overcoming tumor-induced immune escape.We have previously shown that the large majority of spontaneous NY-ESO-1–specific CD8⁺ T cells up-regulates programmed death 1 (PD-1) expression (Fourcade et al., 2009), which appears to be associated with T cell exhaustion/dysfunction in chronic viral infections in animals and humans (Barber et al., 2006; Day et al., 2006; Petrovas et al., 2006; Trautmann et al., 2006). We observed that PD-1 up-regulation on spontaneous NY-ESO-1–specific CD8⁺ T cells occurs along with T cell activation and is not directly associated with an inability to produce cytokines ex vivo upon stimulation with cognate antigen. Blockade of the PD-1–programmed death ligand 1 (PD-L1) pathway in combination with prolonged antigen stimulation with PD-L1⁺ APCs or melanoma cells augmented the frequencies of cytokine-producing, proliferating, and total NY-ESO-1–specific CD8⁺ T cells. Our findings are in line with previous studies of PD-1 expression by HIV- and SIV-specific CD8⁺ T cells, demonstrating that PD-1 is a regulator of antigen-specific CD8⁺ T cell expansion in the context of chronic antigen exposure, although it does not exhibit a major impact upon their functionality on a cell-per-cell basis (Petrovas et al., 2006, 2007). To further determine whether other molecular pathways are involved in tumor antigen–specific T cell dysfunction, we studied T cell immunoglobulin and mucin-domain–containing molecule 3 (Tim-3) expression on spontaneous NY-ESO-1–specific CD8⁺ T cells from patients with advanced melanoma and investigated whether Tim-3 up-regulation defines a subgroup of dysfunctional tumor antigen–specific CD8⁺ T cells. Tim-3 is a transmembrane protein constitutively expressed on Th1/Tc1 cells in mice and humans (Monney et al., 2002). Several lines of evidence support the role of Tim-3 as an inhibitory molecule that down-regulates effector Th1/Tc1 cell responses. In mice, blocking the Tim-3–Tim-3L pathway resulted in hyperproliferation of Th1-type cells and abrogated the induction of peripheral and transplantation tolerance (Sabatos et al., 2003; Sánchez-Fueyo et al., 2003). Tim-3 interacts with its ligand galectin-9 to induce cell death in Th1 cells (Zhu et al., 2005). In humans, Tim-3 expression is defective in CD4⁺ T cells producing high levels of IFN-γ, as well as those isolated from cerebrospinal fluid of patients with multiple sclerosis (Koguchi et al., 2006). Recently, Tim-3 up-regulation has been reported in HIV-specific and HCV-specific CD8⁺ T cells in patients with progressive HIV infection and chronic hepatitis C, respectively (Jones et al., 2008; Golden-Mason et al., 2009). Tim-3⁺ HIV- and HCV-specific CD8⁺ T cells were distinct from the PD-1⁺ CD8⁺ T cells and exhibited T cell dysfunction. However, it is unknown whether tumor antigen–specific CD8⁺ T cells in patients with advanced cancers express Tim-3.In this study, we show that a fraction of PD-1⁺ NY-ESO-1–specific CD8⁺ T cells, which represents the large majority of circulating NY-ESO-1–specific CD8⁺ T cells in patients with advanced melanoma, up-regulates Tim-3 expression. Tim-3⁺PD-1⁺ NY-ESO-1–specific CD8⁺ T cells are highly dysfunctional compared with Tim-3⁻PD-1⁺ and Tim-3⁻PD-1⁻ NY-ESO-1–specific CD8⁺ T cells. Tim-3–Tim-3L pathway blockade alone or in combination with PD-1–PD-L1 pathway blockade enhanced NY-ESO-1–specific CD8⁺ T cell numbers and functions. Collectively, our findings support the use of Tim-3–Tim-3L blockade in association with PD-1–PD-L1 blockade to reverse tumor-induced T cell exhaustion/dysfunction in patients with advanced melanoma.     

Proteome-wide analysis of HIV-specific naive and memory CD4+ T cells in unexposed blood donors

The preexisting HIV-1–specific T cell repertoire must influence both the immunodominance of T cells after infection and immunogenicity of vaccines. We directly compared two methods for measuring the preexisting CD4⁺ T cell repertoire in healthy HIV-1–negative volunteers, the HLA-peptide tetramer enrichment and T cell library technique, and show high concordance (r = 0.989). Using the library technique, we examined whether naive, central memory, and/or effector memory CD4⁺ T cells specific for overlapping peptides spanning the entire HIV-1 proteome were detectable in 10 HLA diverse, HIV-1–unexposed, seronegative donors. HIV-1–specific cells were detected in all donors at a mean of 55 cells/million naive cells and 38.9 and 34.1 cells/million in central and effector memory subsets. Remarkably, peptide mapping showed most epitopes recognized by naive (88%) and memory (56%) CD4⁺ T cells had been previously reported in natural HIV-1 infection. Furthermore, 83% of epitopes identified in preexisting memory subsets shared epitope length matches (8–12 amino acids) with human microbiome proteins, suggestive of a possible cross-reactive mechanism. These results underline the power of a proteome-wide analysis of peptide recognition by human T cells for the identification of dominant antigens and provide a baseline for optimizing HIV-1–specific helper cell responses by vaccination.Only one candidate HIV vaccine, a canarypox vectored gp120 with a protein boost, has shown any efficacy (Rerks-Ngarm et al., 2009). The limited protection correlated with induction of nonneutralizing antibodies to the VI/V2 region of the virus Envelope protein (Env; Rerks-Ngarm et al., 2009; Haynes et al., 2012). This modest success has stimulated efforts to design vaccines that generate more efficient neutralizing antibodies, together with potent CD4⁺ T cell responses capable of providing help to B cells and cytotoxic T cells (Burton et al., 2012). Understanding how the magnitude and specificity of these helper T cells can be optimized will be critical to the design of an effective vaccine.Primary immune responses are probably influenced strongly by the preexisting repertoire of B and T cells. However, characterization and quantification of these repertoires is difficult due to the extremely low number of circulating naive precursor cells (Jenkins et al., 2001; Su et al., 2013). Previous studies of naive CD4⁺ T cell repertoires in humans and mice have relied on magnetic beads to enrich MHC tetramer binding cells (Moon et al., 2007; Kwok et al., 2012; Su et al., 2013). However, although this approach gives precise information on responses to particular MHC-peptide epitopes, it does not measure the total repertoire and misses previously unknown epitopes. An alternative T cell library technique requires no prior knowledge of donor HLA type or epitope specificity (Geiger et al., 2009). The method presorts circulating T cells into naive and memory subsets which are seeded at limiting dilution before polyclonal expansion in the presence of PHA, allogeneic feeder cells, and IL-2. Individual cultures are then screened for proliferative responses to a protein or series of peptides representing the pathogen of interest (Geiger et al., 2009). Combined with epitope mapping and the Poisson distribution, the T cell library technique can provide quantitative data on the specificity of the entire preexisting naive and memory repertoire.The existence of HIV-1–specific memory cells in seronegative donors was originally suggested by studies of highly exposed HIV-1 seronegative (HESN) donors. It has been shown that 25–61% of HESNs have demonstrable HIV-1–specific memory cells, probably primed by exposure to the virus. Surprisingly, HIV-1–specific CD4⁺ T cells were also detected in 24–44% of unexposed donors (Ritchie et al., 2011), although it was not clear whether the latter came from cross-reactive memory T cells or naive T cells primed in vitro. More recently, the existence of low frequency (1–10/million) memory CD4⁺ T cells, specific for a known HIV-1 Gag epitope, was demonstrated by HLA DR4 tetramers in 50% of HIV-1 unexposed HLA DR4⁺ adults (Su et al., 2013), but it was not clear how generalizable the HIV-1 result was beyond the single epitope–HLA DR4 combination.The present study first validates the library technique by direct comparison with the tetramer enrichment method for measuring precursor T cell frequencies. We then use the T cell library technique to provide the first proteome-wide analysis of the frequencies and specificities of preexposure HIV-1–specific naive and memory CD4⁺ T cells in a HLA diverse population of HIV-1 unexposed donors.     

A Small Molecule CXCR4 Inhibitor that Blocks T Cell Line–tropic HIV-1 Infection

Several members of the chemokine receptor family have been shown to function in association with CD4 to permit human immunodeficiency virus type 1 (HIV-1) entry and infection. The CXC chemokine receptor CXCR4/fusin is a receptor for pre–B cell growth stimulating factor (PBSF)/stromal cell–derived factor 1 (SDF-1) and serves as a coreceptor for the entry of T cell line–tropic HIV-1 strains. Thus, the development of CXCR4 antagonists or agonists may be useful in the treatment of HIV-1 infection. T22 ([Tyr^5,12,Lys⁷]-polyphemusin II) is a synthesized peptide that consists of 18 amino acid residues and an analogue of polyphemusin II isolated from the hemocyte debris of American horseshoe crabs (Limulus polyphemus). T22 was found to specifically inhibit the ability of T cell line–tropic HIV-1 to induce cell fusion and infect the cell lines transfected with CXCR4 and CD4 or peripheral blood mononuclear cells. In addition, T22 inhibited Ca²⁺ mobilization induced by pre–B cell growth stimulating factor (PBSF)/SDF-1 stimulation through CXCR4. Thus, T22 is a small molecule CXCR4 inhibitor that blocks T cell line–tropic HIV-1 entry into target cells.     

HIV-1 Variation Diminishes CD4 T Lymphocyte Recognition

Effective long-term antiviral immunity requires specific cytotoxic T lymphocytes and CD4⁺ T lymphocyte help. Failure of these helper responses can be a principle cause of viral persistence. We sought evidence that variation in HIV-1 CD4⁺ T helper epitopes might contribute to this phenomenon. To determine this, we assayed fresh peripheral blood mononuclear cells from 43 asymptomatic HIV-1⁺ patients for proliferative responses to HIV-1 antigens. 12 (28%) showed a positive response, and we went on to map dominant epitopes in two individuals, to p24 Gag restricted by human histocompatibility leukocyte antigen (HLA)-DR1 and to p17 Gag restricted by HLA-DRB52c. Nine naturally occurring variants of the p24 Gag epitope were found in the proviral DNA of the individual in whom this response was detected. All variants bound to HLA-DR1, but three of these peptides failed to stimulate a CD4⁺ T lymphocyte line which recognized the index sequence. Antigenic variation was also detected in the p17 Gag epitope; a dominant viral variant present in the patient was well recognized by a specific CD4⁺ T lymphocyte line, whereas several natural mutants were not. Importantly, variants detected at both epitopes also failed to stimulate fresh uncultured cells while index peptide stimulated successfully. These results demonstrate that variant antigens arise in HIV-1⁺ patients which fail to stimulate the T cell antigen receptor of HLA class II–restricted lymphocytes, although the peptide epitopes are capable of being presented on the cell surface. In HIV-1 infection, naturally occurring HLA class II–restricted altered peptide ligands that fail to stimulate the circulating T lymphocyte repertoire may curtail helper responses at sites where variant viruses predominate.     

HIV-specific T Cell Cytotoxicity Mediated by RANTES Via the Chemokine Receptor CCR3

CC chemokines produced by CD8⁺ T cells are known to act as HIV-suppressive factors. We studied the possible role of these chemokines in HIV-1–specific killing of target cells. We found that the activity of cytotoxic T lymphocytes (CTLs) in CTL lines or freshly isolated peripheral blood mononuclear cells from HIV-1–infected individuals is markedly enhanced by RANTES (regulated on activation, normal T cell expressed and secreted) and virtually abolished by an antibody neutralizing RANTES or the RANTES receptor antagonist RANTES(9-68). Lysis was mediated by CD8⁺ major histocompatibility complex class I–restricted T cells and was obtained with target cells expressing epitopes of the HIV-1_LAI proteins Gag, Pol, Env, and Nef. The cytolytic activity observed in the presence or absence of added RANTES could be abolished by pretreatment of the CTLs with pertussis toxin, indicating that the effect is mediated by a G protein–coupled receptor. The chemokines monocyte chemotactic protein (MCP)-3, MCP-4, and eotaxin acted like RANTES, whereas macrophage inflammatory protein (MIP)-1α, MIP-1β, MCP-1, and stromal cell–derived factor 1 were inactive, suggesting a role for the eotaxin receptor, CCR3, and ruling out the involvement of CCR1, CCR2, CCR5, and CXCR4. CTL activity was abrogated by an antibody that blocks CCR3, further indicating that specific lysis is triggered via this chemokine receptor. These observations reveal a novel mechanism for the induction of HIV-1–specific cytotoxicity that depends on RANTES acting via CCR3.     

Coinhibitory receptor PD-1H preferentially suppresses CD4+ T cell–mediated immunity

T cell activation is regulated by the interactions of surface receptors with stimulatory and inhibitory ligands. Programmed death-1 homolog (PD-1H, also called VISTA) is a member of the CD28 family of proteins and has been shown to act as a coinhibitory ligand on APCs that suppress T cell responses. Here, we determined that PD-1H functions as a coinhibitory receptor for CD4⁺ T cells. CD4⁺ T cells in mice lacking PD-1H exhibited a dramatically increased response to antigen stimulation. Furthermore, delivery of a PD-1H–specific agonist mAb directly inhibited CD4⁺ T cell activation both in vitro and in vivo, validating a coinhibitory function of PD-1H. In a murine model of acute hepatitis, administration of a PD-1H agonist mAb suppressed CD4⁺ T cell–mediated acute inflammation. PD-1H–deficient animals were highly resistant to tumor induction in a murine brain glioma model, and depletion of CD4⁺ T cells, but not CD8⁺ T cells, promoted tumor formation. Together, our findings suggest that PD-1H has potential as a target of immune modulation in the treatment of human inflammation and malignancies.