Vertical T cell immunodominance and epitope entropy determine HIV-1 escape

HIV-1 accumulates mutations in and around reactive epitopes to escape recognition and killing by CD8⁺ T cells. Measurements of HIV-1 time to escape should therefore provide information on which parameters are most important for T cell–mediated in vivo control of HIV-1. Primary HIV-1–specific T cell responses were fully mapped in 17 individuals, and the time to virus escape, which ranged from days to years, was measured for each epitope. While higher magnitude of an individual T cell response was associated with more rapid escape, the most significant T cell measure was its relative immunodominance measured in acute infection. This identified subject-level or “vertical” immunodominance as the primary determinant of in vivo CD8⁺ T cell pressure in HIV-1 infection. Conversely, escape was slowed significantly by lower population variability, or entropy, of the epitope targeted. Immunodominance and epitope entropy combined to explain half of all the variability in time to escape. These data explain how CD8⁺ T cells can exert significant and sustained HIV-1 pressure even when escape is very slow and that within an individual, the impacts of other T cell factors on HIV-1 escape should be considered in the context of immunodominance.     

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.     

CCR5-edited CD4+ T cells augment HIV-specific immunity to enable post-rebound control of HIV replication

BackgroundWe conducted a phase I clinical trial that infused CCR5 gene–edited CD4⁺ T cells to determine how these T cells can better enable HIV cure strategies.MethodsThe aim of trial was to develop RNA-based approaches to deliver zinc finger nuclease (ZFN), evaluate the effect of CCR5 gene–edited CD4⁺ T cells on the HIV-specific T cell response, test the ability of infused CCR5 gene–edited T cells to delay viral rebound during analytical treatment interruption, and determine whether individuals heterozygous for CCR5 Δ32 preferentially benefit. We enrolled 14 individuals living with HIV whose viral load was well controlled by antiretroviral therapy (ART). We measured the time to viral rebound after ART withdrawal, the persistence of CCR5-edited CD4⁺ T cells, and whether infusion of 10 billion CCR5-edited CD4⁺ T cells augmented the HIV-specific immune response.ResultsInfusion of the CD4⁺ T cells was well tolerated, with no serious adverse events. We observed a modest delay in the time to viral rebound relative to historical controls; however, 3 of the 14 individuals, 2 of whom were heterozygous for CCR5 Δ32, showed post-viral rebound control of viremia, before ultimately losing control of viral replication. Interestingly, only these individuals had substantial restoration of HIV-specific CD8⁺ T cell responses. We observed immune escape for 1 of these reinvigorated responses at viral recrudescence, illustrating a direct link between viral control and enhanced CD8⁺ T cell responses.ConclusionThese findings demonstrate how CCR5 gene–edited CD4⁺ T cell infusion could aid HIV cure strategies by augmenting preexisting HIV-specific immune responses.REGISTRATIONClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT02388594","term_id":"NCT02388594"}}NCT02388594.FundingNIH funding (R01AI104400, UM1AI126620, U19AI149680, T32AI007632) was provided by the National Institute of Allergy and Infectious Diseases (NIAID), the National Institute on Drug Abuse (NIDA), the National Institute of Mental Health (NIMH), and the National Institute of Neurological Disorders and Stroke (NINDS). Sangamo Therapeutics also provided funding for these studies.     

T cell receptor–targeted immunotherapeutics drive selective in vivo HIV- and CMV-specific T cell expansion in humanized mice

To delineate the in vivo role of different costimulatory signals in activating and expanding highly functional virus-specific cytotoxic CD8⁺ T cells, we designed synTacs, infusible biologics that recapitulate antigen-specific T cell activation signals delivered by antigen-presenting cells. We constructed synTacs consisting of dimeric Fc-domain scaffolds linking CD28- or 4-1BB–specific ligands to HLA-A2 MHC molecules covalently tethered to HIV- or CMV-derived peptides. Treatment of HIV-infected donor PBMCs with synTacs bearing HIV- or CMV-derived peptides induced vigorous and selective ex vivo expansion of highly functional HIV- and/or CMV-specific CD8⁺ T cells, respectively, with potent antiviral activities. Intravenous injection of HIV- or CMV-specific synTacs into immunodeficient mice intrasplenically engrafted with donor PBMCs markedly and selectively expanded HIV-specific (32-fold) or CMV-specific (46-fold) human CD8⁺ T cells populating their spleens. Notably, these expanded HIV- or CMV-specific CD8⁺ T cells directed potent in vivo suppression of HIV or CMV infections in the humanized mice, providing strong rationale for consideration of synTac-based approaches as a therapeutic strategy to cure HIV and treat CMV and other viral infections. The synTac platform flexibility supports facile delineation of in vivo effects of different costimulatory signals on patient-derived virus-specific CD8⁺ T cells, enabling optimization of individualized therapies, including HIV cure strategies.     

CD4+ and CD8+ T cell–dependent antiviral immunity requires STIM1 and STIM2

Calcium signaling is critical for lymphocyte function, and intracellular Ca²⁺ concentrations are regulated by store-operated Ca²⁺ entry (SOCE) through Ca²⁺ release–activated Ca²⁺ (CRAC) channels. In patients, loss-of-function mutations in CRAC channel components ORAI1 and STIM1 abolish SOCE and are associated with recurrent and chronic viral infections. Here, using mice with conditional deletion of Stim1 and its homolog Stim2 in T cells, we determined that both components are required for the maintenance of virus-specific memory CD8⁺ T cells and recall responses following secondary infection. In the absence of STIM1 and STIM2, acute viral infections became chronic. Early during infection, STIM1 and STIM2 were required for the differentiation of naive CD8⁺ T cells into fully functional cytolytic effector cells and mediated the production of cytokines and prevented cellular exhaustion in viral-specific CD8⁺ effector T cells. Importantly, memory and recall responses by CD8⁺ T cells required expression of STIM1 and STIM2 in CD4⁺ T cells. CD4⁺ T cells lacking STIM1 and STIM2 were unable to provide “help” to CD8⁺ T cells due to aberrant regulation of CD40L expression. Together, our data indicate that STIM1, STIM2, and CRAC channel function play distinct but synergistic roles in CD4⁺ and CD8⁺ T cells during antiviral immunity.     

CD4+CD25+ Immunoregulatory T Cells Suppress Polyclonal T Cell Activation In Vitro by Inhibiting Interleukin 2 Production

Peripheral tolerance may be maintained by a population of regulatory/suppressor T cells that prevent the activation of autoreactive T cells recognizing tissue-specific antigens. We have previously shown that CD4⁺CD25⁺ T cells represent a unique population of suppressor T cells that can prevent both the initiation of organ-specific autoimmune disease after day 3 thymectomy and the effector function of cloned autoantigen-specific CD4⁺ T cells. To analyze the mechanism of action of these cells, we established an in vitro model system that mimics the function of these cells in vivo. Purified CD4⁺CD25⁺ cells failed to proliferate after stimulation with interleukin (IL)-2 alone or stimulation through the T cell receptor (TCR). When cocultured with CD4⁺CD25⁻ cells, the CD4⁺CD25⁺ cells markedly suppressed proliferation by specifically inhibiting the production of IL-2. The inhibition was not cytokine mediated, was dependent on cell contact between the regulatory cells and the responders, and required activation of the suppressors via the TCR. Inhibition could be overcome by the addition to the cultures of IL-2 or anti-CD28, suggesting that the CD4⁺CD25⁺ cells may function by blocking the delivery of a costimulatory signal. Induction of CD25 expression on CD25⁻ T cells in vitro or in vivo did not result in the generation of suppressor activity. Collectively, these data support the concept that the CD4⁺CD25⁺ T cells in normal mice may represent a distinct lineage of “professional” suppressor cells.     

Uncovering a novel role of PLCβ4 in selectively mediating TCR signaling in CD8+ but not CD4+ T cells

Because of their common signaling molecules, the main T cell receptor (TCR) signaling cascades in CD4⁺ and CD8⁺ T cells are considered qualitatively identical. Herein, we show that TCR signaling in CD8⁺ T cells is qualitatively different from that in CD4⁺ T cells, since CD8α ignites another cardinal signaling cascade involving phospholipase C β4 (PLCβ4). TCR-mediated responses were severely impaired in PLCβ4-deficient CD8⁺ T cells, whereas those in CD4⁺ T cells were intact. PLCβ4-deficient CD8⁺ T cells showed perturbed activation of peripheral TCR signaling pathways downstream of IP₃ generation. Binding of PLCβ4 to the cytoplasmic tail of CD8α was important for CD8⁺ T cell activation. Furthermore, GNAQ interacted with PLCβ4, mediated double phosphorylation on threonine 886 and serine 890 positions of PLCβ4, and activated CD8⁺ T cells in a PLCβ4-dependent fashion. PLCβ4-deficient mice exhibited defective antiparasitic host defense and antitumor immune responses. Altogether, PLCβ4 differentiates TCR signaling in CD4⁺ and CD8⁺ T cells and selectively promotes CD8⁺ T cell–dependent adaptive immunity.     

BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination

The function of antigen-specific CD8⁺ T cells, which may protect against both infectious and malignant diseases, can be impaired by ligation of their inhibitory receptors, which include CTL-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1). Recently, B and T lymphocyte attenuator (BTLA) was identified as a novel inhibitory receptor with structural and functional similarities to CTLA-4 and PD-1. BTLA triggering leads to decreased antimicrobial and autoimmune T cell responses in mice, but its functions in humans are largely unknown. Here we have demonstrated that as human viral antigen–specific CD8⁺ T cells differentiated from naive to effector cells, their surface expression of BTLA was gradually downregulated. In marked contrast, human melanoma tumor antigen–specific effector CD8⁺ T cells persistently expressed high levels of BTLA in vivo and remained susceptible to functional inhibition by its ligand herpes virus entry mediator (HVEM). Such persistence of BTLA expression was also found in tumor antigen–specific CD8⁺ T cells from melanoma patients with spontaneous antitumor immune responses and after conventional peptide vaccination. Remarkably, addition of CpG oligodeoxynucleotides to the vaccine formulation led to progressive downregulation of BTLA in vivo and consequent resistance to BTLA-HVEM–mediated inhibition. Thus, BTLA activation inhibits the function of human CD8⁺ cancer-specific T cells, and appropriate immunotherapy may partially overcome this inhibition.     

Absence of mouse 2B4 promotes NK cell–mediated killing of activated CD8+ T cells,leading to prolonged viral persistence and altered pathogenesis

Persistent viral infections are often associated with inefficient T cell responses and sustained high-level expression of inhibitory receptors, such as the NK cell receptor 2B4 (also known as CD244), on virus-specific T cells. However, the role of 2B4 in T cell dysfunction is undefined, and it is unknown whether NK cells contribute to regulation of these processes. We show here that persistent lymphocytic choriomeningitis virus (LCMV) infection of mice lacking 2B4 resulted in diminished LCMV-specific CD8⁺ T cell responses, prolonged viral persistence, and spleen and thymic pathologies that differed from those observed in infected wild-type mice. Surprisingly, these altered phenotypes were not caused by 2B4 deficiency in T cells. Rather, the entire and long-lasting pathology and viral persistence were regulated by 2B4-deficient NK cells acting early in infection. In the absence of 2B4, NK cells lysed activated (defined as CD44^hi) but not naive (defined as CD44^lo) CD8⁺ T cells in a perforin-dependent manner in vitro and in vivo. These results illustrate the importance of NK cell self-tolerance to activated CD8⁺ T cells and demonstrate how an apparent T cell–associated persistent infection can actually be regulated by NK cells.     

Mapping and role of T cell response in SARS-CoV-2–infected mice

Virus-specific T cells play essential roles in protection against multiple virus infections, including SARS-CoV and MERS-CoV. While SARS-CoV-2–specific T cells have been identified in COVID-19 patients, their role in the protection of SARS-CoV-2–infected mice is not established. Here, using mice sensitized for infection with SARS-CoV-2 by transduction with an adenovirus expressing the human receptor (Ad5-hACE2), we identified SARS-CoV-2–specific T cell epitopes recognized by CD4⁺ and CD8⁺ T cells in BALB/c and C57BL/6 mice. Virus-specific T cells were polyfunctional and were able to lyse target cells in vivo. Further, type I interferon pathway was proved to be critical for generating optimal antiviral T cell responses after SARS-CoV-2 infection. T cell vaccination alone partially protected SARS-CoV-2–infected mice from severe disease. In addition, the results demonstrated cross-reactive T cell responses between SARS-CoV and SARS-CoV-2, but not MERS-CoV, in mice. Understanding the role of the T cell response will guide immunopathogenesis studies of COVID-19 and vaccine design and validation.     

TAP-independent self-peptides enhance T cell recognition of immune-escaped tumors

Tumor cells frequently escape from CD8⁺ T cell recognition by abrogating MHC-I antigen presentation. Deficiency in processing components, like the transporter associated with antigen processing (TAP), results in strongly decreased surface display of peptide/MHC-I complexes. We previously identified a class of hidden self-antigens known as T cell epitopes associated with impaired peptide processing (TEIPP), which emerge on tumor cells with such processing defects. In the present study, we analyzed thymus selection and peripheral behavior of T cells with specificity for the prototypic TEIPP antigen, the “self” TRH4 peptide/D^b complex. TEIPP T cells were efficiently selected in the thymus, egressed with a naive phenotype, and could be exploited for immunotherapy against immune-escaped, TAP-deficient tumor cells expressing low levels of MHC-I (MHC-I^lo). In contrast, overt thymus deletion and functionally impaired TEIPP T cells were observed in mice deficient for TAP1 due to TEIPP antigen presentation on all body cells in these mice. Our results strongly support the concept that TEIPPs derive from ubiquitous, nonmutated self-antigens and constitute a class of immunogenic neoantigens that are unmasked during tumor immune evasion. These data suggest that TEIPP-specific CD8⁺ T cells are promising candidates in the treatment of tumors that have escaped from conventional immunotherapies.     

Human adenovirus-specific T cells modulate HIV-specific T cell responses to an Ad5-vectored HIV-1 vaccine

Recombinant viruses hold promise as vectors for vaccines to prevent infectious diseases with significant global health impacts. One of their major limitations is that preexisting anti-vector neutralizing antibodies can reduce T cell responses to the insert antigens; however, the impact of vector-specific cellular immunity on subsequent insert-specific T cell responses has not been assessed in humans. Here, we have identified and compared adenovirus-specific and HIV-specific T cell responses in subjects participating in two HIV-1 vaccine trials using a vaccine vectored by adenovirus serotype 5 (Ad5). Higher frequencies of pre-immunization adenovirus-specific CD4⁺ T cells were associated with substantially decreased magnitude of HIV-specific CD4⁺ T cell responses and decreased breadth of HIV-specific CD8⁺ T cell responses in vaccine recipients, independent of type-specific preexisting Ad5-specific neutralizing antibody titers. Further, epitopes recognized by adenovirus-specific T cells were commonly conserved across many adenovirus serotypes, suggesting that cross-reactivity of preexisting adenovirus-specific T cells can extend to adenovirus vectors derived from rare serotypes. These findings provide what we believe to be a new understanding of how preexisting viral immunity may impact the efficacy of vaccines under current evaluation for prevention of HIV, tuberculosis, and malaria.     

Aplastic Anemia Rescued by Exhaustion of Cytokine-secreting CD8+ T Cells in Persistent Infection with Lymphocytic Choriomeningitis Virus

Aplastic anemia may be associated with persistent viral infections that result from failure of the immune system to control virus. To evaluate the effects on hematopoiesis exerted by sustained viral replication in the presence of activated T cells, blood values and bone marrow (BM) function were analyzed in chronic infection with lymphocytic choriomeningitis virus (LCMV) in perforin-deficient (P^0/0) mice. These mice exhibit a vigorous T cell response, but are unable to eliminate the virus. Within 14 d after infection, a progressive pancytopenia developed that eventually was lethal due to agranulocytosis and thrombocytopenia correlating with an increasing loss of morphologically differentiated, pluripotent, and committed progenitors in the BM. This hematopoietic disease caused by a noncytopathic chronic virus infection was prevented by depletion of CD8⁺, but not of CD4⁺, T cells and accelerated by increasing the frequency of LCMV-specific CD8⁺ T cells in T cell receptor (TCR) transgenic (tg) mice. LCMV and CD8⁺ T cells were found only transiently in the BM of infected wild-type mice. In contrast, increased numbers of CD8⁺ T cells and LCMV persisted at high levels in antigen-presenting cells of infected P^0/0 and P^0/0 × TCR tg mice. No cognate interaction between the TCR and hematopoietic progenitors presenting either LCMV-derived or self-antigens on the major histocompatibility complex was found, but damage to hematopoiesis was due to excessive secretion and action of tumor necrosis factor (TNF)/lymphotoxin (LT)-α and interferon (IFN)-γ produced by CD8⁺ T cells. This was studied in double-knockout mice that were genetically deficient in perforin and TNF receptor type 1. Compared with P^0/0 mice, these mice had identical T cell compartments and T cell responses to LCMV, yet they survived LCMV infection and became life-long virus carriers. The numbers of hematopoietic precursors in the BM were increased compared with P^0/0 mice after LCMV infection, although transient blood disease was still noticed. This residual disease activity was found to depend on IFN-γ–producing LCMV-specific T cells and the time point of hematopoietic recovery paralleled disappearance of these virus-specific, IFN-γ–producing CD8⁺ T cells. Thus, in the absence of IFN-γ and/or TNF/LT-α, exhaustion of virus-specific T cells was not hampered.     

TIGIT and PD-1 impair tumor antigen–specific CD8+ T cells in melanoma patients

T cell Ig and ITIM domain (TIGIT) is an inhibitory receptor expressed by activated T cells, Tregs, and NK cells. Here, we determined that TIGIT is upregulated on tumor antigen–specific (TA-specific) CD8⁺ T cells and CD8⁺ tumor-infiltrating lymphocytes (TILs) from patients with melanoma, and these TIGIT-expressing CD8⁺ T cells often coexpress the inhibitory receptor PD-1. Moreover, CD8⁺ TILs from patients exhibited downregulation of the costimulatory molecule CD226, which competes with TIGIT for the same ligand, supporting a TIGIT/CD226 imbalance in metastatic melanoma. TIGIT marked early T cell activation and was further upregulated by T cells upon PD-1 blockade and in dysfunctional PD-1⁺TIM-3⁺ TA-specific CD8⁺ T cells. PD-1⁺TIGIT⁺, PD-1^–TIGIT⁺, and PD-1⁺TIGIT^– CD8⁺ TILs had similar functional capacities ex vivo, suggesting that TIGIT alone, or together with PD-1, is not indicative of T cell dysfunction. However, in the presence of TIGIT ligand–expressing cells, TIGIT and PD-1 blockade additively increased proliferation, cytokine production, and degranulation of both TA-specific CD8⁺ T cells and CD8⁺ TILs. Collectively, our results show that TIGIT and PD-1 regulate the expansion and function of TA-specific CD8⁺ T cells and CD8⁺ TILs in melanoma patients and suggest that dual TIGIT and PD-1 blockade should be further explored to elicit potent antitumor CD8⁺ T cell responses in patients with advanced melanoma.     

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.     

Resting Memory CD8+ T Cells are Hyperreactive to Antigenic Challenge In Vitro

The characteristics of CD8⁺ T cells responsible for memory responses are still largely unknown. Particularly, it has not been determined whether different activation thresholds distinguish naive from memory CD8⁺ T cell populations. In most experimental systems, heterogeneous populations of primed CD8⁺ T cells can be identified in vivo after immunization. These cells differ in terms of cell cycle status, surface phenotype, and/or effector function. This heterogeneity has made it difficult to assess the activation threshold and the relative role of these subpopulations in memory responses. In this study we have used F5 T cell receptor transgenic mice to generate a homogeneous population of primed CD8⁺ T cells. In the F5 transgenic mice, peptide injection in vivo leads to activation of most peripheral CD8⁺ T cells. In vivo BrdU labeling has been used to follow primed T cells over time periods spanning several weeks after peptide immunization. Our results show that the majority of primed CD8⁺ T cells generated in this system are not cycling and express increased levels of CD44 and Ly6C. These cells remain responsive to secondary peptide challenge in vivo as evidenced by short term upregulation of activation markers such as CD69 and CD44. The activation thresholds of naive and primed CD8⁺ T cells were compared in vitro. We found that CD8⁺ T cells from primed mice are activated by peptide concentrations 10–50-fold lower than naive mice. In addition, the kinetics of interleukin 2Rα chain upregulation by primed CD8⁺ T cells differ from naive CD8⁺ T cells. These primed hyperresponsive CD8⁺ T cells might play an important role in the memory response.     

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.     

An Alternate Pathway for T Cell Development Supported by the Bone Marrow Microenvironment: Recapitulation of Thymic Maturation

In the principal pathway of α/β T cell maturation, T cell precursors from the bone marrow migrate to the thymus and proceed through several well-characterized developmental stages into mature CD4⁺ and CD8⁺ T cells. This study demonstrates an alternative pathway in which the bone marrow microenvironment also supports the differentiation of T cell precursors into CD4⁺ and CD8⁺ T cells. The marrow pathway recapitulates developmental stages of thymic maturation including a CD4⁺CD8⁺ intermediary cell and positive and negative selection, and is strongly inhibited by the presence of mature T cells. The contribution of the marrow pathway in vivo requires further study in mice with normal and deficient thymic or immune function.     

Analysis of the Role of ?Variation of Major Histocompatibility Complex Class II Expression on Nonobese Diabetic (NOD) Peripheral T Cell Response

The current paradigm of major histocompatibility complex (MHC) and disease association suggests that efficient binding of autoantigens by disease-associated MHC molecules leads to a T cell–mediated immune response and resultant autoimmune sequelae. The data presented below offer a different model for this association of MHC with autoimmune diabetes. We used several mouse lines expressing different levels of I-A^g7 and I-A^k on the nonobese diabetic (NOD) background to evaluate the role of MHC class II in the previously described NOD T cell autoproliferation. The ratio of I-A^g7 to I-A^k expression correlated with the peripheral T cell autoproliferative phenotype in the mice studied. T cells from the NOD, [NOD × NOD.I-A^null]F1, and NOD I-A^k transgenic mice demonstrated autoproliferative responses (after priming with self-peptides), whereas the NOD.H2^h4 (containing I-A^k) congenic and [NOD × NOD.H2^h4 congenic]F1 mice did not. Analysis of CD4⁺ NOD I-A^k transgenic primed lymph node cells showed that autoreactive CD4⁺ T cells in the NOD I-A^k transgenic mice were restricted exclusively by I-A^g7. Considered in the context of the avidity theory of T cell activation and selection, the reported poor peptide binding capacity of NOD I-A^g7 suggested a new hypothesis to explain the effects of MHC class II expression on the peripheral autoimmune repertoire in NOD mice. This new explanation suggests that the association of MHC with diabetes results from “altered” thymic selection in which high affinity self-reactive (potentially autoreactive) T cells escape negative selection. This model offers an explanation for the requirement of homozygous MHC class II expression in NOD mice (and in humans) in susceptibility to insulin-dependent diabetes mellitus.