Adoptive T cell immunotherapy for cytomegalovirus

Cytomegalovirus (CMV) is a major opportunistic pathogen following allogeneic transplantation, reflecting the inability of depressed host immunity to contain viral replication. Current antiviral drugs are limited by toxicities and lack of efficacy in established CMV disease, making adoptive immunotherapeutic strategies aimed at hastening virus-specific immune reconstitution attractive alternatives. A number of relatively small Phase I-II studies have demonstrated the feasibility of transferring CMV-specific T cells, varying in composition in terms of CD4⁺ and CD8⁺ T cell content. They have all suggested to varying degrees that restoration of CMV-specific immunity can be accelerated without an obvious effect on rates of graft-versus-host disease in those with CMV-seropositive donors. The majority also infer that recovery of laboratory measures of CMV-specific immunity correlate with clinical protection. Direct isolation of cells from donor blood now allows consideration of more widespread availability outside of a few academic centres, and equally importantly the delivery of randomised studies to establish the true efficacy of these strategies.     

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.     

Cytomegalovirus (CMV)-specific CD8+ T cells in individuals with HIV infection: correlation with protection from CMV disease

CD8+ cytotoxic T cells play a key role in immunological protection from clinical cytomegalovirus (CMV) disease. Numbers of CMV-specific CD8+ T cells are increased in untreated and antiretroviral-treated HIV patients compared with healthy controls. Accumulation of CMV-specific CD8+ T cells during HIV infection may reflect persistent reactivation of CMV owing to suboptimal immune control and/or oligoclonal expansion of the limited populations of CMV-specific CD8+ T cells present before antiretroviral therapy (ART). CD8+ T cells directed against the CMV immediate early (IE)-1 protein may play an important role in preventing CMV replication to pathogenic levels. However, immunological protection from CMV disease in HIV-infected individuals on ART does not appear to depend on total numbers of CMV-specific CD8+ T cells but rather on the presence of both effector-memory and effector CMV-specific CD8+ T cells that produce interferon-gamma and/or perforin in response to CMV antigens.     

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.     

Application of a New Paradigm for Cytomegalovirus Disease Prevention in Mayo Clinic’s First Face Transplant

The optimal approach to preventing cytomegalovirus (CMV) disease after face transplant is unknown. We report an individualized hybrid approach, initially using valganciclovir prophylaxis followed by surveillance and preemptive therapy guided by viral and immunologic markers. A 31-year-old man received a near-total face allotransplant for gunshot injury–related severe facial deformities. He was CMV seronegative, and the donor was CMV seropositive (D+/R? mismatch), and he received intravenous ganciclovir followed by oral valganciclovir prophylaxis. Monthly CD8⁺ T-cell immune competence assay revealed no CMV-specific CD8⁺ T-cell immunity development during valganciclovir prophylaxis. Because of severe leukopenia, valganciclovir was discontinued at 7 months after transplant when the patient had recovered and sustained normal global CD8⁺ T-cell function. Weekly CMV polymerase chain reaction testing detected asymptomatic CMV replication (plasma CMV, 549 IU/mL) 3 months later. Short-course preemptive valganciclovir treatment resulted in sustained virologic clearance. The patient had development of CMV-specific CD8⁺ T cells (12 cells/μL) together with CMV IgM and IgG. No rejection or infection relapse was detected 20 months after transplant. In conclusion, viral and immunologic monitoring allowed for an individualized approach to effective CMV disease prevention after face transplant. This case highlights the importance of understanding the interplay between the host immunity and the pathogen in determining the clinical course and outcome of CMV and other infectious disease syndromes.     

Oligoclonal Expansions of CD8+ T Cells in Chronic HIV Infection Are Antigen Specific

Acute HIV infection is associated with a vigorous immune response characterized by the proliferation of selected T cell receptor V beta (BV)-expressing CD8⁺ T cells. These ‘expansions'', which are commonly detected in the peripheral blood, can persist during chronic HIV infection and may result in the dominance of particular clones. Such clonal populations are most consistent with antigen-driven expansions of CD8⁺ T cells. However, due to the difficulties in studying antigen-specific T cells in vivo, it has been hard to prove that oligoclonal BV expansions are actually HIV specific. The use of tetrameric major histocompatibility complex–peptide complexes has recently enabled direct visualization of antigen-specific T cells ex vivo but has not provided information on their clonal composition. We have now made use of these tetrameric complexes in conjunction with anti-BV chain–specific monoclonal antibodies and analysis of cytotoxic T lymphocyte lines/clones to show that chronically clonally expanded CD8⁺ T cells are HIV specific in vivo.     

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.     

NLRP3 inflammasome induces CD4+ T cell loss in chronically HIV-1–infected patients

Chronic HIV-1 infection is generally characterized by progressive CD4⁺ T cell depletion due to direct and bystander death that is closely associated with persistent HIV-1 replication and an inflammatory environment in vivo. The mechanisms underlying the loss of CD4⁺ T cells in patients with chronic HIV-1 infection are incompletely understood. In this study, we simultaneously monitored caspase-1 and caspase-3 activation in circulating CD4⁺ T cells, which revealed that pyroptotic and apoptotic CD4⁺ T cells are distinct cell populations with different phenotypic characteristics. Levels of pyroptosis and apoptosis in CD4⁺ T cells were significantly elevated during chronic HIV-1 infection, and decreased following effective antiretroviral therapy. Notably, the occurrence of pyroptosis was further confirmed by elevated gasdermin D activation in lymph nodes of HIV-1–infected individuals. Mechanistically, caspase-1 activation closely correlated with the inflammatory marker expression and was shown to occur through NLRP3 inflammasome activation driven by virus-dependent and/or -independent ROS production, while caspase-3 activation in CD4⁺ T cells was more closely related to T cell activation status. Hence, our findings show that NLRP3-dependent pyroptosis plays an essential role in CD4⁺ T cell loss in HIV-1–infected patients and implicate pyroptosis signaling as a target for anti–HIV-1 treatment.     

Inflammatory IL-15 is required for optimal memory T cell responses

Due to their ability to rapidly proliferate and produce effector cytokines, memory CD8⁺ T cells increase protection following reexposure to a pathogen. However, low inflammatory immunizations do not provide memory CD8⁺ T cells with a proliferation advantage over naive CD8⁺ T cells, suggesting that cell-extrinsic factors enhance memory CD8⁺ T cell proliferation in vivo. Herein, we demonstrate that inflammatory signals are critical for the rapid proliferation of memory CD8⁺ T cells following infection. Using murine models of viral infection and antigen exposure, we found that type I IFN–driven expression of IL-15 in response to viral infection prepares memory CD8⁺ T cells for rapid division independently of antigen reexposure by transiently inducing cell-cycle progression via a pathway dependent on mTOR complex-1 (mTORC1). Moreover, exposure to IL-15 allowed more rapid division of memory CD8⁺ T cells following antigen encounter and enhanced their protective capacity against viral infection. Together, these data reveal that inflammatory IL-15 promotes optimal responses by memory CD8⁺ T cells.     

Enhanced T cell recovery in HIV-1–infected adults through IL-7 treatment

HIV infection results in CD4⁺ T cell deficiency, but efficient combination antiretroviral therapy (c-ART) restores T cells and decreases morbidity and mortality. However, immune restoration by c-ART remains variable, and prolonged T cell deficiency remains in a substantial proportion of patients. In a prospective open-label phase I/IIa trial, we evaluated the safety and efficacy of administration of the T cell regulator IL-7. The trial included 13 c-ART–treated HIV-infected patients whose CD4⁺ cell counts were between 100 and 400 cells/μl and plasma HIV RNA levels were less than 50 copies/ml. Patients received a total of 8 subcutaneous injections of 2 different doses of recombinant human IL-7 (rhIL-7; 3 or 10 μg/kg) 3 times per week over a 16-day period. rhIL-7 was well tolerated and induced a sustained increase of naive and central memory CD4⁺ and CD8⁺ T cells. In the highest dose group, 4 patients experienced transient increases in viral replication. However, functional assays showed that the expanded T cells responded to HIV antigen by producing IFN-γ and/or IL-2. In conclusion, in lymphopenic HIV-infected patients, rhIL-7 therapy induced substantial functional and quantitative changes in T cells for 48 weeks. Therefore, patients may benefit from intermittent therapy with IL-7 in combination with c-ART.     

Preferential infection and depletion of Mycobacterium tuberculosis-specific CD4 T cells after HIV-1 infection

HIV-1 infection results in the progressive loss of CD4 T cells. In this study, we address how different pathogen-specific CD4 T cells are affected by HIV infection and the cellular parameters involved. We found striking differences in the depletion rates between CD4 T cells to two common opportunistic pathogens, cytomegalovirus (CMV) and Mycobacterium tuberculosis (MTB). CMV-specific CD4 T cells persisted after HIV infection, whereas MTB-specific CD4 T cells were depleted rapidly. CMV-specific CD4 T cells expressed a mature phenotype and produced very little IL-2, but large amounts of MIP-1β. In contrast, MTB-specific CD4 T cells were less mature, and most produced IL-2 but not MIP-1β. Staphylococcal enterotoxin B-stimulated IL-2-producing cells were more susceptible to HIV infection in vitro than MIP-1β-producing cells. Moreover, IL-2 production was associated with expression of CD25, and neutralization of IL-2 completely abrogated productive HIV infection in vitro. HIV DNA was found to be most abundant in IL-2-producing cells, and least abundant in MIP-1β-producing MTB-specific CD4 T cells from HIV-infected subjects with active tuberculosis. These data support the hypothesis that differences in function affect the susceptibility of pathogen-specific CD4 T cells to HIV infection and depletion in vivo, providing a potential mechanism to explain the rapid loss of MTB-specific CD4 T cells after HIV infection.     

Cytotoxic T Lymphocyte Antigen 4 Is Induced in the Thymus upon In Vivo Activation and Its Blockade Prevents Anti-CD3–mediated Depletion of ?Thymocytes

The development of a normal T cell repertoire in the thymus is dependent on the interplay between signals mediating cell survival (positive selection) and cell death (negative selection or death by neglect). Although the CD28 costimulatory molecule has been implicated in this process, it has been difficult to establish a role for the other major costimulatory molecule, cytotoxic T lymphocyte antigen (CTLA)-4. Here we report that in vivo stimulation through the T cell receptor (TCR)–CD3 complex induces expression of CTLA-4 in thymocytes and leads to the association of CTLA-4 with the SH2 domain–containing phosphatase (SHP)-2 tyrosine phosphatase. Moreover, intrathymic CTLA-4 blockade dramatically inhibits anti-CD3–mediated depletion of CD4⁺CD8⁺ double positive immature thymocytes. Similarly, anti-CD3–mediated depletion of CD4⁺CD8⁺ double positive cells in fetal thymic organ cultures could also be inhibited by anti–CTLA-4 antibodies. Thus, our data provide evidence for a role of CTLA-4 in thymic selection and suggest a novel mechanism contributing to the regulation of TCR-mediated selection of T cell repertoires.     

HIV-specific CD8(+) T cells produce antiviral cytokines but are impaired in cytolytic function

The use of peptide-human histocompatibility leukocyte antigen (HLA) class I tetrameric complexes to identify antigen-specific CD8(+) T cells has provided a major development in our understanding of their role in controlling viral infections. However, questions remain about the exact function of these cells, particularly in HIV infection. Virus-specific cytotoxic T lymphocytes exert much of their activity by secreting soluble factors such as cytokines and chemokines. We describe here a method that combines the use of tetramers and intracellular staining to examine the functional heterogeneity of antigen-specific CD8(+) T cells ex vivo. After stimulation by specific peptide antigen, secretion of interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, macrophage inflammatory protein (MIP)-1beta, and perforin is analyzed by FACS((R)) within the tetramer-positive population in peripheral blood. Using this method, we have assessed the functional phenotype of HIV-specific CD8(+) T cells compared with cytomegalovirus (CMV)-specific CD8(+) T cells in HIV chronic infection. We show that the majority of circulating CD8(+) T cells specific for CMV and HIV antigens are functionally active with regards to the secretion of antiviral cytokines in response to antigen, although a subset of tetramer-staining cells was identified that secretes IFN-gamma and MIP-1beta but not TNF-alpha. However, a striking finding is that HIV-specific CD8(+) T cells express significantly lower levels of perforin than CMV-specific CD8(+) T cells. This lack of perforin is linked with persistent CD27 expression on HIV-specific cells, suggesting impaired maturation, and specific lysis ex vivo is lower for HIV-specific compared with CMV-specific cells from the same donor. Thus, HIV-specific CD8(+) T cells are impaired in cytolytic activity.     

Robust expansion of viral antigen-specific CD4+ and CD8+ T cells for adoptive T cell therapy using gene-modified activated T cells as antigen presenting cells

Cytomegalovirus (CMV) reactivation after stem cell transplantation can be treated with CMV-specific T cells, but current in vitro techniques using dendritic cells as antigen-presenting cells are time-consuming and expensive. To simplify the production of clinical grade CMV-specific T cells, we evaluated gene-modified activated T cells [antigen presenting T cells (T-APCs)] as a reliable and easily produced source of APCs to boost CD4+ and CD8+ T-cell responses against the immunodominant CMV antigen pp65. T-APCs expressing the full-length immunodominant CMV pp65 gene were used to stimulate the expansion of autologous T cells. After 10 to 14 days, the T cell lines were tested for antigen specificity by using the flow cytometric intracellular detection of interferon-gamma after stimulation for 6 hours with a pp65 peptide library of 15-mers, overlapping by 11 amino acids. Under optimal conditions, this technique induced a median 766-fold and a 652-fold expansion of pp65-specific CD4+ and CD8+ responder cells, respectively, in 15 T cell lines. In 13 of 15 T cell lines, over 10 antigen-specific CD4+ plus CD8+ T cells were generated starting with only 5x10 peripheral blood mononuclear cells, representing an over 3-log increase. These data indicate that T-APCs efficiently boost pp65-specific CD4+ and CD8+ T cell numbers to clinically useful levels. The approach has the advantage of using a single leukocyte collection from the donor to generate large numbers of CMV-specific T cells within a total 3-week culture period using only one stimulation of antigen.     

CD27 sustains survival of CTLs in virus-infected nonlymphoid tissue in mice by inducing autocrine IL-2 production

Immunity to infections relies on clonal expansion of CD8⁺ T cells, their maintenance as effector CTLs, and their selection into a memory population. These processes rely on delivery of survival signals to activated CD8⁺ T cells. We here reveal the mechanism by which costimulatory CD27-CD70 interactions sustain survival of CD8⁺ effector T cells in infected tissue. By unbiased genome-wide gene expression analysis, we identified the Il2 gene as the most prominent CD27 target gene in murine CD8⁺ T cells. In vitro, CD27 directed IL-2 expression and promoted clonal expansion of primed CD8⁺ T cells exclusively by IL-2–dependent survival signaling. In mice intranasally infected with influenza virus, Cd27^–/– CD8⁺ effector T cells displayed reduced IL-2 production, accompanied by impaired accumulation in lymphoid organs and in the lungs, which constitute the tissue effector site. Reconstitution of Cd27^–/– CD8⁺ T cells with the IL2 gene restored their accumulation to wild-type levels in the lungs, but it did not rescue their accumulation in lymphoid organs. Competition experiments showed that the IL-2 produced under the control of CD27 supported effector CD8⁺ T cell survival in the lungs in an autocrine manner. We conclude that CD27 signaling directs the IL-2 production that is reportedly essential to sustain survival of virus-specific CTLs in nonlymphoid tissue.     

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.     

T cell memory. Long-term persistence of virus-specific cytotoxic T cells

This study documents that virus-specific CTL can persist indefinitely in vivo. This was accomplished by transferring Thy-1.1 T cells into Thy-1.2 recipient mice to specifically identify the donor T cell population and to characterize its antigenic specificity and function by using a virus-specific CTL assay. Thy-1.1+ T cells from mice previously immunized with lymphocytic choriomeningitis virus (LCMV) were transferred into Thy-1.2 mice persistently infected with LCMV. The transferred LCMV-specific CTL (Thy-1.1+ CD8+) eliminate virus from the chronically infected carriers and persist in the recipient mice in small numbers, comprising only a minor fraction of the total T cells. Upon re-exposure to virus, these long-lived "resting" CD8+ T cells proliferate in vivo to become the predominant cell population. These donor CD8+ T cells can be recovered up to a year post-transfer and still retain antigenic specificity and biological function. They kill LCMV infected H-2-matched cells in vitro and can eliminate virus upon transfer into a second infected host. In addition, these long-lived CD8+ T cells appear not to be dependent on help from CD4+ T cells, since depletion of CD4+ T cells has minimal or no effect on their biological properties (proliferation, CTL response, viral clearance). These donor CTL also exhibit an immunodominance over the host-derived LCMV-specific CTL response. When both host and donor T cells are present, the donor CTL response is dominant over the potential CTL response of the cured carrier host. Taken together, these results suggest that virus-specific CTL can persist for the life span of the host as memory cells.