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.     

Natural killer T cell activation inhibits hepatitis B virus replication in vivo

We have previously reported that hepatitis B virus (HBV)-specific CD8(+) cytotoxic T lymphocytes and CD4(+) helper T lymphocytes can inhibit HBV replication in the liver of HBV transgenic mice by secreting interferon (IFN)-gamma when they recognize viral antigen. To determine whether an activated innate immune system can also inhibit HBV replication, in this study we activated natural killer T (NKT) cells in the liver of HBV transgenic mice by a single injection of alpha-galactosylceramide (alpha-GalCer), a glycolipid antigen presented to Valpha14(+)NK1.1(+) T cells by the nonclassical major histocompatibility complex class I-like molecule CD1d. Within 24 h of alpha-GalCer injection, IFN-gamma and IFN-alpha/beta were detected in the liver of HBV transgenic mice and HBV replication was abolished. Both of these events were temporally associated with the rapid disappearance of NKT cells from the liver, presumably reflecting activation-induced cell death, and by the recruitment of activated NK cells into the organ. In addition, prior antibody-mediated depletion of CD4(+) and CD8(+) T cells from the mice did not diminish the ability of alpha-GalCer to trigger the disappearance of HBV from the liver, indicating that conventional T cells were not downstream mediators of this effect. Finally, the antiviral effect of alpha-GalCer was inhibited in mice that are genetically deficient for either IFN-gamma or the IFN-alpha/beta receptor, indicating that most of the antiviral activity of alpha-GalCer is mediated by these cytokines. Based on these results, we conclude that alpha-GalCer inhibits HBV replication by directly activating NKT cells and by secondarily activating NK cells to secrete antiviral cytokines in the liver. In view of these findings, we suggest that, if activated, the innate immune response, like the adaptive immune response, has the potential to control viral replication during natural HBV infection. In addition, the data suggest that therapeutic activation of NKT cells may represent a new strategy for the treatment of chronic HBV infection.     

Effects of CAPE-like compounds on HIV replication in vitro and modulation of cytokines in vivo

OBJECTIVES: Five CAPE-like compounds, namely caffeic acid phenethyl ester (CAPE), methyl caffeate (MC), ethyl 3-(3,4-dihydroxyphenyl)acrylate (EC), phenethyl dimethyl caffeate (PEDMC) and phenethyl 3-(4-bromophenyl)acrylic (BrCAPE) were tested for their anti-HIV replication in vitro and immune modulation effects in vivo. METHODS: Short-term cytotoxicity was assessed by Trypan Blue stain and MTT assay. For antiviral assays, M-tropic (strain JRCSF), T-tropic (strain NL-4-3) and dual tropic (strain 89.6) HIV isolates were used in peripheral blood mononuclear cell (PBMC) culture. RESULTS: None of these CAPE-like compounds showed significant cytotoxicity in the treatment of PBMCs. By P24 EIA tests, CAPE, MC and EC significantly inhibited HIV replication in PBMC cells, but PEDMC and BrCAPE showed only slightly inhibitory effects. The in vivo modulatory effects on six cytokines [interleukin (IL)-2, IL-4, IL-6, interferon (IFN)-gamma, granulocyte-macrophage colony-stimulating factor (GM-CSF) and soluble Fas] were analysed. BALB/c mice treated with different doses or not treated with these CAPE-like chemicals showed that cytokines were increased to different extents by the different treatments. However, the concentrations of IL-6 and GM-CSF were not significantly affected by administration of any of these compounds (P > 0.05). CONCLUSIONS: The different effects of treatments on anti-HIV replication and cytokine modulation suggested that these compounds affect virological and immunological response via different mechanisms. The virological and immunological mechanisms and response to these treatments need to be elaborated in further studies in order to derive the structural features of more effective compounds. Since neither death nor pathological change in the mice were observed in this study, these CAPE-like compounds are worth studying further as potential chemotherapy agents for anti-HIV infection and cytokine modulation.     

Characterization of apoptosis-resistant antigen-specific T cells in vivo

Clonal deletion via activation-induced apoptosis (AIA) of antigen- specific T cells (ASTC) plays a very important role in the induction of peripheral tolerance. However, none of the studies performed so far has shown a complete deletion of ASTC, a small population always persisting in the periphery. The mechanism by which this small population of ASTC escapes AIA has not been determined. Since the existence of these ASTC may influence the outcome of autoimmune diseases and long-term graft survival, we have characterized the properties of these residual ASTC in vivo with the objective of determining mechanisms that may contribute to their persistence. It was found that the resistance of the residual ASTC to AIA is not due to lack of activation or Fas/Fas-L expression. Compared to those susceptible to AIA, the residual ASTC express a high level of Th2-type cytokines that may help them to escape from AIA. Furthermore, they are able to suppress proliferation of other ASTC, suggesting they may, in fact, prolong tolerance in vivo.     

Dynamic T cell-APC interactions sustain chronic inflammation in atherosclerosis

Atherosclerosis is a chronic inflammatory disease of large and medium-sized arteries characterized by leukocyte accumulation in the vessel wall. Both innate and adaptive immune responses contribute to atherogenesis, but the identity of atherosclerosis-relevant antigens and the role of antigen presentation in this disease remain poorly characterized. We developed live-cell imaging of explanted aortas to compare the behavior and role of APCs in normal and atherosclerotic mice. We found that CD4+ T cells were capable of interacting with fluorescently labeled (CD11c-YFP+) APCs in the aortic wall in the presence, but not the absence, of cognate antigen. In atherosclerosis-prone Apoe-/-CD11c-YFP+ mice, APCs extensively interacted with CD4+ T cells in the aorta, leading to cell activation and proliferation as well as secretion of IFN-γ and TNF-α. These cytokines enhanced uptake of oxidized and minimally modified LDL by macrophages. We conclude that antigen presentation by APCs to CD4+ T cells in the arterial wall causes local T cell activation and production of proinflammatory cytokines, which promote atherosclerosis by maintaining chronic inflammation and inducing foam cell formation.     

Elimination in vivo of developing T cells by natural killer cells

Natural killer cells gauge the absence of self class I MHC on susceptible target cells by means of inhibitory receptors such as members of the Ly49 family. To initiate killing by natural killer cells, a lack of inhibitory signals must be accompanied by the presence of activating ligands on the target cell. Although natural killer cell-mediated rejection of class I MHC-deficient bone marrow (BM) grafts is a matter of record, little is known about the targeting in vivo of specific cellular subsets by natural killer cells. We show here that development of class I MHC-negative thymocytes is delayed as a result of natural killer cell toxicity after grafting of a class I MHC-positive host with class I MHC-negative BM. Double positive thymocytes that persist in the presence of natural killer cells display an unusual T cell receptor-deficient phenotype, yet nevertheless give rise to single positive thymocytes and yield mature class I MHC-deficient lymphocytes that accumulate in the class I MHC-positive host. The resulting class I MHC-deficient CD8 T cells are functional and upon activation remain susceptible to natural killer cell toxicity in vivo. Reconstitution of class I MHC-deficient BM precursors with H2-K(b) by retroviral transduction fully restores normal thymic development.     

Regulatory T cells inhibit stable contacts between CD4+ T cells and dendritic cells in vivo

Regulatory T (T reg) cells exert powerful down-modulatory effects on immune responses, but it is not known how they act in vivo. Using intravital two-photon laser scanning microscopy we determined that, in the absence of T reg cells, the locomotion of autoantigen-specific T cells inside lymph nodes is decreased, and the contacts between T cells and antigen-loaded dendritic cells (DCs) are of longer duration. Thus, T reg cells can exert an early effect on immune responses by attenuating the establishment of stable contacts during priming of naive T cells by DCs.     

Activation of a dendritic cell-T cell axis by Ad5 immune complexes creates an improved environment for replication of HIV in T cells

The STEP HIV vaccine trial, which evaluated a replication-defective adenovirus type 5 (Ad5) vector vaccine, was recently stopped. The reasons for this included lack of efficacy of the vaccine and a twofold increase in the incidence of HIV acquisition among vaccinated recipients with increased Ad5-neutralizing antibody titers compared with placebo recipients. To model the events that might be occurring in vivo, the effect on dendritic cells (DCs) of Ad5 vector alone or treated with neutralizing antiserum (Ad5 immune complexes [IC]) was compared. Ad5 IC induced more notable DC maturation, as indicated by increased CD86 expression, decreased endocytosis, and production of tumor necrosis factor and type I interferons. We found that DC stimulation by Ad5 IC was mediated by the Fcγ receptor IIa and Toll-like receptor 9 interactions. DCs treated with Ad5 IC also induced significantly higher stimulation of Ad5-specific CD8 T cells equipped with cytolytic machinery. In contrast to Ad5 vectors alone, Ad5 IC caused significantly enhanced HIV infection in DC–T cell cocultures. The present results indicate that Ad5 IC activates a DC–T cell axis that, together with the possible persistence of the Ad5 vaccine in seropositive individuals, may set up a permissive environment for HIV-1 infection, which could account for the increased acquisition of HIV-1 infection among Ad5 seropositive vaccine recipients.     

In vivo activated peripheral T cells in autoimmune disease

Starting from the observation, if that in patients suffering from inflammatory bowel diseases elevated numbers of activated peripheral immunocytes can be detected in correlation to the activity of the disease, subpopulations of lymphocytes in immune-mediated disorders were analyzed for the expression of activation associated antigens. It was found that in patients with immunovasculitis, sarcoidosis, M. Beh?et, multiple sclerosis, antibody-mediated hemophilia, SLE,--but not in those with scleroderma--, increased numbers of activated immunocytes could be detected during acute exacerbation, whereas, in remission, the population of activated immunocytes was in the upper normal range. Analyses of phenotypes revealed that the majority of activated immunocytes are T cells. However, a variable minority of cells bear B cell associated determinants. As is the case in total peripheral T cells, the T4 to T8 ratio was in a normal range. Only in Beh?et disease and immunovasculitis was the ratio of activated T4 positive to activated T8 positive lymphocytes found to be decreased. In contrast to T cells in patients with inflammatory bowel disease, the majority of activated T cells in the autoimmune disorders under study does not express Fc alpha-receptors. In Beh?et disease and immunovasculitis moreover, the incidence of activated Leu 7 positive (= natural killer) cells is low compared to T cells from patients with Crohn's disease or ulcerative colitis. These experiments lead to the conclusion that the assessment of activated immunocytes may serve as a parameter in the evaluation of the clinical activity of autoimmune diseases.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo instruction of suppressor commitment in naive T cells

The induction of antigen-specific tolerance in the mature immune system of the intact organism has met with limited success. Therefore, nonspecific immunosuppression has been the treatment of choice to prevent unwanted immunity. Here, it is shown that prolonged subcutaneous infusion of low doses of peptide by means of osmotic pumps transforms mature T cells into CD4+25+ suppressor cells that can persist for long periods of time in the absence of antigen and confer specific immunologic tolerance upon challenge with antigen. The described procedure resembles approaches of tolerance induction used decades ago, induces tolerance in the absence of immunity, and holds the promise to become an effective means of inducing antigen-specific tolerance prospectively, whereas its power to suppress already ongoing immune responses remains to be determined.     

Induction of cytotoxic T cell precursors in vivo. Role of T helper cells

Strain AS rats respond with two populations of cytotoxic T lymphocytes to stimulation in vitro by the major histocompatibility complex (MHC)-incompatible strain HL rat tumor (HL-A2T2). One is specific for MHC alloantigens present on both HL-A2T2 and normal HL targets, the other is tumor specific. The activation of these killer cells requires helper T lymphocytes. The tumor-specific helper cells depend on syngeneic radioresistant accessory cells to present the tumor antigens in an immunogenic form. The appropriate helper-accessory cell interaction results in the production of soluble factors which then induce the maturation of precursor cells into effective killer cells. Studies with a procedure for inducing negative selection of T cells in vivo showed that short-term exposure to HL-A2T2 tumor induced selection only for TH but not cytotoxic T lymphocyte precursors (CTLp). Simultaneous injection of supernatants from concanavalin A-activated spleen cell cultures, however, did produce selection of CTLp. These and other findings suggest that under normal circumstances in vivo, both signals (recognition of antigen and acceptance of maturation factors) are provided in the vicinity of an antigen presenting macrophage-like accessory cell.     

Expansion of HIV-specific T follicular helper cells in chronic HIV infection

HIV targets CD4 T cells, which are required for the induction of high-affinity antibody responses and the formation of long-lived B cell memory. The depletion of antigen-specific CD4 T cells during HIV infection is therefore believed to impede the development of protective B cell immunity. Although several different HIV-related B cell dysfunctions have been described, the role of CD4 T follicular helper (TFH) cells in HIV infection remains unknown. Here, we assessed HIV-specific TFH responses in the lymph nodes of treatment-naive and antiretroviral-treated HIV-infected individuals. Strikingly, both the bulk TFH and HIV-specific TFH cell populations were significantly expanded in chronic HIV infection and were highly associated with viremia. In particular, GAG-specific TFH cells were detected at significantly higher levels in the lymph nodes compared with those of GP120-specific TFH cells and showed preferential secretion of the helper cytokine IL-21. In addition, TFH cell expansion was associated with an increase of germinal center B cells and plasma cells as well as IgG1 hypersecretion. Thus, our study suggests that high levels of HIV viremia drive the expansion of TFH cells, which in turn leads to perturbations of B cell differentiation, resulting in dysregulated antibody production.     

Induction of ovalbumin-specific cytotoxic T cells by in vivo peptide immunization

CTL recognize peptide forms of processed, foreign antigens in association with class I molecules encoded by the MHC and are usually directed against endogenously synthesized "cellular antigens," such as those expressed by virus-infected cells. In vitro studies have shown that small exogenous peptides can directly associate with class I molecules on the cell surface and mimic the target complex derived by intracellular processing and presentation. We have recently generated OVA-specific, H-2Kb-restricted CTL by immunizing C57BL/6 mice with a syngeneic tumor line transfected with the OVA cDNA. The CTL recognize the OVA transfectant E.G7-OVA and the synthetic peptide OVA258-276, but fail to recognize the native protein. We reasoned that given the potential for direct peptide/class I association observed in vitro, OVA258-276 may induce CTL after in vivo priming. However, we found that this is not the case. OVA258-276 and peptides of increasing lengths up to OVA242-276 and OVA242-285, which are all able to form the target complex in vitro, are inefficient at priming E.G7-OVA-specific CTL responses after intravenous injection. This is also true for both native and denatured OVA. In contrast to these results the synthetic peptide OVA229-276 corresponding to a peptide in a partial tryptic digestion of OVA can efficiently prime C57BL/6 mice in vivo after intravenous injection. This peptide elicits CTL that appear identical to those derived from animals immunized with syngeneic cells producing OVA endogenously. These results are discussed in terms of separate class I and class II antigen presentation pathways and the ability of only certain, exogenous antigens to enter the cytoplasmic, class I pathway.     

Relative efficacy of human monocytes and dendritic cells as accessory cells for T cell replication

Monocyte-specific monoclonal antibodies (7) were used to compare the efficacy of monocytes and dendritic cells as accessory or stimulator cells for human T cell replication. Both unfractionated and plastic-adherent mononuclear cells were first treated with a cytolytic antimonocyte antibody that kills greater than 95% of monocytes but not dendritic cells. When tested as stimulators of the mixed leukocyte reaction (MLR) and of oxidative mitogenesis (the proliferation of T cells modified with sodium periodate), the monocyte-depleted cells had normal or enhanced stimulatory capacity. Monocyte-depleted mononuclear cells also proliferated normally to soluble antigens (Candida albicans, tetanus toxoid), even under limiting conditions of cell dose, antigen dose, and culture time. Adherent blood mononuclear cells were next separated into monocyte-enriched and -depleted components using fluoresceinated antimonocyte antibody and the cell sorter. The depleted fraction (less than 2% monocytes by esterase staining and by cytology) contained the dendritic cells and exhibited at least 75% of the accessory activity. The monocyte-rich fraction (approximately 97% esterase positive) stimulated the MLR and oxidative mitogenesis weakly, and was comparable in potency to nonadherent cells. Cell-specific antibodies and complement were also used to prepare dendritic cells that were thoroughly depleted of monocytes and lymphocytes. The dendritic cells (70-80% pure) were potent stimulators of the allogeneic MLR, syngeneic MLR, and tetanus toxoid response, being active at stimulator to responder ratios of 1:100 or less. Taken together with previous studies (1, 2), these experiments indicate that the dendritic cell is the major stimulator of T cell replication in man. The contribution of class II products of the major histocompatibility complex (7) was then evaluated with a new monoclonal, 9.3F10. Accessory function was dramatically inhibited if cells bearing class II antigens were killed with 9.3F10 and complement, or if class II molecules were blocked by the addition of 9.3F10 Fab to the culture medium. The expression of 9.3F10 class II products was therefore studied on purified monocytes and dendritic cells. Most if not all cells in both populations reacted with 9.3F10, and each population exhibited approximately 150,000 125I-Fab 9.3F10 binding sites per cell. Since Ia+ dendritic cells are active accessory cells, but Ia+ monocytes are not, class II products are necessary but not sufficient for the stimulation of T cell proliferation in man.     

Human skin is colonized by T cells that recognize CD1a independently of lipid

CD1a-autoreactive T cells contribute to skin disease, but the identity of immunodominant self-lipid antigens and their mode of recognition are not yet solved. In most models, MHC and CD1 proteins serve as display platforms for smaller antigens. Here, we showed that CD1a tetramers without added antigen stained large T cell pools in every subject tested, accounting for approximately 1% of skin T cells. The mechanism of tetramer binding to T cells did not require any defined antigen. Binding occurred with approximately 100 lipid ligands carried by CD1a proteins, but could be tuned upward or downward with certain natural self-lipids. TCR recognition mapped to the outer A′ roof of CD1a at sites remote from the antigen exit portal, explaining how TCRs can bind CD1a rather than carried lipids. Thus, a major antigenic target of CD1a T cell autoreactivity in vivo is CD1a itself. Based on their high frequency and prevalence among donors, we conclude that CD1a-specific, lipid-independent T cells are a normal component of the human skin T cell repertoire. Bypassing the need to select antigens and effector molecules, CD1a tetramers represent a simple method to track such CD1a-specific T cells from tissues and in any clinical disease.     

Multipotent adult progenitor cells sustain function of ischemic limbs in mice

Despite progress in cardiovascular research, a cure for peripheral vascular disease has not been found. We compared the vascularization and tissue regeneration potential of murine and human undifferentiated multipotent adult progenitor cells (mMAPC-U and hMAPC-U), murine MAPC-derived vascular progenitors (mMAPC-VP), and unselected murine BM cells (mBMCs) in mice with moderate limb ischemia, reminiscent of intermittent claudication in human patients. mMAPC-U durably restored blood flow and muscle function and stimulated muscle regeneration, by direct and trophic contribution to vascular and skeletal muscle growth. This was in contrast to mBMCs and mMAPC-VP, which did not affect muscle regeneration and provided only limited and transient improvement. Moreover, mBMCs participated in a sustained inflammatory response in the lower limb, associated with progressive deterioration in muscle function. Importantly, mMAPC-U and hMAPC-U also remedied vascular and muscular deficiency in severe limb ischemia, representative of critical limb ischemia in humans. Thus, unlike BMCs or vascular-committed progenitors, undifferentiated multipotent adult progenitor cells offer the potential to durably repair ischemic damage in peripheral vascular disease patients.     

HIV and T follicular helper cells: a dangerous relationship

HIV infection leads to progressive destruction of infected CD4 T cells, hypergammaglobulinemia, and loss of memory B cells. Germinal centers, which are key to memory B cell formation and protective antibody responses, are major HIV reservoirs in which the virus replicates within T follicular helper (TFH) cells. In this issue of the JCI, the Koup and Streeck groups report that chronic SIV/HIV infection promotes TFH cell accumulation, which may drive B cell dysregulation. Their discoveries suggest that HIV harnesses TFH cells to evade the antibody response.