Victor Tkachev

The IL-33/ST2 axis augments effector T cell responses during acute GVHD

Interleukin (IL)-33 binding to the receptor suppression of tumorigenicity 2 (ST2) produces pro-inflammatory and anti-inflammatory effects. Increased levels of soluble ST2 (sST2) are a biomarker for steroid-refractory graft-versus-host disease (GVHD) and mortality. However, whether sST2 has a role as an immune modulator or only as a biomarker during GVHD was unclear. We show increased IL-33 production by nonhematopoietic cells in the gastrointestinal (GI) tract in mice post-conditioning and patients during GVHD. Exogenous IL-33 administration during the peak inflammatory response worsened GVHD. Conversely, GVHD lethality and tumor necrosis factor-α production was significantly reduced in il33(-/-) recipients. ST2 was upregulated on murine and human alloreactive T cells and sST2 increased as experimental GVHD progressed. Concordantly, st2(-/-) vs wild-type (WT) donor T cells had a marked reduction in GVHD lethality and GI histopathology. Alloantigen-induced IL-18 receptor upregulation was lower in st2(-/-) T cells, and linked to reduced interferon-γ production by st2(-/-) vs WT T cells during GVHD. Blockade of IL-33/ST2 interactions during allogeneic-hematopoietic cell transplantation by exogenous ST2-Fc infusions had a marked reduction in GVHD lethality, indicating a role of ST2 as a decoy receptor modulating GVHD. Together, these studies point to the IL-33/ST2 axis as a novel and potent target for GVHD therapy.

Keap1/Nrf2/ARE redox-sensitive signaling system as a pharmacological target

The redox-sensitive signaling system Keap1/Nrf2/ARE plays a key role in maintenance of cellular homeostasis under stress, inflammatory, carcinogenic, and proapoptotic conditions, which allows us to consider it as a pharmacological target. Here we review the basic regulatory mechanisms of the Keap1/Nrf2/ARE system, key targets for pharmacological intervention, and interconnection of this system with other redox-sensitive transcriptional factors. We also discuss the range of currently available pharmaceuticals. Finally, we promote “indirect” antioxidants as a promising strategy for prevention and treatment of wide range of diseases associated with oxidative stress.

Programmed Death-1 Controls T Cell Survival by Regulating Oxidative Metabolism

The coinhibitory receptor programmed death-1 (PD-1) maintains immune homeostasis by negatively regulating T cell function and survival. Blockade of PD-1 increases the severity of graft-versus-host disease (GVHD), but the interplay between PD-1 inhibition and T cell metabolism is not well studied. We found that both murine and human alloreactive T cells concomitantly upregulated PD-1 expression and increased levels of reactive oxygen species (ROS) following allogeneic bone marrow transplantation. This PD-1HiROSHi phenotype was specific to alloreactive T cells and was not observed in syngeneic T cells during homeostatic proliferation. Blockade of PD-1 signaling decreased both mitochondrial H2O2 and total cellular ROS levels, and PD-1–driven increases in ROS were dependent upon the oxidation of fatty acids, because treatment with etomoxir nullified changes in ROS levels following PD-1 blockade. Downstream of PD-1, elevated ROS levels impaired T cell survival in a process reversed by antioxidants. Furthermore, PD-1–driven changes in ROS were fundamental to establishing a cell’s susceptibility to subsequent metabolic inhibition, because blockade of PD-1 decreased the efficacy of later F1F0-ATP synthase modulation. These data indicate that PD-1 facilitates apoptosis in alloreactive T cells by increasing ROS in a process dependent upon the oxidation of fat. In addition, blockade of PD-1 undermines the potential for subsequent metabolic inhibition, an important consideration given the increasing use of anti–PD-1 therapies in the clinic.

Anaplerotic Metabolism of Alloreactive T Cells Provides a Metabolic Approach To Treat Graft-Versus-Host Disease

T-cell activation requires increased ATP and biosynthesis to support proliferation and effector function. Most models of T-cell activation are based on in vitro culture systems and posit that aerobic glycolysis is employed to meet increased energetic and biosynthetic demands. By contrast, T cells activated in vivo by alloantigens in graft-versus-host disease (GVHD) increase mitochondrial oxygen consumption, fatty acid uptake, and oxidation, with small increases of glucose uptake and aerobic glycolysis. Here we show that these differences are not a consequence of alloactivation, because T cells activated in vitro either in a mixed lymphocyte reaction to the same alloantigens used in vivo or with agonistic anti-CD3/anti-CD28 antibodies increased aerobic glycolysis. Using targeted metabolic 13C tracer fate associations, we elucidated the metabolic pathway(s) employed by alloreactive T cells in vivo that support this phenotype. We find that glutamine (Gln)-dependent tricarboxylic acid cycle anaplerosis is increased in alloreactive T cells and that Gln carbon contributes to ribose biosynthesis. Pharmacological modulation of oxidative phosphorylation rapidly reduces anaplerosis in alloreactive T cells and improves GVHD. On the basis of these data, we propose a model of T-cell metabolism that is relevant to activated lymphocytes in vivo, with implications for the discovery of new drugs for immune disorders.

Programmed death ligand-1 expression on donor T cells drives graft-versus-host disease lethality

Programmed death ligand-1 (PD-L1) interaction with PD-1 induces T cell exhaustion and is a therapeutic target to enhance immune responses against cancer and chronic infections. In murine bone marrow transplant models, PD-L1 expression on host target tissues reduces the incidence of graft-versus-host disease (GVHD). PD-L1 is also expressed on T cells; however, it is unclear whether PD-L1 on this population influences immune function. Here, we examined the effects of PD-L1 modulation of T cell function in GVHD. In patients with severe GVHD, PD-L1 expression was increased on donor T cells. Compared with mice that received WT T cells, GVHD was reduced in animals that received T cells from Pdl1-/- donors. PD-L1-deficient T cells had reduced expression of gut homing receptors, diminished production of inflammatory cytokines, and enhanced rates of apoptosis. Moreover, multiple bioenergetic pathways, including aerobic glycolysis, oxidative phosphorylation, and fatty acid metabolism, were also reduced in T cells lacking PD-L1. Finally, the reduction of acute GVHD lethality in mice that received Pdl1-/- donor cells did not affect graft-versus-leukemia responses. These data demonstrate that PD-L1 selectively enhances T cell-mediated immune responses, suggesting a context-dependent function of the PD-1/PD-L1 axis, and suggest selective inhibition of PD-L1 on donor T cells as a potential strategy to prevent or ameliorate GVHD.

Transcriptome analysis of GVHD reveals aurora kinase A as a targetable pathway for disease prevention

Transcriptomic profiling of primate T cells during acute graft-versus-host disease reveals signaling pathways that when inhibited, ameliorate disease. Dawn of new graft-versus-host disease therapies Hematopoietic stem cell transplant (HCT) is a common therapy for patients with damaged bone marrow or immunodeficiencies. However, HCT has its own risks: In cases where the donor is not a perfect match to the recipient, immune cells derived from the graft can attack their new home. Furlan et al. examined the gene expression profile of nonhuman primate T cells during acute graft-versus-host disease (GVHD). The transcriptomics signatures specific for alloreactive T cells identified pathways altered during acute GVHD that could serve as therapeutic targets. The authors then examined one target in particular, aurora kinase A, and demonstrated that pharmacologic inhibition could improve survival in a mouse model of GVHD. Graft-versus-host disease (GVHD) is the most common complication of hematopoietic stem cell transplant (HCT). However, our understanding of the molecular pathways that cause this disease remains incomplete, leading to inadequate treatment strategies. To address this, we measured the gene expression profile of nonhuman primate (NHP) T cells during acute GVHD. Utilizing microarray technology, we measured the expression profiles of CD3+ T cells from five cohorts: allogeneic transplant recipients receiving (i) no immunoprophylaxis (No Rx), (ii) sirolimus monotherapy (Siro), (iii) tacrolimus-methotrexate (Tac-Mtx), as well as (iv) autologous transplant recipients (Auto) and (v) healthy controls (HC). This comparison allowed us to identify transcriptomic signatures specific for alloreactive T cells and determine the impact of both mTOR (mechanistic target of rapamycin) and calcineurin inhibition on GVHD. We found that the transcriptional profile of unprophylaxed GVHD was characterized by significant perturbation of pathways regulating T cell proliferation, effector function, and cytokine synthesis. Within these pathways, we discovered potentially druggable targets not previously implicated in GVHD, prominently including aurora kinase A (AURKA). Utilizing a murine GVHD model, we demonstrated that pharmacologic inhibition of AURKA could improve survival. Moreover, we found enrichment of AURKA transcripts both in allo-proliferating T cells and in sorted T cells from patients with clinical GVHD. These data provide a comprehensive elucidation of the T cell transcriptome in primate acute GVHD and suggest that AURKA should be considered a target for preventing GVHD, which, given the many available AURKA inhibitors in clinical development, could be quickly deployed for the prevention of GVHD.

Combined OX40L and mTOR blockade controls effector T cell activation while preserving Treg reconstitution after transplant

OX40L blockade synergizes with sirolimus to prevent GVHD by controlling effector T cell activation while supporting Treg reconstitution. Tackling T cells in GVHD Graft-versus-host disease (GVHD) after stem cell transplantation is mediated by effector T cells derived from donor stem cells, but GVHD can also be abrogated by donor-derived regulatory T cells. GVHD prophylaxis ideally should then allow regulatory T cell responses while inhibiting effector T cells. Tkachev et al. now provide very promising results in a nonhuman primate model, which suggest that such therapy is possible. They used mTOR inhibition in combination with OX40L blockade, which resulted in reduced damaging T cell reconstitution but preserved regulatory T cell activity. The combination therapy also led to a considerable survival benefit. These findings support testing of this therapy in patients. A critical question facing the field of transplantation is how to control effector T cell (Teff) activation while preserving regulatory T cell (Treg) function. Standard calcineurin inhibitor–based strategies can partially control Teffs, but breakthrough activation still occurs, and these agents are antagonistic to Treg function. Conversely, mechanistic target of rapamycin (mTOR) inhibition with sirolimus is more Treg-compatible but is inadequate to fully control Teff activation. In contrast, blockade of OX40L signaling has the capacity to partially control Teff activation despite maintaining Treg function. We used the nonhuman primate graft-versus-host disease (GVHD) model to probe the efficacy of combinatorial immunomodulation with sirolimus and the OX40L-blocking antibody KY1005. Our results demonstrate significant biologic activity of KY1005 alone (prolonging median GVHD-free survival from 8 to 19.5 days), as well as marked, synergistic control of GVHD with KY1005 + sirolimus (median survival time, >100 days; P < 0.01 compared to all other regimens), which was associated with potent control of both TH/TC1 (T helper cell 1/cytotoxic T cell 1) and TH/TC17 activation. Combined administration also maintained Treg reconstitution [resulting in an enhanced Treg/Teff ratio (40% over baseline) in the KY1005/sirolimus cohort compared to a 2.9-fold decrease in the unprophylaxed GVHD cohort]. This unique immunologic signature resulted in transplant recipients that were able to control GVHD for the length of analysis and to down-regulate donor/recipient alloreactivity despite maintaining anti–third-party responses. These data indicate that combined OX40L blockade and sirolimus represents a promising strategy to induce immune balance after transplant and is an important candidate regimen for clinical translation.

Preclinical Testing of Antihuman CD28 Fab′ Antibody in a Novel Nonhuman Primate Small Animal Rodent Model of Xenogenic Graft-Versus-Host Disease

Background Graft-versus-host disease (GVHD) is a severe complication of hematopoietic stem cell transplantation. Current therapies to prevent alloreactive T cell activation largely cause generalized immunosuppression and may result in adverse drug, antileukemia and antipathogen responses. Recently, several immunomodulatory therapeutics have been developed that show efficacy in maintaining antileukemia responses while inhibiting GVHD in murine models. To analyze efficacy and better understand immunological tolerance, escape mechanisms, and side effects of clinical reagents, testing of species cross-reactive human agents in large animal GVHD models is critical. Methods We have previously developed and refined a nonhuman primate (NHP) large animal GVHD model. However, this model is not readily amenable to semi-high throughput screening of candidate clinical reagents. Results Here, we report a novel, optimized NHP xenogeneic GVHD (xeno-GVHD) small animal model that recapitulates many aspects of NHP and human GVHD. This model was validated using a clinically available blocking, monovalent anti-CD28 antibody (FR104) whose effects in a human xeno-GVHD rodent model are known. Conclusions Because human-reactive reagents may not be fully cross-reactive or effective in vivo on NHP immune cells, this NHP xeno-GVHD model provides immunological insights and direct testing on NHP-induced GVHD before committing to the intensive NHP studies that are being increasingly used for detailed evaluation of new immune therapeutic strategies before human trials.

Oxidative Stress and Free-Radical Oxidation in BCG Granulomatosis Development

Background. Little is known about the role of free-radical and oxidative stress signaling in granuloma maturation and resolution. We aimed to study the activity of free-radical oxidation processes in the dynamics of BCG-induced generalized granulomatosis in mice. Methods. Chronic granulomatous inflammation was induced in male BALB/c mice by intravenously injecting the BCG vaccine, and the production of oxidative stress (activity of free-radical oxidation processes) and histological changes in the lungs, liver, and peritoneal exudate were measured 3, 30, 60, and 90 days after infection. Results. The tuberculous granuloma numerical density and diameter continuously increased from day 30 to day 90, and the macrophage content within the granulomas progressively diminished with a concomitant elevation in the number of epithelioid cells. The activity of the free-radical oxidation processes in the liver (i.e., the intensity of the homogenate chemiluminescence) reached a maximum at postinfection day 60 and subsequently began to decrease. The peak generation of reactive oxygen species by phagocytes in the peritoneal exudate (measured using flow cytometry) was also shifted in time and fell on day 30. Conclusions. The rise in the steady-state concentration of H2O2 in the liver of mice with BCG-induced granulomatosis is not related to local H2O2 production by phagocytes, and a decrease in the severity of generalized inflammation precedes the resolution of local inflammation.

Chimeric Antigen Receptor T Cell–Mediated Neurotoxicity in Nonhuman Primates

Chimeric antigen receptor (CAR) T-cell immunotherapy has revolutionized the treatment of refractory leukemias and lymphomas, but is associated with significant toxicities, namely cytokine release syndrome (CRS) and neurotoxicity. A major barrier to developing therapeutics to prevent CAR T cell-mediated neurotoxicity is the lack of clinically relevant models. Accordingly, we developed a rhesus macaque (RM) model of neurotoxicity via adoptive transfer of autologous CD20-specific CAR T cells. Following cyclophosphamide lymphodepletion, CD20 CAR T cells expand to 272 to 4,450 cells/μL after 7 to 8 days and elicit CRS and neurotoxicity. Toxicities are associated with elevated serum IL6, IL8, IL1RA, MIG, and I-TAC levels, and disproportionately high cerebrospinal fluid (CSF) IL6, IL2, GM-CSF, and VEGF levels. During neurotoxicity, both CD20 CAR and non-CAR T cells accumulate in the CSF and in the brain parenchyma. This RM model demonstrates that CAR T cell-mediated neurotoxicity is associated with proinflammatory CSF cytokines and a pan-T cell encephalitis.Significance: We provide the first immunologically relevant, nonhuman primate model of B cell-directed CAR T-cell therapy-mediated CRS and neurotoxicity. We demonstrate CAR and non-CAR T-cell infiltration in the CSF and in the brain during neurotoxicity resulting in pan-encephalitis, accompanied by increased levels of proinflammatory cytokines in the CSF. Cancer Discov; 8(6); 750-63. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 663.