Steven Schutt

Inhibition of BTK and ITK with Ibrutinib Is Effective in the Prevention of Chronic Graft-versus-Host Disease in Mice.

Bruton’s Tyrosine Kinase (BTK) and IL-2 Inducible T-cell Kinase (ITK) are enzymes responsible for the phosphorylation and activation of downstream effectors in the B-cell receptor (BCR) signaling and T cell receptor (TCR) signaling pathways, respectively. Ibrutinib is an FDA-approved potent inhibitor of both BTK and ITK that impairs B-cell and T-cell function. CD4 T cells and B cells are essential for the induction of chronic graft-versus-host disease (cGVHD). We evaluated these targets by testing the ability of Ibrutinib to prevent or ameliorate cGVHD, which is one of the major complications for patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). We found that Ibrutinib significantly alleviated cGVHD across four different mouse models, accompanied by increased long-term survival and reduced clinical score. The clinical improvements in Ibrutinib-treated recipients were associated with decreased serum-autoantibodies, costimulatory molecule activation, B-cell proliferation, and glomerulonephritis compared to vehicle controls. Ibrutinib was also able to alleviate the clinical manifestations in acute GVHD (aGVHD), where the recipients were given grafts with or without B cells, suggesting that an inhibitory effect of Ibrutinib on T cells contributes to a reduction in both aGVHD and cGVHD pathogenesis. An effective prophylactic regimen is still lacking to both reduce the incidence and severity of human cGVHD following allo-HSCT. Our study shows that Ibrutinib is an effective prophylaxis against several mouse models of cGVHD with minimal toxicity and could be a promising strategy to combat human cGVHD clinically.

Metabolic reprogramming of alloantigen-activated T cells after hematopoietic cell transplantation

Alloreactive donor T cells are the driving force in the induction of graft-versus-host disease (GVHD), yet little is known about T cell metabolism in response to alloantigens after hematopoietic cell transplantation (HCT). Here, we have demonstrated that donor T cells undergo metabolic reprograming after allogeneic HCT. Specifically, we employed a murine allogeneic BM transplant model and determined that T cells switch from fatty acid β-oxidation (FAO) and pyruvate oxidation via the tricarboxylic (TCA) cycle to aerobic glycolysis, thereby increasing dependence upon glutaminolysis and the pentose phosphate pathway. Glycolysis was required for optimal function of alloantigen-activated T cells and induction of GVHD, as inhibition of glycolysis by targeting mTORC1 or 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) ameliorated GVHD mortality and morbidity. Together, our results indicate that donor T cells use glycolysis as the predominant metabolic process after allogeneic HCT and suggest that glycolysis has potential as a therapeutic target for the control of GVHD.

MicroRNA-17-92 controls T-cell responses in graft-versus-host disease and leukemia relapse in mice

MicroRNAs (miRs) play important roles in orchestrating many aspects of the immune response. The miR-17-92 cluster, which encodes 6 miRs including 17, 18a, 19a, 20a, 19b-1, and 92-1, is among the best characterized of these miRs. The miR-17-92 cluster has been shown to regulate a variety of immune responses including infection, tumor, and autoimmunity, but the role of this cluster in T-cell response to alloantigens has not been previously explored. By using major histocompatibility complex (MHC)-matched, -mismatched, and haploidentical murine models of allogeneic bone marrow transplantation (allo-BMT), we demonstrate that the expression of miR-17-92 on donor T cells is essential for the induction of graft-versus-host disease (GVHD), but dispensable for the graft-versus-leukemia (GVL) effect. The miR-17-92 plays a major role in promoting CD4 T-cell activation, proliferation, survival, and Th1 differentiation, while inhibiting Th2 and iTreg differentiation. Alternatively, miR-17-92 may promote migration of CD8 T cells to GVHD target organs, but has minimal impact on CD8 T-cell proliferation, survival, or cytolytic function, which could contribute to the preserved GVL effect mediated by T cells deficient for miR-17-92. Furthermore, we evaluated a translational approach and found that systemic administration of antagomir to block miR-17 or miR-19b in this cluster significantly inhibited alloreactive T-cell expansion and interferon-γ (IFNγ) production, and prolonged the survival in recipients afflicted with GVHD while preserving the GVL effect. Taken together, the current work provides a strong rationale and demonstrates the feasibility to target miR-17-92 for the control of GVHD while preserving GVL activity after allo-BMT.

T-bet is critical for the development of acute graft-versus-host disease through controlling T cell differentiation and function.

T-bet is a master regulator for IFN-γ production and Th1 differentiation. We evaluated the roles of T-bet and IFN-γ in T cell responses in acute graft-versus-host disease (GVHD) and found that T-bet−/− T cells induced significantly less GVHD compared with wild-type or IFN-γ−/− counterparts in both MHC-mismatched and MHC-matched but minor histocompatibility Ag–mismatched models driven by CD4 T cells. T-bet−/−, but not IFN-γ−/−, CD4 T cells had a markedly reduced ability to cause tissue damage in liver and gut. This distinct outcome is reflected by the differential gene expression on donor CD4 T cells deficient for T-bet or IFN-γ. At mRNA and protein levels, we defined several T-bet–dependent molecules that may account for the impaired ability of T-bet−/− T cells to migrate into target organs and to produce Th1-related cytokines. Moreover, these molecules were independent of either endogenous IFN-γ, such as CXCR3 and programmed death-1, or systematic IFN-γ, such as NKG2D, I-Ab, and granzyme B. Although both T-bet−/− and IFN-γ−/− CD4 T cells are prone to differentiate into Th17 cells, polarized Th17 cells deficient for T-bet but not for IFN-γ had a significantly reduced ability to cause GVHD. Finally, T-bet−/− T cells had a compromised graft-versus-leukemia effect, which could be essentially reversed by neutralization of IL-17 in the recipients. We conclude that T-bet is required for Th1 differentiation and migration, as well as for optimal function of Th17 cells. Thus, targeting T-bet or regulating its downstream effectors independent of IFN-γ may be a promising strategy to control GVHD in the clinic.

CD8+ Tregs promote GVHD prevention and overcome the impaired GVL effect mediated by CD4+ Tregs in mice

ABSTRACT Adoptive natural regulatory T cell (nTreg) therapy has improved the outcome for patients suffering from graft-versus-host disease (GVHD) following allogeneic hematopoietic cell transplantation (Allo-HCT). However, fear of broad immune suppression and subsequent dampening of beneficial graft-versus-leukemia (GVL) responses remains a challenge. To address this concern, we generated alloreactive induced Tregs (iTregs) from resting CD4+ or CD8+ T cells and tested their ability to suppress GVH and maintain GVL responses. We utilized major mismatched and haploidentical murine models of HCT with host-derived lymphoma or leukemia cell lines to evaluate GVH and GVL responses simultaneously. Alloreactive CD4+ iTregs were effective in preventing GVHD, but abrogated the GVL effect against aggressive leukemia. Alloreactive CD8+ iTregs moderately attenuated GVHD while sparing the GVL effect. Hence, we reasoned that using a combination of CD4+ and CD8+ iTregs could achieve the optimal goal of Allo-HCT. Indeed, the combinational therapy was superior to CD4+ or CD8+ iTreg singular therapy in GVHD control; importantly, the combinational therapy maintained GVL responses. Cellular analysis uncovered potent suppression of both CD4+ and CD8+ effector T cells by the combinational therapy that resulted in effective prevention of GVHD, which could not be achieved by either singular therapy. Gene expression profiles revealed alloreactive CD8+ iTregs possess elevated expression of multiple cytolytic molecules compared to CD4+ iTregs, which likely contributes to GVL preservation. Our study uncovers unique differences between alloreactive CD4+ and CD8+ iTregs that can be harnessed to create an optimal iTreg therapy for GVHD prevention with maintained GVL responses.

Ceramide synthesis regulates T cell activity and GVHD development

Allogeneic hematopoietic cell transplantation (allo-HCT) is an effective immunotherapy for a variety of hematologic malignances, yet its efficacy is impeded by the development of graft-versus-host disease (GVHD). GVHD is characterized by activation, expansion, cytokine production, and migration of alloreactive donor T cells. Hence, strategies to limit GVHD are highly desirable. Ceramides are known to contribute to inflammation and autoimmunity. However, their involvement in T-cell responses to alloantigens is undefined. In the current study, we specifically characterized the role of ceramide synthase 6 (CerS6) after allo-HCT using genetic and pharmacologic approaches. We found that CerS6 was required for optimal T cell activation, proliferation, and cytokine production in response to alloantigen and for subsequent induction of GVHD. However, CerS6 was partially dispensable for the T cell-mediated antileukemia effect. At the molecular level, CerS6 was required for efficient TCR signal transduction, including tyrosine phosphorylation, ZAP-70 activation, and PKCθ/TCR colocalization. Impaired generation of C16-ceramide was responsible for diminished allogeneic T cell responses. Furthermore, targeting CerS6 using a specific inhibitor significantly reduced T cell activation in mouse and human T cells in vitro. Our study provides a rationale for targeting CerS6 to control GVHD, which would enhance the efficacy of allo-HCT as an immunotherapy for hematologic malignancies in the clinic.

MicroRNA-17-92 is required for T-cell and B-cell pathogenicity in chronic graft-versus-host disease in mice

Chronic graft-versus-host disease (cGVHD) is characterized as autoimmune-like fibrosis and antibody production mediated by pathogenic T cells and B cells. MicroRNA-17-92 (miR-17-92) influences the survival, differentiation, and function of lymphocytes in cancer, infections, and autoimmunity. To determine whether miR-17-92 regulates T- and B-cell responses in cGVHD, we generated mice conditionally deficient for miR-17-92 in T cells, B cells, or both. Using murine models of allogeneic bone marrow transplantation, we demonstrate that expression of miR-17-92 in donor T and B cells is essential for the induction of both scleroderma and bronchiolitis obliterans in cGVHD. Mechanistically, miR-17-92 expressed in T cells not only enhances the differentiation of pathogenic T helper 1 (Th1) and Th17 cells, but also promotes the generation of follicular Th cells, germinal center (GC) B cells, and plasma cells. In B cells, miR-17-92 expression is required for autoantibody production and immunoglobulin G deposition in the skin. Furthermore, we evaluated a translational approach using antagomirs specific for either miR-17 or miR-19, key members in miR-17-92 cluster. In a lupus-like cGVHD model, systemic administration of anti-miR-17, but not anti-miR-19, alleviates clinical manifestations and proteinuria incidence in recipients through inhibiting donor lymphocyte expansion, B-cell activation, and GC responses. Blockade of miR-17 also ameliorates skin damage by reducing Th17 differentiation in a scleroderma-cGVHD model. Taken together, our work reveals that miR-17-92 is required for T-cell and B-cell differentiation and function, and thus for the development of cGVHD. Furthermore, pharmacological inhibition of miR-17 represents a potential therapeutic strategy for the prevention of cGVHD.

T-bet Promotes Acute Graft-versus-Host Disease by Regulating Recipient Hematopoietic Cells in Mice.

Beyond its critical role in T cells, T-bet regulates the functions of APCs including dendritic cells and B cells, as well as NK cells. Given that recipient APCs are essential for priming allogeneic T cells and recipient NK or T cells are able to reject allogeneic donor cells, we evaluated the role of T-bet on the host in acute graft-versus-host disease (GVHD) using murine models of allogeneic bone marrow transplantation. T-bet−/− recipients developed significantly milder GVHD than their wild type counterparts in MHC-mismatched or CD4-depedent minor histocompatibility Ag–mismatched models. Allogeneic donor T cells, in particular, CD4 subset, significantly reduced IFN-γ production, proliferation and migration, and caused less injury in liver and gut of T-bet−/− recipients. We further observed that T-bet on recipient hematopoietic cells was primarily responsible for the donor T cell response and pathogenicity in GVHD. T-bet−/− dendritic cells expressed higher levels of Trail, whereas they produced lower levels of IFN-γ and IL-12/23 p40, as well as chemokine CXCL9, resulting in significantly higher levels of apoptosis, less priming, and infiltration of donor T cells. Meanwhile, NK cells in T-bet−/− hosts partially contribute to the decreased donor T cell proliferation. Furthermore, although T-bet on hematopoietic cells was required for GVHD development, it was largely dispensable for the graft-versus-leukemia effect. Taken together with our previous findings, we propose that T-bet is a potential therapeutic target for the control of GVHD through regulating donor T cells and recipient hematopoietic cells.

Inducible T-Cell Co-Stimulator Impacts Chronic Graft-Versus-Host Disease by Regulating Both Pathogenic and Regulatory T Cells

The incidence of chronic graft-versus-host disease (cGVHD) is on the rise and still the major cause of morbidity and mortality among patients after allogeneic hematopoietic stem cell transplantation (HCT). Both donor T and B cells contribute to the pathogenesis of cGVHD. Inducible T-cell co-stimulator (ICOS), a potent co-stimulatory receptor, plays a key role in T-cell activation and differentiation. Yet, how ICOS regulates the development of cGVHD is not well understood. Here, we investigated the role of ICOS in cGVHD pathogenesis using mice with germline or regulatory T cell (Treg)-specific ICOS deficiency. The recipients of ICOS−/− donor grafts had reduced cGVHD compared with wild-type controls. In recipients of ICOS−/− donor grafts, we observed significant reductions in donor T follicular helper (Tfh), Th17, germinal center B-cell, and plasma cell differentiation, coupled with lower antibody production. Interestingly, Tregs, including follicular regulatory T (Tfr) cells, were also impaired in the absence of ICOS. Using ICOS conditional knockout specific for Foxp3+ cells, we found that ICOS was indispensable for optimal survival and homeostasis of induced Tregs during cGVHD. Furthermore, administration of anti-ICOS alleviated cGVHD severity via suppressing T effector cells without affecting Treg generation. Taken together, ICOS promotes T- and B-cell activation and differentiation, which can promote cGVHD development; however, ICOS is critical for the survival and homeostasis of iTregs, which can suppress cGVHD. Hence, ICOS balances the development of cGVHD and could offer a potential target after allo-HCT in the clinic.

Inhibition of the IRE-1α/XBP-1 pathway prevents chronic GVHD and preserves the GVL effect in mice

Hematopoietic stem cell transplantation (HCT) is a curative procedure for hematological malignancies, but chronic graft-versus-host disease (cGVHD) remains a major complication after allogeneic HCT. Because donor B cells are essential for cGVHD development and B cells are sensitive to endoplasmic reticulum (ER) stress, we hypothesized that the IRE-1α/XBP-1 pathway is required for B-cell activation and function and for the development of cGVHD. To test this hypothesis, we used conditional knock-out mice deficient of XBP-1 specifically in B cells. Recipients transplanted with donor grafts containing XBP-1-deficient B cells displayed reduced cGVHD compared with controls. Reduction of cGVHD correlated with impaired B-cell functions, including reduced production of anti-double-stranded DNA immunoglobulin G antibodies, CD86, Fas, and GL7 surface expression, and impaired T-cell responses, including reduced interferon-γ production and follicular helper T cells. In a bronchiolitis obliterans cGVHD model, recipients of transplants containing XBP-1-deficient B cells demonstrated improved pulmonary function correlated with reduced donor splenic follicular helper T cells and increased B cells compared with those of wild-type control donor grafts. We then tested if XBP-1 blockade via an IRE-1α inhibitor, B-I09, would attenuate cGVHD and preserve the graft-versus-leukemia (GVL) effect. In a cutaneous cGVHD model, we found that prophylactic administration of B-I09 reduced clinical features of cGVHD, which correlated with reductions in donor T-cell and dendritic cell skin infiltrates. Inhibition of the IRE-1α/XBP-1 pathway also preserved the GVL effect against chronic myelogenous leukemia mediated by allogeneic splenocytes. Collectively, the ER stress response mediated by the IRE-1α/XBP-1 axis is required for cGVHD development but dispensable for GVL activity.