David Bastian

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.

Targeting JAK2 reduces GVHD and xenograft rejection through regulation of T cell differentiation

Significance Graft-versus-host disease (GVHD) remains a major cause of morbidity and mortality among allogeneic stem-cell transplantation recipients. An effort to identify selective immune suppression whereby GVHD is reduced and the antitumor activity of the graft is preserved is key to improving the success of blood and marrow transplantation. Here we demonstrate that inhibition of Janus kinase 2 (JAK2) significantly decreases GVHD and maintains tumor killing by the donor T cells. Pharmacologic blockade of JAK1 and JAK2 in myelofibrosis patients is known to impair human T cell subsets broadly. Conversely, we show that JAK2 inhibition impairs alloreactive T cells yet promotes beneficial regulatory T cell and Th2 differentiation. This study emphasizes the relevance of JAK2 in GVHD pathogenesis and prevention. Janus kinase 2 (JAK2) signal transduction is a critical mediator of the immune response. JAK2 is implicated in the onset of graft-versus-host disease (GVHD), which is a significant cause of transplant-related mortality after allogeneic hematopoietic cell transplantation (allo-HCT). Transfer of JAK2−/− donor T cells to allogeneic recipients leads to attenuated GVHD yet maintains graft-versus-leukemia. Th1 differentiation among JAK2−/− T cells is significantly decreased compared with wild-type controls. Conversely, iTreg and Th2 polarization is significantly increased among JAK2−/− T cells. Pacritinib is a multikinase inhibitor with potent activity against JAK2. Pacritinib significantly reduces GVHD and xenogeneic skin graft rejection in distinct rodent models and maintains donor antitumor immunity. Moreover, pacritinib spares iTregs and polarizes Th2 responses as observed among JAK2−/− T cells. Collectively, these data clearly identify JAK2 as a therapeutic target to control donor alloreactivity and promote iTreg responses after allo-HCT or solid organ transplantation. As such, a phase I/II acute GVHD prevention trial combining pacritinib with standard immune suppression after allo-HCT is actively being investigated (https://clinicaltrials.gov/ct2/show/NCT02891603).

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.

Stabilization of Foxp3 by Targeting JAK2 Enhances Efficacy of CD8 Induced Regulatory T Cells in the Prevention of Graft-versus-Host Disease

CD8+ induced regulatory T cells (iTregs) have been identified to suppress alloreactive immune responses and expressed regulatory T cell (Treg) ontological markers as similar as CD4+ iTregs. However, adoptive transfer of CD8+ iTreg–based therapy is hampered by the instability of Treg specific-transcription factor, Foxp3. As CD8+ iTregs were previously demonstrated to possess superior tumor-killing ability to CD4+ iTregs, adoptive transfer of stabilized CD8+ iTregs would be a potential therapy to prevent tumor relapse during graft-versus-leukemia disease (GVHD) treatment. In the current study, we generated alloantigen reactive CD8+ iTregs from JAK2−/− T cells and adoptively transferred them to MHC-mismatched and haploidentical murine models of allogeneic bone marrow transplantation. JAK2−/− CD8+ iTregs not only attenuated GVHD but also preserved graft-versus-leukemia effect. Mechanistic analysis revealed that JAK2−/− CD8+ iTregs upregulated natural Treg marker (neuropilin-1), and augmented DNA demethylation of CNS2 region within Foxp3 gene. These properties licensed JAK2−/− CD8+ iTregs to retain high Foxp3 expression resulting in less conversion to type 1 CTLs; as a result, JAK2−/− CD8+ iTregs were able to maintain their suppressive and cytolytic function. Thus, our findings provide a strong rationale and means to stabilize CD8+ iTregs by targeting JAK2, and the stabilized CD8+ iTregs exhibit therapeutic potential for alleviating GVHD and preserving the graft-versus-leukemia effect.

PIM-2 protein kinase negatively regulates T cell responses in transplantation and tumor immunity

&NA; PIM kinase family members play a crucial role in promoting cell survival and proliferation via phosphorylation of their target substrates. In this study, we investigated the role of the PIM kinases with respect to T cell responses in transplantation and tumor immunity. We found that the PIM‐2 isoform negatively regulated T cell responses to alloantigen, in contrast to the PIM‐1 and PIM‐3 isoforms, which acted as positive regulators. T cells deficient in PIM‐2 demonstrated increased T cell differentiation toward Th1 subset, proliferation, and migration to target organs after allogeneic bone marrow transplantation, resulting in dramatically accelerated graft‐versus‐host disease (GVHD) severity. Restoration of PIM‐2 expression markedly attenuated the pathogenicity of PIM‐2‐deficient T cells to induce GVHD. On the other hand, mice deficient in PIM‐2 readily rejected syngeneic tumor, which was primarily dependent on CD8+ T cells. Furthermore, silencing PIM‐2 in polyclonal or antigen‐specific CD8+ T cells substantially enhanced their antitumor response in adoptive T cell immunotherapy. We conclude that PIM‐2 kinase plays a prominent role in suppressing T cell responses, and provide a strong rationale to target PIM‐2 for cancer immunotherapy.