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.

Dairy cows are often fed high grain diets to meet the energy demand for high milk production or simply due to a lack of forages at times. As a result, ruminal acidosis, especially subacute ruminal acidosis (SARA), occurs frequently in practical dairy production. When SARA occurs, bacterial endotoxin (or lipopolysaccharide, LPS) is released in the rumen and the large intestine in a large amount. Many other bacterial immunogens may also be released in the digestive tract following feeding dairy cows diets containing high proportions of grain. LPS can be translocated into the bloodstream across the epithelium of the digestive tract, especially the lower tract, due to possible alterations of permeability and injuries of the epithelial tissue. As a result, the concentration of blood LPS increases. Immune responses are subsequently caused by circulating LPS, and the systemic effects include increases in concentrations of neutrophils and the acute phase proteins such as serum amyloid-A (SAA), haptoglobin (Hp), LPS binding protein (LBP), and C-reactive protein (CRP) in blood. Entry of LPS into blood can also result in metabolic alterations. Blood glucose and nonesterified fatty acid concentrations are enhanced accompanying an increase of blood LPS after increasing the amount of grain in the diet, which adversely affects feed intake of dairy cows. As the proportions of grain in the diet increase, patterns of plasma β-hydoxybutyric acid, cholesterol, and minerals (Ca, Fe, and Zn) are also perturbed. The bacterial immunogens can also lead to reduced supply of nutrients for synthesis of milk components and depressed functions of the epithelial cells in the mammary gland. The immune responses and metabolic alterations caused by circulating bacterial immunogens will exert an effect on milk production. It has been demonstrated that increases in concentrations of ruminal LPS and plasma acute phase proteins (CRP, SAA, and LBP) are associated with declines in milk fat content, milk fat yield, 3.5% fat-corrected milk yield, as well as milk energy efficiency.

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).

Histone acetylation and the families of enzymes responsible for controlling these epigenetic marks have been implicated in regulating T-cell maturation and phenotype. Here, we demonstrate a previously undefined role of histone deacetylase 11 (HDAC11) in regulating T-cell effector functions. Using EGFP-HDAC11 transgenic reporter mice, we found that HDAC11 expression was lower in effector relative to naive and central memory T-cell populations, and activation of resting T cells resulted in its decreased expression. Experiments using HDAC11 knockout (KO) mice revealed that T cells from these mice displayed enhanced proliferation, proinflammatory cytokine production, and effector molecule expression. In addition, HDAC11KO T cells had increased expression of and (, transcription factors previously shown to regulate inflammatory cytokine and effector molecule production. Conversely, overexpression of HDAC11 resulted in decreased expression of these genes. Chromatin immunoprecipitation showed the presence of HDAC11 at the and gene promoters in resting T cells, where it rapidly disassociated following T-cell activation. In vivo, HDAC11KO T cells were refractory to tolerance induction. HDAC11KO T cells also mediated accelerated onset of acute graft-versus-host disease (GVHD) in a murine model, characterized by increased proliferation of T cells and expression of interferon-γ, tumor necrosis factor, and EOMES. In addition, adoptive transfer of HDAC11KO T cells resulted in significantly reduced tumor burden in a murine B-cell lymphoma model. Taken together, these data demonstrate a previously unknown role of HDAC11 as a negative epigenetic regulator of T-cell effector phenotype and function.

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.

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.

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.

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.