Jianing Fu

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.

Reducing CD73 Expression by IL1β-Programmed Th17 Cells Improves Immunotherapeutic Control of Tumors

T cells of the T helper (Th)17 subset offer promise in adoptive T-cell therapy for cancer. However, current protocols for ex vivo programming of Th17 cells, which include TGFβ exposure, increase the expression of CD39 and CD73, two cell surface ATP ectonucleotidases that reduce T-cell effector functions and promote immunosuppression. Here, we report that ATP-mediated suppression of IFNγ production by Th17 cells can be overcome by genetic ablation of CD73 or by using IL1β instead of TGFβ to program Th17 cells ex vivo. Th17 cells cultured in IL1β were also highly polyfunctional, expressing high levels of effector molecules and exhibiting superior short-term control of melanoma in mice, despite reduced stem cell-like properties. TGFβ addition at low doses that did not upregulate CD73 expression but induced stemness properties drastically improved the antitumor effects of IL1β-cultured Th17 cells. Effector properties of IL1β-dependent Th17 cells were likely related to their high glycolytic capacity, since ex vivo programming in pyruvate impaired glycolysis and antitumor effects. Overall, we show that including TGFβ in ex vivo cultures used to program Th17 cells blunts their immunotherapeutic potential and demonstrate how this potential can be more fully realized for adoptive T-cell therapy.

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.

c-Rel is an essential transcription factor for the development of acute graft-versus-host disease in mice

Transcription factors of the Rel/NF‐κB family are known to play different roles in immunity and inflammation, although the putative role of c‐Rel in transplant tolerance and graft‐versus‐host disease (GVHD) remains elusive. We report here that T cells deficient for c‐Rel have a dramatically reduced ability to cause acute GVHD after allogeneic bone marrow transplantation using major and minor histocompatibility mismatched murine models. In the study to understand the underlying mechanisms, we found that c‐Rel−/− T cells had a reduced ability to expand in lymphoid organs and to infiltrate in GVHD target organs in allogeneic recipients. c‐Rel−/− T cells were defective in the differentiation into Th1 cells after encountering alloantigens, but were enhanced in the differentiation toward Foxp3+ regulatory T (Treg) cells. Furthermore, c‐Rel−/− T cells had largely preserved activity to mediate graft‐versus‐leukemia response. Taken together, our findings indicate that c‐Rel plays an essential role in T cells in the induction of acute GVHD, and suggest that c‐Rel can be a potential target for therapeutic intervention in allogeneic hematopoietic cell transplantation in the clinic.

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.