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The gastrointestinal (GI) tract is a frequent target organ in acute graft-versus-host disease (aGVHD), which can determine the morbidity and nonrelapse mortality after allogeneic hematopoietic cell transplantation (allo-HCT). Donor T cells recognize allogeneic Ags presented by host APCs, proliferate, and differentiate into Th1 and Th17 cells that drive GVHD pathogenesis. IL-12 has been shown to play an important role in amplifying the allogeneic response in preclinical and clinical studies. This study demonstrates that IL-12Rβ2 expression on recipient nonhematopoietic cells is required for optimal development of aGVHD in murine models of allo-HCT. aGVHD attenuation by genetic depletion of IL-12R signaling is associated with reduced MHC class II expression by intestinal epithelial cells and maintenance of intestinal integrity. We verified IL-12Rβ2 expression on activated T cells and in the GI tract. This study, to our knowledge, reveals a novel function of IL-12Rβ2 in GVHD pathogenesis and suggests that selectively targeting IL-12Rβ2 on host nonhematopoietic cells may preserve the GI tract after allo-HCT., (Copyright © 2023 by The American Association of Immunologists, Inc.)

Graft-versus-host disease (GVHD), manifesting as either acute (aGVHD) or chronic (cGVHD), presents significant life-threatening complications following allogeneic hematopoietic cell transplantation. Here, we investigated Friend virus leukemia integration 1 (Fli-1) in GVHD pathogenesis and validated Fli-1 as a therapeutic target. Using genetic approaches, we found that Fli-1 dynamically regulated different T cell subsets in allogeneic responses and pathogenicity in the development of aGVHD and cGVHD. Compared with homozygous Fli1-deficient or WT T cells, heterozygous Fli1-deficient T cells induced the mildest GVHD, as evidenced by the lowest Th1 and Th17 cell differentiation. Single-cell RNA-Seq analysis revealed that Fli-1 differentially regulated CD4+ and CD8+ T cell responses. Fli-1 promoted the transcription of Th1/Th17 pathways and T cell receptor-inducible (TCR-inducible) transcription factors in CD4+ T cells, while suppressing activation- and function-related gene pathways in CD8+ T cells. Importantly, a low dose of camptothecin, topotecan, or etoposide acted as a potent Fli-1 inhibitor and significantly attenuated GVHD severity, while preserving the graft-versus-leukemia (GVL) effect. This observation was extended to a xenograft model, in which GVHD was induced by human T cells. In conclusion, we provide evidence that Fli-1 plays a crucial role in alloreactive CD4+ T cell activation and differentiation and that targeting Fli-1 may be an attractive strategy for treating GVHD without compromising the GVL effect.

Allogeneic hematopoietic cell transplantation (allo-HCT) is an effective immunotherapy for various hematologic malignancies, predominantly through potent graft-versus-leukemia (GVL) effect. However, the mortality after allo-HCT is because of relapse of primary malignancy and followed by graft-vs-host-disease (GVHD) as a major cause of transplant-related mortality. Hence, strategies to limit GVHD while preserving the GVL effect are highly desirable. Ceramide, which serves a central role in sphingolipid metabolism, is generated by ceramide synthases (CerS1-6). In this study, we found that genetic or pharmacologic targeting of CerS6 prevented and reversed chronic GVHD (cGVHD). Furthermore, specific inhibition of CerS6 with ST1072 significantly ameliorated acute GVHD (aGVHD) while preserving the GVL effect, which differed from FTY720 that attenuated aGVHD but impaired GVL activity. At the cellular level, blockade of CerS6 restrained donor T cells from migrating into GVHD target organs and preferentially reduced activation of donor CD4 T cells. At the molecular level, CerS6 was required for optimal TCR signaling, CD3/PKCθ co-localization, and subsequent N-RAS activation and ERK signaling, especially on CD4 + T cells. The current study provides rationale and means for targeting CerS6 to control GVHD and leukemia relapse, which would enhance the efficacy of allo-HCT as an immunotherapy for hematologic malignancies in the clinic., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Chronic graft-versus-host disease (cGVHD) remains a major obstacle impeding successful allogeneic hematopoietic cell transplantation (HCT). MicroRNAs (miRs) play key roles in immune regulation during acute GVHD development. Preclinical studies to identify miRs that affect cGVHD pathogenesis are required to develop these as potential lifesaving interventions. Using oligonucleotide array, we identified miR-31, which was significantly elevated in allogeneic T cells after HCT in mice. Using genetic and pharmacologic approaches, we demonstrated a key role for miR-31 in mediating donor T-cell pathogenicity in cGVHD. Recipients of miR-31-deficient T cells displayed improved cutaneous and pulmonary cGVHD. Deficiency of miR-31 reduced T-cell expansion and T helper 17 (Th17) cell differentiation but increased generation and function of regulatory T cells (Tregs). MiR-31 facilitated neuropilin-1 downregulation, Foxp3 loss, and interferon-γ production in alloantigen-induced Tregs. Mechanistically, miR-31 was required for hypoxia-inducible factor 1α (HIF1α) upregulation in allogeneic T cells. Therefore, miR-31-deficient CD4 T cells displayed impaired activation, survival, Th17 cell differentiation, and glycolytic metabolism under hypoxia. Upregulation of factor-inhibiting HIF1, a direct target of miR-31, in miR-31-deficient T cells was essential for attenuating T-cell pathogenicity. However, miR-31-deficient CD8 T cells maintained intact glucose metabolism, cytolytic activity, and graft-versus-leukemia response. Importantly, systemic administration of a specific inhibitor of miR-31 effectively reduced donor T-cell expansion, improved Treg generation, and attenuated cGVHD. Taken together, miR-31 is a key driver for T-cell pathogenicity in cGVHD but not for antileukemia activity. MiR-31 is essential in driving cGVHD pathogenesis and represents a novel potential therapeutic target for controlling cGVHD., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Stimulator of interferon genes (STING)-mediated innate immune activation plays a key role in tumor- and self-DNA-elicited antitumor immunity and autoimmunity. However, STING can also suppress tumor immunity and autoimmunity. STING signaling in host nonhematopoietic cells was reported to either protect against or promote graft-versus-host disease (GVHD), a major complication of allogeneic hematopoietic cell transplantation (allo-HCT). Host hematopoietic antigen-presenting cells (APCs) play key roles in donor T-cell priming during GVHD initiation. However, how STING regulates host hematopoietic APCs after allo-HCT remains unknown. We utilized murine models of allo-HCT to assess the role of STING in hematopoietic APCs. STING-deficient recipients developed more severe GVHD after major histocompatibility complex-mismatched allo-HCT. Using bone marrow chimeras, we found that STING deficiency in host hematopoietic cells was primarily responsible for exacerbating the disease. Furthermore, STING on host CD11c + cells played a dominant role in suppressing allogeneic T-cell responses. Mechanistically, STING deficiency resulted in increased survival, activation, and function of APCs, including macrophages and dendritic cells. Consistently, constitutive activation of STING attenuated the survival, activation, and function of APCs isolated from STING V154M knock-in mice. STING-deficient APCs augmented donor T-cell expansion, chemokine receptor expression, and migration into intestinal tissues, resulting in accelerated/exacerbated GVHD. Using pharmacologic approaches, we demonstrated that systemic administration of a STING agonist (bis-(3'-5')-cyclic dimeric guanosine monophosphate) to recipient mice before transplantation significantly reduced GVHD mortality. In conclusion, we revealed a novel role of STING in APC activity that dictates T-cell allogeneic responses and validated STING as a potential therapeutic target for controlling GVHD after allo-HCT.

Allogeneic hematopoietic cell transplantation (allo-HCT) is performed with curative intent for high- risk blood cancers and bone marrow failure syndromes; yet the development of acute and chronic graft-vs.-host disease (GVHD) remain preeminent causes of death and morbidity. The IL-12 family of cytokines is comprised of IL-12, IL-23, IL-27, IL-35, and IL-39. This family of cytokines is biologically distinct in that they are composed of functional heterodimers, which bind to cognate heterodimeric receptor chains expressed on T cells. Of these, IL-12 and IL-23 share a common β cytokine subunit, p40, as well as a receptor chain: IL-12Rβ1. IL-12 and IL-23 have been documented as proinflammatory mediators of GVHD, responsible for T helper 1 (Th1) differentiation and T helper 17 (Th17) stabilization, respectively. The role of IL-27 is less defined, seemingly immune suppressive via IL-10 secretion by Type 1 regulatory (Tr1) cells yet promoting inflammation through impairing CD4 + T regulatory (Treg) development and/or enhancing Th1 differentiation. More recently, IL-35 was described as a potent anti-inflammatory agent produced by regulatory B and T cells. The role of the newest member, IL-39, has been implicated in proinflammatory B cell responses but has not been explored in the context of allo-HCT. This review is directed at discussing the current literature relevant to each IL-12-family cytokine and cognate receptor engagement, as well as the consequential downstream signaling implications, during GVHD pathogenesis. Additionally, we will provide an overview of translational strategies targeting the IL-12 family cytokines, their receptors, and subsequent signal transduction to control GVHD.

Metabolism, including catabolism and anabolism, is a basic cellular process necessary for cell survival. T lymphocytes have a distinct metabolism that can determine both fate and function. T-cell activation depends on glycolysis to obtain materials and energy for proliferation and effector function. Importantly, T cells utilize different metabolic processes under different conditions and diseases. Allogeneic hematopoietic cell transplantation (allo-HCT) is a classic immunotherapy for hematological malignancies; however, the development of graft-versus-host disease (GVHD) is a major factor limiting the success of allo-HCT. T cells in the donor graft drive GVHD by mounting a robust immunological attack against recipient normal tissues. Hence, understanding T-cell metabolism after allo-HCT would provide potential metabolic targets for the control of GVHD and primary tumor relapse. The purpose of the current review is to highlight the key metabolic pathways involved in alloantigen-activated T cells and to discuss how manipulating these pathways can serve as potential new therapeutic strategies to induce immune tolerance after allo-transplantation. We will also summarize the recent progress in regulating T-cell metabolism in bone marrow transplantation by targeting novel metabolic regulators or immune checkpoint molecules.

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.

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.