Anusara Daenthanasanmak

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.

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).

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.

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.