Graft-versus-leukemia Activity Research Articles

Icariin protects against acute graft-versus-host disease while preserving graft-versus-leukemia activity after allogeneic hematopoietic stem cell transplantation

BackgroundAcute graft-versus-host disease (aGVHD), which is mainly mediated by allogeneic T cells, is a decisive factor in the success of allogeneic hematopoietic stem cell transplantation (allo-HCT). Prophylaxis for aGVHD in clinical patients is unsatisfactory, and there is still a huge unmet need for novel approaches. Icariin (ICA) shows potent anti-inflammatory activity and suppresses T cell-mediated immune responses. Thus, ICA is a potential drug for the prevention of aGVHD. However, there is no data assessing the impact of ICA on aGVHD after allo-HCT. PurposeThis study aimed to investigate the protective effect of ICA against aGVHD and its mechanisms. Moreover, the impact of ICA on the graft-versus-leukemia (GVL) effect and engraftment of donor hematopoietic and immune cells were assessed. MethodsDifferent murine models of allo-HCT were developed to study the influence of the ICA on GVHD and GVL effect. Flow cytometry was used to analyze the growth of leukemia cells, alterations in different immune cells, and apoptosis. Cell proliferation was determined using a CCK-8 assay. RNA sequencing and quantitative proteomic analysis were performed to elucidate the underlying mechanisms, which were further verified by polymerase chain reaction or functional experiments. ResultsDifferent concentrations of ICA exhibited opposite effects: low-concentration ICA promoted, while high concentrations suppressed the proliferation and function of T cells. A high dose of ICA administration during days +3 to +5 post-allo-HCT can alleviate murine aGVHD but does not affect the course of chronic GVHD (cGVHD), the GVL effect against both acute myeloid and lymphoblastic leukemia, or the recovery of donor hematological and immune cells. ICA extensively represses the expansion, function, and infiltration of donor alloreactive T cells, while preserving regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSC). Quantitative proteomic analysis showed that downregulation of integrin-linked kinase (ILK) and lymphocyte cytosolic protein 2 (LCP2) expression was possibly associated with ICA-mediated aGVHD protective effects. Furthermore, an inhibitor of ILK, which can alleviate murine aGVHD administered early after allo-HCT. ConclusionThese findings suggest that the bioactivities of ICA are associated with its concentration and that ICA can effectively mitigate aGVHD without losing GVL activity or engraftment of donor hematopoietic and immune cells. Thus, ICA may be a promising drug for preventing aGVHD in clinical settings.

Tetramerization of pyruvate kinase M2 attenuates graft-versus-host disease by inhibition of Th1 and Th17 differentiation.

Lethal graft-versus-host disease (GVHD) is the major complication of allogeneic hematopoietic stem-cell transplantation (Allo-HSCT). Pyruvate kinase M2 (PKM2) is essential for CD4+ T-cell differentiation. Using the well-characterized mouse models of Allo-HSCT, we explored the effects of TEPP-46-induced PKM2 tetramerization on GVHD and graft-versus-leukemia (GVL) activity. TEPP-46 administration significantly improved the survival rate of GVHD. The severity of GVHD and histopathological damage of GVHD-targeted organs were obviously alleviated by PKM2 tetramerization. Additionally, tetramerized PKM2 inhibited the activation of NF-κB pathway and decreased the inflammation level of GVHD mice. PKM2 tetramerization blocked Th1 and Th17 cell differentiation and secretion of pro-inflammatory cytokine (IFN-γ, TNF-α, and IL-17). Meanwhile, differentiation of Treg cells and IL-10 secretion were promoted by tetramerized PKM2. These findings demonstrated that PKM2 enhanced the augment of Th1 and Th17 cells to accelerate the progression of GVHD, and allosteric activation of PKM2 targeted Th1 and Th17 cells attenuated GVHD. Furthermore, we also confirmed that TEPP-46 administration did not compromise GVL activity and resulted in slightly improvement of leukemia-free survive. Thus, targeting Th1 and Th17 cell response with PKM2 allosteric activator may be a promising therapeutic strategy for GVHD prevention while preserving the GVL activity in patients receiving Allo-HSCT.

Donor T cell STAT3 deficiency enables tissue PD-L1-dependent prevention of graft-versus-host disease while preserving graft-versus-leukemia activity.

STAT3 deficiency (STAT3-/-) in donor T cells prevents graft-versus-host disease (GVHD), but the impact on graft-versus-leukemia (GVL) activity and mechanisms of GVHD prevention remains unclear. Here, using murine models of GVHD, we show that STAT3-/- donor T cells induced only mild reversible acute GVHD while preserving GVL effects against nonsusceptible acute lymphoblastic leukemia (ALL) cells in a donor T cell dose-dependent manner. GVHD prevention depended on programmed death ligand 1/programmed cell death protein 1 (PD-L1/PD-1) signaling. In GVHD target tissues, STAT3 deficiency amplified PD-L1/PD-1 inhibition of glutathione (GSH)/Myc pathways that regulate metabolic reprogramming in activated T cells, with decreased glycolytic and mitochondrial ATP production and increased mitochondrial ROS production and dysfunction, leading to tissue-specific deletion of host-reactive T cells and prevention of GVHD. Mitochondrial STAT3 deficiency alone did not reduce GSH expression or prevent GVHD. In lymphoid tissues, the lack of host-tissue PD-L1 interaction with PD-1 reduced the inhibition of the GSH/Myc pathway despite reduced GSH production caused by STAT3 deficiency and allowed donor T cell functions that mediate GVL activity. Therefore, STAT3 deficiency in donor T cells augments PD-1 signaling-mediated inhibition of GSH/Myc pathways and augments dysfunction of T cells in GVHD target tissues while sparing T cells in lymphoid tissues, leading to prevention of GVHD while preserving GVL effects.

Retention of Donor T Cells in Lymphohematopoietic Tissue and Augmentation of Tissue PD-L1 Protection for Prevention of GVHD While Preserving GVL Activity.

Allogeneic hematopoietic cell transplantation (Allo-HCT) is a curative therapy for hematological malignancies (i.e., leukemia and lymphoma) due to the graft-versus-leukemia (GVL) activity mediated by alloreactive T cells that can eliminate residual malignant cells and prevent relapse. However, the same alloreactive T cells can cause a serious side effect, known as graft-versus-host disease (GVHD). GVHD and GVL occur in distinct organ and tissues, with GVHD occurring in target organs (e.g., the gut, liver, lung, skin, etc.) and GVL in lympho-hematopoietic tissues where hematological cancer cells primarily reside. Currently used immunosuppressive drugs for the treatment of GVHD inhibit donor T cell activation and expansion, resulting in a decrease in both GVHD and GVL activity that is associated with cancer relapse. To prevent GVHD, it is important to allow full activation and expansion of alloreactive T cells in the lympho-hematopoietic tissues, as well as prevent donor T cells from migrating into the GVHD target tissues, and tolerize infiltrating T cells via protective mechanisms, such as PD-L1 interacting with PD-1, in the target tissues. In this review, we will summarize major approaches that prevent donor T cell migration into GVHD target tissues and approaches that augment tolerization of the infiltrating T cells in the GVHD target tissues while preserving strong GVL activity in the lympho-hematopoietic tissues.

Ceramide synthase 6 impacts T-cell allogeneic response and graft-versus-host disease through regulating N-RAS/ERK pathway

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.

Targeting Glycolysis in Alloreactive T Cells to Prevent Acute Graft-Versus-Host Disease While Preserving Graft-Versus-Leukemia Effect.

Alloreactive donor T cells undergo extensive metabolic reprogramming to become activated and induce graft-versus-host disease (GVHD) upon alloantigen encounter. It is generally thought that glycolysis, which promotes T cell growth and clonal expansion, is employed in this process. However, conflicting data have been reported regarding the requirement of glycolysis to induce T cell-mediated GVHD due to the lack of T cell-specific treatments using glycolysis inhibitors. Importantly, previous studies have not evaluated whether graft-versus-leukemia (GVL) activity is preserved in donor T cells deficient for glycolysis. As a critical component affecting the clinical outcome, it is necessary to assess the anti-tumor activity following treatment with metabolic modulators in preclinical models. In the present study, we utilized T cells selectively deficient for glucose transporter 1 (Glut1T-KO), to examine the role of glycolysis exclusively in alloreactive T cells without off-targeting effects from antigen presenting cells and other cell types that are dependent on glycolysis. We demonstrated that transfer of Glut1T-KO T cells significantly improved acute GVHD outcomes through increased apoptotic rates, impaired expansion, and decreased proinflammatory cytokine production. In addition to impaired GVHD development, donor Glut1T-KO T cells mediated sufficient GVL activity to protect recipients from tumor development. A clinically relevant approach using donor T cells treated with a small molecule inhibitor of glycolysis, 2-Deoxy-D-glucose ex vivo, further demonstrated protection from tumor development. These findings indicate that treatment with glycolysis inhibitors prior to transplantation selectively eliminates alloreactive T cells, but spares non-alloreactive T cells including those that protect against tumor growth. The present study has established a definitive role for glycolysis in acute GVHD and demonstrated that acute GVHD can be selectively prevented through targeting glycolysis.

Clonal expansion of CD8+ T cells reflects graft-versus-leukemia activity and precedes durable remission following DLI

AbstractDonor lymphocyte infusion (DLI) is a standard of care for relapse of acute myeloid leukemia after allogeneic hematopoietic stem cell transplantation. Currently it is poorly understood how and when CD8+ αβ T cells exert graft-versus-leukemia (GVL) activity after DLI. Also, there is no reliable biomarker to monitor GVL activity of the infused CD8+ T cells. Therefore, we analyzed the dynamics of CD8+ αβ T-cell clones in patients with DLI. In this prospective clinical study of 29 patients, we performed deep T-cell receptor β (TRB ) sequencing of sorted CD8+ αβ T cells to track patients' repertoire changes in response to DLI. Upon first occurrence of GVL, longitudinal analyses revealed a preferential expansion of distinct CD8+TRB clones (n = 14). This did not occur in samples of patients without signs of GVL (n = 11). Importantly, early repertoire changes 15 days after DLI predicted durable remission for the 36-month study follow-up. Furthermore, absence of clonal outgrowth of the CD8+TRB repertoire after DLI was an early biomarker that predicted relapse at a median time of 11.2 months ahead of actual diagnosis. Additionally, unbiased sample analysis regardless of the clinical outcome revealed that patients with decreasing CD8+TRB diversity at day 15 after DLI (n = 13) had a lower relapse incidence (P = .0040) compared with patients without clonal expansion (n = 6). In conclusion, CD8+TRB analysis may provide a reliable tool for predicting the efficacy of DLI and holds the potential to identify patients at risk for progression and relapse after DLI.

NOTCH Inhibition Separates GVL from GVHD Via Modulating Host Dendritic Cell and Donor T-Cell Activation

Background: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) can eradicate chemorefractory leukemia and reduce the relapse rate of high risk cases through the graft versus-leukemia (GVL) activity. However, graft-versus host disease (GVHD) limited the overall clinical benefit of allo-HSCT. Efforts are made to separate GVL from GVHD but few approaches are confirmed effective. Notch is a highly conserved signaling system important for lymphocyte effector and memory differentiation. Previous studies have shown that inhibition of Notch signaling could reduce aGVHD ex vivo and in vivo by downregulating both activated antigen presenting cells (APCs) and CD8+ T cells. It is less established whether Notch inhibition could also maintain GVL activity while reducing GVHD. The aim of the current work was to further study whether GVL activity could be maintained or enhanced by Notch inhibition.Methods: Murine models of GVHD and GVL were established (C57BL/6→BALB/c). γ-secretase inhibitor N-S-phenyl-glycine-t-butyl ester (DAPT) was used to block Notch1 signaling. The recipients were monitored daily for survival, clinical signs of GVHD and tumor size. GVHD histopathology were systematically examined and evaluated based on the scoring system. In order to study the mechanism of Notch inhibition on both GVHD and GVL, phenotypes of APCs and T cells in recipients were analyzed by flow cytometry. Expression of FasL by PCR, levels of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) by ELISA and cytotoxicity of T cells were compared in recipients with or without Notch inhibition.Results: Reduced GVHD was observed in Notch inhibited recipients, which is similar to the results of previous study. However, in GVL model, both the median time when tumor volume is over 0.5cm3 and the change of tumor volume had no difference with or without Notch inhibition. Suppressed differentiation of CD8+ cytotoxic T cells (CTL) and decreased levels of TNF-α did not result in attenuated GVL activity. Further mechanism studies showed that Notch inhibition up-regulates CD11c+CD80+ DCs and central memory T cells (CD4+CD62L+ and CD8+CD62L+ T cells) while suppressing CD8+ CTL differentiation. We then compared levels of TNF-α and IFN-γ which are important in mediating aGVHD. Levels of TNF-α were decreased but IFN-γ were increased rather than decreased after Notch inhibition. We hypothesized that the contradictory results may ascribe to the difference between TNF-α and IFN-γ in inducing GVHD and GVL. Reduced TNF-α and increased IFN-γ levels could be the causes for down-regulated donor CTL activity. Moreover, FasR-FasL interaction plays a critical role in GVL instead of aGVHD. FasL expression were similar in in DAPT groups and the controls, suggesting similar GVL activity in recipients with or without Notch inhibition.Conclusions: Notch inhibition reduces GVHD and does not impair GVL effect. Increased number of central memory T cells and higher levels of interferon-γ (IFN-γ) could be the causes for enhanced GVL. DisclosuresLi:Guangzhou Science and Technology Project (No. 2015100010211): Research Funding; National Natural Science Foundation of China (91642111): Research Funding; Guangdong Provincial Basic Research Program (No. 2015B020227003: Research Funding; Guangdong Provincial Applied Science and Technology Research & Development Program (No. 2016B020237006): Research Funding.

Targeting JAK2 Kinase Promotes Foxp3 Stability and Function of CD8iTregs in Prevention of Graft-Versus-Host Disease

Graft-versus-host disease (GVHD) is a serious complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Although donor T cells (Teff) have a beneficial effect on graft-versus-leukemia (GVL), they cause mortality and morbidity due to GHVD. Regulatory T cells (Tregs; CD4+Foxp3+) have been demonstrated to efficiently prevent GHVD by suppressing Teff proliferation. In addition to CD4+Foxp3+ Tregs, CD8+Foxp3+induced Tregs (CD8 iTregs) have also been identified not only inhibit Teff function, but also possess the GVL activity. However, instability of CD8 iTregs is an important barrier for GVHD clinical applications. Previous studies have revealed that Janus kinase2 (JAK2) signal transduction is an essential mediator of immune response. JAK2 activation is a positive regulator of Th1 and Th17 differentiation whereas JAK2 acts as a negative regulator in Tregs as its activation leads to Foxp3 instability.In this current study, we attempted to promote Foxp3 stability in CD8 iTregs by targeting JAK2 kinase using JAK2 conditional knockout mice (JAK2fl/flCD4Cre). The enhanced stability of CD8 iTregs could be a potential Treg-based therapy to prevent GVHD onset while maintaining GVL activity. In the generation of alloreactive iTregs in vitro, we found that a significant higher frequency of Foxp3+ cells could be induced from JAK2-/-CD8 T cells than WT CD8 T cells (Fig. 1A). We also observed that JAK2-/- CD8 iTregs exhibited higher levels of canonical Treg markers, Neuropilin-1 (NRP1), which is known to express on natural Tregs (nTregs) and has been proposed to associate with Treg stability (Fig. 1B). We initially examined in vitro stability of JAK2-/- CD8 iTregs in the presence of proinflammatory cytokines. Strikingly, JAK2-/-CD8 iTregs retained much higher Foxp3 than WT CD8 iTregs. Thus, we further investigated the function of JAK2-/- CD8 iTregs in preventing GVHD after allo-HSCT. Upon iTreg transfer into MHC-mismatched C57BL/6 (B6) to BALB/c model, JAK2-/- CD8 iTregs could significantly alleviate GVHD with a marked reduction of recipient's mortality (Fig. 1C).To understand the immune mechanisms of JAK2-/- CD8 iTregs in vivo, spleen, liver and mesenteric lymph nodes from recipient mice received JAK2-/- CD8 iTregs were analyzed on day 7 and 14 after allo-HSCT. JAK2-/- CD8 iTregs (Ly5.2+) remarkably suppressed Teff (Ly5.1+) expansion and maintain higher Foxp3 expression compared to WT counterparts (Fig. 1D). Consistently, lower levels of inflammatory cytokines (IFNγ, TNFα, IL-17 and IL-6) were detected from serum of mice received JAK2-/- CD8 iTregs than those of WT iTregs. For translational application, we evaluated the systemic effect of specific JAK2 kinase inhibitor (Pacritinib; PAC) on CD8 iTregs using a GVL model (B6 to BDF1). Mice transplanted with WT CD8iTregs plus P815 mastocytoma and then treated with Pacritinib could significantly prolong the survival and exhibited the GVL activity (Fig. 1E). In conclusion, our studies provide a strong rationale and means to stabilize CD8 iTregs by targeting JAK2 kinase, and stabilizedCD8 iTregs have a therapeutic potential for controlling GVHD in clinic. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Early Administration of Low-Dose IL-2 Intensify Graft-Versus-Leukemia Effect without Worsening GVHD through Sequential Enhancement of Effector T Cell and CD62L+ Regulatory T Cell Subset

Allogeneic hematopoietic stem cell transplantation (HSCT) is the curative treatment of many hematological malignancies, but the relapse is one of the major causes of treatment failure and mortality. Previous clinical studies have shown that low-dose IL-2 was well tolerated and could lower the relapse rate in human T cell depleted allogeneic BMT. On the other hand, we also demonstrated that low-dose IL-2 could preferentially enhance regulatory T cell (Treg) and improve the symptoms of chronic GVHD (NEJM 2011,Sci Trans Med 2013). In murine bone marrow transplantation (BMT) model showed that Treg can protect from lethal GVHD while preserving graft-versus-leukemia (GVL) effect. Especially, CD62L+ Tregs has a higher capacity in suppressing GVHD than their CD62L- counterpart. Our recent study reported that exogenous low-dose IL-2 induced the expression of PD-1 on Treg preferentially and it was most evident in CD44+CD62L+central-memory Treg (Blood 2016). However, the impact of CD62L+ Treg enhanced by low-dose IL-2 on GVHD and GVL has not been well investigated. In this study, we studied the effect of exogenous IL-2 on the lymphocyte phenotypic profiles including naïve- versus memory-type balance and also examined donor-derived immunity, especially GVHD and GVL, by using leukemia-bearing BMT model. Lethally irradiated B6D2F1 recipients were transplanted spleen cells and BM cells from B6 donors and 5000 IU Teceleukin (recombinant IL-2) was injected once a day for 14 days. The number of CD4+CD25+Foxp3+ Treg, CD4+CD25-Foxp3- conventional T cell (Tcon) and CD8+ T cell was compared between IL2- and vehicle-treated groups. The expressions of CD44, CD62L, CCR4, CCR7, Ki-67, PD-1, CTLA-4, LAG3, GITR, ICOS and TIM-3 in each subset were also examined. Lymphocytes profiling analysis showed that IL-2 treatment in the first 7 days resulted in the dominant expansion of effector T cells without Treg increase (Treg 0.10 vs 0.16,p=0.35, Tcon 2.83 vs 4.20, p=0.01, CD8 T cell 5.52 vs 10.4, p=0.01, million). On the contrary, the extended IL-2 treatment in the next 7 days increased CD62L+ Treg (21.5 vs 48.2 % of total Treg, p<0.001, 0.02 vs 0.07 million, p=0.006) without increasing effector T cells. PD-1 on Treg was very high in BMT-recipients even without receiving IL-2, so further enhancement of PD-1 expression by IL-2 was not observed. However, the expression of other inhibitory molecules including CTLA-4 (MFI, 6.61 vs 9.92, p=<0.001), LAG-3 (1.73 vs 2.17, p=0.04) and ICOS (2.41 vs 3.84, P<0.001) was enhanced by the IL-2 treatment. To elucidate the mechanisms of the increase in CD62L+ Treg, the in vivo violet dye dilution assay was performed. We first sorted out CD62L+ T cells by micro-beads from spleen cells, and then adaptively transferred into recipients. Five days treatment of low-dose IL-2 resulted in the enhancement of the division of CD62L+ Treg (% divided cells; 19.5 vs 29.8 % of Treg, p=0.005, 62.3 vs 108 in number, p=0.002). Interestingly, when CD62L- T cells were transferred, IL-2 treatment also induced the division of the cells and, of note, induced the phenotypic change of CD62L- into CD62L+ Treg (7.63 vs 19.8 % of Treg, p=0.009, 91.3 vs 361, p=0.01). These results suggested that IL-2 have the possibility to enhance the proliferative activity of Treg with the up-regulation of CD62L expression. Lastly, we transplanted lucipherase+P815 leukemia cells into this BMT model and evaluated the clinical impact of low-dose IL-2 on GVL by using in vivo bioluminescence system. To assesse the GVL and GVHD independently, we used two different tumor burden models. In the high tumor burden model, IL-2 injection decreased bioluminescence intensity (BLI) of P815 (BLI at week3, 8.3x10E7 vs 8.1x10E6 photon/second, p<0.001) and significantly extends overall survival. On the other hand, in the low tumor burden model, both treatment groups did not die from leukemia but vehicle-treated control group showed a significant higher clinical GVHD score and died from GVHD. These data demonstrated the clinical benefits on anti-tumor effect of IL-2, which could enhance the GVL activity without the detrimental effect on GVHD. In conclusion, our data indicate multiple effects of IL-2 on post-transplant immunity can coordinate to intensify GVL separately from GVHD on the mechanism of sequential enhancement of effector T cell and CD62L+ Treg in the different phases. These data provide the important information for developing Treg-targeted therapy. DisclosuresMaeda:Toko Pharmaceutical Industries: Other: Investigational drug is provided free of charge.

Microrna-31 Promotes T-Cell Pathogenicity in Chronic Graft-Versus-Host Disease

MicroRNAs (miRs) are short, non-coding RNAs that repress gene expression at post-transcriptional level. Certain miRs play critical roles on T-cell responses in inflammation and autoimmunity. To understand the biology of miRs in T-cell allo-responses, we measured expression profiles of miRs in donor T cells in allogeneic vs. syngeneic recipients following bone marrow transplantation (BMT). We found miR-31 was dramatically up-regulated in T cells under alloantigen-driven vs. homeostatic proliferation in vivo(Fig. A). MiR-31 was shown to regulate autoimmunity and cancer. However, its roles in regulating T-cell response remain largely undetermined. Recent study shows miR-31 represses peripheral Treg differentiation in EAE development. In contrast, miR-31 inhibits CD8 T-cell function in anti-virus response. We hypothesize that miR-31 is required for CD4 T-cell pathogenicity in chronic graft-versus-host disease (cGVHD), but is dispensable for CD8 T-cell cytolytic function in graft-versus-leukemia (GVL) activity.To study how miR-31 regulates T-cell allo-response, we used mice with miR-31 conditional knock out (KO) in T cells (miR-31fl/fl CD4Cre). No differences were observed in the baseline composition of naïve WT (miR-31fl/fl) and miR-31 KO splenocytes. After being transferred into allogeneic recipients, KO T cells had impaired ability to proliferate, reflected by fewer divided (% CFSElow) cells and reduced donor T-cell expansion in recipient spleens (Fig. B). To further evaluate how miR-31 contributes to T-cell pathogenicity in cGVHD, we used a murine model of BMT (B6 → BALB/c), in which low dose spleonocyes can induce scleroderma manifestation. Significant reduction of clinical scores (Fig. C), including improved skin scores and body weight loss, were found in the recipients of miR-31 KO grafts compared to those with WT grafts. This finding was validated in another haploidentical BMT model of scleroderma (B6 → B6D2F1), indicating miR-31 is required for T-cell to induce cGVHD. Consistently, the pathological damage was dramatically reduced in skin, gut and lung in the recipients of miR-31 KO grafts (Fig. D).Mechanistically, miR-31 deficiency in donor T cells resulted in significantly reduced numbers of CD4 T cells that produce IFNγ, IL-4/5 or IL-17 in recipient spleen and mesenteric lymph nodes. In absence of miR-31, fewer effector and central memory cells were generated from donor CD4 T cells. Further, T cells that are capable of localizing to B-cell follicles (CXCR5+PD-1hi) were significantlyreduced in spleen (Fig. E) and peripheral lymph nodes in recipients of miR-31 KO grafts. Interestingly, among these cells, more foxp3+ follicular T-regulatory (Tfr) but fewer foxp3- follicular T-helper (Tfh) cells were observed in the recipients of KO grafts (Fig. F), indicating miR-31 contributes to the unbalance of Tfr and Tfh cells during cGVHD development. Consistently with the role of miR-31 in regulating Tfr and Tfh cells, we found Blimp1 is the predicted targets of miR-31, which is critically involved in the homeostasis of Tfr and Tfh cells. On donor B-cell compartment, lower levels of CD86 and MHC-II expression were observed in the recipients of miR-31 KO grafts. In addition, germinal center B cells and plasma cells were also reduced in those recipients, indicating miR-31 indirectly regulates B-cell activation and differentiation through controlling T-cell pathogenicity.For translational purpose, we evaluated how miR-31 impacts T cell-mediated GVL effect. Using B6 → BDF1 BMT model, we consistently found attenuated skin manifestation in recipients of KO T cells. Importantly, in the ongoing experiment, miR-31 KO T cells were capable of controlling P815(mastocytoma) growth as efficiently as WT T cells (Fig. G), suggesting miR-31 may be dispensable for T cells to mediating GVL activity after allogeneic BMT.Taken together, the current work reveals that miR-31 plays pleotropic roles in enhancing CD4 T-cell pathogenicity in cGVHD. MiR-31 represents a promising therapeutic target for the control of cGVHD while sparing GVL effects after allogeneic hematopoietic stem cell transplantation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Tolerogenic anti–IL-2 mAb prevents graft-versus-host disease while preserving strong graft-versus-leukemia activity

AbstractDonor T cells mediate both graft-versus-leukemia (GVL) activity and graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (allo-HCT). Development of methods that preserve GVL activity while preventing GVHD remains a long-sought goal. Tolerogenic anti–interleukin-2 (IL-2) monoclonal antibody (JES6-1) forms anti–IL-2/IL-2 complexes that block IL-2 binding to IL-2Rβ and IL-2Rγ on conventional T cells that have low expression of IL-2Rα. Here, we show that administration of JES6 early after allo-HCT in mice markedly attenuates acute GVHD while preserving GVL activity that is dramatically stronger than observed with tacrolimus (TAC) treatment. The anti–IL-2 treatment downregulated activation of the IL-2-Stat5 pathway and reduced production of granulocyte-macrophage colony-stimulating factor (GM-CSF). In GVHD target tissues, enhanced T-cell programmed cell death protein 1 (PD-1) interaction with tissue–programmed cell death-ligand 1 (PD-L1) led to reduced activation of protein kinase–mammalian target of rapamycin pathway and increased expression of eomesodermin and B-lymphocyte-induced maturation protein-1, increased T-cell anergy/exhaustion, expansion of Foxp3–IL-10–producing type 1 regulatory (Tr1) cells, and depletion of GM-CSF–producing T helper type 1 (Th1)/cytotoxic T cell type 1 (Tc1) cells. In recipient lymphoid tissues, lack of donor T-cell PD-1 interaction with tissue PD-L1 preserved donor PD-1+TCF-1+Ly108+CD8+ T memory progenitors and functional effectors that have strong GVL activity. Anti–IL-2 and TAC treatments have qualitatively distinct effects on donor T cells in the lymphoid tissues, and CD8+ T memory progenitor cells are enriched with anti–IL-2 treatment compared with TAC treatment. We conclude that administration of tolerogenic anti–IL-2 monoclonal antibody early after allo-HCT represents a novel approach for preventing acute GVHD while preserving GVL activity.

Targeting the Complement Alternative Pathway Permits Graft Versus Leukemia Activity while Preventing Graft Versus Host Disease.

Application of allogeneic hematopoietic cell transplantation (allo-HCT) for patients with hematologic disorders is limited by the development of GVHD. Separation of GVHD and graft-versus-leukemia (GVL) remains a great challenge in the field. We investigated the contribution of individual pathways involved in the complement cascade in GVH and GVL responses to identify specific targets by which to separate these two processes. We used multiple preclinical murine and human-to-mouse xenograft models involving allo-HCT recipients lacking components of the alternative pathway (AP) or classical pathway (CP)/lectin pathway (LP) to dissect the role of each individual pathway in GVHD pathogenesis and the GVL effect. For translational purposes, we used the AP-specific complement inhibitor, CR2-fH, which localizes in injured target organs to allow specific blockade of complement activation at sites of inflammation. Complement deposition was evident in intestines of mice and patients with GVHD. In a preclinical setting, ablation of the AP, but not the CP/LP, significantly improved GVHD outcomes. Complement activation through the AP in host hematopoietic cells, and specifically dendritic cells (DC), was required for GVHD progression. AP deficiency in recipients decreased donor T-cell migration and Th1/Th2 differentiation, while increasing the generation of regulatory T cells. This was because of decreased activation and stimulatory activity of recipient DCs in GVHD target organs. Treatment with CR2-fH effectively prevented GVHD while preserving GVL activity. This study highlights the AP as a new therapeutic target to prevent GVHD and tumor relapse after allo-HCT. Targeting the AP by CR2-fH represents a promising therapeutic approach for GVHD treatment.

Targeting Histone Deacetylases to Modulate Graft-Versus-Host Disease and Graft-Versus-Leukemia.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the main therapeutic strategy for patients with both malignant and nonmalignant disorders. The therapeutic benefits of allo-HSCT in malignant disorders are primarily derived from the graft-versus-leukemia (GvL) effect, in which T cells in the donor graft recognize and eradicate residual malignant cells. However, the same donor T cells can also recognize normal host tissues as foreign, leading to the development of graft-versus-host disease (GvHD), which is difficult to separate from GvL and is the most frequent and serious complication following allo-HSCT. Inhibition of donor T cell toxicity helps in reducing GvHD but also restricts GvL activity. Therefore, developing a novel therapeutic strategy that selectively suppresses GvHD without affecting GvL is essential. Recent studies have shown that inhibition of histone deacetylases (HDACs) not only inhibits the growth of tumor cells but also regulates the cytotoxic activity of T cells. Here, we compile the known therapeutic potential of HDAC inhibitors in preventing several stages of GvHD pathogenesis. Furthermore, we will also review the current clinical features of HDAC inhibitors in preventing and treating GvHD as well as maintaining GvL.

Endothelial Protective Effects of Defibrotide Reduce Acute Graft Versus Host Disease after Experimental Allogeneic Hematopoietic Stem Cell Transplantation

Introduction Graft versus host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). The pathophysiology of acute GVHD involves donor T cell- and inflammatory cytokine-mediated injury to the patient's tissues and causes significant morbidity and mortality. Defibrotide is a mixture of single stranded nucleotides that has a number of pharmacological effects that contribute to its endothelial protective properties. We hypothesized that preventive treatment with defibrotide will reduce the incidence and severity of GVHD due to its endothelial protective and anti-inflammatory properties and potentially does not alter graft versus leukemia (GVL) responses. Methods Recipient B10.BR mice received Cyclophosphamide 120 mg/kg per day on day-3 and -2, and received lethal total body irradiation (750cGy) on day 0, followed by the infusion of donor C57BL/6 T-cell depleted bone marrow cells. To induce GVHD, respective groups were co-infused with splenocytes, while non-GVHD controls received BM cells only. Mice were treated with defibrotide at the dose of 800mg/kg per body weight or vehicle control for the first week daily and then three times/week thereafter. For GVL experiments, C57BL/6 recipients were lethally irradiated (12Gy) with TBI and transplanted with C3H.SW donor TCD bone marrow alone or in combination with 3 million T-cells along with C1498-luc and treated with defibrotide or vehicle controls. Results Allogeneic defibrotide treated recipients demonstrated significantly better survival when compared to control group (83.34% vs 54.55% on day +28). Clinical GVHD scores were significantly worse after day +14 in control group when compared to defibrotide treated recipients. Both at day +7 and on day +28 allogeneic defibrotide treated recipients showed significantly less organ GVHD in gut, lung and liver (p Conclusion Defibrotide treatment through its anti-inflammatory and endothelium-protective effects reduces the incidence and severity of acute GVHD, while not impairing GVL activity.

Caspase-11 signaling enhances graft-versus-host disease

Acute graft-versus-host disease (GVHD) remains a major obstacle for the wider usage of allogeneic hematopoietic stem cell transplantation (allo-HSCT), which is an effective therapy for hematopoietic malignancy. Here we show that caspase-11, the cytosolic receptor for bacterial endotoxin (lipopolysaccharide: LPS), enhances GVHD severity. Allo-HSCT markedly increases the LPS-caspase-11 interaction, leading to the cleavage of gasdermin D (GSDMD). Caspase-11 and GSDMD mediate the release of interleukin-1α (IL-1α) in allo-HSCT. Deletion of Caspase-11 or Gsdmd, inhibition of LPS-caspase-11 interaction, or neutralizing IL-1α uniformly reduces intestinal inflammation, tissue damage, donor T cell expansion and mortality in allo-HSCT. Importantly, Caspase-11 deficiency does not decrease the graft-versus-leukemia (GVL) activity, which is essential to prevent cancer relapse. These findings have major implications for allo-HSCT, as pharmacological interference with the caspase-11 signaling might reduce GVHD while preserving GVL activity.

The mechanistic study behind suppression of GVHD while retaining GVL activities by myeloid-derived suppressor cells.

Graft-versus-host disease (GVHD) is a major barrier to the widespread use of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for treating hematologic malignancies. Myeloid-derived suppressor cells (MDSCs) have been recognized as crucial immunosuppressive cells in various pathologic settings. Here, we investigated whether the unique functional properties of MDSCs could be harnessed to control allo-HSCT-associated GVHD. Using multiple murine GVHD/GVL models including both MHC-mismatched and miHA-mismatched, we demonstrated that treatment with CD115+ MDSCs efficiently suppressed GVHD but did not significantly impair graft-versus-leukemia (GVL) activity, leading to 80% and 67% protection in treated mice in GVHD and GVL models, respectively. The mechanism for this dissociation of GVHD from GVL, specifically the emergence of donor-derived NKG2D+ CD8 T cells with a memory phenotype in MDSC-treated recipient mice, was identified. NKG2D expression on donor T cells was required for eradication of allogeneic lymphoma cells. Furthermore, long-term surviving MDSC recipients that exhibited cytolytic activities against allogeneic leukemia cells had a significantly increased percentage of T regulatory cells and, more importantly, NKG2D+ CD8 T cells. These findings indicate that MDSCs can be used as a novel cell-based therapy to suppress GVHD while maintaining GVL activities through selective induction of NKG2D+ CD8 memory T cells.

Regulation of GVHD and GVL Activity via PD-L1 Interaction With PD-1 and CD80.

Allogeneic hematopoietic cell transplantation (HCT) is a curative therapy for hematological malignancies (i.e. leukemia and lymphoma), because graft-versus-leukemia (GVL) activity mediated by alloreactive T cells can eliminate residual malignant cells and prevent relapse. However, the same alloreactive T cells also mediate a severe side effect, graft-versus-host disease (GVHD), and prevention of GVHD while preserving GVL activity remains an elusive goal. The immune checkpoint molecule PD-L1 and its interaction with PD-1 receptor in regulating cancer immunity is under intensive and wide-spread study, but knowledge about this interaction in regulating GVHD and GVL activity is very limited. In this review, we summarize the literature exploring how PD-L1 interaction with its receptors PD-1 and CD80 regulate GVHD and GVL activities, how PD-L1 signaling regulates T cell metabolic profiles, and how a differential role of PD-L1 interaction with PD-1, CD80 or both may provide a novel avenue to prevent GVHD while preserving strong GVL effects.

Vitamin C Stabilizes CD8iTregs and Enhances Their Therapeutic Potential in Controlling GvHD and Leukemia Relapse

Graft-versus-host disease (GvHD) remains a leading cause of non-relapse mortality after allogeneic hematopoietic cell transplantation (allo-HCT). The imbalance between T effector (Teffs) and regulatory cells (Tregs) is a defined characteristic of GvHD. Therefore, promoting Tregs for controlling GvHD has drawn an intense interest in preclinical and clinical studies of allo-HCT. Although CD4 Tregs (CD4+Foxp3+) have been shown to effectively prevent GvHD, they impaired graft-versus-leukemia (GVL) effect. Our lab has previously demonstrated that CD8 iTregs (CD8+Foxp3+) not only suppressed allogeneic T-cell responses, but also possessed the GVL activity themselves. However, instability of Foxp3 in CD8 iTregs is a major obstacle to translate this Treg population into clinic application.Vitamin C has been reported to promote Foxp3 stability by accelerating its demethylation. In the current study, we asked whether vitamin C could stabilize CD8 iTregs and enhance their therapeutic potential in controlling GvHD. Addition of vitamin C in the culture significantly increased the generation of allo-reactive CD8 iTregs in vitro (Fig. 1A). These CD8 iTregs generated with vitamin C had strikingly increased demethylation on Foxp3 CpGs (Fig. 1B) and superior suppressive activity (data not shown) compared to control-treated CD8 iTregs. We further evaluated the capability of vitamin C-treated CD8 iTregs in preventing GvHD in allo-HCT. Using a MHC-mismatched murine BMT model, vitamin C-treated CD8 iTregs could significantly alleviate GvHD reflected by a marked reduction of recipient mortality (Fig. 1C) and clinical scores (data not shown).To determine the Foxp3 stability in vivo, we analyzed the transferred CD8 iTregs and allogeneic Teffs in recipient spleens 7 days after allo-HCT. Vitamin C-treated CD8 iTregs (Ly5.2+) remarkably suppressed Teff (Ly5.1+) expansion and maintained higher Foxp3 expression compared to the control iTregs (Fig. 1D). To address a critical question whether vitamin C-treated CD8 iTregs can preserve the GVL activity, we performed a haploidentical transplant with a leukemia relapse model, and found that vitamin C-treated CD8 iTregs not only attenuated GvHD severity, but also prevented recipients from tumor relapse (Fig. 1E). In conclusion, our studies provide a rationale and mean to use stabilized CD8 iTregs with vitamin C for controlling GvHD and leukemia relapse in the clinic. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Notch inhibition enhances graft-versus-leukemia while reducing graft-versus-host disease

Graft-versus host disease (GVHD) remains the most significant complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Dissociation of graft versus-leukemia (GVL) activity from GVHD has yet to be achieved. In this study, we used γ-secretase inhibitor (GSIs, DAPT) to inhibit Notch signaling in GVHD and GVL murine model. We found that CD11c+CD80+ dendritic cells (DCs) were up-regulated but did not enhance GVHD. Regulatory T cells (Tregs) and central memory T cells that express high levels of CD62L and CD44 had an expansion after Notch inhibition. Reduced Tumor Necrosis Factor-α and increased Interferon-γ production were found, which might be ascribed to the expansion of Tregs and central memory T cells, and result in increased sensitivity of tumor cells to cytotoxic T lymphocyte activity. Fas Receptor-Fas Ligand interaction plays a critical role in GVL instead of aGVHD. Fas Ligand expressions were similar in recipients with or without Notch inhibition, suggesting that GVL activity was maintained.We showed that Notch inhibition could enhances GVL while reducing GVHD via modulating host DCs and donor T cell activity, and the production of inflammatory cytokines.