Transfer of Cell-Surface Antigens by Scavenger Receptor CD36 Promotes Thymic Regulatory T Cell Receptor Repertoire Development and Allo-tolerance.

The development of T cell tolerance in the thymus requires the presentation of host proteins by multiple antigen-presenting cell (APC) types. However, the importance of transferring host antigens from transcription factor AIRE-dependent medullary thymic epithelial cells (mTECs) to bone marrow (BM) APCs is unknown. We report that antigen was primarily transferred from mTECs to CD8α+ dendritic cells (DCs) and showed that CD36, a scavenger receptor selectively expressed on CD8α+ DCs, mediated the transfer of cell-surface, but not cytoplasmic, antigens. The absence of CD8α+ DCs or CD36 altered thymic T cell selection, as evidenced by TCR repertoire analysis and the loss of allo-tolerance in murine allogeneic BM transplantation (allo-BMT) studies. Decreases in these DCs and CD36 expression in peripheral blood of human allo-BMT patients correlated with graft-versus-host disease. Our findings suggest that CD36 facilitates transfer of mTEC-derived cell-surface antigen on CD8α+ DCs to promote tolerance to host antigens during homeostasis and allo-BMT.

S100A9 upregulated by IFNGR signaling blockade functions as a novel GVHD suppressor without compromising GVL in mice.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative treatment for both malignant and nonmalignant hematologic disorders. However, graft-versus-host disease (GVHD) and malignant relapse limit its therapeutic success. We previously demonstrated that the blockade of interferon-gamma receptor (IFNGR) signaling in donor T cells resulted in a reduction in GVHD while preserving graft-versus-leukemia (GVL) effects. However, the underlying molecular mechanisms remain inconclusive. In this study, we found that S100A9 is a novel GVHD suppressor upregulated when IFNGR is blocked in T cells. Both Ifngr1-/- and S100a9-overexpressing T cells significantly reduced GVHD without compromising GVL, altering donor T-cell trafficking to GVHD target organs in our mouse model of allo-HSCT. In addition, in vivo administration of recombinant murine S100A9 proteins prolongs the overall survival of recipient mice. Furthermore, in vivo administration of anti-human IFNGRα neutralizing antibody (αhGR-Nab) significantly upregulates the expression of S100A9 in human T cells and improved GVHD in our mouse model of xenogeneic human peripheral blood mononuclear cell transplantation. Consistent with S100a9-overexpressing T cells in our allo-HSCT model, αhGR-Nab reduced human T-cell trafficking to the GVHD target organs. Taken together, S100A9, a downstream molecule suppressed by IFNGR signaling, functions as a novel GVHD suppressor without compromising GVL.

Novel JAK Inhibitors to Reduce Graft-Versus-Host Disease after Allogeneic Hematopoietic Cell Transplantation in a Preclinical Mouse Model.

Allogeneic hematopoietic cell transplantation (allo-HCT) is a highly effective, well-established treatment for patients with various hematologic malignancies and non-malignant diseases. The therapeutic benefits of allo-HCT are mediated by alloreactive T cells in donor grafts. However, there is a significant risk of graft-versus-host disease (GvHD), in which the donor T cells recognize recipient cells as foreign and attack healthy organs in addition to malignancies. We previously demonstrated that targeting JAK1/JAK2, mediators of interferon-gamma receptor (IFNGR) and IL-6 receptor signaling, in donor T cells using baricitinib and ruxolitinib results in a significant reduction in GvHD after allo-HCT. Furthermore, we showed that balanced inhibition of JAK1/JAK2 while sparing JAK3 is important for the optimal prevention of GvHD. Thus, we have generated novel JAK1/JAK2 inhibitors, termed WU derivatives, by modifying baricitinib. Our results show that WU derivatives have the potential to mitigate GvHD by upregulating regulatory T cells and immune reconstitution while reducing the frequencies of antigen-presenting cells (APCs) and CD80 expression on these APCs in our preclinical mouse model of allo-HCT. In addition, WU derivatives effectively downregulated CXCR3 and T-bet in primary murine T cells. In summary, we have generated novel JAK inhibitors that could serve as alternatives to baricitinib or ruxolitinib.

S100A8 and S100A9 in Hematologic Malignancies: From Development to Therapy.

S100A8 and S100A9 are multifunctional proteins that can initiate various signaling pathways and modulate cell function both inside and outside immune cells, depending on their receptors, mediators, and molecular environment. They have been reported as dysregulated genes and proteins in a wide range of cancers, including hematologic malignancies, from diagnosis to response to therapy. The role of S100A8 and S100A9 in hematologic malignancies is highlighted due to their ability to work together or as antagonists to modify cell phenotype, including viability, differentiation, chemosensitivity, trafficking, and transcription strategies, which can lead to an oncogenic phase or reduced symptoms. In this review article, we discuss the critical roles of S100A8, S100A9, and calprotectin (heterodimer or heterotetramer forms of S100A8 and S100A9) in forming and promoting the malignant bone marrow microenvironment. We also focus on their potential roles as biomarkers and therapeutic targets in various stages of hematologic malignancies from diagnosis to 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.

Azacitidine Mitigates Graft-versus-Host Disease via Differential Effects on the Proliferation of T Effectors and Natural Regulatory T Cells In Vivo.

Azacitidine (AzaC) mitigates graft-versus-host disease (GvHD) in both murine preclinical transplant models and in human clinical trials while maintaining a robust graft-versus-leukemia effect. Previous studies have failed to investigate the role of natural regulatory T cells (nTregs) on the mitigation of GvHD by AzaC, instead focusing on the generation of suppressive Tregs (CD4+CD25+FOXP3+) through the in vivo conversion of alloreactive donor T effectors (Teffs; CD4+CD25-FOXP3-) and the direct antiproliferative effects of AzaC on allogeneic T cells. Using B6.Foxp3DTR/GFP mice in which Tregs can be specifically ablated through administration of diphtheria toxin, we demonstrate that natural Tregs are required in the donor graft for AzaC to optimally protect against GvHD and that nTregs, unlike Teffs (CD3+FOXP3-), are resistant to the antiproliferative effects of AzaC. Gene expression analysis identified the potent cell cycle inhibitor, p21, was significantly upregulated in Teffs but not nTregs after treatment with AzaC. Furthermore, we demonstrate that Teffs deficient in p21 are less sensitive to the antiproliferative effects of AzaC. These results demonstrate that nTregs are essential for AzaC to fully protect against GvHD and have important clinical implications for future clinical trials testing AzaC as a novel method of GvHD prophylaxis in man.

The Role of Janus Kinase Signaling in Graft-Versus-Host Disease and Graft Versus Leukemia.

For patients with hematologic malignancies, allogeneic hematopoietic cell transplantation (alloHCT) offers a potential curative treatment option, primarily due to an allogeneic immune response against recipient tumor cells (ie, graft-versus-leukemia [GVL] activity). However, many recipients of alloHCT develop graft-versus-host disease (GVHD), in which allogeneic immune responses lead to the damage of healthy tissue. GVHD is a leading cause of nonrelapse mortality and a key contributor to morbidity among patients undergoing alloHCT. Therefore, improving alloHCT outcomes will require treatment strategies that prevent or mitigate GVHD without disrupting GVL activity. Janus kinases (JAKs) are intracellular signaling molecules that are well positioned to regulate GVHD. A variety of cytokines that signal through the JAK signaling pathways play a role in regulating the development, proliferation, and activation of several immune cell types important for GVHD pathogenesis, including dendritic cells, macrophages, T cells, B cells, and neutrophils. Importantly, despite JAK regulation of GVHD, preclinical evidence suggests that JAK inhibition preserves GVL activity. Here we provide an overview of potential roles for JAK signaling in the pathogenesis of acute and chronic GVHD as well as effects on GVL activity. We also review preclinical and clinical results with JAK inhibitors in acute and chronic GVHD settings, with added focus on those actively being evaluated in patients with acute and chronic GVHD.

Maintenance Therapy with Decitabine after Allogeneic Stem Cell Transplantation for Acute Myelogenous Leukemia and Myelodysplastic Syndrome.

Decitabine is a hypomethylating agent that irreversibly inhibits DNA methyltransferase I, inducing leukemic differentiation and re-expression of epigenetically silenced putative tumor antigens. We assessed safety and efficacy of decitabine maintenance after allogeneic transplantation for acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Decitabine maintenance may help eradicate minimal residual disease, decrease the incidence of graft-versus-host disease (GVHD), and facilitate a graft-versus-leukemia effect by enhancing the effect of T regulatory lymphocytes. Patients with AML/MDS in complete remission (CR) after allotransplantation started decitabine between day +50 and +100. We investigated 4 decitabine doses in cohorts of 4 patients: 5, 7.5, 10, and 15 mg/m(2)/day × 5 days every 6 weeks, for a maximum 8 cycles. The maximum tolerated dose (MTD) was defined as the maximum dose at which ≤ 25% of people experience dose-limiting toxicities during the first cycle of treatment. Twenty-four patients were enrolled and 22 were evaluable. All 4 dose levels were completed and no MTD was reached. Overall, decitabine maintenance was well tolerated. Grade 3 and 4 hematological toxicities were experienced by 75% of patients, including all patients treated at the highest dose level. Nine patients completed all 8 cycles and 8 of them remain in CR. Nine patients died from relapse (n = 4), infectious complications (n = 3), and GVHD (n = 2). Most occurrences of acute GVHD were mild and resolved without interruption of treatment; 1 patient died of acute gut GVHD. Decitabine maintenance did not clearly impact the rate of chronic GVHD. Although there was a trend of increased FOXP3 expression, results were not statistically significant. In conclusion, decitabine maintenance is associated with acceptable toxicities when given in the post-allotransplantation setting. Although the MTD was not reached, the dose of 10 mg/m(2) for 5 days every 6 weeks appeared to be the optimal dose rather than 15 mg/m(2), where most hematological toxicities occurred.

IFNγR signaling mediates alloreactive T-cell trafficking and GVHD.

The clinical goal of allogeneic hematopoietic stem cell transplantation (allo-HSCT) is to minimize GVHD while maintaining GvL. Here, we show that interferon γ receptor-deficient (IFNγR(-/-)) allogeneic Tconv, which possess normal alloreactivity and cytotoxicity, induce significantly less GVHD than wild-type (WT) Tconv. This effect is mediated by altered trafficking of IFNγR(-/-) Tconv to GVHD target organs, especially the gastrointestinal (GI) tract. We show that the chemokine receptor CXCR3 is induced via IFNγR-mediated signaling and partially contributes to the trafficking of WT Tconv to GVHD target organs. Indeed, CXCR3(-/-) Tconv recapitulate the reduced GVHD potential of IFNγR(-/-) Tconv in a minor-mismatched GVHD model. Most importantly, IFNγR(-/-) (and CXCR3(-/-)) Tconv mediate a robust and beneficial GvL effect. In addition, we show that IFNγR(-/-) regulatory T cells (Tregs) are fully suppressive in vitro although defective in suppressor function in vivo and that WT Tregs suppress GVHD in vivo only when allogeneic Tconv produce interferon γ (IFNγ), suggesting that the IFNγR signaling pathway is the major mechanism for both Tregs and Tconv to migrate to GVHD target organs. Finally, pharmacologic inhibition of IFNγR signaling with inhibitors of JAK1/JAK2, which are mediators of IFNγR signaling, results in the decreased expression of CXCR3 and reduced GVHD and improved survival after allo-HSCT and this effect is mediated by altered trafficking of Tconv to GVHD target organs.

HSPA9/mortalin inhibition disrupts erythroid maturation through a TP53-dependent mechanism in human CD34+ hematopoietic progenitor cells.

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell malignancies characterized by abnormal hematopoietic cell maturation, increased apoptosis of bone marrow cells, and anemia. They are the most common myeloid blood cancers in American adults. The full complement of gene mutations that contribute to the phenotypes or clinical symptoms in MDS is not fully understood. Around 10%-25% of MDS patients harbor an interstitial heterozygous deletion on the long arm of chromosome 5 [del(5q)], creating haploinsufficiency for a large set of genes, including HSPA9. The HSPA9 gene encodes for the protein mortalin, a highly conserved heat shock protein predominantly localized in mitochondria. Our prior study showed that knockdown of HSPA9 induces TP53-dependent apoptosis in human CD34+ hematopoietic progenitor cells. In this study, we explored the role of HSPA9 in regulating erythroid maturation using human CD34+ cells. We inhibited the expression of HSPA9 using gene knockdown and pharmacological inhibition and found that inhibition of HSPA9 disrupted erythroid maturation as well as increased expression of p53 in CD34+ cells. To test whether the molecular mechanism of HSPA9 regulating erythroid maturation is TP53-dependent, we knocked down HSPA9 and TP53 individually or in combination in human CD34+ cells. We found that the knockdown of TP53 partially rescued the erythroid maturation defect induced by HSPA9 knockdown, suggesting that the defect in cells with reduced HSPA9 expression is TP53-dependent. Collectively, these findings indicate that reduced levels of HSPA9 may contribute to the anemia observed in del(5q)-associated MDS patients due to the activation of TP53.

A Multicenter Phase 2 Clinical Trial of 10-Day Decitabine, Dose-Escalated Donor Lymphocyte Infusion, and Ruxolitinib for Relapsed Acute Myeloid Leukemia and Myelodysplastic Syndromes after Allogeneic Hematopoietic Cell Transplantation.

Post-transplantation relapse of acute myeloid leukemia and myelodysplastic syndromes has a poor prognosis. Donor lymphocyte infusion (DLI) is one treatment approach. However, efficacy is limited, and toxicity, mostly in the form of acute graft-versus-host disease (GVHD), is frequent. We tested a novel approach using 10-day decitabine, dose-escalated DLI, and ruxolitinib in a multicenter phase 2 trial aimed at increasing the efficacy of DLI and reducing its toxicity. Up to four 28-day cycles were administered. The primary endpoint was 6-month overall survival (OS). Of the 14 patients who started cycle 1, 13 received 1 DLI, 6 received 2 DLIs, and 1 received 3 4 DLIs. A preplanned interim analysis after enrolling 14 patients suggested futility, and the trial was closed to accrual. The final analysis showed a 6-month OS of 36% (95% confidence interval [CI], 18 to 72), a 1-year progression-free survival of 7% (95% CI, 1% to 47%), a 6-month cumulative incidence of grade II-IV acute GVHD of 57% (95% CI, 26% to 80%), and a 1-year nonrelapse mortality of 14% (95% CI, 2% to 38%). The combined modality treatment studied in this trial was ineffective and did not reduce DLI toxicity.

The adrenal stress response is an essential host response against therapy-induced lethal immune activation.

Cytokine release syndrome (CRS) is a systemic inflammatory syndrome associated with infection- or drug-induced T cell activation and can cause multiple organ failure and even death. Because current treatments are ineffective in some patients with severe CRS, we set out to identify risk factors and mechanisms behind severe CRS that might lead to preventive therapies and better clinical outcomes in patients. In mice, we found that deficiency in the adrenal stress response-with similarities to such in patients called relative adrenal insufficiency (RAI)-conferred a high risk for lethal CRS. Mice treated with CD3 antibodies were protected against lethal CRS by the production of glucocorticoids (GC) induced by the adrenal stress response in a manner dependent on the scavenger receptor B1 (SR-BI), a receptor for high-density lipoprotein (HDL). Mice with whole-body or adrenal gland-specific SR-BI deficiency exhibited impaired GC production, more severe CRS, and increased mortality in response to CD3 antibodies. Pretreatment with a low dose of GC effectively suppressed the development of CRS and rescued survival in SR-BI-deficient mice without compromising T cell function through apoptosis. Our findings suggest that RAI may be a risk factor for therapy-induced CRS and that pretreating RAI patients with GC may prevent lethal CRS.

Baricitinib with cyclosporine eliminates acute graft rejection in fully mismatched skin and heart transplant models.

Solid organ transplant represents a potentially lifesaving procedure for patients suffering from end-stage heart, lung, liver, and kidney failure. However, rejection remains a significant source of morbidity and immunosuppressive medications have significant toxicities. Janus kinase (JAK) inhibitors are effective immunosuppressants in autoimmune diseases and graft versus host disease after allogeneic hematopoietic cell transplantation. Here we examine the role of JAK inhibition in preclinical fully major histocompatibility mismatched skin and heart allograft models. Baricitinib combined with cyclosporine A (CsA) preserved fully major histocompatibility mismatched skin grafts for the entirety of a 111-day experimental period. In baricitinib plus CsA treated mice, circulating CD4+T-bet+ T cells, CD8+T-bet+ T cells, and CD4+FOXP3+ regulatory T cells were reduced. Single cell RNA sequencing revealed a unique expression profile in immune cells in the skin of baricitinib plus CsA treated mice, including decreased inflammatory neutrophils and increased CCR2- macrophages. In a fully major histocompatibility mismatched mismatched heart allograft model, baricitinib plus CsA prevented graft rejection for the entire 28-day treatment period compared with 9 days in controls. Our findings establish that the combination of baricitinib and CsA prevents rejection in allogeneic skin and heart graft models and supports the study of JAK inhibitors in human solid organ transplantation.

Phase I-II Trial of Early Azacitidine after Matched Unrelated Donor Hematopoietic Cell Transplantation.

Graft-versus-host disease (GVHD) is a major complication after allogeneic hematopoietic cell transplantation (allo-HCT). The hypomethylating agent azacitidine (AZA) has been shown to be effective in preclinical and clinical studies for the prevention of acute GVHD (aGVHD). We sought to determine the maximum tolerated dose (MTD) of AZA when given on days 1 to 5 of a 28-day cycle for 4 cycles, starting on day +7 after allo-HCT, as well as its impact on aGVHD and chronic GVHD (cGVHD), relapse, and overall survival (OS) in patients undergoing matched unrelated donor allo-HCT. This study was a single-arm, single-center, open-label phase I-II study with a total of 15 and 38 patients enrolled in the phase I and II portions of the trial, respectively. A standard 3+3 study design was used in phase I, and all patients in phase II received AZA at the MTD determined in phase I. The MTD of AZA starting at day +7 post-transplantation was 45 mg/m2. Phase II of the study was halted after enrolling 38 of the planned 46 patients following an interim analysis that suggested futility. Overall, AZA at 45 mg/m2 exhibited a side effect profile consistent with prior reports and had a minimal impact on engraftment. The cumulative incidence of clinically significant aGVHD by day +180 was 39.9% (95% confidence interval [CI], 22% to 53.7%). The incidence of all-grade cGVHD was 61.4% (95% CI, 40.3% to 75%). At 1 year, OS was 73.7% (95% CI, 60.9% to 89.1%), and the disease relapse rate was 11.4% (95% CI, .2% to 21.3%). Our results suggest that early post-allo-HCT AZA has limited efficacy in preventing aGVHD and cGVHD but could have a beneficial effect in preventing disease relapse.

AFF-1, a FOS-1-regulated fusogen, mediates fusion of the anchor cell in C. elegans.

Cell fusion is fundamental for reproduction and organ formation. Fusion between most C. elegans epithelial cells is mediated by the EFF-1 fusogen. However, fusion between the anchor cell and the utse syncytium that establishes a continuous uterine-vulval tube proceeds normally in eff-1 mutants. By isolating mutants where the anchor-cell fails to fuse, we identified aff-1. AFF-1 ectopic expression results in fusion of cells that normally do not fuse in C. elegans. The fusogen activity of AFF-1 was further confirmed by its ability to fuse heterologous cells. AFF-1 and EFF-1 differ in their fusogenic activity and expression patterns but share eight conserved predicted disulfide bonds in their ectodomains, including a putative TGF-beta-type-I-Receptor domain. We found that FOS-1, the Fos transcription factor ortholog that controls anchor-cell invasion during nematode development, is a specific activator of aff-1-mediated anchor-cell fusion. Thus, FOS-1 links cell invasion and fusion in a developmental cascade.

In vivo administration of hypomethylating agents mitigate graft-versus-host disease without sacrificing graft-versus-leukemia.

Regulatory T cells (Tregs) suppress graft-versus-host disease (GVHD) while preserving a beneficial graft-versus-leukemia (GVL) effect. Thus, their use in allogeneic stem cell transplantation (SCT) provides a promising strategy to treat GVHD. However, 3 obstacles prevent their routine use in human clinical trials: (1) low circulating number of Tregs in peripheral blood, (2) loss of suppressor function after in vitro expansion, and (3) lack of Treg-specific surface markers necessary for efficient purification. FOXP3 is exclusively expressed in Tregs and forced expression in CD4(+)CD25(-) T cells can convert these non-Tregs into Tregs with functional suppressor function. Here, we show that the FDA-approved hypomethylating agents, decitabine (Dec) and azacitidine (AzaC), induce FOXP3 expression in CD4(+)CD25(-) T cells both in vitro and in vivo. Their suppressor function is dependent on direct contact, partially dependent on perforin 1 (Prf1), but independent of granzyme B (GzmB), and surprisingly, Foxp3. Independence of Foxp3 suggests that genes responsible for the suppressor function are also regulated by DNA methylation. We have identified 48 candidate genes for future studies. Finally, AzaC treatment of mice that received a transplant of major histocompatibility complex mismatched allogeneic bone marrow and T cells mitigates GVHD while preserving GVL by peripheral conversion of alloreactive effector T cells into FOXP3(+) Tregs and epigenetic modulation of genes downstream of Foxp3 required for the suppressor function of Tregs.

Mouse models of graft-versus-host disease.

Transplantation of allogeneic hematopoietic stem and progenitor cells (allo-HCT) allows for cure of life-limiting malignant and non-malignant hematologic diseases. Crossing the human leukocyte antigen (HLA) barrier, however, comes at the cost of graft-versus-host disease (GVHD), a life-threatening syndrome mediated in part by the same donor T-lymphocytes that eliminate malignant cells. Acute GVHD occurs in the skin, gut, and/or liver in 25-55% of patients with a mortality rate of 15-40%, while chronic GVHD develops in 30-65% of patients who survive at least 3 months following allo-HCT and is highly debilitating in its extensive form, with a 30-50% 5year mortality rate stemming in part from immune dysregulation and opportunistic infections. Knowledge gaps remain in understanding the pathogenesis and in developing novel and effective treatments for the acute and chronic GVHD, which have distinct biology and yet are both treated with front line systemic corticosteroids. Novel and informative mouse models remain the primary means by which these diseases are studied and drugs initially developed prior to testing in humans. In this chapter, we describe allo-HCT mouse models and protocols using these mouse models by which to study acute and chronic GVHD with the goal of improving prevention and therapy.