ALBERT

Description: Dendritic cells (DCs) are important for primary T cell responses, and cytokines produced by DCs are thought to be essential for promoting T helper (Th)1 and Th17 differentiation. However, DCs can drive effector differentiation independent of cytokines. In mouse models of graft-versus-host-disease (GVHD), which is a life-threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT), we found that DC-derived Notch ligand Dll4 was important for CD4+ Th1 and Th17 cell differentiation. Blocking Dll4 led to decreased production of IFN-g and IL-17 in mice receiving allo-HSCT and inhibition of GVHD. However, the human counterparts of murine DLL4+ DCs and their function in alloreactive T cells have never been investigated. We report here the identification of human DLL4+ DCs and the critical role of DLL4 in DC-regulation of Th1 and Th17 cell differentiation. Flow cytometric analysis revealed that CD1c+ DCs and plasmacytoid DCs (pDCs) from the peripheral blood (PB) of healthy donors (n=18) did not express DLL4. However, 24 hours after stimulation with Toll-like receptor (TLR) agonists, PB DCs from healthy donors produced high levels of DLL4 on their surface. Pam3 (TLR1/2 stimulus), Poly I:C (TLR3 stimulus), LPS (TLR4 stimulus) and R848 (TLR7/8 stimulus) induced high levels of DLL4 expression on the surface of 50% to 80% of CD1c+ DCs. CpG oligodeoxynucleotides (TLR9 agonists) did not increase DLL4 in CD1c+ DCs, likely due to their lacking of TLR9. pDCs increased DLL4 expression when activated by R848 (16.0% ± 2.7%) and to a less extent by CpG oligodeoxynucleotides (8.6% ± 0.8%). Thus, activation of TLR signaling induces high levels of DLL4 in CD1c+ DCs and pDCs, with R848 being the most potent stimulus. Functional analysis using mixed lymphocyte reaction revealed that R848-activated CD1c+ DCs and pDCs induced greater proliferation of allogeneic CD4+ T cells and production of more IFN-g- and IL-17-producing effector cells compared to unstimulated CD1c+ DCs and pDCs. Blocking DLL4 using a neutralizing antibody decreased Notch signaling in T cells stimulated with activated DCs and led to production of 2- and 3-fold less Th1 cells and Th17 cells compared to IgG control, suggesting the importance of DLL4 in DC-regulation of effector differentiation. Molecular mechanism investigation revealed that SATAT3 and NFkB were crucial for inducing DLL4 in human DCs. Inhibiting STAT3 alone using its specific inhibitor S31-201 dramatically decreased DLL4 expression in activated PB DCs. Promoter reporter assays showed that STAT3 was required for activating DLL4 transcription. Inhibiting NFkB using its inhibitor PDTC also decreased the expression of DLL4 on the surface of R848-stimulated PB DCs. However, DCs derived from monocytes induced by GM-CSF and IL-4, which had activation of NFkB but did not express active STAT3 following stimulation by R848 + LPS, were DLL4 negative despite their upregulation of costimulatory molecules (e.g., CD40, CD80, CD83, and CD86). Thus, activation of STAT3 is critical for inducing DLL4 in human DCs, whereas active NFkB is important but not sufficient for inducing DLL4 in PB DCs. Finally, given the importance of alloreactive Th1 and Th17 cells in mediating GVHD in human allogeneic HSCT recipients, we further obtained PB from patients (n=7) undergoing allo-HSCT between 21 and 39 days after transplantation when these patients were fully engrafted and no longer pancytopenic. As compared to healthy donors, HSCT recipients had an averaged 12-fold higher frequency of DLL4+ CD1c+ DCs. These results indicate that upregulation of DLL4 on the surface of DCs is associated with alloreactive inflammatory conditions in HSCT patients. In summary, our findings show that DLL4 surface expression on human DCs is critical for the priming of human Th1 and Th17 responses and may have significant implication in better understanding of T cell-mediated inflammatory conditions such as chronic infection, autoimmune diseases, tumor rejection and GVHD after allo-HSCT. Disclosures No relevant conflicts of interest to declare.

Description: Leukemia emergence, maintenance, relapse and/or progression are causally linked to the presence of drug-resistant leukemia-initiating cells and impaired natural killer (NK) cell anti-tumor immune-response. Bone marrow microenvironment (BMM)- and/or leukemia-derived signals induce aberrant non-coding RNA expression and inhibit protein phosphatase 2A (PP2A) tumor suppressor activity. PP2A loss-of-function is essential for NK cell activity and leukemic but not normal stem and progenitor cell proliferation and survival. The human MIR300 gene is an intergenic miRNA that belongs to the 14q32.31 DLK1-DIO3 genomic-imprinted tumor suppressor miRNA cluster B. MIR300 was found involved in loss-of heterozygosity, inhibited in several tumor types with high mitotic index and during epithelial-to-mesenchymal transition (EMT), and associated with a cancer stem cell phenotype. By using primary cells from Philadelphia-positive (Ph+) chronic myelogenous leukemia (CML) in chronic (CP) and blastic (BC) phase, and complex karyotype (CK) acute myeloid leukemia (AML) patients, as paradigmatic examples of stem cell-derived neoplasms characterized by constitutive expression of oncogenic kinases, PP2A loss-of-function, altered microRNA expression and impaired NK cell proliferation and cytotoxicity, we found that MIR300 is a cell context-independent tumor suppressor with anti-proliferative and PP2A-dependent pro-apoptotic activities which are sequentially activated in a MIR300 dose-dependent manner through inhibition of CCND2/CDK6 and SET (PP2A inhibitor), respectively. To prevent PP2A-induced apoptosis, MIR300 is inhibited by oncogenic signals in CD34+CML (CP and BC) and CK-AML progenitors. Conversely, tumor-naïve BMM-induced C/EBPbeta-mediated signals (hypoxia and MSC exosomes) markedly upregulate MIR300 expression in primary CML and AML CD34+CFSEmax leukemic stem (LSC) and CD56+CD3-NK cells to induce/maintain quiescence (increased CD34+leukemic blasts in G0) and impair immune-response (suppression of NK cell proliferation and cytotoxic activity toward CD34+ leukemic blasts and CFSEmaxCD34+ CML-BC quiescent LSCs), respectively. Inhibition of MIR300 expression/activity rescues NK cell proliferation and anti-tumor cytotoxicity and prevented MSC- and hypoxia-induced growth-suppression of CD34+leukemic blasts by inhibiting degradation of MIR300 targets (e.g. SET, CCND2). We found that CML and AML LSCs escape MIR300-induced PP2A-mediated apoptosis through the hypoxia- and tumor-dependent TGFb1-FoxM1-mediated upregulation of TUG1 lncRNA. TUG1 is an oncogenic lncRNA described as a MIR300sponge and found upregulated in solid tumors, in which it has strong diagnostic, prognostic and therapeutic relevance and is associated with cancer stem cell maintenance and EMT. In quiescent CML and AML LSCs, TUG1 uncouples and limits MIR300 tumor suppressor functions to cytostasis by maintaining unbound MIR300 at levels sufficient to inhibit CCND2 and CDK6 but not SET expression. Exposure to clinically-relevant CpG-modified oligonucleotides modulating MIR300levels and/or inhibiting TUG1 MIR300-sponging activity, restores NK cell proliferation and cytotoxic activity, and suppresses human leukemic but not normal hematopoiesis by eradicating nearly all (〉 95% reduction) CFSEmaxCD34+ and CD45+CD34+CD38-CD90+ LSCs and CD34+leukemic CML (CP and BC) and CK-AML blasts in vitro (CFCs, LTC-IC, and CFSEmaxCD34+cell tracking) and/or in NRG-SGM3 PDX mouse models of acute and chronic myeloid leukemias. Altogether, this work highlights the therapeutic importance of altering MIR300 expression in anti-LSC and NK cell-based approaches for myeloid leukemias, and indicates that tumor-naïve BMM-induced MIR300 tumor suppressor anti-proliferative and PP2A-activating functions may support leukemogenesis by promoting the formation and initial expansion of the quiescent LSC pool through the induction of LSC dormancy and inhibition of quiescent LSC killing by cytokine-activated NK cells, respectively. (G.S. and R.T. equally contributed to this work) Disclosures Stagno: BMS: Honoraria; Incyte: Honoraria; Pfizer: Honoraria; Novartis: Honoraria. Deininger:TRM: Consultancy; Sangoma: Consultancy; Incyte: Honoraria; Novartis: Honoraria; Sangamo: Consultancy; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Humana: Honoraria; Ascentage Pharma: Consultancy, Honoraria; Blueprint: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Research Funding; Fusion Pharma: Consultancy; Adelphi: Consultancy. Milojkovic:BMS: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Incyte: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau. Apperley:Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Baer:Takeda: Research Funding; Incyte: Research Funding; Kite: Research Funding; Forma: Research Funding; AI Therapeutics: Research Funding; Abbvie: Research Funding; Astellas: Research Funding.

Description: Introduction:The spread of SARS-CoV-2 virus continues to pose a major public health threat. Patients with cancer are thought to be at increased risk from SARS-COV-2 infection due to the immunodeficiency that results from the underlying neoplasm and treatment. The immune response to this infection has been the subject of great interest, with an extreme variation in clinical severity between infected individuals. Variation in the immune cell response (B, T, and NK lymphocytes, monocytes, and myeloid derived suppressor cells (MDSCs), among others) and their function have been hypothesized to be responsible for this range of presentation. Methods:Two patients with a history of hematologic malignancies were matched with three non-cancer patients with similar baseline clinical characteristics and severity of COVID related illness. The critical group (CG) was defined as those requiring mechanical ventilation (MV) due to COVID related respiratory failure and the non-critical group (NCG) were hospitalized but did not require MV. All samples studied were obtained from peripheral blood and processed within 4-hours of collection. Peripheral blood mononuclear cell (PBMC) were isolated using ficoll density gradient separation. Flowcytometric analysis using CytekTM Aurora was done on fresh PBMC samples. Thirty antibody-based flow markers were used to identify 54 distinct immune cell populations. IRB approval was obtained. Results: Critical Group (CG):The CG included case 1, a 47 year-old (y.o.) female (F) with a history (hx) of acute myeloid leukemia and had an matched related donor allogeneic hematopoietic stem cell transplant (alloHSCT) 10-years prior remaining in remission, with hematologic recovery, and off immunosuppressants treated with remdesivir and coritcosteroids for COIVD directed therapy; and case 2, a 55 y.o. F with a hx of HIV treated with corticosteroids for COIVD directed therapy (see Figure 1a). Non-Critical Group (NCG):The NCG included case 3, a 73 y.o male (M) with hx of relapsed/refractory Philadelphia chromosome negative Acute Lymphoblastic Leukemia with loss of CD19 and CD22 expression following treatment with blinatumumab and inotuzumab, and most recently treated with decitabine/venetoclax; case 4, a 66 y.o. M with hx of cardiomyopathy; and case 6, a 54 y.o. M with hx of obesity. None of the NCG cases were treated with COVID directed therapy. See Table 1 for further clinical information. Immunophenotypic expression:Flow cytometry gating strategy done as outlined in Fig 1a. Case 1 had a high proportion of B-cells, CD8+ T-cells, and cells with exhaustion markers (CD8+CD94+ T-cells, CD4+PD1+ T-cells, CD4+PD1+CD94+ T-cells, PD1-CD94+ NK T-cells, Lag3+Cd11b- non-TB leukocytes) and MDSC immunophenotypes compared with matched case 2. Case 3 also had a high proportion of exhaustion markers (Lag3+CD39 low B-cells, CD8+PD1+ T-cells, CD8+CD94+PD1+ T-cells, CD4+PD1+CD94+ T-cells, PD1+CD94+ NK T-cells, PD1-CD94+ NK T-cells, Lag3+CD11b+, Lag3+CD11b- non-TB leukocytes) and high expression of immunosuppressive Treg and all MDSC; although high expression of granulocytic MDSC. Case 2 had a significant number of exhaustion and immunosuppressive cells as well. Cases 4 and 5 had a higher predominance of all T-cell subtypes and also had variable expression of exhaustion and immunosuppressive immunophenotypes (See Fib 1b). Conclusion:In our study of one critical and one non-critical patient with a history of hematologic malignancy matched with three non-cancer patients we demonstrate the high predominance of exhaustion markers (Lag3,PD1,CD94) and immunosuppressive cell types (Treg, granulocytic and monocytic MDSC). These findings are consistent with the fact that both CG and NCG, as hospitalized patients, represent the most severely ill COVID patient cohort. Of notable interest to the cancer population, cases 1 and 3 had a significant number of exhaustion and immunosuppressive immunophenotypes, suggestive of baseline exhaustion following alloHSCT even years after engraftment in case 1 and attenuated functional immunity in a patient undergoing active treatment in case 3. Interestingly, case 3 had lower expression of all MDSC, a known treatment effect of decitabine. Paired cytokine measurement and its effect on immunophenotype is underway. Additionally, we plan to present an atlas of the peripheral immune cell response on fifteen additional non-cancer COVID patients. Disclosures No relevant conflicts of interest to declare.