Postinfusion PD-1+ CD8+ CAR T cells identify patients responsive to CD19 CAR T-cell therapy in non-Hodgkin lymphoma.

ABSTRACT: Chimeric antigen receptor (CAR) T-cell therapy has revolutionized treatment for relapsed/refractory B-cell non-Hodgkin lymphoma (NHL). Robust biomarkers and a complete understanding of CAR T-cell function in the postinfusion phase remain limited. Here, we used a 37-color spectral flow cytometry panel to perform high dimensional single-cell analysis of postinfusion samples in 26 patients treated with CD28 costimulatory domain containing commercial CAR T cells for NHL and focused on computationally gated CD8+ CAR T cells. We found that the presence of postinfusion Programmed cell death protein 1 (PD-1)+ CD8+ CAR T cells at the day 14 time point highly correlated with the ability to achieve complete response (CR) by 6 months. Further analysis identified multiple subtypes of CD8+ PD-1+ CAR T cells, including PD-1+ T cell factor 1 (TCF1)+ stem-like CAR T cells and PD-1+ T-cell immunoglobulin and mucin-domain containing-3 (TIM3)+ effector-like CAR T cells that correlated with improved clinical outcomes such as response and progression-free survival. Additionally, we identified a subset of PD-1+ CD8+ CAR+ T cells with effector-like function that was increased in patients who achieved a CR and was associated with grade 3 or higher immune effector cell-associated neurotoxicity syndrome. Here, we identified robust biomarkers of response to CD28 CAR T cells and highlight the importance of PD-1 positivity in CD8+ CAR T cells after infusion in achieving CR.

A novel CAR-T cell product targeting CD74 is an effective therapeutic approach in preclinical mantle cell lymphoma models.

BACKGROUND: Mantle cell lymphoma (MCL) is a rare B-cell non-Hodgkin lymphoma subtype which remains incurable despite multimodal approach including chemoimmunotherapy followed by stem cell transplant, targeted approaches such as the BTK inhibitor ibrutinib, and CD19 chimeric antigen receptor (CAR) T cells. CD74 is a nonpolymorphic type II integral membrane glycoprotein identified as an MHC class II chaperone and a receptor for macrophage migration inhibitory factor. Our group previously reported on CD74's abundant expression in MCL and its ability to increase via pharmacological inhibition of autophagosomal degradation. Milatuzumab, a fully humanized anti-CD74 monoclonal antibody, demonstrated significant activity in preclinical lymphoma models but failed to provide meaningful benefits in clinical trials mainly due to its short half-life. We hypothesized that targeting CD74 using a CAR-T cell would provide potent and durable anti-MCL activity. METHODS: We engineered a second generation anti-CD74 CAR with 4-1BB and CD3ζ signaling domains (74bbz). Through in silico and rational mutagenesis on the scFV domain, the 74bbz CAR was functionally optimized for superior antigen binding affinity, proliferative signaling, and cytotoxic activity against MCL cells in vitro and in vivo. RESULTS: Functionally optimized 74bbz CAR-T cells (clone 42105) induced significant killing of MCL cell lines, and primary MCL patient samples including one relapse after commercial CD19 CAR-T cell therapy with direct correlation between antigen density and cytotoxicity. It significantly prolonged the survival of an animal model established in NOD-SCIDγc-/- (NSG) mice engrafted with a human MCL cell line Mino subcutaneously compared to controls. Finally, while CD74 is also expressed on normal immune cell subsets, treatment with 74bbz CAR-T cells resulted in minimal cytotoxicity against these cells both in vitro and in vivo. CONCLUSIONS: Targeting CD74 with 74bbz CAR-T cells represents a new cell therapy to provide a potent and durable and anti-MCL activity.

Follicular Helper and Regulatory T Cells Drive the Development of Spontaneous Epstein-Barr Virus Lymphoproliferative Disorder.

Epstein-Barr virus (EBV) is a ubiquitous herpes virus associated with various cancers. EBV establishes latency with life-long persistence in memory B-cells and can reactivate lytic infection placing immunocompromised individuals at risk for EBV-driven lymphoproliferative disorders (EBV-LPD). Despite the ubiquity of EBV, only a small percentage of immunocompromised patients (~20%) develop EBV-LPD. Engraftment of immunodeficient mice with peripheral blood mononuclear cells (PBMCs) from healthy EBV-seropositive donors leads to spontaneous, malignant, human B-cell EBV-LPD. Only about 20% of EBV+ donors induce EBV-LPD in 100% of engrafted mice (High-Incidence, HI), while another 20% of donors never generate EBV-LPD (No-Incidence, NI). Here, we report HI donors to have significantly higher basal T follicular helper (Tfh) and regulatory T-cells (Treg), and depletion of these subsets prevents/delays EBV-LPD. Transcriptomic analysis of CD4+ T cells from ex vivo HI donor PBMC revealed amplified cytokine and inflammatory gene signatures. HI vs. NI donors showed a marked reduction in IFNγ production to EBV latent and lytic antigen stimulation. In addition, we observed abundant myeloid-derived suppressor cells in HI donor PBMC that decreased CTL proliferation in co-cultures with autologous EBV+ lymphoblasts. Our findings identify potential biomarkers that may identify individuals at risk for EBV-LPD and suggest possible strategies for prevention.

PRMT5 inhibition drives therapeutic vulnerability to combination treatment with BCL-2 inhibition in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is an incurable B-cell malignancy that comprises up to 6% of non-Hodgkin lymphomas diagnosed annually and is associated with a poor prognosis. The average overall survival of patients with MCL is 5 years, and for most patients who progress on targeted agents, survival remains at a dismal 3 to 8 months. There is a major unmet need to identify new therapeutic approaches that are well tolerated to improve treatment outcomes and quality of life. The protein arginine methyltransferase 5 (PRMT5) enzyme is overexpressed in MCL and promotes growth and survival. Inhibition of PRMT5 drives antitumor activity in MCL cell lines and preclinical murine models. PRMT5 inhibition reduced the activity of prosurvival AKT signaling, which led to the nuclear translocation of FOXO1 and modulation of its transcriptional activity. Chromatin immunoprecipitation and sequencing identified multiple proapoptotic BCL-2 family members as FOXO1-bound genomic loci. We identified BAX as a direct transcriptional target of FOXO1 and demonstrated its critical role in the synergy observed between the selective PRMT5 inhibitor, PRT382, and the BCL-2 inhibitor, venetoclax. Single-agent and combination treatments were performed in 9 MCL lines. Loewe synergy scores showed significant levels of synergy in most MCL lines tested. Preclinical, in vivo evaluation of this strategy in multiple MCL models showed therapeutic synergy with combination venetoclax/PRT382 treatment with an increased survival advantage in 2 patient-derived xenograft models (P ≤ .0001, P ≤ .0001). Our results provide mechanistic rationale for the combination of PRMT5 inhibition and venetoclax to treat patients with MCL.

PRMT5 supports multiple oncogenic pathways in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is an incurable B-cell malignancy with an overall poor prognosis, particularly for patients that progress on targeted therapies. Novel, more durable treatment options are needed for patients with MCL. Protein arginine methyltransferase 5 (PRMT5) is overexpressed in MCL and plays an important oncogenic role in this disease via epigenetic and posttranslational modification of cell cycle regulators, DNA repair genes, components of prosurvival pathways, and RNA splicing regulators. The mechanism of targeting PRMT5 in MCL remains incompletely characterized. Here, we report on the antitumor activity of PRMT5 inhibition in MCL using integrated transcriptomics of in vitro and in vivo models of MCL. Treatment with a selective small-molecule inhibitor of PRMT5, PRT-382, led to growth arrest and cell death and provided a therapeutic benefit in xenografts derived from patients with MCL. Transcriptional reprograming upon PRMT5 inhibition led to restored regulatory activity of the cell cycle (p-RB/E2F), apoptotic cell death (p53-dependent/p53-independent), and activation of negative regulators of B-cell receptor-PI3K/AKT signaling (PHLDA3, PTPROt, and PIK3IP1). We propose pharmacologic inhibition of PRMT5 for patients with relapsed/refractory MCL and identify MTAP/CDKN2A deletion and wild-type TP53 as biomarkers that predict a favorable response. Selective targeting of PRMT5 has significant activity in preclinical models of MCL and warrants further investigation in clinical trials.

Role of IL-22 in intestinal microenvironment and potential targeted therapy through diet.

IL-22 is a type 2 receptor cytokine in IL-10 family. IL-22 is usually secreted by innate and adaptive immune cells and takes its effects on non-hematopoietic cells. Through activate STAT3 pathway, IL-22 plays an important role in infection clearance and tissue regeneration, which is critical for barrier integrate and homeostasis. Abnormal activation of IL-22 signal was observed in inflammation diseases, autoimmune diseases, and cancers. We review the recent discoveries about the mechanism and regulation of IL-22 signal pathway from the perspective of intestinal micro-environment. Diet-based IL-22 target therapeutic strategies and their potential clinical significance will also be discussed.

A sumoylation program is essential for maintaining the mitotic fidelity in proliferating mantle cell lymphoma cells.

BACKGROUND: Mantle cell lymphoma (MCL) is a rare, highly heterogeneous type of B-cell non-Hodgkin's lymphoma. The sumoylation pathway is known to be upregulated in many cancers including lymphoid malignancies. However, little is known about its oncogenic role in MCL. METHODS: Levels of sumoylation enzymes and sumoylated proteins were quantified in MCL cell lines and primary MCL patient samples by scRNA sequencing and immunoblotting. The sumoylation enzyme SAE2 was genetically and pharmacologically targeted with shRNA and TAK-981 (subasumstat). The effects of SAE2 inhibition on MCL proliferation and cell cycle were evaluated using confocal microscopy, live-cell microscopy, and flow cytometry. Immunoprecipitation and orbitrap mass spectrometry were used to identify proteins targeted by sumoylation in MCL cells. RESULTS: MCL cells have significant upregulation of the sumoylation pathway at the level of the enzymes SAE1 and SAE2 which correlated with poor prognosis and induction of mitosis associated genes. Selective inhibition of SAE2 with TAK-981 results in significant MCL cell death in vitro and in vivo with mitotic dysregulation being an important mechanism of action. We uncovered a sumoylation program in mitotic MCL cells comprised of multiple pathways which could be directly targeted with TAK-981. Centromeric localization of topoisomerase 2A, a gene highly upregulated in SAE1 and SAE2 overexpressing MCL cells, was lost with TAK-981 treatment likely contributing to the mitotic dysregulation seen in MCL cells. CONCLUSIONS: This study not only validates SAE2 as a therapeutic target in MCL but also opens the door to further mechanistic work to uncover how to best use desumoylation therapy to treat MCL and other lymphoid malignancies.

Targeting DNA Damage Repair Functions of Two Histone Deacetylases, HDAC8 and SIRT6, Sensitizes Acute Myeloid Leukemia to NAMPT Inhibition.

PURPOSE: Nicotinamide phosphoribosyltransferase (NAMPT) inhibitors (NAMPTi) are currently in development, but may be limited as single-agent therapy due to compound-specific toxicity and cancer metabolic plasticity allowing resistance development. To potentially lower the doses of NAMPTis required for therapeutic benefit against acute myeloid leukemia (AML), we performed a genome-wide CRISPRi screen to identify rational disease-specific partners for a novel NAMPTi, KPT-9274. EXPERIMENTAL DESIGN: Cell lines and primary cells were analyzed for cell viability, self-renewal, and responses at RNA and protein levels with loss-of-function approaches and pharmacologic treatments. In vivo efficacy of combination therapy was evaluated with a xenograft model. RESULTS: We identified two histone deacetylases (HDAC), HDAC8 and SIRT6, whose knockout conferred synthetic lethality with KPT-9274 in AML. Furthermore, HDAC8-specific inhibitor, PCI-34051, or clinical class I HDAC inhibitor, AR-42, in combination with KPT-9274, synergistically decreased the survival of AML cells in a dose-dependent manner. AR-42/KPT-9274 cotreatment attenuated colony-forming potentials of patient cells while sparing healthy hematopoietic cells. Importantly, combined therapy demonstrated promising in vivo efficacy compared with KPT-9274 or AR-42 monotherapy. Mechanistically, genetic inhibition of SIRT6 potentiated the effect of KPT-9274 on PARP-1 suppression by abolishing mono-ADP ribosylation. AR-42/KPT-9274 cotreatment resulted in synergistic attenuation of homologous recombination and nonhomologous end joining pathways in cell lines and leukemia-initiating cells. CONCLUSIONS: Our findings provide evidence that HDAC8 inhibition- or shSIRT6-induced DNA repair deficiencies are potently synergistic with NAMPT targeting, with minimal toxicity toward normal cells, providing a rationale for a novel-novel combination-based treatment for AML.

Transducin ß-like protein 1 controls multiple oncogenic networks in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common Non-Hodgkin's lymphoma and is characterized by a remarkable heterogeneity with diverse variants that can be identified histologically and molecularly. Large-scale gene expression profiling studies have identified the germinal center B-cell (GCB-) and activated B-cell (ABC-) subtypes. Standard chemo-immunotherapy remains standard front line therapy, curing approximately two thirds of patients. Patients with refractory disease or those who relapse after salvage treatment have an overall poor prognosis highlighting the need for novel therapeutic strategies. Transducin ß-like protein 1 (TBL1) is an exchange adaptor protein encoded by the TBL1X gene and known to function as a master regulator of the Wnt signalling pathway by binding to ß-CATENIN and promoting its downstream transcriptional program. Here, we show that, unlike normal B-cells, DLBCL cells express abundant levels of TBL1 and its overexpression correlates with poor clinical outcome regardless of DLBCL molecular subtype. Genetic deletion of TBL1 and pharmacological approach using tegavivint, a first-in-class small molecule targeting TBL1 (Iterion Therapeutics), promotes DLBCL cell death in vitro and in vivo. Through an integrated genomic, biochemical, and pharmacologic analyses, we characterized a novel, ß-CATENIN independent, post-transcriptional oncogenic function of TBL1 in DLBCL where TBL1 modulates the stability of key oncogenic proteins such as PLK1, MYC, and the autophagy regulatory protein BECLIN-1 through its interaction with a SKP1-CUL1-F-box (SCF) protein supercomplex. Collectively, our data provide the rationale for targeting TBL1 as a novel therapeutic strategy in DLBCL.

A CS1-NKG2D Bispecific Antibody Collectively Activates Cytolytic Immune Cells against Multiple Myeloma.

Multiple myeloma (MM) is an incurable hematologic malignancy of plasma cells, with an estimated 30,000 new cases diagnosed each year in the United States, signifying the need for new therapeutic approaches. We hypothesized that targeting MM using a bispecific antibody (biAb) to simultaneously engage both innate and adaptive cytolytic immune cells could present potent antitumor activity. We engineered a biAb by fusing an anti-CS1 single-chain variable fragment (scFv) and an anti-NKG2D scFv (CS1-NKG2D biAb). Although NKG2D is a potent activation receptor ubiquitously expressed on mostly cytolytic immune cells including NK cells, CD8+ T cells, γδ T cells, and NKT cells, the CS1 tumor-associated antigen on MM represents a promising target. CS1-NKG2D biAb engaged human MM cell lines and NKG2D+ immune cells, forming immune synapses. In effector cells, CS1-NKG2D biAb triggered the phosphorylation of AKT, a downstream protein kinase of the activated NKG2D-DAP10 complex. The EC50 values of CS1-NKG2D biAb for CS1high and for CS1low MM cell lines with effector PBMCs were 10-12 and 10-9 mol/L, respectively. CS1-NKG2D biAb acted through multiple types of immune cells, and this induced cytotoxicity was both CS1- and NKG2D-specific. In vivo, survival was significantly prolonged using CS1-NKG2D biAb in a xenograft NOD-SCIDIL2γc-/- (NSG) mouse model engrafted with both human PBMCs and MM cell lines. Collectively, we demonstrated that the CS1-NKG2D biAb facilitated an enhanced immune synapse between CS1+ MM cells and NKG2D+ cytolytic innate and antigen-specific effector cells, which, in turn, activated these immune cells for improved clearance of MM. Cancer Immunol Res; 6(7); 776-87. ©2018 AACR.

Epigenetic and Posttranscriptional Regulation of CD16 Expression during Human NK Cell Development.

The surface receptor FcγRIIIA (CD16a) is encoded by the FCGR3A gene and is acquired by human NK cells during maturation. NK cells bind the Fc portion of IgG via CD16a and execute Ab-dependent cell-mediated cytotoxicity, which is critical for the effectiveness of several antitumor mAb therapies. The role of epigenetic regulatory mechanisms controlling transcriptional and posttranscriptional CD16 expression in NK cells is unknown. In this study, we compared specific patterns of DNA methylation and expression of FCGR3A with FCGR3B, which differ in cell type-specific expression despite displaying nearly identical genomic sequences. We identified a sequence within the FCGR3A promoter that selectively exhibits reduced methylation in CD16a+ NK cells versus CD16a- NK cells and neutrophils. This region contained the transcriptional start site of the most highly expressed CD16a isoform in NK cells. Luciferase assays revealed remarkable cell-type specificity and methylation-dependent activity of FCGR3A- versus FCGR3B-derived sequences. Genomic differences between FCGR3A and FCGR3B are enriched at CpG dinucleotides, and mutation of variant CpGs reversed cell-type specificity. We further identified miR-218 as a posttranscriptional negative regulator of CD16a in NK cells. Forced overexpression of miR-218 in NK cells knocked down CD16a mRNA and protein expression. Moreover, miR-218 was highly expressed in CD16a- NK cells compared with CD16a+ NK cells. Taken together, we propose a system of FCGR3A regulation in human NK cells in which CpG dinucleotide sequences and concurrent DNA methylation confer developmental and cell type-specific transcriptional regulation, whereas miR-218 provides an additional layer of posttranscriptional regulation during the maturation process.

IL-18 Drives ILC3 Proliferation and Promotes IL-22 Production via NF-κB.

Group 3 innate lymphoid cells (ILC3s) are important regulators of the immune system, maintaining homeostasis in the presence of commensal bacteria, but activating immune defenses in response to microbial pathogens. ILC3s are a robust source of IL-22, a cytokine critical for stimulating the antimicrobial response. We sought to identify cytokines that can promote proliferation and induce or maintain IL-22 production by ILC3s and determine a molecular mechanism for this process. We identified IL-18 as a cytokine that cooperates with an ILC3 survival factor, IL-15, to induce proliferation of human ILC3s, as well as induce and maintain IL-22 production. To determine a mechanism of action, we examined the NF-κB pathway, which is activated by IL-18 signaling. We found that the NF-κB complex signaling component, p65, binds to the proximal region of the IL22 promoter and promotes transcriptional activity. Finally, we observed that CD11c+ dendritic cells expressing IL-18 are found in close proximity to ILC3s in human tonsils in situ. Therefore, we identify a new mechanism by which human ILC3s proliferate and produce IL-22, and identify NF-κB as a potential therapeutic target to be considered in pathologic states characterized by overproduction of IL-18 and/or IL-22.

Genome-wide single-nucleotide polymorphism analysis revealed SUFU suppression of acute graft-versus-host disease through downregulation of HLA-DR expression in recipient dendritic cells.

Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). To identify recipient risk factors, a genome-wide study was performed including 481,820 single-nucleotide polymorphisms (SNPs). Two GVHD susceptibility loci (rs17114803 and rs17114808) within the SUFU gene were identified in the discovery cohort (p = 2.85 × 10(-5)). The incidence of acute GVHD among patients homozygous for CC at SUFU rs17114808 was 69%, which was significantly higher than the 8% rate observed in CT heterozygous patients (p = 0.0002). In an independent validation cohort of 100 patients, 50% of the patients with the CC genotype developed GVHD compared to 8% of the patients with either CT or TT genotype (p = 0.01). In comparison to CC dendritic cells, those from CT expressed higher levels of SUFU mRNA and protein, had lower levels of surface HLA-DR, and induced less allogeneic mixed leukocyte response (MLR). Ectopic expression of SUFU in THP-1 derived DCs reduced HLA-DR expression and suppressed MLR, whereas silencing of SUFU enhanced HLA-DR expression and increased MLR. Thus our findings provide novel evidence that recipient SUFU germline polymorphism is associated with acute GVHD and is a novel molecular target for GVHD prevention and treatment.

Multiplex and genome-wide analyses reveal distinctive properties of KIR+ and CD56+ T cells in human blood.

Killer cell Ig-like receptors (KIRs) on NK cells have been linked to a wide spectrum of health conditions such as chronic infections, autoimmune diseases, pregnancy complications, cancers, and transplant failures. A small subset of effector memory T cells also expresses KIRs. In this study, we use modern analytic tools including genome-wide and multiplex molecular, phenotypic, and functional assays to characterize the KIR(+) T cells in human blood. We find that KIR(+) T cells primarily reside in the CD56(+) T population that is distinctively DNAM-1(high) with a genome-wide quiescent transcriptome, short telomere, and limited TCR excision circles. During CMV reactivation in bone marrow transplant recipients, KIR(+)CD56(+) T cells rapidly expanded in real-time but not KIR(+)CD56(-) T cells or KIR(+) NK cells. In CMV(+) asymptomatic donors, as much as 50% of CD56(+) T cells are KIR(+), and most are distinguishably KIR2DL2/3(+)NKG2C(+)CD57(+). Functionally, the KIR(+)CD56(+) T cell subset lyses cancer cells and CMVpp65-pulsed target cells in a dual KIR-dependent and TCR-dependent manner. Analysis of metabolic transcriptome confirms the immunological memory status of KIR(+)CD56(+) T cells in contrast to KIR(-)CD56(+) T cells that are more active in energy metabolism and effector differentiation. KIR(-)CD56(+) T cells have >25-fold higher level of expression of RORC than the KIR(+) counterpart and are a previously unknown producer of IL-13 rather than IL-17 in multiplex cytokine arrays. Our data provide fundamental insights into KIR(+) T cells biologically and clinically.

PSP activates monocytes in resting human peripheral blood mononuclear cells: immunomodulatory implications for cancer treatment.

Polysaccharopeptide (PSP), from Coriolus versicolor, has been used as an adjuvant to chemotherapy, and has demonstrated anti-tumor and immunomodulating effects. However its mechanism remains unknown. To elucidate how PSP affects immune populations, we compared PSP treatments both with and without prior incubation in phytohaemagglutinin (PHA) - a process commonly used in immune population experimentation. We first standardised a capillary electrophoresis fingerprinting technique for PSP identification and characterisation. We then established the proliferative capability of PSP on various immune populations in peripheral blood mononuclear cells, using flow cytometry, without prior PHA treatment. It was found that PSP significantly increased the number of monocytes (CD14(+)/CD16(-)) compared to controls without PHA. This increase in monocytes was confirmed using another antibody panel of CD14 and MHCII. In contrast, proliferations of T-cells, NK, and B-cells were not significantly changed by PSP. Thus, stimulating monocyte/macrophage function with PSP could be an effective therapeutic intervention in targeting tumors.

Ex vivo activation of CD56(+) immune cells that eradicate neuroblastoma.

Despite the use of intensive contemporary multimodal therapy, the overall survival of patients with high-risk neuroblastoma is still less than 50%. Therefore, immunotherapy without cross-resistance and overlapping toxicity has been proposed. In this study, we report the development of a novel strategy to specifically activate and expand human CD56(+) (NCAM1) natural killer (NK) immune cells from normal donors and patients with neuroblastoma. Enriched CD56(+) cells from peripheral blood were mixed with CD56(-) fraction at 1:1 ratio and cultured in the presence of OKT3, interleukin (IL)-2, and -15 for five days and then without OKT3 for 16 more days. The final products contained more than 90% CD56(+) cells and could kill neuroblastoma cells effectively that were originally highly resistant to nonprocessed NK cells. Mechanistically, cytolysis of neuroblastoma was mediated through natural cytotoxicity receptor (NCR), DNAX accessory molecule-1 (DNAM-1; CD226), perforin, and granzyme B. Successful clinical scale-up in a good manufacturing practices (GMP)-compliant bioreactor yielded effector cells that in a neuroblastoma xenograft model slowed tumor growth and extended survival without GVHD. Investigation of CD56(+) cells from patients with neuroblastoma revealed a similar postactivation phenotype and lytic activity. Our findings establish a novel and clinically expedient strategy to generate allogeneic or autologous CD56(+) cells that are highly cytotoxic against neuroblastoma with minimal risk of GVHD.

Antibody-dependent cell-mediated cytotoxicity overcomes NK cell resistance in MLL-rearranged leukemia expressing inhibitory KIR ligands but not activating ligands.

PURPOSE: Leukemias with MLL gene rearrangement are associated with a poor prognosis. Natural killer (NK) cell therapy is a potential treatment, but leukemia cells may be resistant. Here, we sought to determine the susceptibility of MLL-rearranged leukemia cells to NK cell lysis and to develop a novel immunotherapeutic approach to optimize NK cell therapy, including the use of an antibody against leukemia-associated antigen and the elimination of killer-cell immunoglobulin-like receptor (KIR)-mediated inhibition. EXPERIMENTAL DESIGN: Three MLL-rearranged leukemia cell lines (RS4;11, SEM, and MV4-11) and primary leukemia blasts were assessed for surface phenotype and susceptibility to NK cell lysis with or without antibodies against CD19 (XmAb5574), CD33 (lintuzumab), or KIR ligands. RESULTS: All three cell lines were resistant to NK cell lysis, had some inhibitory KIR ligands and protease inhibitor-9, and expressed low levels of NKG2D activating ligands and adhesion molecules. After treatment with XmAb5574 or lintuzumab, MLL-rearranged leukemia cells were efficiently killed by NK cells. The addition of pan-major histocompatibility complex class I antibody, which blocked inhibitory KIR-HLA interaction, further augmented degranulation in all three KIR2DL1, KIR2DL2/3, and KIR3DL1 subsets of NK cells based on the rule of missing-self recognition. A mouse model showed a decreased rate of leukemia progression in vivo as monitored by bioluminescence imaging and longer survival after antibody treatment. CONCLUSION: Our data support the use of a triple immunotherapy approach, including an antibody directed against tumor-associated antigen, KIR-mismatched NK cell transplantation, and inhibitory KIR blockade, for the treatment of NK cell-resistant MLL-rearranged leukemias.

Brazilian green propolis and its constituent, Artepillin C inhibits allogeneic activated human CD4 T cells expansion and activation.

ETHNOPHARMACOLOGICAL RELEVANCE: Propolis has long been used as a popular folk medicine by various ethnic groups due to its wide spectrum of alleged biological and pharmaceutical properties including anti-microbial, anti-cancer and anti-inflammatory functions. All these can be linked to the modulation of immune function. Therefore, it will be relevant for us to find out whether there is any novel compound that can account for such action and the mechanism involved. AIM OF THE STUDY: We investigated the immune modulating effect of Brazilian green propolis (PBrazil) and its constituent Artepillin C (Art-C) by using mixed leukocytes reaction. MATERIALS AND METHODS: The cytotoxic effect of Art-C on non-tumorigenic human liver cell line miHA and non-tumorigenic human kidney cell line HK-2 as well as human peripheral blood mononuclear cells (PBMCs) were measured by XTT cell proliferation assay. The effect of PBrazil and Art-C on T cell proliferation and activation were determined by using carboxyfluorescein succinimidyl ester (CFSE) and by CD25 expression, respectively. Cytokines including tumor necrosis factor-α (TNF-α), interferon-gamma (IFN-γ), interleukins such as IL-2, IL-17 were measured by intracellular cytokine staining and IL-10 was measured by ELISA. The effect of PBrazil and Art-C on regulatory T cells (Treg) induction was determined by the Foxp3 expression. The apoptotic effect of these compounds on CFSE labeled alloreactive T cells was measured by using Annexin V. RESULTS: Using mixed leukocytes reaction we demonstrated for the first time that both Art-C and PBrazil significantly inhibited the alloreactive CD4 T cell proliferation, activation, and suppressed the expressions of IL-2, IFN-γ and IL-17 in these alloreactive CD4 T cells. The inhibitions of Art-C and PBrazil on CD4 T cells were not due to direct cytotoxic effect on PBMC or inducing regulatory T cells differentiation. Both Art-C and PBrazil were found to selectively induce apoptosis in proliferating T cells. The anti-proliferative effect of Art-C and PBrazil were reversible and were also applied to the activated T cells. CONCLUSIONS: In conclusion, our results indicated that Art-C and PBrazil can suppress alloreactive CD4 T cell responses in vitro, suggesting that Art-C could be used as a potential immunosuppressant, either solely or as adjunct agent in treating graft versus host disease.

Significant functional heterogeneity among KIR2DL1 alleles and a pivotal role of arginine 245.

Killer immunoglobulin-like receptors (KIRs) play an essential role in the regulation of natural killer cell functions. KIR genes are highly polymorphic in nature, showing both haplotypic and allelic variations among people. We demonstrated in both in vitro and in vivo models a significant heterogeneity in function among different KIR2DL1 alleles, including their ability to inhibit YT-Indy cells from degranulation, interferon gamma production, and cytotoxicity against target cells expressing the HLA-Cw6 ligand. Subsequent experiments showed that the molecular determinant was an arginine residue at position 245 (R245) in its transmembrane domain that mechanistically affects both the efficiency of inhibitory signaling and durability of surface expression. Specifically, in comparison with R245-negative alleles, KIR2DL1 that included R245 recruited more Src-homology-2 domain-containing protein tyrosine phosphatase 2 and beta-arrestin 2, showed higher inhibition of lipid raft polarization at immune synapse, and had less down-regulation of cell-surface expression upon interaction with its ligand. Thus, our findings provide novel insights into the molecular determinant of KIR2DL1 and conceivably a fundamental understanding of KIR2DL1 allelic polymorphism in human disease susceptibility, transplant outcome, and donor selection.

The effects of beta-glucan on human immune and cancer cells.

Non-prescriptional use of medicinal herbs among cancer patients is common around the world. The alleged anti-cancer effects of most herbal extracts are mainly based on studies derived from in vitro or in vivo animal experiments. The current information suggests that these herbal extracts exert their biological effect either through cytotoxic or immunomodulatory mechanisms. One of the active compounds responsible for the immune effects of herbal products is in the form of complex polysaccharides known as beta-glucans. beta-glucans are ubiquitously found in both bacterial or fungal cell walls and have been implicated in the initiation of anti-microbial immune response. Based on in vitro studies, beta-glucans act on several immune receptors including Dectin-1, complement receptor (CR3) and TLR-2/6 and trigger a group of immune cells including macrophages, neutrophils, monocytes, natural killer cells and dendritic cells. As a consequence, both innate and adaptive response can be modulated by beta-glucans and they can also enhance opsonic and non-opsonic phagocytosis. In animal studies, after oral administration, the specific backbone 1-->3 linear beta-glycosidic chain of beta-glucans cannot be digested. Most beta-glucans enter the proximal small intestine and some are captured by the macrophages. They are internalized and fragmented within the cells, then transported by the macrophages to the marrow and endothelial reticular system. The small beta-glucans fragments are eventually released by the macrophages and taken up by other immune cells leading to various immune responses. However, beta-glucans of different sizes and branching patterns may have significantly variable immune potency. Careful selection of appropriate beta-glucans is essential if we wish to investigate the effects of beta-glucans clinically. So far, no good quality clinical trial data is available on assessing the effectiveness of purified beta-glucans among cancer patients. Future effort should direct at performing well-designed clinical trials to verify the actual clinical efficacy of beta-glucans or beta-glucans containing compounds.