ALBERT

Description: Key PointsTargeting APCs to enhance GVT. CD8+ DCs are important for optimizing antitumor responses after allogeneic bone marrow transplantation.

Description: While miRNAs are increasingly linked to various immune responses, whether they can be targeted for regulating in vivo inflammatory processes such as endotoxin-induced Gram-negative sepsis is not known. Production of cytokines by the dendritic cells (DCs) plays a critical role in response to endotoxin, lipopolysaccharide (LPS). We profiled the miRNA and mRNA of CD11c+ DCs in an unbiased manner and found that at baseline, miR-142-3p was among the most highly expressed endogenous miRs while IL-6 was among the most highly expressed mRNA after LPS stimulation. Multiple computational algorithms predicted the IL-6 3′ untranslated region (UTR) to be a target of miR-142-3p. Studies using luciferase reporters carrying wild-type (WT) and mutant IL-6 3′UTR confirmed IL-6 as a target for miR-142-3p. In vitro knockdown and overexpression studies demonstrated a critical and specific role for miR142-3p in regulating IL-6 production by the DCs after LPS stimulation. Importantly, treatment of only WT but not the IL-6–deficient (IL-6−/−) mice with locked nucleic acid (LNA)–modified phosphorothioate oligonucleotide complementary to miR 142-3p reduced endotoxin-induced mortality. These results demonstrate a critical role for miR-142-3p in regulating DC responses to LPS and provide proof of concept for targeting miRs as a novel strategy for treatment of endotoxin-induced mortality.

Description: Elderly patients (pts) with acute myelogenous leukemia (AML) and high-risk myelodysplastic syndrome (MDS) particularly who are ineligible for allogeneic hematopoietic stem cell transplantation (allo-HSCT) and have disease relapse after allo-HSCT still have a poor prognosis. Thus, the development of novel treatment option providing higher response rate and prolonged survival time for those patients still remains an unmet need. Because not a few clinical and preclinical studies have described a promising value of Wilms Tumor 1 (WT1), a leukemia-associated antigen as a therapeutic target of antileukemia immunotherapy, we conducted a clinical study of novel adoptive immunotherapy using gene-modified autologous lymphocytes expressing WT1-specific T-cell receptor (TCR) for the treatment of refractory AML and high-risk MDS. A multicenter phase 1 study was conducted to assess feasibility, safety and preliminary antileukemia reactivity of patient-derived gene-modified lymphocytes expressing WT1-specific TCR. Antigen-specific TCR-gene transfer may cause a serious autoimmune disease mediated by mispaired TCR between introduced and endogenous TCR α/β chains. To avoid that, we established a retroviral vector system encoding siRNAs for endogenous TCR genes (siTCR vector). We conducted a first-in-human clinical trial employing this siTCR vector. After given written informed consents, mononuclear cells were collected from at most 200ml of peripheral blood (PB) from each patient. Then, proliferating lymphocytes pre-cultured with IL-2, anti-CD3 antibody and RetroNectinTM were infected with a retroviral vector, MS3-WT1-siTCR composed of DNAs encoding WT1235-243/HLA-A*24:02 complex specific TCR-α/β chains and siRNAs against endogenous TCR genes. Expanded gene-modified lymphocytes (WT1-siTCR/T cells) in additional culture for 13-14 more days were harvested and frozen until use. Eligibility included HLA-A*24:02 positive pts with refractory AML or high-risk MDS, 〉 20 y.o, ineligible for allo-HSCT and performance status of 0 to 2. WT1-siTCR/T cells were intravenously infused twice on days 0 and 28. Heteroclitic WT1235-243 ninemer peptide (300mg) emulsified with MontanideTM was given subcutaneously on day 2 and 16 after the second infusion. Besides safety assays, kinetics of WT1-siTCR/T cells in PB, immunological responses and residual leukemia burden determined by qRT-PCR for WT1 mRNA were serially measured until day 58 since the first infusion. Among 12 pts enrolled, 8 pts (5 AML, 3 MDS) with a median age of 68.5 y. received study treatment. Three pts received 2x108 cells/ infusion (cohort 1), 3 received 1x109 cells/ infusion (cohort 2), and 2 received extra-cohort doses. Median follow-up time after the first infusion was 257 days (as June 13, 2016). At the first infusion, all pts contracted progressive disease. Circulatory WT1-siTCR/T cells retaining the target reactivity appeared immediately after each infusion, peaked between 1 to 3 days, and declined thereafter. WT1 peptide vaccination did not seem to affect the transition of infused cells. Values of WT1 mRNA in PB were transiently suppressed in all pts and declined in 4 pts thereafter. Clinical outcomes included one with stable disease and 2 pts with partial remission (PR). In one with PR, the epitope-spreading phenomenon was suggested. In all pts, no serious adverse events associated with infused WT1-siTCR/T cells were observed. Adoptive transfer of autologous WT1-siTCR/T cells was feasible and safe. Although the persistence of infused WT1-siTCR/T cells was limited, infused WT1-siTCR/T cells at least seemed to be involved in the antileukemia reactivity. Disclosures Tawara: Astellas: Honoraria. Akatsuka:Takara Bio Inc.: Consultancy. Nukaya:Takara Bio Inc.: Employment. Takesako:Takara Bio Inc.: Employment.

Description: Extracorporeal photopheresis (ECP), a technique that exposes isolated white blood cells to photoactivatable 8-methoxypsoralen and ultraviolet A radiation, is used clinically to treat cutaneous T-cell lymphoma and immune-mediated diseases such as graft-versus-host disease (GVHD). ECP is thought to control these diseases in part through direct induction of lymphocyte apoptosis, but its effects on the immune system beyond apoptosis remain poorly characterized. We have developed a novel method for incorporating ECP treatment into well-established and clinically relevant murine models of GVHD to examine its effects during an ongoing immune response. We demonstrate that the transfer of cells treated with ECP reverses established GVHD by increasing donor regulatory T cells and indirectly reducing the number of donor effector lymphocytes that themselves had never been exposed to psoralen and ultraviolet A radiation.

Description: Key Points HDAC inhibition reduced proinflammatory cytokines and increased regulatory T-cell number and function after allo-HCT. HDAC inhibition enhanced signal transducer and activator of transcription 3 acetylation and induced indoleamine-2,3-dioxygenase after allo-HCT.

Description: NLRP6 (NOD-like receptor family pyrin domain containing 6) is an important inflammasome component and is highly expressed in intestinal epithelial and in immune cells. NLRP6 mediated inflammasome activation plays a critical role in response to intestinal infection and preventing dysbiosis of gut microbiota through the secretion of IL-18 and mucus. However, we recently found that NLRP6 plays a pathogenic role in GVHD that is independent of microbial dysbiosis, which is in contrast to its well-appreciated microbiome-dependent protective role in intestinal colitis and tumorigenesis. Interestingly, we also found that activated T cells increased NLRP6 expression, but the T cell autonomousrole of NLRP6 in regulating T cell responses is unknown. Because NLRP6 is an important regulator of GVH responses, we tested the hypothesis that NLRP6 deficiency in donor T cells would ameliorate GVHD. To test our hypothesis, we first performed adetailed phenotypic analysis of various T cell subsets and activation markers in naïve NLRP6-/-and wild-type (WT) B6 animals and found a similar distribution of naïve, memory, effector and regulatory T cells. In order to examine whether the absence of NLRP6 in donors affects GVHD, WT-BALB/canimals were lethally irradiated (700cGy) and transplanted on day 0 with 5x106bone marrow and 1.0x106 splenic CD90+T cells from either syngeneic WT-BALB/c, allogeneic MHC-mismatched WT-B6 or NLRP6-/-animals. Contrary to our hypothesis, the recipients receiving donor T cells from NLRP6-/-animals showed a significantly worse survival compared to allogeneic WT-B6 animals (p

Description: Introduction: Fibrocytes are derived from a subset of monocytes and express mesenchymal markers such as collagen type I (Col I) and hematopoietic markers such as CD45 and CD11b. They also express several chemokine receptors such as CC chemokine receptor (CCR)-1, CCR2, CCR5, CCR7, and CXC chemokine receptor type 4 (CXCR4). They circulate in the peripheral blood (PB) and can be isolated from many fibrotic tissues. Fibrocytes participate in both physiological wound healing and various pathological fibrosis including myelofibrosis, hypertrophic scar, systemic sclerosis, idiopathic pulmonary fibrosis, liver cirrhosis, and progressive kidney disease. In murine and human colitis, fibrocytes are also reported to be associated with the colon fibrosis. It has been described that migration of fibrocytes to the injured sites involves CC chemokine ligand 2 (CCL2)/CCR2 axis in the liver and kidney and CXC chemokine ligand 12 (CXCL12)/CXCR4 axis in the lung. However, there are few reports concerning the role of fibrocytes and their expression of chemokine receptors related to the induction of colon fibrosis. Methods: We generated bone marrow (BM) chimeric mice by transplantation of BM total-nucleated cells, which were isolated from enhanced green fluorescent protein (EGFP)-transgenic mice or CCR2 knockout (KO) mice, into lethally irradiated C57BL/6J-Ly5.1 mice. Two months after BM transplantation, BM chimeric mice were treated with a single intraperitoneal injection of azoxymethane (10 mg/kg body weight) followed by 3 cycles of 1% dextran sulfate sodium (DSS) in the drinking water. We assessed the level of fibrosis in the colon using Sirius red staining and analyzed the presence of BM-derived CD45+CD11b+Col I+ fibrocytes in the colon lamina propria (LP) using immunofluorescence staining and flow cytometry. Furthermore, we investigated the expressions of Col I, transforming growth factor-ß (TGF- ß), matrix metalloproteinases (MMPs), and tissue inhibitor of MMPs (TIMP)-1 in the colon tissues and fibrocytes sorted from colon LP cells after chronic DSS treatment using quantitative real-time RT-PCR. Results: During chronic inflammation, infiltration of CCR2+ BM-derived monocytes and fibrocytes and production of CCL2 in the colon were particularly increased and colon fibrosis was developed in EGFP BM chimeric mice. Two types of fibrocytes, CCR2+CXCR4+Ly6C-F4/80+ fibrocytes and CCR2-CXCR4+Ly6ChighF4/80- fibrocytes, were identified in the colon LP, whereas only the latter fibrocytes were detected in the PB. Adoptive transferred CCR2+Ly6ChighCol I- monocytes migrated to the injured colon and a part of them differentiated into CCR2+Col I+ fibrocytes. In CCR2KO BM chimeric mice, the numbers of monocytes and fibrocytes in the colon LP were significantly decreased and colon fibrosis was attenuated. However, there was no difference in the mRNA expressions of Col I, TGF-ß, and MMPs (MMP-1a, MMP-8, and MMP-13, known as collagenases) in colon tissues between EGFP BM chimeric mice and CCR2KO BM chimeric mice. Improvement of colon fibrosis in CCR2KO BM chimeric mice was associated with the decreased expression of Timp1 mRNA in colon tissues. We analyzed the expression of Timp1 mRNA in CCR2+ cells and CCR2- cells sorted from colon LP cells and found a high expression of Timp1 in CCR2+ monocytes/macrophages and fibrocytes. Conclusions: Circulating CCR2+ monocytes migrate into the inflamed colon via CCL2/CCR2 axis and differentiate into CCR2+Ly6C-F4/80+ fibrocytes, which inhibit collagen degradation and contribute to the development of colon fibrosis by the production of TIMP-1. Disclosures Masuya: Kyowa Hakko Kirin Co., Ltd.: Research Funding. Katayama:Ono Pharmaceutical: Research Funding; Novo Nordisk: Honoraria, Research Funding; Chugai Pharma: Honoraria, Research Funding; Toyama Chemical Co: Research Funding; Sysmex: Honoraria; Mochida Pharmaceutical Co. Ltd.,: Research Funding; Bristol-Myers Squibb: Honoraria; Astellas Pharma: Honoraria, Research Funding; Daiichi Sankyo: Research Funding; Takeda: Honoraria, Research Funding; Teijin Pharma: Research Funding; Eisai: Research Funding; Sumitomo Group: Honoraria, Research Funding; Nippon Shinyaku: Honoraria, Research Funding; Shire: Honoraria; Alexion Pharmaceuticals: Honoraria; Celgene: Honoraria; Taisho Toyama Pharma: Honoraria; Pfizer: Honoraria, Research Funding; Shionogi Pharmaceutical: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Janssen: Research Funding; Kyowa Hakko Kirin: Honoraria, Research Funding.

Description: Background: Rituximab with cyclophosphamide, doxorubicin, vincristine (VCR), and prednisone (R-CHOP) chemotherapy is the standard regimen for newly diagnosed diffuse large B-cell lymphoma (DLBCL). VCR has been widely used in the treatment of lymphoid malignancies and is included in R-CHOP. However, VCR causes peripheral neuropathy (PN), which is one of major toxicities that reduce quality of life. VCR-induced PN (VIPN) is associated with the VCR dose, vincristine cumulative dose, age, and comorbidities (e.g., impaired glucose tolerance). During the last decade, more than 10 gene polymorphisms have been reported to be associated with VIPN in studies of children with acute lymphoblastic leukemia (ALL) who were treated with VCR-containing regimens. Among these polymorphisms, CEP72 rs924607 TT, MTNR1B rs12786200, and ETAA1 rs17032980 were associated with VIPN in both children and adult patients (pts) with ALL in a North American cohort. In a few studies of children with ALL outside the US, the CEP72 rs924607 TT genotype was not associated with VIPN, suggesting an ethnic deviation in the association. Recent studies including children with ALL have also reported that CYP3A5 rs776746, rs7963521, and rs1045644 are associated with VIPN. Little is known about the association between these gene polymorphisms and VIPN in adult pts with B-cell lymphoma. The present study aimed to elucidate the relation between VIPN in adult pts with lymphoma and the CEP72 rs924607, MTNR1B rs12786200, ETAA1 rs17032980, CYP3A5rs776746, rs7963521, and rs1045644 polymorphisms. Methods: This study included 36 pts with mature B-cell lymphoma diagnosed according to the 2008 WHO classification between 2003 and 2017 at Mie University Hospital and who received R-CHOP-like chemotherapy as a first-line therapy. VIPN was graded according to the Common Terminology Criteria for Adverse Events ver. 3.0 from 2003 to 2011 and ver. 4.0 from 2012 to 2017. The specimens used in this study were obtained from the oral mucosa or lymphoma tissue of the pts. Mutation analysis was performed by direct sequencing. CEP72 rs924607, MTNR1B rs12786200, ETAA1 rs17032980, CYP3A5rs776746, rs7963521, and rs1045644 were analyzed in this study. Results: The median age of pts was 64 years (range, 30-78). All pts were Japanese. Five pts had impaired glucose tolerance with no compliant of PN before chemotherapy. Twenty-two pts were diagnosed as having DLBCL, 13 had follicular lymphoma, and one had marginal zone B-cell lymphoma. All pts with the exception of one were treated with R-CHOP. In the remaining patient who had atrial fibrillation, pirarubicin was used instead of doxorubicin in R-CHOP. The total number of cycles of chemotherapy was 6 in 16 pts, 7 in one patient, and 8 in 19 pts. The median cumulative dose of VCR was 12 mg/m2 (range, 2-16). In all 36 pts, 24 pts (67%) experienced any grade of VIPN during chemotherapy and 4 (11%) developed grade 2-4 VIPN. Grade 1 VIPN was observed in 20 pts (56%). Age, impaired glucose tolerance, and the VCR cumulative dose were not associated with the incidence of VIPN. The number of cycles of chemotherapy was significantly associated with the incidence of any grade VIPN (P = 0.04). The CEP72 TT genotype was detected in 7 pts (19%). Four (57%) of 7 pts with the CEP72 TT genotype and 20 (69%) of 29 pts with the CEP72 CT or CC genotype experienced any grade VIPN (P = 0.7). Frequencies of the other gene genotype were as follows: MTNR1B rs12786200 CC in 12 (33%), CT in 18 (50%), and TT in 6 (17%); ETAA1 rs17032980 AA in 22 (61%), AG in 12 (33%), and GG in 2 (6%); CYP3A5rs776746 AA in 4 (11%), AG in 15 (42%), and GG in 17 (47%); rs7963521 CC in 3 (8%), CT in 9 (25%), and TT in 24 (67%); and rs1045644 CC in 21 (58%), CG in 15 (42%), and GG in 0 (0%) of 36 pts. There was no significant association between the incidence of VIPN and these five gene polymorphisms. Conclusions: Our results suggest that the CEP72, MTNR1B, ETAA1, CYP3A5, rs7963521, and rs1045644 polymorphisms analyzed in this study are not associated with the incidence of VIPN in adult pts with mature B-cell lymphoma who received R-CHOP-like chemotherapy. To reduce VIPN in pts who receive R-CHOP, a strategy that devises a VCR administration may be more effective than targeting genetic polymorphisms analyzed in this study. Gene polymorphisms associated with VIPN need to be identified in adult pts with lymphoma. Disclosures Sawaki: Kyowa Hakko Kirin: Research Funding; Ono Pharmaceutical: Research Funding; Astellas Pharma: Research Funding; Chugai: Honoraria. Miyazaki:Kyowa Hakko Kirin: Honoraria, Research Funding; Chugai: Honoraria; Ono Pharmaceutical: Research Funding; Celgene: Honoraria; Eisai: Honoraria; SymBio Pharmaceuticals: Honoraria; Takeda: Honoraria; Nippon Shinyaku: Honoraria; Astellas Pharma: Research Funding; Janssen Pharmaceutical: Honoraria. Imai:Chugai: Honoraria. Tawara:Astellas Pharma: Research Funding; Ono Pharmaceutical: Research Funding; Kyowa Hakko Kirin: Honoraria, Research Funding. Yamaguchi:Ono Pharmaceutical: Research Funding; Astrazeneca: Membership on an entity's Board of Directors or advisory committees; Sorrento: Membership on an entity's Board of Directors or advisory committees; Sumitomo Dainippon Pharma: Honoraria; Takeda: Honoraria; Celgene: Honoraria; Teijin Pharma: Honoraria; MSD: Honoraria; Meiji Seika Kaisha: Honoraria; Janssen: Honoraria; Kyowa Hakko Kirin: Honoraria, Research Funding; Eisai: Honoraria; Chugai: Honoraria, Research Funding; Astellas Pharma: Research Funding. Katayama:Taisho Toyama Pharma: Honoraria; Sysmex: Honoraria; Celgene: Honoraria; Pfizer: Honoraria; Alexion Pharmaceuticals: Honoraria; Chugai: Honoraria; Nippon Shinyaku: Honoraria, Research Funding; Sumitomo Dainippon Pharma: Honoraria; Ono Pharmaceutical: Research Funding; Novo Nordisk: Honoraria; Shionogi Pharmaceutical: Honoraria; Shire: Honoraria; Novartis: Honoraria; Astellas Pharma: Honoraria, Research Funding; Takeda: Honoraria; Bristol-Myers Squibb: Honoraria; kyowa hakko kirin: Honoraria, Research Funding.

Description: It is currently thought that acute GVHD cannot be elicited in the absence of Ag presentation by radiosensitive host hematopoietic-derived APCs after allogeneic BM transplantation. Because clinical data suggest that sex-mismatched H-Y Ags may be important minor histocompatibility Ags for GVH responses, we directly tested their relevance and ability to initiate GVHD when presented by either the hematopoietic- (host or donor) or the nonhematopoietic-derived APCs. H-Y minor Ag incompatibility elicited both CD4+ and CD8+ T-cell driven GVHD lethality. Studies with various well-established BM chimera recipients, in contrast to the current views, have reported that in the absence of functional radiosensitive host hematopoietic-derived APCs, H-Y Ag presentation by either the donor hematopoietic-derived or the host nonhematopoietic-derived APCs is sufficient for inducing GVHD. Our data further suggest that infusion of sufficient numbers of alloreactive donor T cells will induce GVHD in the absence of radiosensitive host hematopoietic-derived APCs.

Description: Little is known about the role of active immunization in suppressing undesirable immune responses. Because CD8α+ dendritic cells (DCs) suppress certain immune responses, we tested the hypothesis that immunization of donors with host-derived CD8α+ DCs will reduce host-specific donor T-cell responses. BALB/c T cells from the animals that were immunized with B6 CD8α+ DCs demonstrated, in vitro and in vivo, significantly reduced proliferation and secretion of inflammatory cytokines but showed enhanced secretion of interleukin-10 (IL-10). The responses against third-party and model antigens were preserved demonstrating antigen specificity. The in vivo relevance was further demonstrated by the reduction on graft-versus-host disease (GVHD) in both a major histocompatibility complex–mismatched clinically relevant BALB/c → B6 model and major histocompatibility complex–matched, minor-mismatched C3H.SW → B6 model of GVHD. Immunization of the donors that were deficient in IL-10 (IL-10−/−) or with CD8α+ DCs from B6 class II (class II−/−) failed to reduce T-cell responses, demonstrating (1) a critical role for secretion of IL-10 by donor T cells and (2) a direct contact between the T cells and the CD8α+ DCs. Together, these data may represent a novel strategy for reducing GVHD and suggest a broad counterintuitive role for vaccination strategies in mitigating undesirable immune responses in an antigen-specific manner.