技术资料

PURPOSE Targeting nonspecific,tumor-associated antigens (TAA) with chimeric antigen receptors (CAR) requires specific attention to restrict possible detrimental on-target/off-tumor effects. A reduced affinity may direct CAR-engineered T (CAR-T) cells to tumor cells expressing high TAA levels while sparing low expressing normal tissues. However,decreasing the affinity of the CAR-target binding may compromise the overall antitumor effects. Here,we demonstrate the prime importance of the type of intracellular signaling on the function of low-affinity CAR-T cells. EXPERIMENTAL DESIGN We used a series of single-chain variable fragments (scFv) with five different affinities targeting the same epitope of the multiple myeloma-associated CD38 antigen. The scFvs were incorporated in three different CAR costimulation designs and we evaluated the antitumor functionality and off-tumor toxicity of the generated CAR-T cells in vitro and in vivo. RESULTS We show that the inferior cytotoxicity and cytokine secretion mediated by CD38 CARs of very low-affinity (Kd {\textless} 1.9 × 10-6 mol/L) bearing a 4-1BB intracellular domain can be significantly improved when a CD28 costimulatory domain is used. Additional 4-1BB signaling mediated by the coexpression of 4-1BBL provided the CD28-based CD38 CAR-T cells with superior proliferative capacity,preservation of a central memory phenotype,and significantly improved in vivo antitumor function,while preserving their ability to discriminate target antigen density. CONCLUSIONS A combinatorial costimulatory design allows the use of very low-affinity binding domains (Kd {\textless} 1 mumol/L) for the construction of safe but also optimally effective CAR-T cells. Thus,very-low-affinity scFvs empowered by selected costimulatory elements can enhance the clinical potential of TAA-targeting CARs. View Publication

The bone marrow microenvironment (BMME) contributes to the regulation of hematopoietic stem cell (HSC) function,though its role in age-associated lineage skewing is poorly understood. Here we show that dysfunction of aged marrow macrophages (Mphis) directs HSC platelet-bias. Mphis from the marrow of aged mice and humans exhibited an activated phenotype,with increased expression of inflammatory signals. Aged marrow Mphis also displayed decreased phagocytic function. Senescent neutrophils,typically cleared by marrow Mphis,were markedly increased in aged mice,consistent with functional defects in Mphi phagocytosis and efferocytosis. In aged mice,Interleukin 1B (IL1B) was elevated in the bone marrow and caspase 1 activity,which can process pro-IL1B,was increased in marrow Mphis and neutrophils. Mechanistically,IL1B signaling was necessary and sufficient to induce a platelet bias in HSCs. In young mice,depletion of phagocytic cell populations or loss of the efferocytic receptor Axl expanded platelet-biased HSCs. Our data support a model wherein increased inflammatory signals and decreased phagocytic function of aged marrow Mphis induce the acquisition of platelet bias in aged HSCs. This work highlights the instructive role of Mphis and IL1B in the age-associated lineage-skewing of HSCs,and reveals the therapeutic potential of their manipulation as antigeronic targets. View Publication

The types and magnitude of Ag-specific immune responses can be determined by the functional plasticity of dendritic cells (DCs). However,how DCs display functional plasticity and control host immune responses have not been fully understood. In this study,we report that ligation of DC-asialoglycoprotein receptor (DC-ASGPR),a C-type lectin receptor (CLR) expressed on human DCs,resulted in rapid activation of Syk,followed by PLCgamma2 and PKCdelta engagements. However,different from other Syk-coupled CLRs,including Dectin-1,signaling cascade through DC-ASGPR did not trigger NF-kappaB activation. Instead,it selectively activated MAPK ERK1/2 and JNK. Rapid and prolonged phosphorylation of ERK1/2 led to sequential activation of p90RSK and CREB,which consequently bound to IL10 promoter and initiated cytokine expression. In addition,DC-ASGPR ligation activated Akt,which differentially regulated the activities of GSK-3alpha/beta and beta-catenin and further contributed to IL-10 expression. Our observations demonstrate that DC-ASGPR induces IL-10 expression via an intrinsic signaling pathway,which provides a molecular explanation for DC-ASGPR-mediated programing of DCs to control host immune responses. View Publication

Emerging evidence suggests an immunosuppressive role of altered tumor glycosylation due to downregulation of innate immune responses via immunoregulatory Siglecs. In contrast,human T cells,a major anticancer effector cell,only rarely express Siglecs. However,here,we report that the majority of intratumoral,but not peripheral blood,cytotoxic CD8+ T cells expressed Siglec-9 in melanoma. We identified Siglec-9+ CD8+ T cells as a subset of effector memory cells with high functional capacity and signatures of clonal expansion. This cytotoxic T-cell subset was functionally inhibited in the presence of Siglec-9 ligands or by Siglec-9 engagement by specific antibodies. TCR signaling pathways and key effector functions (cytotoxicity,cytokine production) of CD8+ T cells were suppressed by Siglec-9 engagement,which was associated with the phosphorylation of the inhibitory protein tyrosine phosphatase SHP-1,but not SHP-2. Expression of cognate Siglec-9 ligands was observed on the majority of tumor cells in primary and metastatic melanoma specimens. Targeting the tumor-restricted,glycosylation-dependent Siglec-9 axis may unleash this intratumoral T-cell subset,while confining T-cell activation to the tumor microenvironment. View Publication

The maintenance of homeostasis in the gut is a major challenge for the immune system. Here we demonstrate that the transcription factor MAF plays a central role in T cells for the prevention of gastro-intestinal inflammation. Conditional knock out mice lacking Maf in all T cells developed spontaneous late-onset colitis,correlating with a decrease of FOXP3+RORgammat+ T cells proportion,dampened IL-10 production in the colon and an increase of inflammatory TH17 cells. Strikingly,FOXP3+ specific conditional knock out mice for MAF did not develop colitis and demonstrated normal levels of IL-10 in their colon,despite the incapacity of regulatory T cells lacking MAF to suppress colon inflammation in Rag1-/- mice transferred with na{\{i}}ve CD4+ T cells. We showed that one of the cellular sources of IL-10 in the colon of these mice are TH17 cells. Thus MAF is critically involved in the maintenance of the gut homeostasis by regulating the balance between Treg and TH17 cells either at the level of their differentiation or through the modulation of their functions." View Publication

Acute inflammatory bowel disease (AIBD) is a wide clinical entity including severe gastrointestinal pathologies with common histopathological basis. Epidemiologically increasing diseases,such as necrotizing enterocolitis (NEC),gastrointestinal graft versus host disease (GVHD),and the primary acute phase of chronic inflammatory bowel disease (CIBD),exhibit a high necessity for new therapeutic strategies. Mesenchymal stem cell (MSC) cellular therapy represents a promising option for the treatment of these diseases. In our study,we comparatively assess the efficacy of human MSCs derived from bone marrow (BM),umbilical cord blood (UCB),human embryonic stem cells (ESCs),or human-induced pluripotent stem cells (iPSCs) in a mouse model of chemically induced acute enterocolitis. The laboratory animals were provided ad libitum potable dextrane sulfate sodium solution (DSS) in order to reproduce an AIBD model and then individually exposed intraperitoneally to MSCs derived from BM (BM-MSCs),UCB (UCB-MSCs),ESCs (ESC-MSCs),or iPSCs (iPSC-MSCs). The parameters used to evaluate the cellular treatment efficacy were the animal survival prolongation and the histopathological-macroscopic picture of bowel sections. Although all categories of mesenchymal stem cells led to statistically significant survival prolongation compared to the control group,significant clinical and histopathological improvement was observed only in mice receiving BM-MSCs and UCB-MSCs. Our results demonstrated that the in vivo anti-inflammatory effect of ESC-MSCs and iPSC-MSCs was inferior to that of UCB-MSCs and BM-MSCs. Further investigation will clarify the potential of ESCs and iPSC-derived MSCs in AIBD treatment. View Publication

Deregulation of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway is found in cancer with STAT5A/B controlling leukemic cell survival and disease progression. As mutations in STAT5B,but not STAT5A,have been frequently described in hematopoietic tumors,we used BCR/ABL as model systems to investigate the contribution of STAT5A or STAT5B for leukemogenesis. The absence of STAT5A decreased cell survival and colony formation. Even more drastic effects were observed in the absence of STAT5B. STAT5B-deficient cells formed BCR/ABL+ colonies or stable cell lines at low frequency. The rarely evolving Stat5b-/- cell lines expressed enhanced levels of BCR/ABL oncoprotein compared to wild-type cells. In line,Stat5b-/- leukemic cells induced leukemia with a significantly prolonged disease onset,whereas Stat5a-/- cells rapidly caused a fatal disease superimposable to wild-type cells. RNA-sequencing (RNA-seq) profiling revealed a marked enhancement of interferon (IFN)-alpha and IFN-gamma signatures in Stat5b-/- cells. Inhibition of IFN responses rescued BCR/ABL+ colony formation of Stat5b-/--deficient cells. A downregulated IFN response was also observed in patients suffering from leukemia carrying STAT5B mutations. Our data define STAT5B as major STAT5 isoform driving BCR/ABL+ leukemia. STAT5B enables transformation by suppressing IFN-alpha/gamma,thereby facilitating leukemogenesis. Our findings might help explain the high frequency of STAT5B mutations in hematopoietic tumors. View Publication

Human pluripotent cell lines hold enormous promise for the development of cell-based therapies. Safety,however,is a crucial prerequisite condition for clinical applications. Numerous groups have attempted to eliminate potentially harmful cells through the use of suicide genes1,but none has quantitatively defined the safety level of transplant therapies. Here,using genome-engineering strategies,we demonstrate the protection of a suicide system from inactivation in dividing cells. We created a transcriptional link between the suicide gene herpes simplex virus thymidine kinase (HSV-TK) and a cell-division gene (CDK1); this combination is designated the safe-cell system. Furthermore,we used a mathematical model to quantify the safety level of the cell therapy as a function of the number of cells that is needed for the therapy and the type of genome editing that is performed. Even with the highly conservative estimates described here,we anticipate that our solution will rapidly accelerate the entry of cell-based medicine into the clinic. View Publication

The flow cytometric crossmatch (FCXM) assay,which detects the presence of donor specific HLA antibodies in patient sera,is a cornerstone of HLA compatibility testing. Since relatively long FCXM assay turnaround times may contribute to transplant delays and increased graft ischemia time,we developed and validated two modified crossmatch procedures,namely the Halifax and Halifaster FCXM protocols. These protocols reduce FCXM assay time >60{\%} and simplify their set-up without compromising quality or sensitivity. Optimization of the FCXM (the Halifax protocol) includes a 96-well tray platform,reduced wash times,increased serum to cell suspension volume ratio,shortened incubations and higher incubation temperature. The Halifaster protocol is a further modification,employing methods that improve lymphocyte purity compared to density gradient centrifugation (96 ± 2.63{\%} vs 69 ± 19.06{\%}),reduce cell isolation time (by ∼40{\%}) and conserve FCXM assay reagents. Importantly,linear regression analysis of the median channel fluorescence shift (MCFS) values revealed excellent concordance (R2 of 0.98-0.99) among all three FCXM protocols (standard vs Halifax vs Halifaster). Finally,a retrospective review of 2013 crossmatches performed using the Halifax protocol demonstrated excellent correlation with the virtual crossmatch (95.7{\%} and 96.8{\%} specificity and sensitivity,respectively) regarding the identification of donor specific antibodies (HLA-A/B/DR) assigned based on the single antigen bead (SAB) assay testing with a 2000 mean fluorescence intensity (MFI) cutoff. Implementation of the Halifax or Halifaster protocols will expedite pre-transplantation work-up and improve patient care. View Publication

Antagonists of the 5-hydroxytryptamine (serotonin) 3 receptor (5-HT3R) have anti-inflammatory and anti-apoptotic activities,but the detailed,underlying mechanisms are not well understood. We focused on anti-apoptotic activities via 5-HT3R signaling to clarify the underlying mechanisms. Mice were administered 5-fluorouracil (5-FU),which induced apoptosis in intestinal epithelial cells. Coadministration with 5-HT3R antagonists or agonists tended to decrease or increase the number of apoptotic cells,respectively. In serotonin 3A receptor (5-HT3AR) null (HTR3A-/-) mice,the number of apoptotic cells induced by 5-FU was decreased compared with that in wild-type (WT) mice. Bone marrow (BM) transplantation was performed to determine if BM-derived immune cells regulated 5-FU-induced apoptosis,but they were found to be unrelated to this process. Data from 5-HT3AR/enhanced green fluorescent protein reporter mice revealed that 50{\%} of enterochromaffin (EC) cells expressed 5-HT3AR,but the number of apoptotic cells induced by 5-FU in the intestinal crypt organoids of HTR3A-/- mice was not altered compared with WT mice. In contrast,plasma 5-HT concentrations in WT mice but not in HTR3A-/- mice administered 5-FU were increased significantly. In conclusion,5-HT3R signaling may enhance 5-HT release,possibly from EC cells intravascularly,or paracrine,resulting in increases in plasma 5-HT concentration,which in turn,enhances apoptotic activities induced by 5-FU.-Mikawa,S.,Kondo,M.,Kaji,N.,Mihara,T.,Yoshitake,R.,Nakagawa,T.,Takamoto,M.,Nishimura,R.,Shimada,S.,Ozaki,H.,Hori,M. Serotonin 3 receptor signaling regulates 5-fluorouracil-mediated apoptosis indirectly via TNF-alpha production by enhancing serotonin release from enterochromaffin cells. View Publication

Mesenchymal stromal cells (MSCs) are multipotent stem cells that participate in tissue repair and posses considerable immunomodulatory potential. MSCs have been shown to promote allograft survival,yet the mechanisms behind this phenomenon have not been fully defined. Here,we investigate the capacity of MSCs to suppress the allogeneic immune response by secreting the pleiotropic molecule hepatocyte growth factor (HGF). Using an in vivo mouse model of corneal transplantation,we report that MSCs promote graft survival in an HGF-dependent manner. Moreover,our data indicate that topically administered recombinant HGF (1) suppresses antigen-presenting cell maturation in draining lymphoid tissue,(2) limits T-helper type-1 cell generation,(3) decreases inflammatory cell infiltration into grafted tissue,and (4) is itself sufficient to promote transplant survival. These findings have potential translational implications for the development of HGF-based therapeutics. Stem Cells Translational Medicine 2019. View Publication

Immune activation may underlie the pathogenesis of irritable bowel syndrome (IBS),but the evidence is conflicting. We examined whether peripheral CD4+ T-cells from IBS patients demonstrated immune activation and changes in cytokine production. To gain mechanistic insight,we examined whether immune activation correlated with psychological stress and changing symptoms over time. IBS patients (n = 29) and healthy volunteers (HV; n = 29) completed symptom and psychological questionnaires. IBS patients had a significant increase in CD4+ T-cells expressing the gut homing marker integrin beta7 (p = 0.023) and lymphoid marker CD62L (p = 0.026) compared to HV. Furthermore,phytohaemagglutinin stimulated CD4+ T-cells from IBS-D patients demonstrated increased TNFalpha secretion when compared to HV (p = 0.044). Increased psychological scores in IBS did not correlate with TNFalpha production,while stress hormones inhibited cytokine secretion from CD4+ T-cells of HV in vitro. IBS symptoms,but not markers of immune activation,decreased over time. CD4+ T-cells from IBS-D patients exhibit immune activation,but this did not appear to correlate with psychological stress measurements or changing symptoms over time. This could suggest that immune activation is a surrogate of an initial trigger and/or ongoing parallel peripheral mechanisms. View Publication