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Chronic lymphocytic leukaemia (CLL) is characterised by a heterogeneous clinical course. Such heterogeneity is associated with a number of markers,including TP53 gene inactivation. While TP53 gene alterations determine resistance to chemotherapy,it is not clear whether they can influence early disease progression. To clarify this issue,TP53 mutations and deletions of the corresponding locus [del(17p)] were evaluated in 469 cases from the O-CLL1 observational study that recruited a cohort of clinically and molecularly characterised Binet stage A patients. Twenty-four cases harboured somatic TP53 mutations [accompanied by del(17p) in 9 cases],2 patients had del(17p) only,and 5 patients had TP53 germ-line variants. While del(17p) with or without TP53 mutations was capable of significantly predicting the time to first treatment,a reliable measure of disease progression,TP53 mutations were not. This was true for cases with high or low variant allele frequency. The lack of predictive ability was independent of the functional features of the mutant P53 protein in terms of transactivation and dominant negative potential. TP53 mutations alone were more frequent in patients with mutated IGHV genes,whereas del(17p) was associated with the presence of adverse prognostic factors,including CD38 positivity,unmutated-IGHV gene status,and NOTCH1 mutations. View Publication

Mutations in nucleotide-binding oligomerization domain-containing protein 2 (NOD2) cause Blau syndrome,an inflammatory disorder characterized by uveitis. The antimicrobial functions of Nod2 are well-established,yet the cellular mechanisms by which dysregulated Nod2 causes uveitis remain unknown. Here,we report a non-conventional,T cell-intrinsic function for Nod2 in suppression of Th17 immunity and experimental uveitis. Reconstitution of lymphopenic hosts with Nod2-/- CD4+ T cells or retina-specific autoreactive CD4+ T cells lacking Nod2 reveals a T cell-autonomous,Rip2-independent mechanism for Nod2 in uveitis. In naive animals,Nod2 operates downstream of TCR ligation to suppress activation of memory CD4+ T cells that associate with an autoreactive-like profile involving IL-17 and Ccr7. Interestingly,CD4+ T cells from two Blau syndrome patients show elevated IL-17 and increased CCR7. Our data define Nod2 as a T cell-intrinsic rheostat of Th17 immunity,and open new avenues for T cell-based therapies for Nod2-associated disorders such as Blau syndrome. View Publication

Small molecules that promote the metabolic activity of the pyruvate kinase isoform PKM2,such as TEPP-46 and DASA-58,limit tumorigenesis and inflammation. To understand how these compounds alter T cell function,we assessed their therapeutic activity in a mouse model of T cell-mediated autoimmunity that mimics multiple sclerosis (MS). TH17 cells are believed to orchestrate MS pathology,in part,through the production of two proinflammatory cytokines: interleukin-17 (IL-17) and GM-CSF. We found that both TEPP-46 and DASA-58 suppressed the development of IL-17-producing TH17 cells but increased the generation of those producing GM-CSF. This switch redirected disease pathology from the spinal cord to the brain. In addition,we found that activation of PKM2 interfered with TGF-$\beta$1 signaling,which is necessary for the development of TH17 and regulatory T cells. Collectively,our data clarify the therapeutic potential of PKM2 activators in MS-like disease and how these agents alter T cell function. View Publication

Type III interferon (interferon lambda [IFN-$\lambda$]) is known to be a potential immune modulator,but the mechanisms behind its immune-modulatory functions and its impact on plasmablast differentiation in humans remain unknown. Human B cells and their subtypes directly respond to IFN-$\lambda$. Using B cell transcriptome profiling,we investigate the immune-modulatory role of IFN-$\lambda$ in B cells. We find that IFN-$\lambda$-induced gene expression in B cells is steady,prolonged,and importantly,cell type specific. Furthermore,IFN-$\lambda$ enhances the mTORC1 (mammalian/mechanistic target of rapamycin complex 1) pathway in B cells activated by the B cell receptor (BCR/anti-IgM). Engagement of mTORC1 by BCR and IFN-$\lambda$ induces cell-cycle progress in B cells. Subsequently,IFN-$\lambda$ boosts the differentiation of naive B cells into plasmablasts upon activation,and the cells gain effector functions such as cytokine release (IL-6 and IL-10) and antibody production. Our study shows how IFN-$\lambda$ systematically boosts the differentiation of naive B cells into plasmablasts by enhancing the mTORC1 pathway and cell-cycle progression in activated B cells. View Publication

Immunotherapy is innovating clinical cancer management. Nevertheless,only a small fraction of patient's benefit from current immunotherapies. To improve clinical management of cancer immunotherapy,it is critical to develop strategies for response monitoring and prediction. In this study,we describe inducible T-cell costimulator (ICOS) as a conserved mediator of immune response across multiple therapy strategies. ICOS expression was evaluated by flow cytometry,89Zr-DFO-ICOS mAb PET/CT imaging was performed on Lewis lung cancer models treated with different immunotherapy strategies,and the change in tumor volume was used as a read-out for therapeutic response. ImmunoPET imaging of ICOS enabled sensitive and specific detection of activated T cells and early benchmarking of immune response. A STING (stimulator of interferon genes) agonist was identified as a promising therapeutic approach in this manner. The STING agonist generated significantly stronger immune responses as measured by ICOS ImmunoPET and delayed tumor growth compared with programmed death-1 checkpoint blockade. More importantly,ICOS ImmunoPET enabled early and robust prediction of therapeutic response across multiple treatment regimens. These data show that ICOS is an indicator of T-cell-mediated immune response and suggests ICOS ImmunoPET as a promising strategy for monitoring,comparing,and predicting immunotherapy success in cancer. SIGNIFICANCE: ICOS ImmunoPET is a promising strategy to noninvasively predict and monitor immunotherapy response.See related commentary by Choyke,p. 2975. View Publication

Neutrophils are short-lived cells that play important roles in both health and disease. Neutrophils and monocytes originate from the granulocyte monocyte progenitor (GMP) in bone marrow; however,unipotent neutrophil progenitors are not well defined. Here,we use cytometry by time of flight (CyTOF) and single-cell RNA sequencing (scRNA-seq) methodologies to identify a committed unipotent early-stage neutrophil progenitor (NeP) in adult mouse bone marrow. Importantly,we found a similar unipotent NeP (hNeP) in human bone marrow. Both NeP and hNeP generate only neutrophils. NeP and hNeP both significantly increase tumor growth when transferred into murine cancer models,including a humanized mouse model. hNeP are present in the blood of treatment-naive melanoma patients but not of healthy subjects. hNeP can be readily identified by flow cytometry and could be used as a biomarker for early cancer discovery. Understanding the biology of hNeP should allow the development of new therapeutic targets for neutrophil-related diseases,including cancer. View Publication

Neurologic melioidosis occurs in both human and animals; however,the mechanism by which the pathogen Burkholderia pseudomallei invades the central nervous system (CNS) remains unclear. B. pseudomallei-loaded Ly6C cells have been suggested as a putative portal; however,during melioidosis,lipopolysaccharide (LPS) can drive disruption of the blood-brain barrier (BBB). This study aims to test whether the Trojan horse-like mechanism occurs during endotoxemia. The expression levels of cerebral cytokines,chemokines and cell adhesion molecules; the activation of astrocytes,microglia and endothelial cells; and the increased vascular permeability and brain-infiltrating leukocytes were evaluated using B. pseudomallei,B. thailandensis,B. cenocepacia and B. multivorans LPS-induced brains. Accordingly,different degrees of BBB damage in those brains with endotoxemia were established. The B. multivorans LPS-induced brain exhibited the highest levels of disruptive BBB according to the above mediators/indicators. Into these distinct groups of endotoxemic mice,B. pseudomallei-loaded Ly6C cells or free B. pseudomallei were adoptively transferred at equal bacterial concentrations (103 CFU). The bacterial load and number of cases of meningeal neutrophil infiltration in the brains of animals treated with B. pseudomallei-loaded Ly6C cells were higher than those in brains induced by free B. pseudomallei in any of the endotoxemic groups. In particular,these results were reproducible in B. multivorans LPS-induced brains. We suggest that B. pseudomallei-loaded cells can act as a Trojan horse and are more effective than free B. pseudomallei in invading the CNS under septic or endotoxemic conditions even when there is a high degree of BBB disruption. View Publication

Human mucosal tissues and skin contain two distinct types of dendritic cell (DC) subsets,epidermal Langerhans cells (LCs) and dermal DCs,which can be distinguished by the expression of C-type lectin receptors,Langerin and DC-SIGN,respectively. Although peripheral blood monocytes differentiate into these distinct subsets,monocyte-derived LCs (moLCs) induced by coculture with GM-CSF,IL-4,and TGF-$\beta$1 coexpress both Langerin and DC-SIGN,suggesting that the environmental cues remain unclear. In this study,we show that LC differentiation is TGF-$\beta$1 dependent and that cofactors such as IL-4 and TNF-$\alpha$ promote TGF-$\beta$1-dependent LC differentiation into Langerin+DC-SIGN- moLCs but continuous exposure to IL-4 blocks differentiation. Steroids such as dexamethasone greatly enhanced TNF-$\alpha$-induced moLC differentiation and blocked DC-SIGN expression. Consistent with primary LCs,dexamethasone-treated moLCs express CD1a,whereas monocyte-derived DCs (moDCs) express CD1b,CD1c,and CD1d. moDCs but not moLCs produced inflammatory cytokines after stimulation with CD1b and CD1d ligands mycolic acid and $\alpha$-galactosylceramide,respectively. Strikingly,CD1a triggering with squalene on moLCs but not moDCs induced strong IL-22-producing CD4+ helper T cell responses. As IL-22 is an important cytokine in the maintenance of skin homeostasis,these data suggest that CD1a on LCs is involved in maintaining the immune barrier in the skin. View Publication

Effective humoral immunity requires class switch recombination (CSR) catalyzed by activation-induced cytidine deaminase (AID). In response to T cell-dependent (TD) Ags,CSR can be induced by CD40 signaling in B cells. TNFR-associated factors 2 and 3 (TRAF2/TRAF3) function as adaptors of the CD40 signaling pathway. B cell-intrinsic TRAF2 or TRAF3 (B-TRAF2 or B-TRAF3) knockout mice were previously reported to have indistinguishable phenotypes in gene expression,B cell survival and development,and enlarged peripheral lymphoid organs. However,it remains unknown whether deficiency of B-TRAF2 or B-TRAF3 differentially affects TD humoral immune responses and CD40-induced CSR. In this article,we show that B-TRAF2 is essential for optimal isotype switching induced by in vivo TD Ag immunization or by engaging CD40 in vitro. Our data clarify the controversial role of B-TRAF3 and confirm its dispensability in CD40-induced CSR. Mechanistically,CD40-induced AID expression was markedly impaired by B-TRAF2,but not B-TRAF3,deficiency. Moreover,B-TRAF2 deficiency causes defective activation of the NF-$\kappa$B1 complex in a CD40-autonomous manner,and restoring CD40-induced NF-$\kappa$B1 activation in TRAF2-deficient B cells rescues AID expression and CSR. We conclude that TRAF2 is essential but TRAF3 is dispensable for TD humoral immunity and CD40-induced CSR. Our studies provide significant biological bases for optimizing treatment of B cell-associated immune disorders by targeting CD40 signaling. View Publication

Indoleamine 2,3-dioxygenase 2 (Ido2) is a recently identified catalytic enzyme in the tryptophan-kynurenine pathway that is expressed primarily in monocytes and dendritic cells. To elucidate the biological role of Ido2 in immune function,we introduced lipopolysaccharide (LPS) endotoxin shock to Ido2 knockout (Ido2 KO) mice,which led to higher mortality than that in the wild type (WT) mice. LPS-treated Ido2 KO mice had increased production of inflammatory cytokines (including interleukin-6; IL-6) in serum and signal transducer and activator of transcription 3 (stat3) phosphorylation in the spleen. Moreover,the peritoneal macrophages of LPS-treated Ido2 KO mice produced more cytokines than did the WT mice. By contrast,the overexpression of Ido2 in the murine macrophage cell line (RAW) suppressed cytokine production and decreased stat3 expression. Finally,RAW cells overexpressing Ido2 did not alter nuclear factor $\kappa$B (NF-$\kappa$B) or stat1 expression,but IL-6 and stat3 expression decreased relative to the control cell line. These results reveal that Ido2 modulates IL-6/stat3 signalling and is induced by LPS,providing novel options for the treatment of immune disorders. View Publication

Despite showing success in treating melanoma and haematological malignancies,adoptive cell therapy (ACT) has generated only limited effects in solid tumors. This is,in part,due to a lack of specific antigen targets,poor trafficking/infiltration and immunosuppression in the tumor microenvironment. In this study,we combined ACT with oncolytic virus vaccines (OVV) to drive expansion and tumor infiltration of transferred antigen-specific T cells,and demonstrated that the combination is highly potent for the eradication of established solid tumors. Consistent with other successful immunotherapies,this approach elicited severe autoimmune consequence when the antigen targeted was a self-protein. However,modulation of IFN$\alpha$/$\beta$ signaling,either by functional blockade or rational choice of an OVV backbone,ameliorated autoimmune side effects without compromising antitumor efficacy. Our study uncovers a pathogenic role for IFN$\alpha$/$\beta$ in facilitating autoimmune toxicity during cancer immunotherapy and offers a safe and powerful combinatorial regimen with immediate translational applications. View Publication

The costimulation of immune cells using first-generation anti-4-1BB monoclonal antibodies (mAbs) has demonstrated anti-tumor activity in human trials. Further clinical development,however,is restricted by significant off-tumor toxicities associated with Fc$\gamma$R interactions. Here,we have designed an Fc-free tumor-targeted 4-1BB-agonistic trimerbody,1D8N/CEGa1,consisting of three anti-4-1BB single-chain variable fragments and three anti-EGFR single-domain antibodies positioned in an extended hexagonal conformation around the collagen XVIII homotrimerization domain. The1D8N/CEGa1 trimerbody demonstrated high-avidity binding to 4-1BB and EGFR and a potent in vitro costimulatory capacity in the presence of EGFR. The trimerbody rapidly accumulates in EGFR-positive tumors and exhibits anti-tumor activity similar to IgG-based 4-1BB-agonistic mAbs. Importantly,treatment with 1D8N/CEGa1 does not induce systemic inflammatory cytokine production or hepatotoxicity associated with IgG-based 4-1BB agonists. These results implicate Fc$\gamma$R interactions in the 4-1BB-agonist-associated immune abnormalities,and promote the use of the non-canonical antibody presented in this work for safe and effective costimulatory strategies in cancer immunotherapy. View Publication