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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

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

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

Hematopoiesis provides an accessible system for studying the principles underlying cell-fate decisions in stem cells. Proposed models of hematopoiesis suggest that quantitative changes in lineage-specific transcription factors (LS-TFs) underlie cell-fate decisions. However,evidence for such models is lacking as TF levels are typically measured via RNA expression rather than by analyzing temporal changes in protein abundance. Here,we used single-cell mass cytometry and absolute quantification by mass spectrometry to capture the temporal dynamics of TF protein expression in individual cells during human erythropoiesis. We found that LS-TFs from alternate lineages are co-expressed,as proteins,in individual early progenitor cells and quantitative changes of LS-TFs occur gradually rather than abruptly to direct cell-fate decisions. Importantly,upregulation of a megakaryocytic TF in early progenitors is sufficient to deviate cells from an erythroid to a megakaryocyte trajectory,showing that quantitative changes in protein abundance of LS-TFs in progenitors can determine alternate cell fates. View Publication

MALT1 paracaspase is central for lymphocyte antigen-dependent responses including NF-kappaB activation. We discovered nanomolar,selective allosteric inhibitors of MALT1 that bind by displacing the side chain of Trp580,locking the protease in an inactive conformation. Interestingly,we had previously identified a patient homozygous for a MALT1 Trp580-to-serine mutation who suffered from combined immunodeficiency. We show that the loss of tryptophan weakened interactions between the paracaspase and C-terminal immunoglobulin MALT1 domains resulting in protein instability,reduced protein levels and functions. Upon binding of allosteric inhibitors of increasing potency,we found proportionate increased stabilization of MALT1-W580S to reach that of wild-type MALT1. With restored levels of stable MALT1 protein,the most potent of the allosteric inhibitors rescued NF-kappaB and JNK signaling in patient lymphocytes. Following compound washout,MALT1 substrate cleavage was partly recovered. Thus,a molecular corrector rescues an enzyme deficiency by substituting for the mutated residue,inspiring new potential precision therapies to increase mutant enzyme activity in other deficiencies. View Publication

During metazoan development,immune surveillance and cancer dissemination,cells migrate in complex three-dimensional microenvironments1-3. These spaces are crowded by cells and extracellular matrix,generating mazes with differently sized gaps that are typically smaller than the diameter of the migrating cell4,5. Most mesenchymal and epithelial cells and some-but not all-cancer cells actively generate their migratory path using pericellular tissue proteolysis6. By contrast,amoeboid cells such as leukocytes use non-destructive strategies of locomotion7,raising the question how these extremely fast cells navigate through dense tissues. Here we reveal that leukocytes sample their immediate vicinity for large pore sizes,and are thereby able to choose the path of least resistance. This allows them to circumnavigate local obstacles while effectively following global directional cues such as chemotactic gradients. Pore-size discrimination is facilitated by frontward positioning of the nucleus,which enables the cells to use their bulkiest compartment as a mechanical gauge. Once the nucleus and the closely associated microtubule organizing centre pass the largest pore,cytoplasmic protrusions still lingering in smaller pores are retracted. These retractions are coordinated by dynamic microtubules; when microtubules are disrupted,migrating cells lose coherence and frequently fragment into migratory cytoplasmic pieces. As nuclear positioning in front of the microtubule organizing centre is a typical feature of amoeboid migration,our findings link the fundamental organization of cellular polarity to the strategy of locomotion. View Publication

Specialized immune cell subsets are involved in autoimmune disease,cancer immunity,and infectious disease through a diverse range of functions mediated by overlapping pathways and signals. However,subset-specific responses may not be detectable in analyses of whole blood samples,and no efficient approach for profiling cell subsets at high throughput from small samples is available. We present a low-input microfluidic system for sorting immune cells into subsets and profiling their gene expression. We validate the system's technical performance against standard subset isolation and library construction protocols and demonstrate the importance of subset-specific profiling through in vitro stimulation experiments. We show the ability of this integrated platform to identify subset-specific disease signatures by profiling four immune cell subsets in blood from patients with systemic lupus erythematosus (SLE) and matched control subjects. The platform has the potential to make multiplexed subset-specific analysis routine in many research laboratories and clinical settings. View Publication

Purpose: We determined whether elimination of CD105+ cells in the tumor microenvironment (TME) with anti-CD105 antibodies enhanced anti-disialoganglioside (GD2) antibody dinutuximab therapy of neuroblastoma when combined with activated natural killer (aNK) cells.Experimental Design: The effect of MSCs and monocytes on antibody-dependent cellular cytotoxicity (ADCC) mediated by dinutuximab with aNK cells against neuroblastoma cells was determined in vitro. ADCC with anti-CD105 mAb TRC105 and aNK cells against MSCs,monocytes,and endothelial cells,which express CD105,was evaluated. Anti-neuroblastoma activity in immunodeficient NSG mice of dinutuximab with aNK cells without or with anti-CD105 mAbs was determined using neuroblastoma cell lines and a patient-derived xenograft.Results: ADCC mediated by dinutuximab with aNK cells against neuroblastoma cells in vitro was suppressed by addition of MSCs and monocytes,and dinutuximab with aNK cells was less effective against neuroblastomas formed with coinjected MSCs and monocytes in NSG mice than against those formed by tumor cells alone. Anti-CD105 antibody TRC105 with aNK cells mediated ADCC against MSCs,monocytes,and endothelial cells. Neuroblastomas formed in NSG mice by two neuroblastoma cell lines or a patient-derived xenograft coinjected with MSCs and monocytes were most effectively treated with dinutuximab and aNK cells when anti-human (TRC105) and anti-mouse (M1043) CD105 antibodies were added,which depleted human MSCs and murine endothelial cells and macrophages from the TME.Conclusions: Immunotherapy of neuroblastoma with anti-GD2 antibody dinutuximab and aNK cells is suppressed by CD105+ cells in the TME,but suppression is overcome by adding anti-CD105 antibodies to eliminate CD105+ cells. View Publication

The multikinase inhibitors sunitinib,sorafenib,and axitinib have an impact not only on tumor growth and angiogenesis,but also on the activity and function of immune effector cells. In this study,a comparative analysis of the growth inhibitory properties and apoptosis induction potentials of tyrosine kinase inhibitors on T cells was performed. Tyrosine kinase inhibitor treatment resulted in a dramatic decrease in T cell proliferation along with distinct impacts on the cell cycle progression. This was at least partially associated with an enhanced induction of apoptosis although triggered by distinct apoptotic mechanisms. In contrast to sunitinib and sorafenib,axitinib did not affect the mitochondrial membrane potential but resulted in an induction or stabilization of the induced myeloid leukemia cell differentiation protein (Mcl-1),leading to an irreversible arrest in the G2/M cell cycle phase and delayed apoptosis. Furthermore,the sorafenib-mediated suppression of immune effector cells,in particular the reduction of the CD8(+) T cell subset along with the down-regulation of key immune cell markers such as chemokine CC motif receptor 7 (CCR7),CD26,CD69,CD25,and CXCR3,was not observed in axitinib-treated immune effector cells. Therefore,axitinib rather than sorafenib seems to be suitable for implementation in complex treatment regimens of cancer patients including immunotherapy. View Publication

The macrophages from Crohn's Disease (CD) patients are defective to control the replication of CD-associated adherent-invasive E. coli (AIEC). We aimed to identify the host factors associated with AIEC replication focusing on polymorphisms related to autophagy. Peripheral blood monocyte-derived macrophages (MDM),obtained from 95 CD patient,30 ulcerative colitis (UC) patients and 15 healthy subjects,were genotyped for several CD-associated polymorphisms. AIEC bacteria survival increased within MDM from CD patients compared to UC (p = 0.0019). AIEC bacteria survival increased in patients with CD-associated polymorphism IRGM (p = 0.05) and reduced in those with CD-associated polymorphisms XBP-1 (p = 0.026) and ULK-1 (p = 0.033). AIEC infection led to an increase of pro-inflammatory cytokines IL-1$\beta$ (p {\textless} 0.0001) and TNF-$\alpha$ (p {\textless} 0.0001) in CD macrophages. ULK-1 expression increased in AIEC-infected MDM from CD patients compared to MDM from UC patients or healthy subjects (p = 0.0056) and correlated with AIEC survival (p = 0.0013). Moreover,the expression of ULK-1 phosphorylation on Serine 757 decreased following to AIEC infection (p {\textless} 0.0001). Short-term silencing of ULK-1 and IRGM genes restricted and promote,respectively,AIEC survival within MDM (p = 0.0018 and p = 0.0291). In conclusion,the macrophage defect to mediate AIEC clearance in CD patients is linked to polymorphisms related to autophagy such as IRGM and ULK-1. View Publication

Filoviruses of the genus Ebolavirus include five species with marked differences in their ability to cause disease in humans. From the highly virulent Ebola virus to the seemingly nonpathogenic Reston virus,case-fatality rates can range between 0-90{\%}. In order to understand the molecular basis of these differences it is imperative to establish disease models that recapitulate human disease as faithfully as possible. Non-human primates are the gold-standard models for filovirus pathogenesis,but comparative studies are skewed by the fact that Reston virus infection can be lethal for NHP. Here we have used HLA-A2 transgenic,NOD-scid-interleukin 2$\gamma$ receptor knockout (NSG-A2) mice reconstituted with human hematopoiesis to compare Ebola virus and Reston virus pathogenesis in a human-like environment. While significantly less pathogenic than Ebola virus,Reston virus killed 20{\%} of infected mice,a finding that was linked to exacerbated inflammation and viral replication in the liver. In addition,'humanized' mice recapitulated the case-fatality ratios of different Ebolavirus species in humans. Our findings point out at humanized mice as a putative model to test the pathogenicity of newly discovered filoviruses,and warrants further investigations on Reston virus pathogenesis in humans. View Publication

Myeloid-derived suppressor cells (MDSCs) can subdue inflammation. In mice with acute graft-versus-host disease (GVHD),donor MDSC infusion enhances survival that is only partial and transient because of MDSC inflammasome activation early posttransfer,resulting in differentiation and loss of suppressor function. Here we demonstrate that conditioning regimen-induced adenosine triphosphate (ATP) release is a primary driver of MDSC dysfunction through ATP receptor (P2x7R) engagement and NLR pyrin family domain 3 (NLRP3) inflammasome activation. P2x7R or NLRP3 knockout (KO) donor MDSCs provided significantly higher survival than wild-type (WT) MDSCs. Although in vivo pharmacologic targeting of NLRP3 or P2x7R promoted recipient survival,indicating in vivo biologic effects,no synergistic survival advantage was seen when combined with MDSCs. Because activated inflammasomes release mature interleukin-1$\beta$ (IL-1$\beta$),we expected that IL-1$\beta$ KO donor MDSCs would be superior in subverting GVHD,but such MDSCs proved inferior relative to WT. IL-1$\beta$ release and IL-1 receptor expression was required for optimal MDSC function,and exogenous IL-1$\beta$ added to suppression assays that included MDSCs increased suppressor potency. These data indicate that prolonged systemic NLRP3 inflammasome inhibition and decreased IL-1$\beta$ could diminish survival in GVHD. However,loss of inflammasome activation and IL-1$\beta$ release restricted to MDSCs rather than systemic inhibition allowed non-MDSC IL-1$\beta$ signaling,improving survival. Extracellular ATP catalysis with peritransplant apyrase administered into the peritoneum,the ATP release site,synergized with WT MDSCs,as did regulatory T-cell infusion,which we showed reduced but did not eliminate MDSC inflammasome activation,as assessed with a novel inflammasome reporter strain. These findings will inform future clinical using MDSCs to decrease alloresponses in inflammatory environments. View Publication