Upper and/or Lower Respiratory Tract Infection Caused by Human Metapneumovirus After Allogeneic Hematopoietic Stem Cell Transplantation.

BACKGROUND: Human metapneumovirus (hMPV) epidemiology, clinical characteristics and risk factors for poor outcome after allogeneic stem cell transplantation (allo-HCT) remain a poorly investigated area. METHODS: This retrospective multicenter cohort study examined the epidemiology, clinical characteristics, and risk factors for poor outcomes associated with human metapneumovirus (hMPV) infections in recipients of allo-HCT. RESULTS: We included 428 allo-HCT recipients who developed 438 hMPV infection episodes between January 2012 and January 2019. Most recipients were adults (93%). hMPV infections were diagnosed at a median of 373 days after allo-HCT. The infections were categorized as upper respiratory tract disease (URTD) or lower respiratory tract disease (LRTD), with 60% and 40% of cases, respectively. Patients with hMPV LRTD experienced the infection earlier in the transplant course and had higher rates of lymphopenia, neutropenia, corticosteroid use, and ribavirin therapy. Multivariate analysis identified lymphopenia and corticosteroid use (>30 mg/d) as independent risk factors for LRTD occurrence. The overall mortality at day 30 after hMPV detection was 2% for URTD, 12% for possible LRTD, and 21% for proven LRTD. Lymphopenia was the only independent risk factor associated with day 30 mortality in LRTD cases. CONCLUSIONS: These findings highlight the significance of lymphopenia and corticosteroid use in the development and severity of hMPV infections after allo-HCT, with lymphopenia being a predictor of higher mortality in LRTD cases.

A Novel Heterozygous Variant in AICDA Impairs Ig Class Switching and Somatic Hypermutation in Human B Cells and is Associated with Autosomal Dominant HIGM2 Syndrome.

B cells and their secreted antibodies are fundamental for host-defense against pathogens. The generation of high-affinity class switched antibodies results from both somatic hypermutation (SHM) of the immunoglobulin (Ig) variable region genes of the B-cell receptor and class switch recombination (CSR) which alters the Ig heavy chain constant region. Both of these processes are initiated by the enzyme activation-induced cytidine deaminase (AID), encoded by AICDA. Deleterious variants in AICDA are causal of hyper-IgM syndrome type 2 (HIGM2), a B-cell intrinsic primary immunodeficiency characterised by recurrent infections and low serum IgG and IgA levels. Biallelic variants affecting exons 2, 3 or 4 of AICDA have been identified that impair both CSR and SHM in patients with autosomal recessive HIGM2. Interestingly, B cells from patients with autosomal dominant HIGM2, caused by heterozygous variants (V186X, R190X) located in AICDA exon 5 encoding the nuclear export signal (NES) domain, show abolished CSR but variable SHM. We herein report the immunological and functional phenotype of two related patients presenting with common variable immunodeficiency who were found to have a novel heterozygous variant in AICDA (L189X). This variant led to a truncated AID protein lacking the last 10 amino acids of the NES at the C-terminal domain. Interestingly, patients' B cells carrying the L189X variant exhibited not only greatly impaired CSR but also SHM in vivo, as well as CSR and production of IgG and IgA in vitro. Our findings demonstrate that the NES domain of AID can be essential for SHM, as well as for CSR, thereby refining the correlation between AICDA genotype and SHM phenotype as well as broadening our understanding of the pathophysiology of HIGM disorders.

Gut microbiome alterations at acute myeloid leukemia diagnosis are associated with muscle weakness and anorexia.

The gut microbiota makes critical contributions to host homeostasis, and its role in the treatment of acute myeloid leukaemia (AML) has attracted attention. We investigated whether the gut microbiome is affected by AML, and whether such changes are associated with cachectic hallmarks. Biological samples and clinical data were collected from 30 antibiotic-free AML patients at diagnosis and matched volunteers (1:1) in a multicenter cross-sectional prospective study. The composition and functional potential of the faecal microbiota were analyzed using shotgun metagenomics. Faecal, blood, and urine metabolomics analyses were performed. AML patients displayed muscle weakness, anorexia, signs of altered gut function, and glycaemic disorders. The composition of the faecal microbiota differed between patients with AML and control subjects, with an increase in oral bacteria. Alterations in bacterial functions and faecal metabolome support an altered redox status in the gut microbiota, which may contribute to the altered redox status observed in patients with AML. Eubacterium eligens, reduced 3-fold in AML patients, was strongly correlated with muscle strength and citrulline, a marker of enterocyte mass and function. Blautia and Parabacteroides, increased in patients with AML, were correlated with anorexia. Several bacterial taxa and metabolites (e.g. Blautia, Prevotella, phenylacetate, and hippurate) previously associated with glycaemic disorders were altered. Our work revealed important perturbations in the gut microbiome of AML patients at diagnosis, which are associated with muscle strength, altered redox status, and anorexia. These findings pave the way for future mechanistic work to explore the function and therapeutic potential of the bacteria identified in this study.

Longevity of the humoral and cellular responses after SARS-CoV-2 booster vaccinations in immunocompromised patients.

We assessed the humoral and cellular immune responses after two booster mRNA vaccine administrations [BNT162b2 (Pfizer-BioNTech vaccine)] in cohorts of immunocompromised patients (n = 199) and healthy controls (HC) (n = 54). All patients living with HIV (PLWH) and chronic kidney disease (CKD) patients and almost all (98.2%) of the primary immunodeficiency (PID) patients had measurable antibodies 3 and 6 months after administration of the third and fourth vaccine dose, comparable to the HCs. In contrast, only 53.3% and 83.3% of the multiple sclerosis (MS) and rheumatologic patients, respectively, developed a humoral immune response. Cellular immune response was observed in all PLWH after administration of four vaccine doses. In addition, cellular immune response was positive in 89.6%, 97.8%, 73.3% and 96.9% of the PID, MS, rheumatologic and CKD patients, respectively. Unlike the other groups, only the MS patients had a significantly higher cellular immune response compared to the HC group. Administration of additional vaccine doses results in retained or increased humoral and cellular immune response in patients with acquired or inherited immune disorders.

Baseline chest computed tomography for diagnosis of invasive aspergillosis in patients with acute myeloid leukaemia treated with intensive chemotherapy: A retrospective single-centre cohort study.

BACKGROUND: Invasive pulmonary aspergillosis (IPA) is a relatively common infection in patients with acute myeloid leukaemia (AML), and is associated with high mortality rates. Optimising early detection is key to reduce the burden of IPA in this population. In this retrospective cohort study, we evaluated the added value of baseline chest CT before start of classical induction chemotherapy. METHODS: Adult patients receiving first-line intensive chemotherapy for AML were included if a baseline chest CT scan was available (±7 days). Data were collected from the electronic health record. IPA was classified using the EORTC/MSGERC 2020 consensus definitions. RESULTS: Between 2015 and 2019, 99 patients were included. During first-line treatment, 29/99 (30%) patients developed a probable IPA. Baseline chest CT was abnormal in 61/99 (62%) and 14/61 (23%) patients had typical radiological signs for IPA. An abnormal scan showed a trend towards higher risk for IPA (hazard ratio (HR): 2.12; 95% CI 0.95-4.84). Ground glass opacities were a strong predictor for developing IPA (HR 3.35: 95% CI 1.61-7.00). No probable/proven IPA was diagnosed at baseline; however, a bronchoalveolar lavage (BAL) at baseline was only performed in seven patients. Twelve-week mortality was higher in patients with IPA (7/26, 27% vs. 5/59, 8%; p = .024). CONCLUSION: Baseline chest CT scan could be an asset in the early diagnosis of IPA and contribute to risk estimation for IPA. In patients with an abnormal baseline CT, performing a BAL should be considered more frequently, and not only in patients with radiological findings typical for IPA.

Infrared Spectroscopy: A New Frontier in Hematological Disease Diagnosis.

Hematological diseases, due to their complex nature and diverse manifestations, pose significant diagnostic challenges in healthcare. The pressing need for early and accurate diagnosis has driven the exploration of novel diagnostic techniques. Infrared (IR) spectroscopy, renowned for its noninvasive, rapid, and cost-effective characteristics, has emerged as a promising adjunct in hematological diagnostics. This review delves into the transformative role of IR spectroscopy and highlights its applications in detecting and diagnosing various blood-related ailments. We discuss groundbreaking research findings and real-world applications while providing a balanced view of the potential and limitations of the technique. By integrating advanced technology with clinical needs, we offer insights into how IR spectroscopy may herald a new era of hematological disease diagnosis.

Mutations in RNU7-1 Weaken Secondary RNA Structure, Induce MCP-1 and CXCL10 in CSF, and Result in Aicardi-Goutières Syndrome with Severe End-Organ Involvement.

BACKGROUND: Aicardi-Goutières syndrome (AGS) is a type I interferonopathy usually characterized by early-onset neurologic regression. Biallelic mutations in LSM11 and RNU7-1, components of the U7 small nuclear ribonucleoprotein (snRNP) complex, have been identified in a limited number of genetically unexplained AGS cases. Impairment of U7 snRNP function results in misprocessing of replication-dependent histone (RDH) pre-mRNA and disturbance of histone occupancy of nuclear DNA, ultimately driving cGAS-dependent type I interferon (IFN-I) release. OBJECTIVE: We performed a clinical, genetic, and immunological workup of 3 unrelated patients with uncharacterized AGS. METHODS: Whole exome sequencing (WES) and targeted Sanger sequencing of RNU7-1 were performed. Primary fibroblasts were used for mechanistic studies. IFN-I signature and STAT1/2 phosphorylation were assessed in peripheral blood. Cytokines were profiled on serum and cerebrospinal fluid (CSF). Histopathology was examined on brain and kidney tissue. RESULTS: Sequencing revealed compound heterozygous RNU7-1 mutations, resulting in impaired RDH pre-mRNA processing. The 3' stem-loop mutations reduced stability of the secondary U7 snRNA structure. A discrete IFN-I signature in peripheral blood was paralleled by MCP-1 (CCL2) and CXCL10 upregulation in CSF. Histopathological analysis of the kidney showed thrombotic microangiopathy. We observed dysregulated STAT phosphorylation upon cytokine stimulation. Clinical overview of all reported patients with RNU7-1-related disease revealed high mortality and high incidence of organ involvement compared to other AGS genotypes. CONCLUSIONS: Targeted RNU7-1 sequencing is recommended in genetically unexplained AGS cases. CSF cytokine profiling represents an additional diagnostic tool to identify aberrant IFN-I signaling. Clinical follow-up of RNU7-1-mutated patients should include screening for severe end-organ involvement including liver disease and nephropathy.

The transcription factor ETS1 is an important regulator of human NK cell development and terminal differentiation.

Natural killer (NK) cells are important in the immune defense against tumor cells and pathogens, and they regulate other immune cells by cytokine secretion. Although murine NK cell biology has been extensively studied, knowledge about transcriptional circuitries controlling human NK cell development and maturation is limited. By generating ETS1-deficient human embryonic stem cells and by expressing the dominant-negative ETS1 p27 isoform in cord blood hematopoietic progenitor cells, we show that the transcription factor ETS1 is critically required for human NK cell differentiation. Genome-wide transcriptome analysis determined by RNA-sequencing combined with chromatin immunoprecipitation-sequencing analysis reveals that human ETS1 directly induces expression of key transcription factors that control NK cell differentiation (ie, E4BP4, TXNIP, TBET, GATA3, HOBIT, BLIMP1). In addition, ETS1 regulates expression of genes involved in apoptosis and NK cell activation. Our study provides important molecular insights into the role of ETS1 as an important regulator of human NK cell development and terminal differentiation.

Distinct Notch1 and BCL11B requirements mediate human γδ/αß T cell development.

γδ and αß T cells have unique roles in immunity and both originate in the thymus from T-lineage committed precursors through distinct but unclear mechanisms. Here, we show that Notch1 activation is more stringently required for human γδ development compared to αß-lineage differentiation and performed paired mRNA and miRNA profiling across 11 discrete developmental stages of human T cell development in an effort to identify the potential Notch1 downstream mechanism. Our data suggest that the miR-17-92 cluster is a Notch1 target in immature thymocytes and that miR-17 can restrict BCL11B expression in these Notch-dependent T cell precursors. We show that enforced miR-17 expression promotes human γδ T cell development and, consistently, that BCL11B is absolutely required for αß but less for γδ T cell development. This study suggests that human γδ T cell development is mediated by a stage-specific Notch-driven negative feedback loop through which miR-17 temporally restricts BCL11B expression and provides functional insights into the developmental role of the disease-associated genes BCL11B and the miR-17-92 cluster in a human context.

GATA2 deficiency and haematopoietic stem cell transplantation: challenges for the clinical practitioner.

GATA2 deficiency, first described in 2011, is a bone marrow failure disorder resulting in a complex haematological and immunodeficiency syndrome characterised by cytopenias, severe infections, myelodysplasia and leukaemia. The only curative treatment is allogeneic haematopoietic stem cell transplantation (HSCT). Although knowledge on this syndrome has greatly expanded, in clinical practice many challenges remain. In particular, guidelines on optimal donor and stem cell source and conditioning regimens regarding HSCT are lacking. Additionally, genetic analysis of GATA2 is technically cumbersome and could easily result in false-negative results. With this report, we wish to raise awareness of these pitfalls amongst physicians dealing with haematological malignancies and primary immunodeficiencies.

TARP is an immunotherapeutic target in acute myeloid leukemia expressed in the leukemic stem cell compartment.

Immunotherapeutic strategies targeting the rare leukemic stem cell compartment might provide salvage to the high relapse rates currently observed in acute myeloid leukemia (AML). We applied gene expression profiling for comparison of leukemic blasts and leukemic stem cells with their normal counterparts. Here, we show that the T-cell receptor γ chain alternate reading frame protein (TARP) is over-expressed in de novo pediatric (n=13) and adult (n=17) AML sorted leukemic stem cells and blasts compared to hematopoietic stem cells and normal myeloblasts (15 healthy controls). Moreover, TARP expression was significantly associated with a fms-like tyrosine kinase receptor-3 internal tandem duplication in pediatric AML. TARP overexpression was confirmed in AML cell lines (n=9), and was found to be absent in B-cell acute lymphocytic leukemia (n=5) and chronic myeloid leukemia (n=1). Sequencing revealed that both a classical TARP transcript, as described in breast and prostate adenocarcinoma, and an AML-specific alternative TARP transcript, were present. Protein expression levels mostly matched transcript levels. TARP was shown to reside in the cytoplasmic compartment and showed sporadic endoplasmic reticulum co-localization. TARP-T-cell receptor engineered cytotoxic T-cells in vitro killed AML cell lines and patient leukemic cells co-expressing TARP and HLA-A*0201. In conclusion, TARP qualifies as a relevant target for immunotherapeutic T-cell therapy in AML.

Human Thymic CD10+ PD-1+ Intraepithelial Lymphocyte Precursors Acquire Interleukin-15 Responsiveness at the CD1a- CD95+ CD28- CCR7- Developmental Stage.

Human thymic CD8αα+ CD10+ PD-1+ αß T cells selected through early agonist selection have been proposed as the putative thymic precursors of the human CD8αα+ intestinal intraepithelial lymphocytes (IELs). However, the progeny of these thymic precursor cells in human blood or tissues has not yet been characterized. Here, we studied the phenotypical and transcriptional differentiation of the thymic IEL precursor (IELp) lineage upon in vitro exposure to cytokines prominent in the peripheral tissues such as interleukin-15 (IL-15) and the inflammatory cytokines interleukin-12 (IL-12) and interleukin-18 (IL-18). We showed that only the CD1a- fraction of the CD10+ PD-1+ IELp population was able to proliferate with IL-15, suggesting that this subset had acquired functionality. These cells downregulated PD-1 expression and completely lost CD10 expression, whereas other surface markers such as CD95 and CXCR3 remained highly expressed. RNA-seq analysis of the IL-15-cultured cells clearly showed induction of innate-like and effector genes. Induction of the cytotoxic machinery by the CD10+ PD-1+ population was acquired in the presence of IL-15 and was further augmented by inflammatory cytokines. Our data suggest that only the CD1a- CD10+ PD-1+ population exits the thymus and survives in the periphery. Furthermore, PD-1 and CD10 expression is not an intrinsic property of this lineage, but rather characterizes a transient stage in differentiation. CD95 and CXCR3 expression combined with the absence of CD28, CCR7, and CD6 expression might be more powerful markers to define this lineage in the periphery.

Rapid and Effective Generation of Nanobody Based CARs using PCR and Gibson Assembly.

Recent approval of chimeric antigen receptor (CAR) T cell therapy by the European Medicines Agency (EMA)/Federal and Drug Administration (FDA) and the remarkable results of CAR T clinical trials illustrate the curative potential of this therapy. While CARs against a multitude of different antigens are being developed and tested (pre)clinically, there is still a need for optimization. The use of single-chain variable fragments (scFvs) as targeting moieties hampers the quick generation of functional CARs and could potentially limit the efficacy. Instead, nanobodies may largely circumvent these difficulties. We used an available nanobody library generated after immunization of llamas against Cluster of Differentiation (CD) 20 through DNA vaccination or against the ectodomain of CD33 using soluble protein. The nanobody specific sequences were amplified by PCR and cloned by Gibson Assembly into a retroviral vector containing two different second-generation CAR constructs. After transduction in T cells, we observed high cell membrane nanoCAR expression in all cases. Following stimulation of nanoCAR-expressing T cells with antigen-positive cell lines, robust T cell activation, cytokine production and tumor cell lysis both in vitro and in vivo was observed. The use of nanobody technology in combination with PCR and Gibson Assembly allows for the rapid and effective generation of compact CARs.

The eGVHD App has the potential to improve the accuracy of graft-versus-host disease assessment: a multicenter randomized controlled trial.

Graft-versus-host disease (GvHD) assessment has been shown to be a challenge for healthcare professionals, leading to the development of the eGVHD App (www.uzleuven.be/egvhd). In this study, we formally evaluated the accuracy of using the App compared to traditional assessment methods to assess GvHD. Our national multicenter randomized controlled trial involved seven Belgian transplantation centers and 78 healthcare professionals selected using a 2-stage convenience sampling approach between January and April 2017. Using a 1:1 randomization stratified by profession, healthcare professionals were assigned to use either the App ("APP") or their usual GvHD assessment aids ("No APP") to assess the diagnosis and severity score of 10 expert-validated clinical vignettes. Our main outcome measure was the difference in accuracy for GvHD severity scoring between both groups. The odds of being correct were 6.14 (95%CI: 2.83-13.34) and 6.29 (95%CI: 4.32-9.15) times higher in favor of the "APP" group for diagnosis and scoring, respectively (P<0.001). App-assisted GvHD severity scoring was significantly superior for both acute and chronic GvHD, with an Odds Ratio of 17.89 and 4.34 respectively (P<0.001) and showed a significantly increased inter-observer agreement compared to standard practice. Despite a mean increase of 24 minutes (95%CI: 20.45-26.97) in the time needed to score the whole GvHD test package in the "APP" group (P<0.001), usability feedback was positive. The eGVHD App shows superior GvHD assessment accuracy compared to standard practice and has the potential to improve the quality of outcome data registration in allogeneic stem cell transplantation.

Hereditary polycystic kidney disease is characterized by lymphopenia across all stages of kidney dysfunction: an observational study.

Background: Polycystic kidney disease (PKD) is characterized by urinary tract infections and extrarenal abnormalities such as an increased risk of cancer. As mutations in polycystin-1 and -2 are associated with decreased proliferation of immortalized lymphoblastoid cells in PKD, we investigated whether lymphopenia could be an unrecognized trait of PKD. Methods: We studied 700 kidney transplant recipients with (n = 126) or without PKD at the time of kidney transplantation between 1 January 2003 and 31 December 2014 at Ghent University Hospital. We also studied 204 patients with chronic kidney disease (CKD) with PKD and 204 matched CKD patients without PKD across comparable CKD strata with assessment between 1 January 1999 and 1 February 2016 at three renal outpatient clinics. We compared lymphocyte counts with multiple linear regression analysis to adjust for potential confounders. We analysed flow cytometric immunophenotyping data and other haematological parameters. Results: Lymphocyte counts were 264/µL [95% confidence interval (CI) 144-384] and 345/µL (95% CI 245-445) (both P < 0.001) lower in the end-stage kidney disease (ESKD) and CKD cohort, respectively, after adjustment for age, sex, ln(C-reactive protein) and estimated glomerular filtration rate (in the CKD cohort only). In particular, CD8+ T and B lymphocytes were significantly lower in transplant recipients with versus without PKD (P < 0.001 for both). Thrombocyte and monocyte counts were lower in patients with versus without PKD in both cohorts (P < 0.001 for all analyses except P = 0.01 for monocytes in the ESKD cohort). Conclusion: PKD is characterized by distinct cytopenias and especially lymphopenia, independent of kidney function. This finding has the potential to alter our therapeutic approach to patients with PKD.

Nanobody Based Dual Specific CARs.

Recent clinical trials have shown that adoptive chimeric antigen receptor (CAR) T cell therapy is a very potent and possibly curative option in the treatment of B cell leukemias and lymphomas. However, targeting a single antigen may not be sufficient, and relapse due to the emergence of antigen negative leukemic cells may occur. A potential strategy to counter the outgrowth of antigen escape variants is to broaden the specificity of the CAR by incorporation of multiple antigen recognition domains in tandem. As a proof of concept, we here describe a bispecific CAR in which the single chain variable fragment (scFv) is replaced by a tandem of two single-antibody domains or nanobodies (nanoCAR). High membrane nanoCAR expression levels are observed in retrovirally transduced T cells. NanoCARs specific for CD20 and HER2 induce T cell activation, cytokine production and tumor lysis upon incubation with transgenic Jurkat cells expressing either antigen or both antigens simultaneously. The use of nanobody technology allows for the production of compact CARs with dual specificity and predefined affinity.

The immunophenotypic fingerprint of patients with primary antibody deficiencies is partially present in their asymptomatic first-degree relatives.

The etiology of primary antibody deficiencies is largely unknown. Beside rare monogenic forms, the majority of cases seem to have a more complex genetic basis. Whereas common variable immunodeficiency has been investigated in depth, there are only a few reports on milder primary antibody deficiencies such as idiopathic primary hypogammaglobulinemia and IgG subclass deficiency. We performed flow cytometric immunophenotyping in 33 patients with common variable immunodeficiency, 23 with idiopathic primary hypogammaglobulinemia and 21 with IgG subclass deficiency, as well as in 47 asymptomatic first-degree family members of patients and 101 unrelated healthy controls. All three groups of patients showed decreased memory B- and naïve T-cell subsets and decreased B-cell activating factor receptor expression. In contrast, circulating follicular helper T-cell frequency and expression of inducible T-cell co-stimulator and chemokine receptors were only significantly altered in patients with common variable immunodeficiency. Asymptomatic first-degree family members of patients demonstrated similar, albeit intermediate, alterations in naïve and memory B- and T-cell subsets. About 13% of asymptomatic relatives had an abnormal peripheral B-cell composition. Furthermore, asymptomatic relatives showed decreased levels of CD4+ recent thymic emigrants and increased central memory T cells. Serum IgG and IgM levels were also significantly lower in asymptomatic relatives than in healthy controls. We conclude that, in our cohort, the immunophenotypic landscape of primary antibody deficiencies comprises a spectrum, in which some alterations are shared between all primary antibody deficiencies whereas others are only associated with common variable immunodeficiency. Importantly, asymptomatic first-degree family members of patients were found to have an intermediate phenotype for peripheral B- and T-cell subsets.

The role of Ly49E receptor expression on murine intraepithelial lymphocytes in intestinal cancer development and progression.

Ly49E is a member of the Ly49 family of NK receptors and is distinct from other members of this family on the basis of its structural properties, expression pattern and ligand recognition. Importantly, Ly49E receptor expression is high on small intestinal and colonic intraepithelial lymphocytes (IELs). Intestinal IELs are regulators of the mucosal immune system and contribute to front-line defense at the mucosal barrier, including anti-tumor immune response. Whereas most Ly49 receptors have MHC class-I ligands, we showed that Ly49E is instead triggered by urokinase plasminogen activator (uPA). uPA has been extensively implicated in tumor development, where increased uPA expression correlates with poor prognosis. As such, we investigated the role of Ly49E receptor expression on intestinal IELs in the anti-tumor immune response. For this purpose, we compared Ly49E wild-type mice to Ly49E knockout mice in two established tumor models: ApcMin/+-mediated and azoxymethane-induced intestinal cancer. Our results indicate that Ly49E expression on IELs does not influence the development or progression of intestinal cancer.

Notch3 activation is sufficient but not required for inducing human T-lineage specification.

Although the role for the individual Notch receptors in early hematopoiesis have been thoroughly investigated in mouse, studies in human have been mostly limited to the use of pan-Notch inhibitors. However, such studies in human are important to predict potential side effects of specific Notch receptor blocking reagents because these are currently being considered as therapeutic tools to treat various Notch-dependent diseases. In this study, we studied the individual roles of Notch1 and Notch3 in early human hematopoietic lineage decisions, particularly during T-lineage specification. Although this process in mice is solely dependent on Notch1 activation, we recently reported Notch3 expression in human uncommitted thymocytes, raising the possibility that Notch3 mediates human T-lineage specification. Although expression of a constitutive activated form of Notch3 (ICN3) results in the induction of T-lineage specification in human CD34(+) hematopoietic progenitor cells, similar to ICN1 overexpression, loss-of-function studies using blocking Abs reveal that only Notch1, but not Notch3, is critical in this process. Blocking of Notch1 activation in OP9-DLL4 cocultures resulted in a complete block in T-lineage specification and induced monocytic and plasmacytoid dendritic cell differentiation instead. In fetal thymus organ cultures, impeded Notch1 activation resulted in B and dendritic cell development. In contrast, Notch3 blocking Abs only marginally affected T-lineage specification and hematopoietic differentiation with a slight increase in monocyte development. No induction of B or dendritic cell development was observed. Thus, our results unambiguously reveal a nonredundant role for Notch1 in human T-lineage specification, despite the expression of other Notch receptors.