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SARS-CoV-2, although not being a circulatory virus, spread from the respiratory tract resulting in multiorgan failures and thrombotic complications, the hallmarks of fatal COVID-19. A convergent contributor could be platelets that beyond hemostatic functions can carry infectious viruses. Here, we profiled 52 patients with severe COVID-19 and demonstrated that circulating platelets of 19 out 20 non-survivor patients contain SARS-CoV-2 in robust correlation with fatal outcome. Platelets containing SARS-CoV-2 might originate from bone marrow and lung megakaryocytes (MKs), the platelet precursors, which were found infected by SARS-CoV-2 in COVID-19 autopsies. Accordingly, MKs undergoing shortened differentiation and expressing anti-viral IFITM1 and IFITM3 RNA as a sign of viral sensing were enriched in the circulation of deadly COVID-19. Infected MKs reach the lung concomitant with a specific MK-related cytokine storm rich in VEGF, PDGF and inflammatory molecules, anticipating fatal outcome. Lung macrophages capture SARS-CoV-2-containing platelets in vivo. The virus contained by platelets is infectious as capture of platelets carrying SARS-CoV-2 propagates infection to macrophages in vitro, in a process blocked by an anti-GPIIbIIIa drug. Altogether, platelets containing infectious SARS-CoV-2 alter COVID-19 pathogenesis and provide a powerful fatality marker. Clinical targeting of platelets might prevent viral spread, thrombus formation and exacerbated inflammation at once and increase survival in COVID-19.

International audience; Abstract The cellular prion protein PrP C partners with caveolin-1 (CAV1) in neurodegenerative diseases but whether this interplay occurs in cancer has never been investigated. By leveraging patient and cell line datasets, we uncover a molecular link between PrP C and CAV1 across cancer. Using cell-based assays, we show that PrP C regulates the expression of and interacts with CAV1. PrP C additionally controls the expression of the amyloid precursor protein APP and of the Aβ generating enzyme BACE1, and regulates the levels of Aβ, whose accumulation is a central event in Alzheimer’s disease. We further identify DKK1 and DKK3, involved in both Alzheimer’s disease and cancer progression, as targets of the PrP C -dependent axis. Finally, we establish that antibody-mediated blocking of the Aβ-PrP C interaction delays the growth of prostate cancer cell line-derived xenografts and prevents the development of metastases. Our data additionally support an enrichment of the Aβ-PrP C -dependent pathway in the basal subtype of prostate cancer, associated with anti-hormonal therapy resistance, and in mesenchymal colon cancer, associated with poor prognosis. Thus, based on a parallel with neurodegenerative diseases, our results bring to light an Aβ-PrP C axis and support the potential of targeting this pathway in patients with selected subtypes of prostate and colon cancer.

Immunological non-responders (InRs) are HIV-infected individuals in whom the administration of combination antiretroviral therapy (cART), although successful in suppressing viral replication, cannot properly reconstitute patient circulating CD4

International audience; Pembrolizumab, a PD1 immune checkpoint inhibitor (ICI), was recently reported to be very effective in patients with microsatellite instable/deficient mismatch repair metastatic colorectal cancer (MSI/dMMR mCRC), unlike patients with microsatellite stable/proficient MMR (MSS/pMMR) mCRC, in whom ICIs are generally ineffective. However, about 15% of MSS/pMMR CRCs are highly infiltrated by tumour infiltrating lymphocytes. In addition, both oxaliplatin and bevacizumab have been shown to have immunomodulatory properties that may increase the efficacy of an ICI. We formulated the hypothesis that patients with MSS/pMMR mCRC with a high immune infiltrate can be sensitive to ICI plus oxalipatin and bevacizumab-based chemotherapy. POCHI is a multicenter, open-label, single-arm phase II trial to evaluate efficacy of Pembrolizumab with Capox Bevacizumab as first-line treatment of MSS/pMMR mCRC with a high immune infiltrate for which we plan to enrol 55 patients. Primary endpoint is progression-free survival (PFS) at 10 months, which is expected greater than 50%, but a 70% rate is hoped for. Main secondary objectives are overall survival, secondary resection rate and depth of response. Patients must have been resected of their primary tumour so as to evaluate two different immune scores (Immunoscore® and TuLIS) and are eligible if one score is "high";. The first patient was included on April 20, 2021.

Introduction Monocytosis is a frequent trigger for blood smear review in a routine hematology laboratory whereas chronic myelomonocytic leukemia (CMML) is infrequent and arises mostly in elderly patients. In order to define the best workflow for monocytosis, we studied three diagnostic approaches: the classical morphology approach (blood smear review), the flow cytometry assay (quantification of monocyte subsets as described by Selimoglu‐Buet et al in 2015), and the “mono‐dysplasia‐score” also referred to as “Monoscore (as described by our team in 2018 using the structural parameters of the Sysmex XN™ analyzers). Methods Studying a multicentric cohort of 196 nonclonal monocytoses and CMML patients aged over 50 years, we compared the diagnostic performance of the three approaches alone and in combination to propose a diagnostic decision tree. Results In patients presenting with additional criteria for slide review to monocytosis (37% of our cohort), we propose to sequentially combine morphology, Monoscore, and flow cytometry. On the contrary, for patients with isolated monocytosis (63%), slide review is not mandatory and we suggest performing flow cytometry depending on the Monoscore value. Using the proposed algorithm, 98% of CMML patients would have been correctly identified, slide review rate drastically reduced, and flow cytometry would have been carried out in 44% of patients. Conclusion We have shown that implementation of Monoscore is a useful input filter to significantly reduce slide reviews without losing sensitivity and that flow cytometry is a performant technique in the second step of the diagnostic workup of CMML.

Cohen syndrome (CS) is a rare genetic disorder due to mutations in VPS13B gene. Among various clinical and biological features, CS patients suffer from inconsistent neutropenia, which is associated with recurrent but minor infections. We demonstrate here that this neutropenia results from an exaggerate rate of neutrophil apoptosis. Besides this increased cell death, which occurs in the absence of any endoplasmic reticulum stress or defect in neutrophil elastase (ELANE) expression or localization, all neutrophil functions appeared to be normal. We showed a disorganization of the Golgi apparatus in CS neutrophils precursors, that correlates with an altered glycosylation of ICAM-1 in these cells, as evidenced by a migration shift of the protein. Furthermore, a striking decrease in the expression of SERPINB1 gene, which encodes a critical component of neutrophil survival, was detected in CS neutrophils. These abnormalities may account for the excessive apoptosis of neutrophils leading to neutropenia in CS. KEY MESSAGES: Cohen syndrome patients' neutrophils display normal morphology and functions. Cohen syndrome patients' neutrophils have an increased rate of spontaneous apoptosis compared to healthy donors' neutrophils. No ER stress or defective ELA2 expression or glycosylation was observed in Cohen syndrome patients' neutrophils. SerpinB1 expression is significantly decreased in Cohen syndrome neutrophils as well as in VPS13B-deficient cells.

Background Immune checkpoint inhibitors (ICI) are very effective in deficient DNA mismatch-repair system (dMMR)/microsatellite instable (MSI) metastatic colorectal cancer (mCRC). About 15% of MSS/pMMR CRCs are highly infiltrated by tumor infiltrating lymphocyte (TIL) with a good prognosis. Some immune scores based on CD3+ and/or CD8+ T-cells infiltration are validated and reproducible, especially TuLIS1 and Immunoscore®.2 No data are available concerning efficacy of ICI in this subpopulation of mCRC. Pembrolizumab, an anti-PD1 (programmed death-1) monoclonal antibody has been recently reported very effective in patients with MSI/dMMR mCRC. Immunogenic cell death induced by chemotherapy, such as oxaliplatin, could increase the efficacy of ICI. We formulated the hypothesis that patients with a pMMR mCRC with a high immune infiltrate can be sensitive to ICI plus oxalipatin-based chemotherapy. Methods POCHI is a multicenter, open-label, single-arm phase II trial to evaluate efficacy of pembrolizumab in combination with chemotherapy as first-line treatment of pMMR mCRC with a high immune infiltrate. Primary objective is PFS at 10 months, i.e. number of patients alive and without radiological and/or clinical progression at 10 months evaluated by the investigator. Main secondary objectives are overall survival, secondary resection rate, depth of response and early tumour shrinkage. Main inclusion criteria are pMMR mCRC untreated for metastatic disease and with at least one measurable metastatic target according to RECIST v1.1 criteria. Patients must have resected primary tumor to evaluate two different immune scores (Immunoscore® and TuLIS) and patients are eligible if one score is ‘high’. Patients will receive combination of pembrolizumab (200 mg), bevacizumab (7.5 mg/kg), oxaliplatin (130 mg/m²) and capecitabine (2000 mg/m²/day, on day 1 to 14). Treatment will be repeated every 3 weeks until disease progression or unacceptable toxicity. The clinical hypotheses are to increase PFS at 10 months from 50% to 70%. With a one-sided type error of 5%, power of 85%, 10% rate of patients lost to follow-up or not evaluable, 55 patients have to be included. If 32 patients or more are alive and without progression at 10 months, the treatment will be considered as effective. Thus, with 15% ‘high’ immune score, about 400 patients must be tested in order to include 55 patients in POCHI trial. The ancillary study will consist to identify predictive biomarkers of response and included expression of PD-L1, circulating lymphocytes circulating tumour DNA, mutational load and gut microbiota. Inclusions will start in September 2020 and theoretical end of recruitment is 2023. Results N/A Conclusions N/A Acknowledgements We thank all the cooperative groups (FFCD – UNICANCER GI– GERCOR) for their contribution and participation to the present trial. We thank MSD and HalioDX for their support. Trial Registration NCT04262687 Ethics Approval This study was approved by ‘Agence Nationale de Securite du Medicament et des produits de sante (ANSM)’ on 24/03/2020; approval number MEDAECNAT-2020-01-00038_2019-002407-18.’ Consent N/A References Emile JF, et al., Prospective validation of a lymphocyte infiltration prognostic test in stage III colon cancer patients treated with adjuvant FOLFOX. Eur J Cancer 2017 Sep;82:16–24 Pages, et al. International validation of the consensus Immunoscore for the classification of colon cancer: a prognostic and accuracy study. Lancet 2018 May 26;391(10135):2128–2139.

In addition to hemostasis, human platelets have several immune functions and interact with infectious pathogens including HIV in vitro. Here, we report that platelets from HIV-infected individuals on combined antiretroviral drug therapy (ART) with low blood CD4+ T cell counts (

Aneuploidy is a common feature of cancer cells and may contribute to cellular transformation and cancer development. In this study, we found that significant down-regulation of CDKN2A, CHEK2, CDCA8, TP53BP1, and CCNDBP1 led to chromosome imbalances in two diploid non-immortalized human cell lines; however, only CDKN2A inhibition enhanced cell proliferation and additionally up-regulated three cell cycle control genes: CDCA8, AURKA, and CCND. These results confirm that CDKN2A is a tumor suppressor gene driving human cancer development by inducing cell aneuploidy and cell cycle up-regulation.

Intratumoral injection of biocompatible gels is increasingly used for the sustained delivery of drugs and vaccines to enhance the anti-cancer immune response. Granulocyte-macrophage colony stimulating factor (GM-CSF) has become an attractive adjuvant thanks to its ability to boost the antitumor immune response by inducing proliferation, maturation and migration of the dendritic-cells (DCs) and the differentiation of lymphocytes. Killed Mycobacteria, such as Heat-killed Mycobacterium tuberculosis (HKMT) have been used in several studies as TLR-2 agonist to increase maturation of DCs. In this study, we designed a mucoadhesive thermosensitive formulation for the local delivery of GM-CSF and HKMT in order to enhance DCs activation and improve the local antitumor immune response. This formulation was selected based on its elastic and mucoadhesive properties obtained thanks to rheological studies. More importantly, intratumoral residence time of the labelled gel and protein were evidenced by means of MRI and non invasive in vivo optical imaging. Then, the efficacy of the combination of immunomodulators loaded thermogel was demonstated in vitro and in vivo. The selected thermogel exhibits rheological properties which confer a good elasticity and increased residence time of the immunostimulatory agents in the tumor, thus increasing the recruitment of DCs and T cytotoxic CD8+ lymphocytes.

Human immunodeficiency virus type 1 (HIV-1) eradication is prevented by the establishment on infection of cellular HIV-1 reservoirs that are not fully characterized, especially in genital mucosal tissues (the main HIV-1 entry portal on sexual transmission). Here, we show, using penile tissues from HIV-1-infected individuals under suppressive combination antiretroviral therapy, that urethral macrophages contain integrated HIV-1 DNA, RNA, proteins and intact virions in virus-containing compartment-like structures, whereas viral components remain undetectable in urethral T cells. Moreover, urethral cells specifically release replication-competent infectious HIV-1 following reactivation with the macrophage activator lipopolysaccharide, while the T-cell activator phytohaemagglutinin is ineffective. HIV-1 urethral reservoirs localize preferentially in a subset of polarized macrophages that highly expresses the interleukin-1 receptor, CD206 and interleukin-4 receptor, but not CD163. To our knowledge, these results are the first evidence that human urethral tissue macrophages constitute a principal HIV-1 reservoir. Such findings are determinant for therapeutic strategies aimed at HIV-1 eradication. Suppressive combination antiretroviral therapy fails to eradicate HIV-1 latent reservoirs in poorly characterized cell compartments. Here, urethral macrophages, but not urethral T cells, are shown to contain integrated HIV-1 DNA and to be able to release infectious HIV-1 following reactivation with lipopolysaccharide.

In addition to hemostasis, human platelets have several immune functions and interact with infectious pathogens including HIV in vitro. Here, we report that platelets from HIV-infected individuals on combined antiretroviral drug therapy (ART) with low blood CD4

The effect of oxygen on virus replication is complex, and the role of hypoxia-inducible factor 1α (HIF-1α) in the metabolism of virus-infected cells remains uncertain. Solid tumours are hypoxic, and some viruses use this low oxygen tension level to facilitate their replication in tumour cells, thereby causing cell lysis. In addition, the interactions between viruses and HIF-1α may stimulate a trained immunity. However, the evolutionary basis for the oxygen regulatory mechanism of virus replication is ill-defined and requires further investigation.

The role of oxygen tension level is a well-known phenomenon that has been studied in oncology and radiotherapy since about 60 years. Oxygen tension may inhibit or stimulate propagation of viruses in vitro as well as in vivo. In turn modulating oxygen metabolism may constitute a novel approach to treat viral infections as an adjuvant therapy. The major transcription factor which regulates oxygen tension level is hypoxia-inducible factor-1 alpha (HIF-1α). Down-regulating the expression of HIF-1α is a possible method in the treatment of chronic viral infection such as human immunodeficiency virus infection, chronic hepatitis B and C viral infections and Kaposi sarcoma in addition to classic chemotherapy. The aim of this review is to supply an updating concerning the influence of oxygen tension level in human viral infections and to evoke possible new therapeutic strategies regarding this environmental condition.

Gain of function mutations of KIT are frequent in some human tumors, and are sensible to tyrosine kinase inhibitors. In most tumors, oncogenic mutations are heterozygous, however most in vitro data of KIT activation have been obtained with hemizygous mutation. This study aimed to investigate the maturation and activation of wild‐type (WT) and mutant (M) forms of KIT in hemizygous and heterozygous conditions. WT and two types of exon 11 deletions M forms of human KIT were expressed in NIH3T3 cell lines. Membrane expression of KIT was quantified by flow cytometry. Quantification of glycosylated forms of KIT and phosphorylated forms of AKT and ERK were performed by western blot. Simultaneous activation of WT KIT and treatment with endoplasmic reticulum (ER) inhibitors, tunicamycin or brefeldin A induced a complete inhibition of membrane expression of the 145 kDa form of KIT. By contrast activation or ER inhibitors alone, only partly inhibited this form. ER inhibitors also inhibited KIT activation‐dependent phosphorylation of AKT and ERK1/2. Brefeldin A induced a complete down regulation of the 145 kDa form in hemizygous M, and induced an intra‐cellular accumulation of the 125 kDa form in WT but not in hemizygous M. Heterozygous cells had glycosylation and response to ER inhibitors patterns more similar to WT than to hemizygous M. Phosphorylated AKT was reduced in hemizygous cells in comparison to WT KIT cells and heterozygous cells, and in the presence of brefeldin A in all cell lines. Effects of ER inhibitors are significantly different in hemizygous and heterozygous mutants. Differences in intra‐cellular trafficking of KIT forms result in differences in downstream signaling pathways, and activation of PI3K/AKT pathway appears to be tied to the presence of the mature 145 kDa form of KIT at the membrane surface.

Objective LYL-1 is a transcription factor containing a basic helix-loop-helix motif closely related to SCL/TAL-1, a regulator of erythroid differentiation. Because LYL-1 is expressed in erythroid cell populations, we addressed its role in erythropoiesis using knockin mice. Materials and Methods Erythropoiesis of LYL-1 −/− mice was studied by progenitor assays, flow cytometry, reconstitution assays, and functional tests. Expression of LYL-1, SCL , and GATA-1 was assessed at messenger RNA level by quantitative reverse transcription polymerase chain reaction. Results LYL-1 −/− mice displayed decreased erythropoiesis with a partial arrest in differentiation, and enhanced apoptosis associated with decreased Bcl-x L expression in the bone marrow (BM). In addition, LYL-1 −/− BM cells were severely impaired in their abilities to reconstitute the erythroid lineage in competitive assays, suggesting a cell autonomous abnormality of erythropoiesis. In parallel, erythroid progenitor and precursor cells were significantly increased in the spleen of LYL-1 −/− mice. Expression of LYL-1 was differentially regulated during maturation of erythroblasts and strikingly different between spleen- and BM-derived erythroblasts. Expression of LYL-1 decreased during erythroid differentiation in the spleen whereas it increased in the BM to reach the same level in mature erythroblasts as in the soleen. Loss of Lyl-1 expression was accompanied with an increase of SCL / TAL-1 and GATA-1 transcripts in spleen but not in BM-derived erythroblasts. Furthermore, phenylhydrazine-induced stress erythropoiesis was elevated in LYL-1 −/− mice and mutant BM and spleen erythroid progenitors were hypersensitive to erythropoietin. Conclusions Taken together, these results suggest that LYL-1 plays a definite role in erythropoiesis, albeit with different effects in BM specifically regulating basal erythropoiesis, and spleen, controlling stress-induced erythropoiesis.

Background MPN-associated myelofibrosis (MF) is a condition characterized by splenomegaly, anemia, bone marrow (BM) fibrosis and debilitating symptoms. About 80% of patients (pts) harbor a driver mutations in JAK2, CALR or MPL genes that can be used as biomarkers for minimal residual disease assessment. Ruxolitinib (Rux) is a JAK inhibitor approved in intermediate or high risk (HR) MF to improve symptoms and splenomegaly but with little impact on the malignant clone and fibrosis. Interferon alpha (IFNa) can reduce mutant allele burden and fibrosis but is often poorly tolerated in highly symptomatic pts. The RUXOPEG study was designed to assess the efficacy and safety of the combination of Rux + IFNa in MF (NCT02742324). Methods RUXOPEG is a multi-center Bayesian Phase 1/2 adaptive trial. Phase 1 includes up to 9 cohorts of 3 pts with increasing doses of both drugs. Tested doses of Rux and IFNa are 10, 15 and 20 mg BID, and 45, 90 and 135 mcg/week, respectively. Phase 2 will randomize between the 2 best dose combinations selected from phase 1. Primary objective: identify the most efficacious dose combination that also satisfies safety requirements. Primary tolerance criterion is the occurrence of dose limiting toxicities (DLT) within 45 days; primary efficacy criterion is >50% reduction in spleen length within 6 months. Secondary objectives include molecular response, reduction of BM fibrosis, quality of life and symptoms evolution, event-free and overall survival. The planned total enrollment is 42 pts. Key inclusion criteria are: diagnosis of MF (WHO criteria), intermediate or HR (IPSS), need of active therapy, presence of a driver mutation. Key exclusion criteria: prior treatment (or contra-indication) with Rux or IFNa, eligibility for stem cell transplantation, inadequate liver, cardiac or renal function, autoimmune disease, history of depression. Enrolment in 5 cohorts was completed in June 2018, and the last cohort for phase 1 will be opened in August. This abstract reports the current available data for the 5 cohorts who have completed the primary endpoint, but the presentation will provide the detailed analysis of primary and secondary endpoints of phase 1, which will be available in October 2018. Results Among the 15 pts currently enrolled in phase 1, 6 were females, mean age was 60.9 years (range: 38-72), 8 had primary MF, 5 post ET and 2 post PV MF. Median spleen size was 6 cm (range 0 - 18) by palpation and 18 cm (range 10-25) by imaging. Mean (range) blood counts were: hemoglobin 12 g/dL (8.5 - 13.8), WBC 18.3 G/L (8 - 35.5), platelets 457 G/L (157 - 906) and 6 pts had circulating blasts. 12 pts had JAK2V617F and 2 had CALR mutations; karytotype was normal in 9 pts, abnormal in 5 (very HR in 3). In 10 pts analyzed by NGS so far, 8 had additional mutations (1 in 5 pts, 3 in 1, and 4 in 2) in TET2 (n= 5), ASXL1 (4), DNMT3A (2), TP53 (2), SF3B1 (1) and SRSF2 (1) genes. Safety: No DLT was observed in the 5 cohorts (primary safety criterion), the highest tested dose combination being Rux 15 mg BID + IFNa 135 mcg/week. The last cohort will test Rux 20 mg BID + IFNa 135 mcg/week. 4 serious adverse events have been reported: 1 AML transformation (very HR cytogenetics, 3% circulating blasts at baseline), 1 thrombotic event, 1 squamous cell carcinoma and 1 aggravation of Raynaud's phenomenon. Efficacy: preliminary data show a clear decrease in spleen size at 6 months (median 0 cm by palpation, range 0-9; 12.1 cm by imaging, range 10-21) and improvement in blood counts (mean, range): hemoglobin 10.5 g/dL (9.7 - 12.5), WBC 8.6 G/L (5.4 - 11.1), platelets 267 G/L (80 - 486). According to IWG criteria, all the 10 pts evaluable at time of abstract preparation responded (3 partial response, 7 hematological improvement). JAK2V617F allele burden decreased from a mean of 75% (range 43- 96) at baseline to 46% (range 24 - 84) at 6 months. Encouraging results were also found in a patient with 5 mutations (figure1) with a clear decrease in JAK2V617F, ASXL1, DNMT3A and EZH2 mutations after 12 months of treatment. Conclusion RUXOPEG is the first study to formally assess the safety and efficacy of Rux + IFNa combination in MF patients never exposed to either drugs before. The first 5 dose combinations tested showed no DLT, confirming that this combination was generally well tolerated. Preliminary efficacy results are encouraging, including in patients who received very low doses of both drugs. Full results of the 6 cohorts tested in phase 1 and doses selected for phase 2 will be presented. Disclosures Kiladjian: Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; AOP Orphan: Membership on an entity's Board of Directors or advisory committees, Research Funding. Giraudier:Novartis: Research Funding. Cassinat:Novartis: Research Funding; AOP Orphan: Research Funding.

Animal evidence that platelet production occurs in the lungs is growing. We have investigated whether there is evidence to support pulmonary platelet production from studies using human conditions. We documented the presence of megakaryocytes (MKs) in the human pulmonary circulation and analyzed the role of the vascular microenvironment on MK function. Our results suggest that the endothelial microenvironment favors platelet formation and that von Willebrand factor combined with appropriate physical forces in flowing blood are determinant for platelet release. We also demonstrate that MKs have the potential to change ploidy as they circulate. These findings demonstrate a new pathophysiological environment affecting platelet production and provide new targets for therapeutic intervention.

e15562Background: After radiofrequency ablation (RFA), cancer cells death releasing tumor antigens and expression of danger signals activate a specific T-cell response however inadequate to avoid r...

102 Background: Radiofrequency ablation (RFA) efficiency of liver tumors is compromised by high rates of relapse. Death of cancer cells by hyperthermia induced tumor antigen releasing, expression of danger signals that activate a specific T-cell response. This effect is ineffective to avoid recurrence. We propose to combine RFA with priming of a strong immune antitumor response as curative treatment of an aggressive colorectal cancer (CRC) in immunocompetent mouse. Methods: RFA was used to treat a CT26- luc tumor as primary lesion. In two distinct clinical situations, macroscopic or microscopic distant tumors were established as secondary lesions. The immune response was modulated by the injection, in the treated area, of a thermo-reversible hydrogel loaded by GM-CSF and BCG, targeting recruitment and maturation of dendritic cells. In mice with far large lesions, this strategy was combined with PD1checkpoint inhibition. The efficiency was assessed on survival, evolution of distant lesions, characterization of tumoral lymphocyte infiltration TNF-α and IFN-y expression in peripheral T lymphocytes. Results: The in situ immunogel injection after RFA resulted in prolonged survival of mice. Regression of distant lesions was related to the induction of a strong systemic antitumor immune response and a great improvement of tumor infiltration by CD3+ T lymphocytes. In adjuvant situation, the use of immunogel induced a complete cure of microscopic secondary lesions without another treatment. Immune escape of large secondary lesions was reversed by association of the RFA-immunogel vaccination with a systemic immune checkpoint inhibition, separately ineffective. Conclusions: Validation of this strategy, combining RFA of macroscopic lesions and activation of a strong immune response controlling the residual disease, could result in the design of a clinical assay including this approach within a standard treatment of colorectal liver metastases. The synergy between in situ immunomodulation as priming process and checkpoint blockade, ineffective alone in metastatic microsatellite stable CRC or after single RFA, allows reconsidering the use of checkpoint inhibitors in CRC.

The TAL-1/SCL and LYL-1 genes encode two closely related basic helix-loop-helix transcription factors involved in child T-acute lymphoblastic leukemia through chromosomal rearrangements and transcriptional deregulation. During ontogeny, Tal-1/SCL is required for hematopoietic cell generation, both in the yolk sac, where erythro-myeloid cells are first produced, then in the intra-embryonic compartment, where hematopoietic stem cells independently arise. We describe here the expression pattern of lyl-1 in mouse embryos from 7 to 14 days post coitus using in situ hybridization, as well as beta-Galactosidase (beta-Gal) expression in lyl-1-lacZ knock-in embryos, which express a C-terminally truncated Lyl-1 protein fused to the beta-Galactosidase (Lyl-1Delta/beta-Gal). In addition, we compare lyl-1 expression pattern with that of tal-1/scl. Similar to Tal-1/SCL, Lyl-1 mRNA expression occurs in the developing cardiovascular and hematopoietic systems. However, contrary to tal-1/scl, lyl-1 is not expressed in the developing nervous system. In lyl-1-lacZ knock-in heterozygous and homozygous embryos, beta-Gal expression completely correlates with Lyl-1 mRNA expression in the intra-embryonic compartment and is present: (1) in the developing hematopoietic system, precisely where hematopoietic stem cells emerge, and thereafter in the fetal liver; (2) in the developing vascular system; and (3) in the endocardium. In contrast, whereas Lyl-1 mRNA is expressed in yolk sac-derived endothelial and hematopoietic cells, Lyl-1Delta/beta-Gal is either absent or poorly expressed in these cell types, thus differing from Tal-1/SCL, which is highly expressed there at both mRNA and protein levels.

Hematopoietic stem cells (HSCs) arise, self-renew, or give rise to all hematopoietic lineages through the effects of transcription factors activated by signaling cascades. Lyl-1 encodes a transcription factor containing a basic helix-hoop-helix (bHLH) motif closely related to scl/tal, which controls numerous decisions in embryonic and adult hematopoiesis. We report here that Lyl-1 null mice are viable and display normal blood cell counts, except for a reduced number of B cells resulting from a partial block after the pro-B stage. Nevertheless, the deletion of Lyl-1 results in a diminution in the frequency of immature progenitors (Lin–, CD34–, sca-1+, c-kit+ [LSK], and LSK-side population [LSK-SP]) and in S12 colony-forming unit (CFU-S12) and long-term culture-initiating cell (LTC-IC) content in embryonic day 14 fetal liver (E14 FL) and adult bone marrow (BM). More important, Lyl-1–/– E14 FL cells and BM are severely impaired in their competitive reconstituting abilities, especially with respect to B and T lineage reconstitution. Thus, ablation of Lyl-1 quantitatively and functionally affects HSCs, a cell population that transcribes Lyl-1 more actively than their differentiated progenies. Our results demonstrate for the first time that Lyl-1 functions are important for HSC properties and B-cell differentiation and that they are largely distinct from scl functions.

A defect in cell trafficking and chemotaxis plays an important role in the immune deficiency observed in Wiskott-Aldrich syndrome (WAS). In this report, we show that marrow cells from WAS protein (WASP)–deficient mice also have a defect in chemotaxis. Serial transplantation and competitive reconstitution experiments demonstrated that marrow cells, including hematopoietic progenitors and stem cells (HSCs), have decreased homing capacities that were associated with a defect in adhesion to collagen. During development, HSCs migrate from the liver to the marrow and the spleen, prompting us to ask if a defect in HSC homing during development may explain the skewed X-chromosome inactivation in WAS carriers. Preliminary evidence has shown that, in contrast to marrow progenitor cells, fetal liver progenitor cells from heterozygous females had a random X-chromosome inactivation. When fetal liver cells from WASP-carrier females were injected into irradiated recipients, a nonrandom inactivation of the X-chromosome was found at the level of hematopoietic progenitors and HSCs responsible for the short- and long-term hematopoietic reconstitution. Therefore, the mechanism of the skewed X-chromosomal inactivation observed in WAS carriers may be related to a migration defect of WASP-deficient HSCs.

The purpose of this work was to identify the principal human cytochrome P450s (CYPs) involved in the metabolism of the retinoic acid (RA) isomers, 9-cis- and 13-cis-RA, by using a combination of techniques including human liver microsomes (correlation of activity and inhibition), and lymphoblast microsomes expressing a single CYP. Concerning the 9-cis-RA, 4-OH- and 4-oxo-9-cis-RA were formed with human liver microsomes, and their formation correlated with activities linked to CYPs 3A4/5, 2B6, 2C8, 2A6, and 2C9. The use of lymphoblast microsomes expressing a single human CYP identified CYPs 2C9>2C8>3A7 as the most active in the formation of 4-OH-9-cis-RA. With regard to 13-cis-RA, specific P450 activities linked to CYPs 2B6, 2C8, 3A4/5, and 2A6 were correlated with the formation of 4-OH- and 4-oxo-13-cis-RA. Microsomes expressing a single CYP identified CYPs 3A7, 2C8, 4A11, 1B1, 2B6, 2C9, 2C19, 3A4 (decreasing activity) in the formation of 4-OH-13-cis-RA. The use of CYP-specific inhibitors in human liver microsomes disclosed that the formation of the 4-OH-9-cis-RA was best inhibited by sulfaphenazole (72%) and quercetin (66%), whereas ketoconazole and troleandomycin inhibited its formation by 55 and 38%, respectively; the formation of 4-OH-13-cis-RA was best inhibited by troleandomycin (54%) and ketoconazole (46%), whereas quercetin was a weak inhibitor (14%). In conclusion, adult human CYPs 2C9, 2C8, 3A4 have been identified as active in the 9-cis-RA metabolism, whereas CYPs 3A4 and 2C8 were active in 13-cis-RA metabolism. The fetal form CYP3A7 was also identified as very active in either 9-cis- or 13-cis-RA metabolism. The role of these human CYPs in the biological response or resistance to RA isomers remains to be determined.

e14540 Background: Results of radiofrequency ablation (RFA), increasingly used to treat liver tumors, are compromised by local and systemic relapse. Hyperthermia related cancer cells death, release of tumor antigens and expression of danger signals activate a tumor-specific T-cells response. This effect remains ineffective to avoid recurrence. Therefore we propose to combine RFA with an activation of a solid immune antitumor response as curative treatment of a colorectal (CRC) metastatic disease in immunocompetent mouse. Methods: RFA was used to treat a CT26- luc tumor. In two distinct clinical situations, distant macroscopic or microscopic tumors were established as metastases before or at the time of RFA. Immune response was modulated by an injection in situof a thermo-reversible hydrogel loaded by GM-CSF and BCG, targeting dendritic cells. In the group of mice with large far lesions this strategy was combined with immune checkpoint inhibition. The efficiency was assessed on survival, evolution of distant lesions, characterization of lymphocyte infiltration in tumors and systemic immunity through specific TNF- α and IFN-y expression in spleen and draining lymph nodes. Results: The in situ immunogel injection after RFA resulted in a prolonged survival of mice. Regression of distant lesions was related to a strong systemic antitumor immune response and a great improvement of tumor infiltration by specific cytotoxic lymphocytes. In adjuvant situation, the use of immunogel induced a complete cure of microscopic secondary lesions without any treatment. Immune escape of large secondary lesions was reversed by association of RFA-immunogel vaccination with a systemic check point blockade, separately ineffective. Conclusions: Validation of this strategy, combining RFA of liver metastases and activation of a strong immune response controlling the residual disease, could result in a clinical assay including this approach within the standard treatment of CRC. Furthermorethe powerful synergy between RFA-in situ immunomodulation as a starter treatment and checkpoint blockade ineffective alone in CRC or after single RFA, allows reconsidering the use of immune checkpoint inhibitors in metastatic microsatellite stable CRC.

We report the first identified mutation in the gene encoding human cytochrome c (CYCS). Glycine 41, invariant throughout eukaryotes, is substituted by serine in a family with autosomal dominant thrombocytopenia caused by dysregulated platelet formation. The mutation yields a cytochrome c variant with enhanced apoptotic activity in vitro. Notably, the family has no other phenotypic indication of abnormal apoptosis, implying that cytochrome c activity is not a critical regulator of most physiological apoptosis.

Background Both the human immunodeficiency virus (HIV) and hepatitis C virus (HCV), either alone or as coinfections, persist in their hosts by destroying and/or escaping immune defenses, with high morbidity as consequence. In some cases, however, a balance between infection and immunity is reached, leading to prolonged asymptomatic periods. We report a case of such an indolent co-infection, which could be explained by the development of a peculiar subset of Natural Killer (NK) cells. Results Persistently high peripheral levels of CD56+ NK cells were observed in a peculiar hemophiliac HIV/HCV co-infected patient with low CD4 counts, almost undetectable HIV viral load and no opportunistic infections. Thorough analysis of NK-subsets allowed to identify a marked increase in the CD56bright/dim cell ratio and low numbers of CD16+/CD56- cells. These cells have high levels of natural cytotoxicity receptors but low NCR2 and CD69, and lack both CD57 and CD25 expression. The degranulation potential of NK-cells which correlates with target cytolysis was atypically mainly performed by CD56bright NK-cells, whereas no production of interferon γ (IFN-γ) was observed following NK activation by K562 cells. Conclusions These data suggest that the expansion and lytic capacity of the CD56bright NK subset may be involved in the protection of this « rare » HIV/HCV co-infected hemophiliac A patient from opportunistic infections and virus-related cancers despite very low CD4+ cell counts.

Background Cancer gene therapy by retroviral vectors is mainly limited by the level of transduction. Retroviral gene transfer requires target cell division. Cell synchronization, obtained by drugs inducing a reversible inhibition of DNA synthesis, could therefore be proposed to precondition target cells to retroviral gene transfer. We tested whether drug-mediated cell synchronization could enhance the transfer efficiency of a retroviral-mediated gene encoding herpes simplex virus thymidine kinase (HSV-tk) in two colon cancer cell lines, DHDK12 and HT29. Methods Synchronization was induced by methotrexate (MTX), aracytin (ara-C) or aphidicolin. Gene transfer efficiency was assessed by the level of HSV-TK expression. Transduced cells were driven by ganciclovir (GCV) towards apoptosis that was assessed using annexin V labeling by quantitative flow cytometry. Results DHDK12 and HT29 cells were synchronized in S phase with MTX but not ara-C or aphidicolin. In synchronized DHDK12 and HT29 cells, the HSV-TK transduction rates were 2 and 1.5-fold higher than those obtained in control cells, respectively. Furthermore, the rate of apoptosis was increased two-fold in MTX-treated DHDK12 cells after treatment with GCV. Conclusions Our findings indicate that MTX-mediated synchronization of target cells allowed a significant improvement of retroviral HSV-tk gene transfer, resulting in an increased cell apoptosis in response to GCV. Pharmacological control of cell cycle may thus be a useful strategy to optimize the efficiency of retroviral-mediated cancer gene therapy.

Transforming growth factor-β1 (TGF-β1) is a pleiotropic cytokine with major in vitro effects on hematopoietic stem cells (HSCs) and lymphocyte development. Little is known about hematopoiesis from mice with constitutive TGF-β1 inactivation largely because of important embryonic lethality and development of a lethal inflammatory disorder in TGF-β1−/− pups, making these studies difficult. Here, we show that no sign of the inflammatory disorder was detectable in 8- to 10-day-old TGF-β1−/− neonates as judged by both the number of T-activated and T-regulator cells in secondary lymphoid organs and the level of inflammatory cytokines in sera. After T-cell depletion, the inflammatory disease was not transplantable in recipient mice. Bone marrow cells from 8- to 10-day-old TGF-β1−/− neonates showed strikingly impaired short- and long-term reconstitutive activity associated with a parallel decreased in vivo homing capacity of lineage negative (Lin−) cells. In addition an in vitro–reduced survival of immature progenitors (Lin− Kit+ Sca+) was observed. Similar defects were found in liver cells from TGF-β1−/− embryos on day 14 after vaginal plug. These data indicate that TGF-β1 is a critical regulator for in vivo homeostasis of the HSCs, especially for their homing potential.

Platelets originate from megakaryocytes (MKs) by cytoplasmic elongation into proplatelets. Direct platelet release is not seen in bone marrow hematopoietic islands. It was suggested that proplatelet fragmentation into platelets can occur intravascularly, yet evidence of its dependence on hydrodynamic forces is missing. Therefore, we investigated whether platelet production from MKs could be up-regulated by circulatory forces. Human mature MKs were perfused at a high shear rate on von Willebrand factor. Cells were observed in real time by videomicroscopy, and by confocal and electron microscopy after fixation. Dramatic cellular modifications followed exposure to high shear rates: 30% to 45% adherent MKs were converted into proplatelets and released platelets within 20 minutes, contrary to static conditions that required several hours, often without platelet release. Tubulin was present in elongated proplatelets and platelets, thus ruling out membrane tethers. By using inhibitors, we demonstrated the fundamental roles of microtubule assembly and MK receptor GPIb. Secretory granules were present along the proplatelet shafts and in shed platelets, as shown by P-selectin labeling. Platelets generated in vitro were functional since they responded to thrombin by P-selectin expression and cytoskeletal reorganization. In conclusion, MK exposure to high shear rates promotes platelet production via GPIb, depending on microtubule assembly and elongation.

Tumor growth promotes the expansion of CD4+CD25+ regulatory T (T reg) cells that counteract T cell–mediated immune responses. An inverse correlation between natural killer (NK) cell activation and T reg cell expansion in tumor-bearing patients, shown here, prompted us to address the role of T reg cells in controlling innate antitumor immunity. Our experiments indicate that human T reg cells expressed membrane-bound transforming growth factor (TGF)–β, which directly inhibited NK cell effector functions and down-regulated NKG2D receptors on the NK cell surface. Adoptive transfer of wild-type T reg cells but not TGF-β−/− T reg cells into nude mice suppressed NK cell–mediated cytotoxicity, reduced NKG2D receptor expression, and accelerated the growth of tumors that are normally controlled by NK cells. Conversely, the depletion of mouse T reg cells exacerbated NK cell proliferation and cytotoxicity in vivo. Human NK cell–mediated tumor recognition could also be restored by depletion of T reg cells from tumor-infiltrating lymphocytes. These findings support a role for T reg cells in blunting the NK cell arm of the innate immune system.

Objective Osteoprotegerin (OPG) is a soluble member of the tumor necrosis factor receptor superfamily critically involved in the regulation of bone resorption. Within the bone microenvironment, OPG is abundantly produced by osteoblast/stromal cells, and its expression is regulated by transforming growth factor-β 1 (TGF-β 1 ). However, OPG expression and regulation in primary hematopoietic cells have not been fully investigated. Materials and methods Opg mRNA was studied in murine hematopoietic cells by semiquantitative reverse transcriptase-polymerase chain reaction. The OPG protein was identified by immunofluorescence labeling and secretion was assessed by enzyme-linked immunosorbent assay. Results Opg transcripts were detected in platelets, megakaryocytes (MK), monocytes, and B lymphocytes, but not in erythroblasts, neutrophils, and T lymphocytes. Mature MK and proplatelets exhibited strong immunostaining for OPG outside the storage α-granules, and secretion was detected in the conditioned medium. To analyze whether opg transcription in MK was influenced by TGF-β 1 , the opg/GpIIb mRNA ratio was compared in cultured MK derived from TGF-β 1 null mutants and wild-type littermates without or after the addition of bioactive TGF-β 1 . No difference was seen, indicating that opg expression in MK was not modulated by TGF-β 1 . However, mRNA levels were increased when thrombopoietin was present in the culture medium, suggesting that MK maturation was correlated with enhanced opg expression. Conclusions With these results we document for the first time that murine MK and platelets express OPG. This suggests a novel role for MK in bone homeostasis, in addition to its role in vascular homeostasis.

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation in the peripheral blood, secondary lymphoid tissues and bone marrow of functionally defective clonal B lymphocytes with prolonged survival in vivo. Despite therapeutic achievements have been accomplished in the management of this disease, however CLL remains incurable partially because of the resistance to apoptosis of CLL B-cells and of the altered immune system of CLL patients. CLL is hetereogenous, but its evolution is mostly slow, probably linked to some level of immune control of the leukemic cells. Indeed, clinical observations have been reported of spontaneous remissions associated with the intense immunological activity following a viral infection. Clinical responses have also been observed after treatment by immunomodulating cytokines and long-term survival is described, without disease, after allogeneic stem cells transplantation. All these data suggest that immunotherapy could be useful in the treatment of CLL, possibly as an adjuvant therapy after classical immunochemotherapy schedules. Toll like receptors (TLR) are proteins of the innate immune system belonging to the family of Pattern Recognition receptors (PRRs). Recognition of their ligands by the TLR present on neutrophils, macrophages, dendritic cells and B-cells are important in the initiation of adaptive immune responses. TLR-7 and 9 are expressed by CLL B-cells (Grandjenette, Hematologica, 2007). Previous studies have shown that stimulation of TLR-9 by CpG ODN (oligodinucleotides) induces an activation of CLL B-cells while triggering TLR-7 increases in vitro apoptosis of these cells. Here we show that these effects of TLR-9 and TLR-7 stimulation differ depending on the clinical form (stable or aggressive) of the disease and on the mutational status of CLL B-cells. In vitro stimulation with three doses of Imiquimod R837 or ODN CpG M362 was carried out for three days on cells from 40 patients (22 stable, 18 aggressive, mutational status known for 35, 18 IgVH mutated, 17 unmutated). Flow cytometry was used to measure apoptosis, proliferation after carboxyfluorescein succinimidyl ester (CFSE) labeling and modulation of surface differentiation antigens. Signaling pathways after incubation were further studied by antibody arrays and western blot. Spontaneous apoptosis occurring in vitro was demonstrated to involve the mitochondrial pathway. CLL B-cells were also confirmed to proliferate strongly and produce large amounts of IL-6 and IL-8 upon triggering by phorbol myrsistate (positive control), this compound almost completely aborting in vitro apoptosis. Cells from patients with a stable disease were significantly more prone to rapid apoptosis after TLR-7 triggering with Imiquimod, compared to cells from patients with an aggressive disease which displayed only spontaneous apoptosis. This rapid apoptosis in stable patients involved the p38 MAP-Kinase pathway. It was concomitant to an important production of IL-8 and IL-6. Conversely, CpG ODN conferred a protection against apoptosis to CLL B-cells from patients with an aggressive disease. This was accompanied by the activation of numerous anti-apoptotic proteins in the cells. CpG ODN also significantly increased CLL B-cells proliferation, concomitantly to the phosphorylation of Erk and Akt proteins. ODN finally increased the expression of CD20 and CD19 on the cells’surface. The same differences in reactivity were observed comparing mutated (∼stable) and unmutated (∼aggressive) cases. These data indicate that CLL B-cells from patients with a stable or aggressive (mutated/unmutated) disease answer differently when triggered through their surface TLRs. This might have an incidence on the behavior of these cells in vivo, in answer to stimulations by microbial compounds naturally binding these structures. These properties could also be used to develop adjuvant immunotherapies by loading CpG ODN-activated CLL B-cells with autologous apoptotic fragments issued from stimulation of part of the same cells with Imiquimod. Disclosures: No relevant conflicts of interest to declare.

Abstract 3411 It is well established that megakaryocytes (MK), in order to achieve terminal maturation in the bone marrow, migrate from the osteoblastic niche to the vascular niche, close to medullary sinusoids. It is also admitted that, in order to release platelets, MK extend cytoplasmic extensions in the lumen of the sinusoid followed by platelet release. Therefore, we have investigated the role of endothelial cells combined with shear stress on MK late differentiation steps and platelet production. To do so, human MK were grown from umbilical cord blood or bone marrow CD34+ cells, in the presence of Stem Cell Factor (SCF) and thrombopoietin for 10 days. Then they were co-cultured with human endothelial cells (HUVEC) for 3 days and analysed by video-microscopy, flow cytometry, immunofluorescence and electron microscopy. In these conditions, most MK (>50%) extended numerous and prominent proplatelets: immunofluorescence showed virtually complete unwinding of their cytoplasm, which extended in the form of long proplatelets; this rarely occurred in control MK. Electron microscopy showed that these MK were formed by a coarse chromatin nucleus surrounded by a thin cytoplasmic ring and surrounded by platelet-size territories displaying alpha-granules, mitochondria and canalicular system which coincided to the sections of the proplatelet swellings and to the unwinding of the demarcation membrane system. Another type of experiment of co-culture MK/endothelial cells was conducted in transwells and led to similar results, ie, increase of MK cytoplasmic maturation and proplatelet formation indicating that cell/cell contact was not necessary for up-regulating proplatelet production, rather that (a) soluble product(s) was(ere) secreted from endothelial cells. Flow cytometry, at this step, failed to demonstrate significant changes in the platelet production rate. We then submitted MK co-cultured with endothelial cells to shear stress and examined them by flow cytometry and video microscopy. We could then demonstrate that platelet release was strongly increased (× 3.8 ± 0.9, n=3) when MK had been in contact with endothelial cells compared to control MK. The released platelet-size particles expressed CD41 and, when stimulated by thrombin, were also able to express CD62P. Video-microscopy confirmed that proplatelet and platelet shedding occurred after exposition of mature MK to shear stress. When examined by video-microscopy, live MK whose DNA had been stained with the fluorescent dye Hoechst 33342 showed that nuclear lobes separated under high shear stress: indeed they became located at opposite poles of the cell, while the cytoplasmic volume extended and elongated, becoming organized in proplatelets which exhibited a succession of platelet size subunits; eventually cytoplasmic scission occurred in parallel with MK nuclear lobe segregation in distinct cell fractions, each carrying proplatelets; proplatelets subsequently fractionated and were then released from the cell core containing the fluorescent nuclear lobe. Thus shear stress leads both to cytoplasm and nucleus fragmentation. This is a dynamic explanation to the fact that entire MK nuclei are rarely found in the human bone marrow. This observation also gives sense to the unique phenomenon of MK polyploidy. In conclusion, this study indicates that endothelial microenvironment combined with circulatory shear forces are determinant up-regulating factors which increase platelet production. It also shows that shear stress is able to induce nuclear as well as cytoplasmic MK fragmentation, leading to a new anatomical concept of circulating platelet shedding MK subunits. Disclosures: No relevant conflicts of interest to declare.

TGF-β1 is a cytokine with pleiotropic effects. It has been considered that TGF-β1plays a major role on hematopoietic stem cells (HSC) based on in vitro experiment. Achieving in vivo experiments proved to be difficult because constitutive TGF-β1 knock-out (KO) in mice leads to lethality during the first 4 weeks of life from a wasting syndrome related to tissue infiltration by activated T cells and macrophages. For this reason, hematopoiesis of TGF-β1−/− mice has not been studied in details. In contrast the role of TGF-β1 has been recently extensively studied in conditional TGF-β type I receptor (TβRI) KO mice. No clear effect was observed on HSC functions, suggesting that TGF-β1 was not a key physiological regulator of hematopoiesis in the adult. However, these experiments have some limitations. They do not exclude a putative role for TGF-β1 during fetal hematopoiesis and they do not specifically address the role of TGF-β1 on hematopoiesis because KO of TGF-β receptor leads to signaling arrest for all TGF-βs. In addition, other receptors may be involved in TGF-β1 signaling. For these reasons, we have investigated the hematopoiesis of constitutive TGF-β1 KO mice with a mixed Sv129 × CF-1 genetic background allowing the birth of a high proportion of homozygotes. In 2 week-old neonate mice, we have shown a decrease of bone marrow (BM) and spleen progenitors and a decrease of immature progenitors colony forming unit of the spleen (CFU-s). Moreover this was associated with a loss in reconstitutive activity of TGF-β1−/− HSC from BM. However, although asymptomatic, these mice had an excess of activated lymphocytes and an augmentation of Sca-1 antigen on hematopoietic cells suggesting an excess of γ-interferon release. Thus we studied hematopoiesis of 7 to 10 days-old neonate mice, before phenotypic modification and inflammatory cytokine release. Similar results were observed with a decrease in the number of progenitors and in the proliferation of TGF-β1−/− BM cells along with an increased differentiation but without an augmentation in apoptosis. Moreoever, a loss of long term reconstitutive capacity of BM Lineage negative (Lin−) TGF-β1−/− cells along with a diminution of homing of TGF-β1−/− progenitors was found. These results demonstrate that TGF-β1 may play a major role on the HSC/Progenitor compartment in vivo and that this defect does not seem to be linked to the immune disease. To completely overpass the risk of the inflammatory syndrome, we analyzed hematopoiesis of fetal liver (FL) of TGF-β1−/− mice and still found a decrease in progenitors, a profound defect in the proliferative capacities, in long term reconstitutive activity and homing potential of primitive FL hematopoietic cells. Our results demonstrate that TGF-β1 plays an important role during hematopoietic embryonic development. Altogether these findings suggest that TGF-β1 is a potent positive regulator for the in vivo homeostasis of the HSC compartment.