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CXCR4 est un récepteur de chimiokine qui joue un rôle central dans la migration cellulaire mais également dans d’autres mécanismes essentiels, tels que le développement du système immunitaire. De concert avec son ligand naturel, la chimiokine CXCL12, cet axe de signalisation joue un rôle important dans la biologie des lymphocytes B, des stades précoces de différenciation dans la moelle osseuse à leur activation et différenciation en cellules sécrétrices d’anticorps, aussi appelées plasmocytes. Des mutations gain de fonction de CXCR4 sont retrouvées dans une immunodéficience rare, le Syndrome WHIM. Ces mutations affectent le mécanisme de désensibilisation du récepteur et entraînent un gain de fonction en réponse à CXCL12. Cette revue résume le rôle de CXCR4 dans la réponse immune humorale et, à travers l’étude du Syndrome WHIM, souligne le rôle régulateur essentiel de la désensibilisation de CXCR4 dans ces processus. Des travaux récents rapportent en effet qu’une signalisation correcte de CXCR4 est essentielle pour limiter la réponse immune dite « extra-folliculaire » et pour permettre une protection au long terme assurée par les anticorps.

Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is an ultra-rare combined primary immunodeficiency disease caused by heterozygous gain-of-function mutations in the chemokine receptor CXCR4. WHIM patients typically present with recurrent acute infections associated with myelokathexis (severe neutropenia due to bone marrow retention of mature neutrophils). Severe lymphopenia is also common, but the only associated chronic opportunistic pathogen is human papillomavirus and mechanisms are not clearly defined. In this study, we show that WHIM mutations cause more severe CD8 than CD4 lymphopenia in WHIM patients and WHIM model mice. Mechanistic studies in mice revealed selective and WHIM allele dose-dependent accumulation of mature CD8 single-positive cells in thymus in a cell-intrinsic manner due to prolonged intrathymic residence, associated with increased CD8 single-positive thymocyte chemotactic responses in vitro toward the CXCR4 ligand CXCL12. In addition, mature WHIM CD8+ T cells preferentially home to and are retained in the bone marrow in mice in a cell-intrinsic manner. Administration of the specific CXCR4 antagonist AMD3100 (plerixafor) in mice rapidly and transiently corrected T cell lymphopenia and the CD4/CD8 ratio. After lymphocytic choriomeningitis virus infection, we found no difference in memory CD8+ T cell differentiation or viral load between wild-type and WHIM model mice. Thus, lymphopenia in WHIM syndrome may involve severe CXCR4-dependent CD8+ T cell deficiency resulting in part from sequestration in the primary lymphoid organs, thymus, and bone marrow.

The magnitude and quality of the germinal center (GC) response decline with age, resulting in poor vaccine-induced immunity in older individuals. A functional GC requires the co-ordination of multiple cell types across time and space, in particular across its two functionally distinct compartments: the light and dark zones. In aged mice, there is CXCR4-mediated mislocalization of T follicular helper (TFH) cells to the dark zone and a compressed network of follicular dendritic cells (FDCs) in the light zone. Here we show that TFH cell localization is critical for the quality of the antibody response and for the expansion of the FDC network upon immunization. The smaller GC and compressed FDC network in aged mice were corrected by provision of TFH cells that colocalize with FDCs using CXCR5. This demonstrates that the age-dependent defects in the GC response are reversible and shows that TFH cells support stromal cell responses to vaccines.

Despite the essential role of plasma cells in health and disease, the cellular mechanisms controlling their survival and secretory capacity are still poorly understood. Here, we identified the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) Sec22b as a unique and critical regulator of plasma cell maintenance and function. In the absence of Sec22b, plasma cells were hardly detectable and serum antibody titers were dramatically reduced. Accordingly, Sec22b -deficient mice fail to mount a protective immune response. At the mechanistic level, we demonstrated that Sec22b contributes to efficient antibody secretion and is a central regulator of plasma cell maintenance through the regulation of their transcriptional identity and of the morphology of the endoplasmic reticulum and mitochondria. Altogether, our results unveil an essential and nonredundant role for Sec22b as a regulator of plasma cell fitness and of the humoral immune response.

The extrafollicular immune response is essential to generate a rapid but transient wave of protective antibodies during infection. Despite its importance, the molecular mechanisms controlling this first response are poorly understood. Here, we demonstrate that enhanced Cxcr4 signaling caused by defective receptor desensitization leads to exacerbated extrafollicular B-cell response. Using a mouse model bearing a gain-of-function mutation of Cxcr4 described in 2 human hematologic disorders, warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome and Waldenström macroglobulinemia, we demonstrated that mutant B cells exhibited enhanced mechanistic target of rapamycin signaling, cycled more, and differentiated more potently into plasma cells than wild-type B cells after Toll-like receptor (TLR) stimulation. Moreover, Cxcr4 gain of function promoted enhanced homing and persistence of immature plasma cells in the bone marrow, a phenomenon recapitulated in WHIM syndrome patient samples. This translated in increased and more sustained production of antibodies after T-independent immunization in Cxcr4 mutant mice. Thus, our results establish that fine-tuning of Cxcr4 signaling is essential to limit the strength and length of the extrafollicular immune response.

Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome immunodeficiency is caused by autosomal dominant gain-of-function CXCR4 mutations that promote severe panleukopenia caused by bone marrow retention of mature leukocytes. Consequently, WHIM patients develop recurrent bacterial infections; however, sepsis is uncommon. To study this clinical dichotomy, we challenged WHIM model mice with LPS. The LD50 was similar in WHIM and wild-type (WT) mice, and LPS induced acute lymphopenia in WT mice that was Cxcr4 independent. In contrast, in WHIM mice, LPS did not affect circulating T cell levels, but the B cell levels anomalously increased because of selective, cell-intrinsic, and Cxcr4 WHIM allele–dependent emergence of Cxcr4high late pre-B cells, a pattern that was phenocopied by Escherichia coli infection. In both WT and WHIM mice, the CXCR4 antagonist AMD3100 rapidly increased circulating lymphocyte levels that then rapidly contracted after subsequent LPS treatment. Thus, LPS-induced lymphopenia is CXCR4 independent, and a WHIM mutation does not increase clinical LPS sensitivity. Anomalous WT Cxcr4-independent, but Cxcr4 WHIM-dependent, promobilizing effects of LPS on late pre-B cell mobilization reveal a distinct signaling pathway for the variant receptor.

Dendritic cells (DCs) encompass several cell subsets that collaborate to initiate and regulate immune responses. Proper DC localization determines their function and requires the tightly controlled action of chemokine receptors. All DC subsets express CXCR4, but the genuine contribution of this receptor to their biology has been overlooked. We addressed this question using natural CXCR4 mutants resistant to CXCL12-induced desensitization and harboring a gain of function that cause the warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome (WS), a rare immunodeficiency associated with high susceptibility to the pathogenesis of human papillomavirus (HPV). We report a reduction in the number of circulating plasmacytoid DCs (pDCs) in WHIM patients, whereas that of conventional DCs is preserved. This pattern was reproduced in an original mouse model of WS, enabling us to show that the circulating pDC defect can be corrected upon CXCR4 blockade and that pDC differentiation and function are preserved, despite CXCR4 dysfunction. We further identified proper CXCR4 signaling as a critical checkpoint for Langerhans cell and DC migration from the skin to lymph nodes, with corollary alterations of their activation state and tissue inflammation in a model of HPV-induced dysplasia. Beyond providing new hypotheses to explain the susceptibility of WHIM patients to HPV pathogenesis, this study shows that proper CXCR4 signaling establishes a migration threshold that controls DC egress from CXCL12-containing environments and highlights the critical and subset-specific contribution of CXCR4 signal termination to DC biology.

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WHIM Syndrome (WS) is a rare immunodeficiency caused by gain-of-function CXCR4 mutations. Here we report for the first time a substantial decrease in bone mineral density in 25% of WS patients and bone defects leading to osteoporosis in a WS mouse model. Reduction in bone content involved impaired CXCR4 desensitization that disrupts cell cycle progression and osteogenic specification of mouse bone marrow (BM)-residing skeletal stromal/stem cells (SSCs). This was also evidenced in BM stromal cells from WS patients. Consistent with this, chronic treatment with the CXCR4 antagonist AMD3100 normalized in vitro osteogenic fate of mutant SSCs and reversed in vivo loss in skeletal cells, thus demonstrating that proper CXCR4 desensitization is required for the osteogenic specification of BM SSCs. Our study provides novel mechanistic insights into how CXCR4 signaling regulates the osteogenic fate of BM SSCs.

Despite the essential role of plasma cells in health and disease, the cellular mechanisms controlling their survival and secretory capacity are still poorly understood. Here, we identified the SNARE Sec22b as a unique and critical regulator of plasma cell maintenance and function. In absence of Sec22b, plasma cells were barely detectable and serum antibody titres were dramatically reduced. Accordingly, Sec22b deficient mice fail to mount a protective immune response. At the mechanistic level, we demonstrated that Sec22b is indispensable for efficient antibody secretion but also for plasma cell fitness through the regulation of the morphology of the endoplasmic reticulum and mitochondria. Altogether, our results unveil a critical role for Sec22b-mediated regulation of plasma cell biology through the control of organelle dynamics.

Neutrophils are essential immune cells that defend the host against pathogenic microbial agents. Neutrophils are produced in the bone marrow and are retained there through CXCR4-CXCL12 signaling. However, patients with the Warts, Hypogammaglobulinemia, Infections, and Myelokathexis (WHIM) syndrome are prone to infections due to increased accumulation of neutrophils in the bone marrow leading to low numbers of circulating neutrophils. How neutrophils accumulate in the bone marrow in this condition is poorly understood. To better understand factors involved in neutrophil accumulation in the bone marrow, neutrophils from wildtype and WHIM mouse models were characterized in their response to CXCL12 stimulation. WHIM neutrophils were found to exert stronger traction forces, formed significantly more lamellipodia-type protrusions and migrated with increased speed and displacement upon CXCL12 stimulation as compared to wildtype cells. Migration speed of WHIM neutrophils showed a larger initial increase upon CXCL12 stimulation, which decayed over a longer time period as compared to wildtype cells. We proposed a computational model based on the chemotactic behavior of neutrophils that indicated increased CXCL12 sensitivity and prolonged CXCR4 internalization adaptation time in WHIM neutrophils as being responsible for increased accumulation in the bone marrow. These findings provide a mechanistic understanding of bone marrow neutrophil accumulation in WHIM condition and novel insights into restoring neutrophil regulation in WHIM patients.

B cell development is a stepwise process encompassing several B cell precursor stages that can be phenotypically distinguished, and that is taking place in the bone marrow in adults. Interestingly, within the bone marrow B cell precursors coexist with the most differentiated actors of this lineage, the plasma cells. In this chapter, we describe a method to recover cells from the bone marrow and a flow cytometric approach to identify each subpopulation of the B cell lineage that resides within the bone marrow compartment. This protocol focuses on membrane markers to discriminate all the B cell subpopulations in order to preserve cell integrity during experimentation and for further analyses.

Hematopoietic stem cells (HSCs) are responsible for replenishing immune cells and reside in bone marrow (BM) niches, which provide all cellular and molecular components required for their lifelong maintenance and differentiation. Although HSCs have been extensively analyzed and characterized, their ex vivo expansion, which constitutes a promising approach for therapeutic development in regenerative medicine, remains challenging. Here, we describe an original in vitro system allowing to quantify by flow cytometry the differentiation of mouse HSCs into lineage-primed multipotent hematopoietic progenitors (MPPs) in a cytokine-supplemented feeder-free medium.

Mesenchymal stromal cells (MSC) are a rare, heterogeneous and multipotent population that can be isolated from several tissues. MSC were originally discovered in the bone marrow and studied for their capacity to maintain hematopoietic cells. We will describe here methods to isolate, culture, and bank MSC from human bone marrow. Then, characterization protocols by flow cytometry, clonogenic assays and doubling time evaluation will be developed. Finally, in vitro MSC culture and differentiation into osteoblasts, adipocytes, and chondrocytes will be explained. Thus, this chapter will detail all bases to work on MSC with consensus and clear methods and protocols.

Aberrant CXCR4 activity has been implicated in lymphoma pathogenesis, disease progression, and resistance to therapies. Using a mouse model with a gain-of-function CXCR4 mutation (CXCR4C1013G) that hyperactivates CXCR4 signaling, we identified CXCR4 as a crucial activator of multiple key oncogenic pathways. CXCR4 hyperactivation resulted in an expansion of transitional B1 lymphocytes, which represent the precursors of chronic lymphocytic leukemia (CLL). Indeed, CXCR4 hyperactivation led to a significant acceleration of disease onset and a more aggressive phenotype in the murine Eµ-TCL1 CLL model. Hyperactivated CXCR4 signaling cooperated with TCL1 to cause a distinct oncogenic transcriptional program in B cells, characterized by PLK1/FOXM1-associated pathways. In accordance, Eµ-TCL1;CXCR4C1013G B cells enriched a transcriptional signature from patients with Richter’s syndrome, an aggressive transformation of CLL. Notably, MYC activation in aggressive lymphoma was associated with increased CXCR4 expression. In line with this finding, additional hyperactive CXCR4 signaling in the Eµ-Myc mouse, a model of aggressive B-cell cancer, did not impact survival. In summary, we here identify CXCR4 hyperactivation as a co-driver of an aggressive lymphoma phenotype.

Activating mutations of MYD88 (MYD88L265P being the far most frequent) are found in most cases of Waldenström macroglobulinemia (WM) as well as in various aggressive B-cell lymphoma entities with features of plasma cell (PC) differentiation, such as activated B-cell type diffuse large B-cell lymphoma (DLBCL). To understand how MYD88 activation exerts its transformation potential, we developed a new mouse model in which the MYD88L252P protein, the murine ortholog of human MYD88L265P, is continuously expressed in CD19 positive B-cells together with the Yellow Fluorescent Protein (Myd88L252P mice). In bone marrow, IgM B and plasma cells were expanded with a CD138 expression continuum from IgMhigh CD138low to IgMlow CD138high cells and the progressive loss of the B220 marker. Serum protein electrophoresis (SPE) longitudinal analysis of 40 Myd88L252P mice (16 to 56 weeks old) demonstrated that ageing was first associated with serum polyclonal hyper gammaglobulinemia (hyper Ig) and followed by a monoclonal immunoglobulin (Ig) peak related to a progressive increase in IgM serum levels. All Myd88L252P mice exhibited spleen enlargement which was directly correlated with the SPE profile and was maximal for monoclonal Ig peaks. Myd88L252P mice exhibited very early increased IgM PC differentiation. Most likely due to an early increase in the Ki67 proliferation index, IgM lymphoplasmacytic (LP) and plasma cells continuously expanded with age being first associated with hyper Ig and then with monoclonal Ig peak. This peak was consistently associated with a spleen LP-like B-cell lymphoma. Clonal expression of both membrane and secreted µ chain isoforms was demonstrated at the mRNA level by high throughput sequencing. The Myd88L252P tumor transcriptomic signature identified both proliferation and canonical NF-κB p65/RelA activation. Comparison with MYD88L265P WM showed that Myd88L252P tumors also shared the typical lymphoplasmacytic transcriptomic signature of WM bone marrow purified tumor B-cells. Altogether these results demonstrate for the first time that continuous MYD88 activation is specifically associated with clonal transformation of differentiating IgM B-cells. Since MYD88L252P targets the IgM PC differentiation continuum, it provides an interesting preclinical model for development of new therapeutic approaches to both WM and aggressive MYD88 associated DLBCLs.

Mesenchymal stromal cells (MSC) are a rare, heterogeneous and multipotent population that can be isolated from several tissues. MSC were originally discovered in the bone marrow and studied for their capacity to maintain hematopoietic cells. We will describe here methods to isolate, culture, and bank MSC from human bone marrow. Then, characterization protocols by flow cytometry, clonogenic assays and doubling time evaluation will be developed. Finally, in vitro MSC culture and differentiation into osteoblasts, adipocytes, and chondrocytes will be explained. Thus, this chapter will detail all bases to work on MSC with consensus and clear methods and protocols.

Hematopoietic stem cells (HSCs) are responsible for replenishing immune cells and reside in bone marrow (BM) niches, which provide all cellular and molecular components required for their lifelong maintenance and differentiation. Although HSCs have been extensively analyzed and characterized, their ex vivo expansion, which constitutes a promising approach for therapeutic development in regenerative medicine, remains challenging. Here, we describe an original in vitro system allowing to quantify by flow cytometry the differentiation of mouse HSCs into lineage-primed multipotent hematopoietic progenitors (MPPs) in a cytokine-supplemented feeder-free medium.

B cell development is a stepwise process encompassing several B cell precursor stages that can be phenotypically distinguished, and that is taking place in the bone marrow in adults. Interestingly, within the bone marrow B cell precursors coexist with the most differentiated actors of this lineage, the plasma cells. In this chapter, we describe a method to recover cells from the bone marrow and a flow cytometric approach to identify each subpopulation of the B cell lineage that resides within the bone marrow compartment. This protocol focuses on membrane markers to discriminate all the B cell subpopulations in order to preserve cell integrity during experimentation and for further analyses.

Simple Summary Chemokines present in the tumor microenvironment are essential for the control of tumor progression. We show here that the knock-down of Cxcr2 in PyMT animals led to an increased growth of the primary tumor and lung metastasis. The analysis of tumor content of PyMT-Cxcr2−/− animals highlighted an increased infiltration of tumor associated neutrophils (TANs), mirrored by a decreased recruitment of tumor associated macrophages (TAMs) compared to PyMT animals. Analysis of PyMT-Cxcr2−/− TANs revealed that they lost their killing ability compared to PyMT-Cxcr2+/+ TANs and that they had a more pronounced pro-tumor TAN2 profile compared to PyMT TANs. PyMT-Cxcr2−/− TANs displayed an up-regulation of the pathways involved in reactive oxygen species (ROS) production and angiogenesis and factors favoring metastasis, but reduced apoptosis. In summary, our data reveal that a lack of Cxcr2 provides TANs with pro-tumor effects. Abstract Chemokines present in the tumor microenvironment are essential for the control of tumor progression. We show here that several ligands of the chemokine receptor Cxcr2 were up-regulated in the PyMT (polyoma middle T oncogene) model of breast cancer. Interestingly, the knock-down of Cxcr2 in PyMT animals led to an increased growth of the primary tumor and lung metastasis. The analysis of tumor content of PyMT-Cxcr2−/− animals highlighted an increased infiltration of tumor associated neutrophils (TANs), mirrored by a decreased recruitment of tumor associated macrophages (TAMs) compared to PyMT animals. Analysis of PyMT-Cxcr2−/− TANs revealed that they lost their killing ability compared to PyMT-Cxcr2+/+ TANs. The transcriptomic analysis of PyMT-Cxcr2−/− TANs showed that they had a more pronounced pro-tumor TAN2 profile compared to PyMT TANs. In particular, PyMT-Cxcr2−/− TANs displayed an up-regulation of the pathways involved in reactive oxygen species (ROS) production and angiogenesis and factors favoring metastasis, but reduced apoptosis. In summary, our data reveal that a lack of Cxcr2 provides TANs with pro-tumor effects.

Mesenchymal stem/stromal cells (MSCs) are multipotent adult cells that are present in several tissues including the bone marrow (BM), in which they can differentiate in a variety of cell types such as osteoblasts, chondrocytes and adipocytes. The isolation of MSCs has been carried out by many studies that aim to control their differentiation into cartilaginous and bone cells in vitro in order to use this technology in the repair of damaged tissues. Here we describe the minimum requirements and an efficient method for isolation, expansion of mouse bone-derived multipotent mesenchymal stromal cells and their differentiation into osteoblasts, responsible for the bone matrix synthesis and mineralization.

Microbiota and chronic infections can affect not only immune status, but also the overall physiology of animals. Here, we report that chronic infections dramatically modify the phenotype ofCxcr2KO mice, impairing in particular, their reproduction ability. We show that exposure ofCxcr2KO females to multiple types of chronic infections prevents their ability to cycle, reduces the development of the mammary gland and alters the morphology of the uterus due to an impairment of ovary function. Mammary gland and ovary transplantation demonstrated that the hormonal contexture was playing a crucial role in this phenomenon. This was further evidenced by alterations to circulating levels of sex steroid and pituitary hormones. By analyzing at the molecular level the mechanisms of pituitary dysfunction, we showed that in the absence of Cxcr2, bystander infections affect leukocyte migration, adhesion, and function, as well as ion transport, synaptic function behavior, and reproduction pathways. Taken together, these data reveal that a chemokine receptor plays a direct role in pituitary function and reproduction in the context of chronic infections.

MEK1 and MEK2, the only known activators of ERK, are attractive therapeutic candidates for both cancer and autoimmune diseases. However, how MEK signaling finely regulates immune cell activation is only partially understood. To address this question, we specifically delete Mek1 in hematopoietic cells in the Mek2 null background. Characterization of an allelic series of Mek mutants reveals the presence of distinct degrees of spontaneous B cell activation, which are inversely proportional to the levels of MEK proteins and ERK activation. While Mek1 and Mek2 null mutants have a normal lifespan, 1Mek1 and 1Mek2 mutants retaining only one functional Mek1 or Mek2 allele in hematopoietic cell lineages die from glomerulonephritis and lymphoproliferative disorders, respectively. This establishes that the fine-tuning of the ERK/MAPK pathway is critical to regulate B and T cell activation and function and that each MEK isoform plays distinct roles during lymphocyte activation and disease development.

Blood production is a tightly regulated process that starts with hematopoietic stem cells (HSCs). In adults, HSCs are unique in their capacity to self-renew and replenish the entire blood system through production of a series of increasingly committed progenitor cells within the bone marrow (BM) microenvironment. HSCs form a rare, quiescent population that displays a metabolism skewed towards anaerobic glycolysis at the expense of mitochondrial oxidative phosphorylation (OXPHOS) to preserve its quiescent state and long-term reconstitution capacity. However, when HSCs differentiate, they undergo a metabolic switch from anaerobic glycolysis to mitochondrial OXPHOS, a process that is in part mediated by the metabolic sensor mTOR. It is well-established that HSCs in the BM adapt the production of myeloid and lymphoid cells depending on the needs of the body and that metabolic plasticity is a critical driver of HSC fate decisions. This has never been assessed for multipotent progenitors (MPPs) which constitute the stage at which the major divergence of lymphoid and myeloid lineages occurs. In mice, common lymphoid progenitors (CLPs) and common myeloid progenitors (CMPs) are generated from phenotypically and functionally distinct subpopulations of lineage-biased MPPs, i.e. MPP2 and MPP3 are reported as distinct myeloid-biased MPP subsets that operate together with lymphoid-primed MPP4 to control blood leukocyte production. This question is thus of paramount importance to understand how the lympho-myeloid specification process is regulated. Signaling by the G protein-coupled receptor CXCR4 on MPPs in response to stimulation by its natural ligand, the chemokine CXCL12, produced by BM perivascular stromal cells constitutes a key pathway through which the niches and MPPs communicate. However, the mechanisms whereby CXCR4 signaling regulates MPP specification are still unknown. We addressed this point using BM samples of patients with WHIM Syndrome (WS), a rare immunodeficiency caused by inherited heterozygous autosomal gain-of-CXCR4-function mutations affecting desensitization of CXCR4 and characterized by chronic lympho-neutropenia, as well as a unique WS mouse model which phenocopies severe pan-leukopenia. We unraveled myeloid skewing of the hematopoietic stem and progenitor cell (HSPC) compartment in BM of patients with WS and of WS mice. This relied on CXCR4 signaling strength that controls the output of the lymphoid and myeloid lineages by coordinating the composition and molecular identity of the MPP compartment. The fate of the lymphoid-biased MPP4 subset was central in such a process. Indeed, CXCR4 signaling termination was required for efficient generation and maintenance of the MPP4 pool, while regulating intrinsically their cell cycle status and lymphoid-myeloid gene landscape. In fact, we demonstrated for the first time that enhanced mTOR signaling, accumulation of damaged mitochondria and overactive OXPHOS-driven metabolism promoted cell-autonomous molecular changes that reprogram mutant MPP4 away from lymphoid differentiation. Consistent with this, in vivo chronic treatment with the CXCR4 antagonist AMD3100/Plerixafor or the mTOR inhibitor Rapamycin normalized mitochondrial metabolism and MPP4 differentiation. Thus, our study shows that CXCR4 signaling acts through the mTOR pathway as an essential gatekeeper for integrity of the mitochondrial machinery, which in turn controls lymphoid potential of MPP4. Disclosures No relevant conflicts of interest to declare.

Introduction: Congenital neutropenia (CN) is characterized by chronic neutropenia caused by a constitutional genetic defect and can be considered an orphan disease. Nationwide estimations of its incidence and prevalence are poorly documented but would provide key information to better follow-up of CN patients. Notably, orphan-drug status also is accorded based on such epidemiological parameters. Methods: The French Severe Chronic Neutropenia Registry (FSCNR) has prospectively enrolled CN patients since 1993, with multiple source verifications in France of that information: pediatric and adult hemato-immunology units, diagnostic labs... We also actively collect all cases followed in France, regardless of the healthcare facility monitoring the patient. To calculate incidence at birth, we considered subjects born between 1/1/1995 and 12/31/2017, because information completeness has been validated for this 22-year period. Number of births per year was provided by the French National Institute of Statistics and Economic Studies (INSEE). We used American College of Medical Genetics class 4 and 5 variants for genetic classification and the overall CN classification developed elsewhere.1 To estimate expected prevalence, we assumed 50-year life expectancy for these patients and compared ongoing enrolment to the prevalence estimation and calculated FNSCR coverage. A Poisson distribution was assumed. Results: On 15 July 2020, the FSCNR had identified 3205 patients. Reasons for non-enrolment of 2096 were, mainly: autoimmune neutropenia (n=501), foreign residency (n=214), other diagnosis (n=882) and diagnostic work-up not completed (n=249). Among the 1109 patients who fulfilled Chronic Neutropenia criteria, 242 had idiopathic neutropenia2 and 867 patients were considered to have CN1. Global results are presented in Table 1. In France, the CN incidence at birth (all subtypes combined) was 2.6×10-5 (95% CI: 2.04-2.8×10-5), which represents a mean of 23 new cases/year in a country with ~870,000 births/year. For all CN combined, the expected prevalence, assuming 50-year life expectancy, would be 1131 cases in a country of 65×106 inhabitants while the FCSNR currently has 867 cases enrolled or an estimated 77% nationwide coverage. Based on our results and our assumptions for life expectancy, estimated prevalence of CN for 10 millions inhabitants is therefore 174 CN. Genetic subtype representation is as follows: 20% SBDS, 17% ELANE (8% cyclic, 9% permanent), 9% GATA2, 7% SLC37A4, ~4-5% each of TAZ and CXCR4 and VPS13B, while the other subtypes are even rarer. At present, no cause has been identified for 25% of the cases. Conclusion: The results of this analysis provide an estimation of the major CN-descriptive epidemiological parameters and the relative frequencies of several subtypes. Despite the FSCNR's quite large registry, we estimate that about a quarter of the prevalent cases in France were missed, mainly those followed as adults. References 1 Donadieu J, Beaupain B, Fenneteau O, Bellanne-Chantelot C. Congenital neutropenia in the era of genomics: classification, diagnosis, and natural history. Br.J.Haematol. 2017; 179(4): 557-574. 2 Sicre De Fontbrune F, Moignet A, Beaupain B et al. Severe chronic primary neutropenia in adults: report on a series of 108 patients. Blood 2015; 126(14): 1643-1650. Acknowledgments: The French SCN registry is supported by grants from Amgen, Chugai, Prolong Pharma, X4 Pharma, Inserm, the Association 111 les Arts, the Association RMHE, the Association Sportive de Saint Quentin Fallavier. The authors thank the association IRIS and Mrs Grosjean and Mr Gonnot(ASSQF), the association Barth France for their support. Disclosures Sicre de Fontbrune: Alexion Pharmaceuticals Inc.: Honoraria, Research Funding. cohen Beaussant:X4 Pharmaceuticals, Inc.: Current Employment.

Introduction: Approximately 20% of Myelodysplastic Syndromes (MDS) and Chronic Myelomonocytic Leukemia (CMML) are associated with SIAD, but pathophysiological mechanisms underlying this association remain largely unknown and data on somatic mutations are very scarce. Autoreactive T lymphocytes play a major role in SIAD development. The main objective of this study was to investigate the mutational landscape of MDS/CMML associated with SIAD, and its impact on the immunological phenotype of T lymphocytes. Patients and Methods: This retrospective study was conducted in the hematology department of Hôpital Saint-Louis, Paris, in all MDS/CMML patients diagnosed between 2012 and 2017, and with a molecular analysis by NGS targeting a panel of 80 genes. MDS/CMML patients with associated SIAD (n= 85, whose diagnosis was based on usual international criteria) were compared with MDS/CMML patients without SIAD (n=319) who constituted the control cohort. Flow cytometry was performed on peripheral blood samples from 28 MDS/CMML patients (of whom 12/28 had SIAD) and 18 healthy controls, on a BD Fortessa X20 to study CD8+T lymphocytes subsets and the expression of immune checkpoints. Analysis was performed with FlowAI R v1.14.0 and Cytobank website for T cells clustering with unsupervised Citrus algorithm. Results: We included 404 MDS/CMML patients (323 MDS, 81 CMML), of whom 85 (21%) had SIAD diagnoses including 35 (34%) inflammatory arthritis, 20 (19%) systemic vasculitis, 16 (15%) autoimmune cytopenias, 15 (14%) connective tissue diseases, 9 (9%) neutrophilic dermatosis, 6 (6%) inflammatory bowel disease, and 3 (3%) unclassified SIAD. Both SIAD and control cohorts were similar in terms of age, levels of cytopenias, MDS/CMML subtypes and cytogenetic features. There was a preponderance of low risk MDS/CMML (85.9% and 82.8% in SIAD and control cohort), and the number of patients with CMML did not differ between the 2 groups (p=0.36). Median follow-up was 32.2 months in the whole cohort. Median overall survival was 95.7 months [60.7 - not estimable] and 101.0 months [70.8 - not estimable] in the control and SIAD cohort respectively (p=0.91). Figure A represents the mutational landscape of MDS/CMML patients for the recurrent mutated genes. Median number of mutations was 2 [1-4] in both cohorts (ns). TET2mutations were found in 39/85 (46%) and 108/319 (34%) patients in the SIAD and control cohorts respectively (p=0.04), and IDH1/2 mutationsin 12/85 (14%) patients in the SIAD cohort vs 14/319 (4%) in the control group (p SRSF2 mutations were also more frequent in the SIAD cohort (26/85 (31%) vs 47/319 (15%), p Flow cytometry analysis of CD8+ T cells from TET2/IDHmut patients (17/28, 60.7%) showed an increase of the terminally differenciated effector memory T cells (p Discussion Our study provides an extended mutational landscape of MDS/CMML associated with SIAD, and finds a correlation between TET2/IDH mutations, T lymphocyte imbalance and association with SIAD in those diseases. This correlation could suggest common mechanisms underlying both SIAD and MDS/CMML, and reinforces our observations of a frequent positive effect of Azacytidine (a drug with better response in patients with TET2mutation) on both MDS/CMML and SIAD in patients with both disorders (Fraison, Leuk Res 2016). Further analysis of the functional role of T lymphocytes in those patients is underway. Disclosures Clappier: Amgen: Honoraria, Research Funding. Itzykson:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Research Funding; Oncoethix (now Merck): Research Funding; Astellas: Honoraria; Sanofi: Honoraria; BMS (Celgene): Honoraria; Daiichi Sankyo: Honoraria; Jazz Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria; Amgen: Membership on an entity's Board of Directors or advisory committees; Otsuka Pharma: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Stemline: Membership on an entity's Board of Directors or advisory committees. Mékinian:LFB: Honoraria; CELGENE: Honoraria; CELGENE: Honoraria; SANOFI: Honoraria. Fenaux:BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Jazz: Honoraria, Research Funding. Ades:jazz: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; novartis: Research Funding; Celgene/BMS: Research Funding.

This study explores the effects of microbiota on reproductive function of Cxcr2 knockout animals. Cxcr2 is involved in the control of pituitary action and the subsequent development of mammary gland, uterus and ovary., Microbiota and chronic infections can affect not only immune status, but also the overall physiology of animals. Here, we report that chronic infections dramatically modify the phenotype of Cxcr2 KO mice, impairing in particular, their reproduction ability. We show that exposure of Cxcr2 KO females to multiple types of chronic infections prevents their ability to cycle, reduces the development of the mammary gland and alters the morphology of the uterus due to an impairment of ovary function. Mammary gland and ovary transplantation demonstrated that the hormonal contexture was playing a crucial role in this phenomenon. This was further evidenced by alterations to circulating levels of sex steroid and pituitary hormones. By analyzing at the molecular level the mechanisms of pituitary dysfunction, we showed that in the absence of Cxcr2, bystander infections affect leukocyte migration, adhesion, and function, as well as ion transport, synaptic function behavior, and reproduction pathways. Taken together, these data reveal that a chemokine receptor plays a direct role in pituitary function and reproduction in the context of chronic infections.

The signaling axis formed by the chemokine CXCL12 and its receptor CXCR4 plays an important role in B cell development and activation and is finely regulated by a process termed desensitization. Mutations leading to a truncation of the C-terminus tail of CXCR4 and thus to a defective desensitization have been reported in two diseases, a rare immunodeficiency called the WHIM syndrome and a B cell plasmacytoma called Waldenstrom’s Macroglobulinemia (WM). How CXCR4 desensitization may impact B cell activation in the context of a T-independent extra-follicular response is still unknown. Here using a unique mouse model bearing an orthologous gain of function mutation ofCxcr4we report that Cxcr4 desensitization is an essential gatekeeper controlling B lymphocyte entry into cycle, plasma cell differentiation, migration and maturation upon Myd88-dependent signaling. Altogether, our results support an essential role for Cxcr4 desensitization in limiting the depth and width of the B cell extra-follicular response and PC development.

Atypical chemokine receptor 3 (ACKR3), previously known as C-X-C chemokine receptor type 7 (CXCR7), has emerged as a key player in several biologic processes, particularly during development. Its CXCL11 and CXCL12 scavenging activity and atypical signaling properties, together with a new array of other nonchemokine ligands, have established ACKR3 as a main regulator of physiologic processes at steady state and during inflammation. Here, we present a comprehensive review of ACKR3 expression in mammalian tissues in search of a possible connection with the receptor function. Besides the reported roles of ACKR3 during development, we discuss the potential contribution of ACKR3 to the function of the immune system, focusing on the myeloid lineage.