CXCR4 WHIM syndrome is a cancer predisposition condition for virus-induced malignancies.

Warts, hypogammaglobulinaemia, infections and myelokathexis syndrome (WHIMS) is a rare combined primary immunodeficiency caused by the gain of function of the CXCR4 chemokine receptor. We present the prevalence of cancer in WHIMS patients based on data from the French Severe Chronic Neutropenia Registry and an exhaustive literature review. The median follow-up of the 14 WHIMS 'patients was 28.5 years. A central review and viral evaluation of pathological samples were organized, and we conducted a thorough literature review to identify all reports of WHIMS cases. Six French patients were diagnosed with cancer at a median age of 37.6 years. The 40-year risk of malignancy was 39% (95% confidence interval [CI]: 6%-74%). We observed two human papillomavirus (HPV)-induced vulvar carcinomas, three lymphomas (two Epstein-Barr virus [EBV]-related) and one basal cell carcinoma. Among the 155 WHIMS cases from the literature, 22 cancers were reported in 16 patients, with an overall cancer 40-year risk of 23% (95% CI: 13%-39%). Malignancies included EBV-associated lymphoproliferative disorders and HPV-positive genital and anal cancers as in the French cohort. Worldwide, nine cases of malignancy were associated with HPV and four with EBV. Immunocompromised WHIMS patients appear to be particularly susceptible to developing early malignancy, mainly HPV-induced carcinomas, followed by EBV-related lymphomas.

Transinteractome analysis reveals distinct niche requirements for isotype-based plasma cell subsets in the bone marrow.

Bone marrow (BM) long-lived plasma cells (PCs) are essential for long-term protection against infection, and their persistence within this organ relies on interactions with Cxcl12-expressing stromal cells that are still not clearly identified. Here, using single cell RNAseq and in silico transinteractome analyses, we identified Leptin receptor positive (LepR+ ) mesenchymal cells as the stromal cell subset most likely to interact with PCs within the BM. Moreover, we demonstrated that depending on the isotype they express, PCs may use different sets of integrins and adhesion molecules to interact with these stromal cells. Altogether, our results constitute an unprecedented characterization of PC subset stromal niches and open new avenues for the specific targeting of BM PCs based on their isotype.

Severe CD8+ T Lymphopenia in WHIM Syndrome Caused by Selective Sequestration in Primary Immune Organs.

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.

Spatial dysregulation of T follicular helper cells impairs vaccine responses in aging.

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.

WHIM Syndrome-linked CXCR4 mutations drive osteoporosis.

WHIM Syndrome is a rare immunodeficiency caused by gain-of-function CXCR4 mutations. Here we report a decrease in bone mineral density in 25% of WHIM patients and bone defects leading to osteoporosis in a WHIM mouse model. Imbalanced bone tissue is observed in mutant mice combining reduced osteoprogenitor cells and increased osteoclast numbers. Mechanistically, impaired CXCR4 desensitization disrupts cell cycle progression and osteogenic commitment of skeletal stromal/stem cells, while increasing their pro-osteoclastogenic capacities. Impaired osteogenic differentiation is evidenced in primary bone marrow stromal cells from WHIM patients. In mice, chronic treatment with the CXCR4 antagonist AMD3100 normalizes in vitro osteogenic fate of mutant skeletal stromal/stem cells and reverses in vivo the loss of skeletal cells, demonstrating that proper CXCR4 desensitization is required for the osteogenic specification of skeletal stromal/stem cells. Our study provides mechanistic insights into how CXCR4 signaling regulates the osteogenic fate of skeletal cells and the balance between bone formation and resorption.

Myelodysplastic Syndrome associated TET2 mutations affect NK cell function and genome methylation.

Myelodysplastic syndromes (MDS) are clonal hematopoietic disorders, representing high risk of progression to acute myeloid leukaemia, and frequently associated to somatic mutations, notably in the epigenetic regulator TET2. Natural Killer (NK) cells play a role in the anti-leukemic immune response via their cytolytic activity. Here we show that patients with MDS clones harbouring mutations in the TET2 gene are characterised by phenotypic defects in their circulating NK cells. Remarkably, NK cells and MDS clones from the same patient share the TET2 genotype, and the NK cells are characterised by increased methylation of genomic DNA and reduced expression of Killer Immunoglobulin-like receptors (KIR), perforin, and TNF-α. In vitro inhibition of TET2 in NK cells of healthy donors reduces their cytotoxicity, supporting its critical role in NK cell function. Conversely, NK cells from patients treated with azacytidine (#NCT02985190; https://clinicaltrials.gov/ ) show increased KIR and cytolytic protein expression, and IFN-γ production. Altogether, our findings show that, in addition to their oncogenic consequences in the myeloid cell subsets, TET2 mutations contribute to repressing NK-cell function in MDS patients.

[CXCR4 as a rheostat of humoral response]. / La signalisation de CXCR4, un rhéostat de la réponse immunitaire à médiation humorale.

CXCR4 is a chemokine receptor that plays a central role in cell migration but also in other essential processes such as the development of the immune system. Together with its ligand, the chemokine CXCL12, this signalling axis plays an important role in B lymphocyte biology from their early differentiation in the bone marrow to their activation and differentiation into antibody secreting cells, also called plasma cells. Gain-of-function mutations of CXCR4 are found in a rare immunodeficiency, the WHIM Syndrome. These mutations affect the desensitization of the receptor and lead to a gain of function in response to CXCL12. This review summarizes the role of CXCR4 in the humoral immune responses and using the WHIM Syndrome as a paradigm, highlights the critical regulatory role of CXCR4 desensitization in these processes. Indeed, recent works report that fine-tuning of CXCR4 signalling is essential to limit the extra-follicular immune response and support long term antibody-mediated protection.

Title: La signalisation de CXCR4, un rhéostat de la réponse immunitaire à médiation humorale. Abstract: 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 ¼ et pour permettre une protection au long terme assurée par les anticorps.

Sec22b is a critical and nonredundant regulator of plasma cell maintenance.

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.

CXCR2 intrinsically drives the maturation and function of neutrophils in mice.

Neutrophils play a major role in the protection from infections but also in inflammation related to tumor microenvironment. However, cell-extrinsic and -intrinsic cues driving their function at steady state is still fragmentary. Using Cxcr2 knock-out mice, we have evaluated the function of the chemokine receptor Cxcr2 in neutrophil physiology. We show here that Cxcr2 deficiency decreases the percentage of mature neutrophils in the spleen, but not in the bone marrow (BM). There is also an increase of aged CD62Llo CXCR4hi neutrophils in the spleen of KO animals. Spleen Cxcr2-/- neutrophils display a reduced phagocytic ability, whereas BM neutrophils show an enhanced phagocytic ability compared to WT neutrophils. Spleen Cxcr2-/- neutrophils show reduced reactive oxygen species production, F-actin and α-tubulin levels. Moreover, spleen Cxcr2-/- neutrophils display an altered signaling with reduced phosphorylation of ERK1/2 and p38 MAPK, impaired PI3K-AKT, NF-κB, TGFß and IFNγ pathways. Altogether, these results suggest that Cxcr2 is essential for neutrophil physiology.

Clinical and Hematologic Effects of Endotoxin in Warts, Hypogammaglobulinemia, Infections, and Myelokathexis Syndrome Model Mice.

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.

Fine-tuning of MEK signaling is pivotal for limiting B and T cell activation.

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.

Deciphering Tumor Niches: Lessons From Solid and Hematological Malignancies.

Knowledge about the hematopoietic niche has evolved considerably in recent years, in particular through in vitro analyzes, mouse models and the use of xenografts. Its complexity in the human bone marrow, in particular in a context of hematological malignancy, is more difficult to decipher by these strategies and could benefit from the knowledge acquired on the niches of solid tumors. Indeed, some common features can be suspected, since the bone marrow is a frequent site of solid tumor metastases. Recent research on solid tumors has provided very interesting information on the interactions between tumoral cells and their microenvironment, composed notably of mesenchymal, endothelial and immune cells. This review thus focuses on recent discoveries on tumor niches that could help in understanding hematopoietic niches, with special attention to 4 particular points: i) the heterogeneity of carcinoma/cancer-associated fibroblasts (CAFs) and mesenchymal stem/stromal cells (MSCs), ii) niche cytokines and chemokines, iii) the energy/oxidative metabolism and communication, especially mitochondrial transfer, and iv) the vascular niche through angiogenesis and endothelial plasticity. This review highlights actors and/or pathways of the microenvironment broadly involved in cancer processes. This opens avenues for innovative therapeutic opportunities targeting not only cancer stem cells but also their regulatory tumor niche(s), in order to improve current antitumor therapies.

CXCR4 hyperactivation cooperates with TCL1 in CLL development and aggressiveness.

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.

Mek1 and Mek2 Functional Redundancy in Erythropoiesis.

Several studies have established the crucial role of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase pathway in hematopoietic cell proliferation and differentiation. MEK1 and MEK2 phosphorylate and activate ERK1 and ERK2. However, whether MEK1 and MEK2 differentially regulate these processes is unknown. To define the function of Mek genes in the activation of the ERK pathway during hematopoiesis, we generated a mutant mouse line carrying a hematopoietic-specific deletion of the Mek1 gene function in a Mek2 null background. Inactivation of both Mek1 and Mek2 genes resulted in death shortly after birth with a severe anemia revealing the essential role of the ERK pathway in erythropoiesis. Mek1 and Mek2 functional ablation also affected lymphopoiesis and myelopoiesis. In contrast, mice that retained one functional Mek1 (1Mek1) or Mek2 (1Mek2) allele in hematopoietic cells were viable and fertile. 1Mek1 and 1Mek2 mutants showed mild signs of anemia and splenomegaly, but the half-life of their red blood cells and the response to erythropoietic stress were not altered, suggesting a certain level of Mek redundancy for sustaining functional erythropoiesis. However, subtle differences in multipotent progenitor distribution in the bone marrow were observed in 1Mek1 mice, suggesting that the two Mek genes might differentially regulate early hematopoiesis.

Pivotal Role for Cxcr2 in Regulating Tumor-Associated Neutrophil in Breast Cancer.

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.

Hematopoietic Multipotent Progenitors and Plasma Cells: Neighbors or Roommates in the Mouse Bone Marrow Ecosystem?

The bone marrow is a complex ecosystem in which hematopoietic and non-hematopoietic cells reside. In this review, we discuss the bone marrow niches in mice that facilitate the survival, maintenance, and differentiation of cells of hematopoietic origin based on the recent literature. Our review places a special focus on the hematopoietic multipotent progenitors and on plasma cells, corresponding to the last stage of the B-cell lineage, that play a key role in the humoral memory response. We highlight the similarities between the microenvironments necessary for the establishment and the maintenance of these two immune cell subsets, and how the chemokine CXCL12/CXCR4 signaling axis contributes to these processes. Finally, we bring elements to address the following question: are multipotent progenitors and plasma cells neighbors or roommates within the bone marrow?