Search

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.

WHIM syndrome (warts, hypogammaglobulinemia, infections, myelokathexis) is a rare combined primary immunodeficiency. Here we describe 10 Russian patients with WHIM syndrome that were followed in the Dmitry Rogachev National Medical Research Center оf Pediatric Hematology, Oncology and Immunology. This study is supported by the Independent Ethics Committee and approved by the Academic Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology. Neutropenia and lymphopenia were observed in all 10 patients, hypogammaglobulinemia – in 7/10 patients. In all cases bone marrow analysis demonstrated myelokathexis features (cytoplasmic vacuolization in neutrophils and eosinophils, hyperlobulated pyknotic nuclear lobes connected by long thin strands, hypogranular and hypersegmented neutrophils). All patients were treated with granulocyte colony-stimulating factor and intravenous immunoglobulin. In 3/10 poor disease control was an indication to perform HSCT. In 2 of 3 patients HSTC was successful and all symptoms of the disease resolved. In conclusion, the diagnosis of WHIM syndrome must be considered in patients with early onset of neutro- and lymphopenia in conjunction with morphological features of myelokathexis. Treatment of this disease is still a challenging problem.

Background Warts, hypogammaglobulinemia, recurrent bacterial infections and myelokathexis (WHIM) syndrome is a primary immunodeficiency disease (PID) usually caused by autosomal dominant mutations in the chemokine receptor CXCR4 gene. To date, a total of nine different mutations including eight truncation mutations and one missense mutation (E343K, CXCR4E343K) distributed in the C-terminus of CXCR4 have been identified in humans. Studies have clarified that the loss of phosphorylation sites in the C-terminus of truncated CXCR4 impairs the desensitization process, enhances the activation of G-protein, prolongs downstream signaling pathways and introduces over immune responses, thereby causing WHIM syndrome. So far, there is only one reported case of WHIM syndrome with a missense mutation, CXCR4E343K, which has a full length of C-terminus with entire phosphorylation sites, no change in all potential phosphorylation sites. The mechanism of the missense mutation (CXCR4E343K) causing WHIM syndrome is unknown. This study aimed to characterize the effect of mutation at the 343 site of CXCR4 causing the replacement of arginine/E with glutamic acid/K on the receptor signal transduction, and elucidate the mechanism underling CXCR4E343K causing WHIM in the reported family. Results We completed a series of mutagenesis to generate different mutations at the 343 site of CXCR4 tail, and established a series of HeLa cell lines stably expressing CXCR4WT or CXCR4E343D (glutamic acid/E replaced with aspartic acid/D) or CXCR4E343K (glutamic acid/E replaced with lysine/K) or CXCR4E343R (glutamic acid/E replaced with arginine/R) or CXCR4E343A (glutamic acid/E replaced with alanine/A) and then systematically analyzed functions of the CXCR4 mutants above. Results showed that the cells overexpressing of CXCR4E343D had no functional changes with comparison that of wild type CXCR4. However, the cells overexpressing of CXCR4E343K or CXCR4E343R or CXCR4E343A had enhanced cell migration, prolonged the phosphorylation of ERK1/2, p38, JNK1/2/3, aggravated activation of PI3K/AKT/NF-κB signal pathway, introduced higher expression of TNFa and IL6, suggesting over immune response occurred in CXCR4 mutants with charge change at the 343 site of receptor tail, as a result, causing WHIM syndrome. Biochemical analysis of those mutations at the 343 site of CXCR4 above shows that CXCR4 mutants with no matter positive or neutral charge have aberrant signal pathways downstream of activated mutated CXCR4, only CXVR4 with negative charge residues at the site shows normal signal pathway post activation with stromal-derived factor (SDF1, also known as CXCL12). Conclusion Taken together, our results demonstrated that the negative charge at the 343 site of CXCR4 plays an essential role in regulating the down-stream signal transduction of CXCR4 for physiological events, and residue charge changes, no matter positive or neutral introduce aberrant activities and functions of CXCR4, thus consequently lead to WHIM syndrome.

SignificanceNew imaging-based approaches are incorporating new concepts to our knowledge of biological processes. The analysis of receptor dynamics involved in cell movement using single-particle tracking demonstrates that cells require chemokine-mediated receptor clustering to sense appropriately chemoattractant gradients. Here, we report that this process does not occur in T cells expressing CXCR4R334X, a mutant form of CXCR4 linked to WHIM syndrome (warts, hypogammaglobulinemia, infections, myelokathexis). The underlaying molecular mechanism involves inappropriate actin cytoskeleton remodeling due to the inadequate β-arrestin1 activation by CXCR4R334X, which alters its lateral mobility and spatial organization. These defects, associated to CXCR4R334X expression, contribute to the retention of hematopoietic precursors in bone marrow niches and explain the severe immunological symptoms associated with WHIM syndrome., Proceedings of the National Academy of Sciences of the United States of America, 119 (21), ISSN:0027-8424, ISSN:1091-6490

Chemokine receptor nanoscale organization at the cell membrane is orchestrated by the actin cytoskeleton and influences cell responses. Using single-particle tracking analysis we show that CXCR4R334X, a truncated mutant chemokine receptor linked to WHIM syndrome (warts, hypogammaglobulinemia, infections, myelokathexis), fails to nanoclus terize after CXCL12 stimulation, and alters the lateral mobility and spatial organization of CXCR4 when coexpressed. These findings correlate with multiple phalloidin-positive protrusions in cells expressing CXCR4R334X, and their inability to correctly sense chemo kine gradients. The underlying mechanisms involve inappropriate actin cytoskeleton remodeling due to the inadequate β-arrestin1 activation by CXCR4R334X, which disrupts the equilibrium between activated and deactivated cofilin. Overall, we provide insights into the molecular mechanisms governing CXCR4 nanoclustering, signaling and cell function, and highlight the essential scaffold role of β-arrestin1 to support CXCL12- mediated actin reorganization and receptor clustering. These defects associated with CXCR4R334X expression might contribute to the severe immunological symptoms asso ciated with WHIM syndrome.

C-X-C chemokine receptor type 4 (CXCR4), initially recognized as a co-receptor for HIV, contributes to several disorders, including the WHIM (Warts, Hypogammaglobulinemia, Infections, and Myelokathexis) syndrome. CXCR4 binds to its ligand SDF-1 to make an axis involved in the homing property of stem cells. This study aimed to employ WHIM syndrome pathogenesis as an inspirational approach to reinforce cell therapies. Wild type and WHIM-type variants of the CXCR4 gene were chemically synthesized and cloned in the pCDH-513B-1 lentiviral vector. Molecular cloning of the synthetic genes was confirmed by DNA sequencing, and expression of both types of CXCR4 at the protein level was confirmed by western blotting in HEK293T cells. Human adipose-derived mesenchymal stem cells (Ad-MSCs) were isolated, characterized, and subjected to lentiviral transduction with Wild type and WHIM-type variants of CXCR4. The presence of copGFP-positive MSCs confirmed the high efficiency of transduction. The migration ability of both groups of transduced cells was then assessed by transwell migration assay in the presence or absence of a CXCR4-blocking agent. Our qRT-PCR results showed overexpression of CXCR4 at mRNA level in both groups of transduced MSCs, and expression of WHIM-type CXCR4 was significantly higher than Wild type CXCR4 ( P

WHIM syndrome is a rare combined primary immunodeficiency disease named by acronym for the diagnostic tetrad of warts, hypogammaglobulinemia, infections, and myelokathexis. Myelokathexis is a unique form of non-cyclic severe congenital neutropenia caused by accumulation of mature and degenerating neutrophils in the bone marrow; monocytopenia and lymphopenia, especially B lymphopenia, also commonly occur. WHIM syndrome is usually caused by autosomal dominant mutations in the G protein-coupled chemokine receptor CXCR4 that impair desensitization, resulting in enhanced and prolonged G protein- and β-arrestin-dependent responses. Accordingly, CXCR4 antagonists have shown promise as mechanism-based treatments in phase 1 clinical trials. This review is based on analysis of all 105 published cases of WHIM syndrome and covers current concepts, recent advances, unresolved enigmas and controversies, and promising future research directions.

Background In the warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome, variable phenotypic expression may delay diagnosis. Panleukopenia, malignancy, and chronic lung disease all affect morbidity and mortality risks. Routinely used treatments include immunoglobulins, granulocyte-colony stimulating factor (G-CSF), and antibiotics; recent trials with a target C-X-C chemokine receptor type 4 (CXCR4) antagonist show promising results. Objective We sought to characterize the largest cohort of patients with WHIM and evaluate their diagnostic and therapeutic management. Methods Data were collected from an international cohort of 18 patients with CXCR4 mutations. Results The clinical features manifested at 2.2 ± 2.6 years of age, whereas the disease diagnosis was delayed until 12.5 ± 10.4 years of age. Patients with WHIM commonly presented with a severe bacterial infection (78%). Pneumonia recurrence was observed in 61% of patients and was complicated with bronchiectasis in 27%. Skin warts were observed in 61% of patients at a mean age of 11 years, whereas human papilloma virus (HPV)-related malignancies manifested in 16% of patients. All the patients had severe neutropenia (195 ± 102 cells/mm3 at onset), whereas lymphopenia and hypogammaglobulinemia were detected in 88% and 58% of patients, respectively. Approximately 50% of patients received antibiotic prophylaxis, whereas G-CSF and immunoglobulin treatments were used in 72% and 55% of patients, respectively. Conclusions The WHIM syndrome onsets early in life and should be suspected in patients with chronic neutropenia. Patients with WHIM need careful monitoring and timely intervention for complications, mainly lung disease and HPV-related malignancies. We suggest that immunoglobulin therapy should be promptly considered to control the frequency of bacterial infections and prevent chronic lung damage.

Background Warts Hypogammaglobulinemia Immunodeficiency Myelokathexis (WHIM) syndrome is a primary immunodeficiency characterized by recurrent bacterial infections, severe chronic neutropenia, with lymphopenia, monocytopenia and myelokathexis which is caused by heterozygous gain of functions mutations of the CXC chemokine receptor 4 (CXCR4). WHIM patients display an increased incidence of non-hematopoietic conditions, such as congenital heart disease suggesting that abnormal CXCR4 may put these patients at increased risk of congenital anomalies. Studies conducted on CXCR4 and SDF-1-deficient mice have demonstrated the role of CXCR4 signaling in neuronal cell migration and brain development. In particular, CXCR4 conditional knockout mice display abnormal cerebellar morphology and poor coordination and balance on motor testing. Results In order to evaluate a possible neurological involvement in WHIM syndrome subjects, we performed neurological examination, including International Cooperative Ataxia Rating Scale, cognitive and psychopathological assessment and brain Magnetic Resonance Imaging (MRI) in 6 WHIM patients (age range 8–51 years) with typical gain of functions mutations of CXCR4 (R334X or G336X). In three cases (P3, P5, P6) neurological evaluation revealed fine and global motor coordination disorders, balance disturbances, mild limb ataxia and excessive talkativeness. Brain MRI showed an abnormal orientation of the cerebellar folia involving bilaterally the gracilis and biventer lobules together with the tonsils in four subjects (P3, P4, P5, P6). The neuropsychiatric evaluation showed increased risk of internalizing and/or externalizing problems in four patients (P2, P3, P4, P6). Conclusions Taken together, these observations suggest CXCR4 gain of function mutations can be associated with cerebellar malformation, mild neuromotor and psychopathological dysfunction in WHIM patients.

Purpose WHIM (warts, hypogammaglobulinemia, infections, and myelokathexis) syndrome is a rare disease, caused by CXCR4 gene mutations, which incorporates features of combined immunodeficiency, congenital neutropenia, and a predisposition to human papillomavirus infection. Established conventional treatment for WHIM syndrome does not fully prevent infectious complications in these patients. Only single case reports of hematopoietic stem cell transplantation (HSCT) efficacy in WHIM have been published. Methods To summarize current information on HSCT efficacy in disease treatment, seven pediatric patients with WHIM syndrome who underwent allogeneic HSCT were identified in five centers worldwide. Results All patients presented early after birth with neutropenia. Two of seven patients exhibited severe disease complications: poorly controlled autoimmunity (arthritis and anemia) in one and progressive myelofibrosis with recurrent infections in the other. The remaining patients received HSCT to correct milder disease symptoms (recurrent respiratory infections, progressing thrombocytopenia) and/or to preclude severe disease course in older age. All seven patients engrafted but one developed graft rejection and died of infectious complications after third HSCT. Three other patients experienced severe viral infections after HSCT (including post-transplant lymphoproliferative disease in one) which completely resolved with therapy. At last follow-up (median 6.7 years), all six surviving patients were alive with full donor chimerism. One patient 1.4 years after HSCT had moderate thrombocytopenia and delayed immune recovery; the others had adequate immune recovery and were free of prior disease symptoms. Conclusion HSCT in WHIM syndrome corrects neutropenia and immunodeficiency, and leads to resolution of autoimmunity and recurrent infections, including warts.

PURPOSE: T cell receptor excision circle (TREC) quantification is a recent addition to newborn screening (NBS) programs and is intended to identify infants with severe combined immunodeficiencies (SCID). However, other primary immunodeficiency diseases (PID) have also been identified as the result of TREC screening. We recently reported a newborn with a low TREC level on day 1 of life who was diagnosed with WHIM (warts, hypogammaglobulinemia, infections, myelokathexis) syndrome, a non-SCID primary immunodeficiency caused by mutations in the chemokine receptor CXCR4. METHODS: We have now retrospectively reviewed the birth and clinical histories of all known WHIM infants born after the implementation of NBS for SCID. RESULTS: We identified six infants with confirmed WHIM syndrome who also had TREC quantification on NBS. Three of the six WHIM infants had low TREC levels on NBS. All six patients were lymphopenic. No infant demonstrated a neonatal T cell count below 1,500 cells/μL. The most common clinical manifestation was viral bronchiolitis requiring hospitalization. One infant died of complications related to Tetralogy of Fallot, a known WHIM phenotype. CONCLUSION: The results suggest that WHIM syndrome should be considered in the differential diagnosis of newborns with low NBS TREC levels.

International audience; 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?

Hematopoietic stem and progenitor cells (HSPCs), including the multipotent progenitors (MPPs), are responsible for replenishing immune cells. They reside in bone marrow (BM) endosteal and (peri)-vascular niches, which provide all cellular and molecular components required for their lifelong maintenance and fate. Among them, the CXCL12 chemokine and one of its receptor, CXCR4, exert a dominant role in promoting HSPC retention and quiescence. These processes are deregulated in the WHIM Syndrome (WS), a rare immunodeficiency caused by inherited heterozygous autosomal gain-of-function CXCR4 mutations that affect homologous desensitization of the receptor. Clinically, WS is notably characterized by severe, chronic circulating lymphopenia whose mechanisms remain to be elucidated. Using a mouse model carrying a naturally occurring WS-linked Cxcr4 mutation as well as human BM and blood samples, we explored the possibility that the lymphopenia in WS originates from defects at the HSPC level in BM. We reported that Cxcr4 desensitization is required for lymphoid differentiation of HSPCs and further identified the MPP stage as defective in mutant mice. The divergence between lymphoid and myeloid lineages occurs at the MPP stage, which is composed of distinct subpopulations, 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. Our understanding of how cell-extrinsic niche-related and cell-intrinsic cues drive the lymphoid versus myeloid fate decision of MPPs is still fragmentary. Therefore, my PhD project aimed at determining whether and how CXCR4 signaling regulates bioenergetics demands of MPPs and at understanding how these metabolic pathways shape the lympho-myeloid fate of MPPs. We unraveled a myeloid skewing of the HSPC compartment in BM of WS mice and patients. In mutant mice, this partly relied on the contraction of the MPP4 pool and on cell-autonomous molecular and metabolic changes that reprogramed MPP4 away from lymphoid differentiation. Interestingly, chronic treatment with the CXCR4 antagonist AMD3100 normalized mitochondrial metabolism and fate of MPP4, while correcting circulating lymphopenia in WS mice. This study provides evidence that CXCR4 signaling acts as an essential gatekeeper for integrity of the mitochondrial machinery, which in turn controls lymphoid potential of MPP4.; Les cellules souches et progéniteurs hématopoïétiques (CSPHs), incluant les progéniteurs multipotents (MPPs), sont responsables de la production des cellules immunes circulantes. Ils résident dans la moelle osseuse (MO) au sein de structures spécialisées, les niches endostéale et (péri)-vasculaire, qui régulent la spécification et l'engagement lymphoïde versus myéloïde des CSPHs. Dans la MO, le couple formé par la chimiokine CXCL12 et l’un de ses récepteurs, CXCR4, exerce un rôle clé dans la régulation de la rétention et la quiescence des CSPHs. Ces processus sont dérégulés dans le Syndrome WHIM (SW), une maladie immuno-hématologique rare liée à des mutations autosomiques dominantes du gène codant CXCR4, qui altèrent la désensibilisation du récepteur et conduisent à un gain de fonction en réponse à CXCL12. Cliniquement, le SW se caractérise notamment par une profonde leucopénie circulante qui affecte les lignages lymphoïde et myéloïde et dont les mécanismes restent à déterminer. Grâce à un modèle murin génétiquement modifié du SW et à l'accès à des prélèvements biologiques de patients atteints du SW, nous avons testé l'hypothèse que la lymphopénie circulante associée au SW résultait de défauts hématopoïétiques dans la MO. Nous avons révélé un rôle clé de la désensibilisation de CXCR4 dans la différenciation lymphoïde des CSPHs et identifié les MPPs comme étant le stade défectueux dans le SW. La divergence entre les lignages lymphoïde et myeloïde se produit précisément à ce stade au sein duquel règne une hétérogénéité : les MPP2/3 sont biaisés myéloïde et les MPP4 sont orientés lymphoïde. Notre compréhension de la façon dont les signaux extrinsèques (niches) et intrinsèques aux MPPs déterminent leur devenir lymphoïde versus myéloïde est encore parcellaire. Dans ce contexte, l’objectif de ma thèse a été de déterminer si et comment la signalisation de CXCR4 régule la dépendance énergétique des MPPs et à comprendre comment les voies métaboliques façonnent leur spécification lympho-myéloïde. Dans la MO des souris porteuses de la mutation gain de fonction de Cxcr4, nous avons observé une diminution du nombre de MPP4 qui contrastait avec l'augmentation des MPP2/3. L’analyse de prélèvements médullaires de patients a également permis de rapporter une diminution de la fréquence des progéniteurs lymphoïdes et une augmentation de celle des progéniteurs myéloïdes. Chez la souris mutantes, ce biais myéloïde du compartiment de MPPs s'avèrait associé à une expansion anormale et une reprogrammation moléculaire et métabolique des MPP4. Fait marquant, un traitement chronique par l’AMD3100, un antagoniste de CXCR4, permettait de normaliser le nombre de MPP4 dans la MO, de restaurer leurs propriétés métaboliques, et de corriger la lymphopénie des souris mutantes. Par conséquent, nos résultats suggèrent que l’axe CXCL12/CXCR4 est requis au maintien du potentiel lymphoïde des MPP4 au travers de la modulation de leur activité métabolique mitochondriale.

Studies over the last couple of decades have shown that hematopoietic stem cells (HSCs) are critically dependent on cytokines such as Stem Cell Factor and other signals provided by bone marrow niches comprising of mesenchymal stem and progenitor cells (MSPCs) and endothelial cells (ECs). Because of their critical roles in HSC maintenance the niches formed by MSPCs and ECs are commonly referred to as HSC niches. For the most part, the signals required for HSC maintenance act in a short-range manner, which imposes the necessity for directional and positional cues in order for HSCs to localize and be retained properly in stem cell niches. The chemokine CXCL12 and its Gαi protein coupled receptor CXCR4, besides promoting HSC quiescence directly, also play instrumental roles in enabling HSCs to access bone marrow stem cell niches. Recent studies have revealed, however, that HSC niches also provide a constellation of hematopoietic cytokines that are critical for the production of most, if not all, blood cell types. Some hematopoietic cytokines, namely IL-7 and IL-15 produced by HSC niches, are not only required for lymphopoiesis but are also essential for memory T cell maintenance. Consequently, hematopoietic progenitors and differentiated immune cells, such as memory T cell subsets, also depend on the CXCL12/CXCR4 axis for migration into bone marrow and interactions with MSPCs and ECs. Similarly, subsets of antibody-secreting plasma cells also reside in close association with CXCL12-producing MSPCs in the bone marrow and require the CXCR4/CXCL12 axis for survival and long-term maintenance. Collectively, these studies demonstrate a broad range of key physiological roles, spanning blood cell production and maintenance of immunological memory, that are orchestrated by stem cell niches through a common and simple mechanism: CXCL12/CXCR4-mediated cell recruitment followed by receipt of a maintenance and/or instructive signal. A fundamental flaw of this type of cellular organization is revealed by myeloid and lymphoid leukemias, which target stem cell niches and induce profound transcriptomic changes that result in reduced hematopoietic activity and altered mesenchymal cell differentiation.

Treatment with the CXCR4 antagonist, plerixafor (AMD3100), has been proposed for clinical use in patients with WHIM (warts, hypogammaglobulinemia, infections and myelokathexis) syndrome and in pulmonary fibrosis. However, there is controversy with respect to the impact of plerixafor on neutrophil dynamics in the lung, which may affect its safety profile. In this study we investigated the kinetics of endogenous neutrophils by direct imaging, using confocal intravital microscopy in mouse bone marrow, spleen and lungs. Neutrophils are observed increasing their velocity and exiting the bone marrow following plerixafor administration, with a concomitant increase in neutrophil numbers in the blood and spleen, while the marginated pool of neutrophils in the lung microvasculature remained unchanged in terms of numbers and cell velocity. Use of autologous radiolabeled neutrophils and SPECT/CT imaging in healthy volunteers showed that plerixafor did not affect GM-CSF-primed neutrophil entrapment or release in the lungs. Taken together these data suggest that plerixafor causes neutrophil mobilization from the bone marrow but does not impact on lung marginated neutrophil dynamics and thus is unlikely to compromise respiratory host defense both in humans and mice., This work was funded by a grant provided to JP by the Lung Foundation Netherlands (5.2.14.058JO), the NIHR Cambridge Biomedical Research Centre and NIHR Imperial Biomedical Research Centre. ERC and CS’ laboratories receive grant support from the Medical Research Council, Wellcome Trust, NIHR, GlaxoSmithKline, MedImmune Ltd., and Bristol-Myers Squibb. CLC is supported by Bloodwise (12033), CRUK (C36195/A1183) and European Research Council (ERC) (337066). CP is supported by Bloodwise (12033). The Facility for Imaging by Light Microscopy (FILM) at Imperial College London is part-supported by funding from the Wellcome Trust (grant 104931/Z/14/Z) and BBSRC (grant BB/L015129/1). KDF is supported by funding from the Wellcome Trust (201356/Z/16/Z). LMC is supported by core funding from Cancer Research UK (A23983 and A17196).

Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is a rare primary immunodeficiency caused by gain-of-function mutations in the CXCR4 gene. We report the safety, tolerability, pharmacokinetics, pharmacodynamics, and preliminary efficacy of mavorixafor from a phase 2 open-label dose-escalation and extension study in 8 adult patients with genetically confirmed WHIM syndrome. Mavorixafor is an oral small molecule selective antagonist of the CXCR4 receptor that increases mobilization and trafficking of white blood cells from the bone marrow. Patients received escalating doses of mavorixafor, up to 400 mg once daily. Five patients continued on the extension study for up to 28.6 months. Mavorixafor was well tolerated with no treatment-related serious adverse events. At a median follow-up of 16.5 months, we observed dose-dependent increases in absolute neutrophil count (ANC) and absolute lymphocyte count (ALC). At doses ≥300 mg/d, ANC was maintained at >500 cells per microliter for a median of 12.6 hours, and ALC was maintained at >1000 cells per microliter for up to 16.9 hours. Continued follow-up on the extension study resulted in a yearly infection rate that decreased from 4.63 events (95% confidence interval, 3.3-6.3) in the 12 months prior to the trial to 2.27 events (95% confidence interval, 1.4-3.5) for patients on effective doses. We observed an average 75% reduction in the number of cutaneous warts. This study demonstrates that mavorixafor, 400 mg once daily, mobilizes neutrophil and lymphocytes in adult patients with WHIM syndrome and provides preliminary evidence of clinical benefit for patients on long-term therapy. The trial was registered at www.clinicaltrials.gov as #NCT03005327.

AMD3100 (plerixafor), a CXCR4 antagonist, has opened a variety of avenues for potential therapeutic approaches in different refractory diseases. The CXCL12/CXCR4 axis and its signaling pathways are involved in diverse disorders including HIV-1 infection, tumor development, non-Hodgkin lymphoma, multiple myeloma, WHIM Syndrome, and so on. The mechanisms of action of AMD3100 may relate to mobilizing hematopoietic stem cells, blocking infection of X4 HIV-1, increasing circulating neutrophils, lymphocytes and monocytes, reducing myeloid-derived suppressor cells, and enhancing cytotoxic T-cell infiltration in tumors. Here, we first revisit the pharmacological discovery of AMD3100. We then review monotherapy of AMD3100 and combination use of AMD3100 with other agents in various diseases. Among those, we highlight the perspective of AMD3100 as an immunomodulator to regulate immune responses particularly in the tumor microenvironment and synergize with other therapeutics. All the pre-clinical studies support the clinical testing of the monotherapy and combination therapies with AMD3100 and further development for use in humans.

CXCL12 is a CXC chemokine that traditionally has been classified as a homeostatic chemokine. It contributes to physiological processes such as embryogenesis, hematopoiesis and angiogenesis. In contrast to these homeostatic functions, increased expression of CXCL12 in general, or of a specific CXCL12 splicing variant has been demonstrated in various pathologies. In addition to this increased or differential transcription of CXCL12, also upregulation of its receptors CXC chemokine receptor 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3) contributes to the onset or progression of diseases. Moreover, posttranslational modification of CXCL12 during disease progression, through interaction with locally produced molecules or enzymes, also affects CXCL12 activity, adding further complexity. As CXCL12, CXCR4 and ACKR3 are broadly expressed, the number of pathologies wherein CXCL12 is involved is growing. In this review, the role of the CXCL12/CXCR4/ACKR3 axis will be discussed for the most prevalent pathologies. Administration of CXCL12-neutralizing antibodies or small-molecule antagonists of CXCR4 or ACKR3 delays disease onset or prevents disease progression in cancer, viral infections, inflammatory bowel diseases, rheumatoid arthritis and osteoarthritis, asthma and acute lung injury, amyotrophic lateral sclerosis and WHIM syndrome. On the other hand, CXCL12 has protective properties in Alzheimer's disease and multiple sclerosis, has a beneficial role in wound healing and has crucial homeostatic properties in general.

Chemokines are named and best known for their chemotactic cytokine activity in the hematopoietic system; however, their importance extends far beyond leukocytes, cell movement and immunoregulation. CXCL12, the most protean of chemokines, regulates development in multiple systems, including the hematopoietic, cardiovascular and nervous systems, and regulates diverse cell functions, including differentiation, distribution, activation, immune synapse formation, effector function, proliferation and survival in the immune system alone. The broad importance of CXCL12 is revealed by the complex lethal developmental phenotypes in mice lacking either Cxcl12 or either one of its two known 7-transmembrane domain receptors Cxcr4 and Ackr3, as well as by gain-of-function mutations in human CXCR4, which cause WHIM syndrome, a multisystem and combined immunodeficiency disease and the only Mendelian condition caused by a chemokine system mutation. In addition, wild type CXCR4 is important in the pathogenesis of HIV/AIDS and cancer. Thus, CXCL12 and its receptors CXCR4 and ACKR3 provide extraordinary examples of multisystem multitasking in the chemokine system in both health and disease.

Introduction: WHIM (Warts, Hypogammaglobulinemia, Infections, and Myelokathexis) syndrome is an inborn error of immunity characterized as a primary immunodeficiency with neutropenia-but the acronym does not reflect the broad spectrum of disease manifestations that patients may experience. A WHIM syndrome diagnosis may be confirmed clinically by the presence of myelokathexis, the retention of white blood cells in the bone marrow, or by identification of a known pathogenic gain-of-function mutation in the CXCR4 gene coding for the CXCR4 receptor. Diagnosis of WHIM syndrome is thought to be frequently missed because of low disease awareness, missed identification of myelokathexis, and lack of routine genetic testing (Al Ustwani O, et al. Br J Haematol. 2014:164;15-23; Dotta L, et al. Curr Mol Med. 2011;11:317-325; Heusinkveld L, et al. Exp Opin Orphan Drugs. 2017;5(10):813-825). The prevalence of WHIM syndrome has never been systematically studied and is unknown. Determination of prevalence via insurance claims data is hindered by the absence of an International Classification of Diseases (ICD)-10 code for WHIM syndrome as well as inconsistent coding for key symptoms of WHIM syndrome, which are variably penetrant. This study applied an artificial intelligence (AI)/machine learning (ML) model to estimate the potential prevalence of WHIM syndrome using a large US insurance claims database. Methods: A deidentified, longitudinal, patient-level US claims database of >300 million lives was used for this study. Thirty-two patients with genetically confirmed WHIM syndrome were identified from the claims database by linking deidentified patients to known physicians and matching clinical and demographic features. Using this group as a positive training class, an AI/ML model was deployed to identify patients with WHIM look-alike clinical phenotypes in the database. Patients were further filtered based on clinical features to generate low (presence of warts, history of infections, and hypogammaglobulinemia) and high (presence of warts, history of infections, and coding associated with immunodeficiency) prevalence estimates; a final prevalence number for the US was projected to account for incomplete coverage of the US population in the claims database. Finally, insurance codes for disease symptoms, treatments, and management were analyzed to investigate the burden of disease in patients identified by the model. Results: The model showed a high predictive value for distinguishing patients with known WHIM syndrome from a random sample of age-matched patients in the database (area under the curve [AUC] of receiver operating characteristic [ROC] plot, >0.99) as well as a control group of patients with ICD-10 codes defining immunodeficiency conditions (AUC of ROC plot, 0.99). The model generated estimates ranging from 1803 (low) to 3718 (high) patients with WHIM look-alike phenotype in the US. Analysis of medical history in the high-estimate WHIM look-alike group revealed symptomatic and severe disease, as evidenced by ≥1 instance of use of granulocyte colony-stimulating factor (41%) or intravenous immunoglobulin (46%) therapy (both Conclusions: The methodology used here provides an approach to explore the prevalence of rare diseases that are often mis- or under-diagnosed and are not captured with a unique ICD-10 code. This study estimates a prevalence of 1803 to 3718 WHIM look-alike patients in the US, supporting the possibility that there may be ≤~3700 patients with either diagnosed or undiagnosed WHIM syndrome in the US. An analysis of the medical history of the WHIM look-alike patients revealed a history of symptomatic and severe disease and a high unmet medical need. Since it is not feasible to definitively confirm a WHIM diagnosis in the look-alike group, it is possible that some of these look-alike patients may have diagnosed or undiagnosed WHIM syndrome, while others may have a clinical phenotype consistent with WHIM syndrome without meeting its classic diagnostic criteria. Disclosures Garabedian: X4 Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Neri: X4 Pharmaceuticals: Current Employment. Seng: X4 Pharmaceuticals: Current Employment. Jones: Real Chemistry (Formerly Swoop/IPM): Current Employment, Other: I was paid salary to perform secondary research project work for client "X4" which resulted in this publication.

Background: WHIM (Warts, Hypogammaglobulinemia, Infections, and Myelokathexis) syndrome is a rare primary immunodeficiency associated with broad cytopenia, including neutropenia. It is caused by gain-of-function mutations in C XCR4, leading to dysregulated immune cell trafficking with retention of neutrophils, lymphocytes, and monocytes in the bone marrow and in some cases, hypogammaglobulinemia. As a result, patients with WHIM syndrome have recurrent bacterial and viral infections, and unusual susceptibility to human papillomavirus infection predisposing individuals to recalcitrant warts and malignancy (McDermott D, et al. Immunol Rev. 2019;91-102). Therapeutic options are limited and do not address the underlying pathogenic mechanism of WHIM syndrome. The investigational oral CXCR4 antagonist mavorixafor directly targets the underlying cause of disease and has been shown to increase absolute neutrophil, lymphocyte, and monocyte counts, and to decrease annualized infection rate, and reduce cutaneous wart burden in a phase 2 trial of adults with WHIM syndrome (NCT03005327; Dale D, et al. Blood. 2020;136: 2994-3003). Findings from an ongoing long-term extension of this study support a sustained clinical benefit of long-term mavorixafor treatment in patients with WHIM syndrome. Here, we describe the design of a global phase 3 registrational trial evaluating the safety and efficacy of mavorixafor in WHIM syndrome in participants aged ≥12 years while reporting on preliminary baseline characteristics of the enrolled population. Methods: This phase 3 trial (4WHIM; NCT03995108) is a randomized, double-blind, placebo-controlled study with open-label extension (OLE) and planned enrollment of 18 to 28 patients from sites in Asia, Australia, Europe, Israel, and the United States. Patients aged ≥12 years with a confirmed CXCR4 mutation consistent with WHIM syndrome phenotype and a screening absolute neutrophil count (ANC) ≤400 cells/µL without clinical evidence of active systemic infection are eligible for enrollment. Patients are randomized 1:1 to receive mavorixafor (400 mg in adults, and adolescents weighing >50 kg; 200 mg in adolescents weighing ≤50 kg) or matching placebo once daily for a total of 52 weeks (≥9 patients per group). Patients who complete the randomized period or are granted early release due to recurrent infections requiring treatment (≤2 requiring hospitalization or 4 requiring intravenous antibiotic or granulocyte colony-stimulating factor) are eligible to enroll in the OLE and receive mavorixafor until commercial availability or study termination. The primary end point is the number of hours above ANC threshold of 500 cells/µL over a 24-hour period, assessed prior to treatment, and 4 times (every 3 months) over the 52-week randomized period. Secondary end points include infection rates adjudicated by a blinded, independent committee, change from baseline in cutaneous warts also with blinded assessment, number of hours above absolute lymphocyte count (ALC) of 1000 cells/µL over a 24-hour period, and patient-reported outcomes such as work/school absence and quality of life assessment using age-appropriate questionnaires. Patient Demographics and Characteristics: As of July 15 2021, the first 18 patients have enrolled from 10 countries and 15 sites. Of these, 56% are pediatric patients, 56% are males, 56% of the patients have warts, 94% have nonsense and 6% have frameshift CXCR4 mutations. All patients had severe neutropenia (ANC ≤400 cells/μL) and majority had significant lymphopenia (ALC ≤1000 cells/μL). Conclusions: Evidence supporting efficacy of current therapies in WHIM syndrome is lacking, and approved therapies targeting the underlying molecular mechanisms and hence, able to address the full clinical spectrum of this condition, are needed. The 4WHIM study is the first double-blind, placebo-controlled, randomized trial in patients with this syndrome and is an important next step in the clinical development of a novel, orally bioavailable targeted therapy for WHIM syndrome and potentially for other individuals with related cellular immunodeficiencies. This robust study will build on the findings of the phase 2 trial that suggested a clinical benefit of mavorixafor for WHIM syndrome by incorporating a broader, global population of patients observed for a longer period of time. Full phase 3 trial results are anticipated in late 2022. Disclosures Dale: X4 Pharmaceuticals: Consultancy, Honoraria, Research Funding. Azar: X4 Pharmaceuticals: Research Funding. Badolato: Angelini: Consultancy; X4 Pharmaceuticals: Consultancy; Janssen: Consultancy; SOBI (IDMC): Other. Bhandari: X4 Pharmaceuticals: Current Employment. Belschner: X4 Pharmaceuticals: Current Employment. Cadavid: X4 Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Dickerson: Bluebird Bio: Membership on an entity's Board of Directors or advisory committees. Hoffman: Zomagen: Research Funding; Novartis: Consultancy, Speakers Bureau; Takeda: Research Funding; Jecure: Research Funding. Jiang: X4 Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Kang: Cartexell: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Korea: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen Korea: Membership on an entity's Board of Directors or advisory committees, Research Funding. Kulagin: Roche: Speakers Bureau; Sanofi: Speakers Bureau; Generium: Speakers Bureau; Biocad: Research Funding; Apellis: Research Funding; Alexion: Research Funding; X4 Pharmaceuticals: Research Funding; Novartis: Speakers Bureau; Johnson & Johnson: Speakers Bureau; Pfizer: Speakers Bureau. Langguth: RCPA: Membership on an entity's Board of Directors or advisory committees. MacLeod: X4 Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Sharathkumar: Amgen: Research Funding; BMS: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Membership on an entity's Board of Directors or advisory committees, Research Funding; Shire: Membership on an entity's Board of Directors or advisory committees, Research Funding; X4 Pharmaceuticals: Research Funding; NIH Trials of Pediatric VTE: Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees. Shcherbina: X4 Pharmaceuticals: Speakers Bureau. Tang: X4 Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Vossen: X4 Pharmaceuticals: Other: PI in WHIM Trial; Brothers of St. John: Consultancy; Austrian National Bank: Research Funding; Menarini: Honoraria; Gilead: Honoraria; Astro Pharma: Honoraria.

Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is a genetic disease characterized by neutropenia, lymphopenia, susceptibility to infections, and myelokathexis, which describes degenerative changes of mature neutrophils and hyperplasia of bone marrow myeloid cells. Some patients present with hypogammaglobulinemia and/or refractory warts of skin and genitalia. Congenital cardiac defects constitute uncommon manifestations of the disease. The disorder, which is inherited as an autosomal dominant trait, is caused by heterozygous mutations of the chemokine receptor CXCR4. These mutations lead to an increased sensitivity of neutrophils and lymphocytes to the unique ligand CXCL12 and to an increased accumulation of mature neutrophils in the bone marrow. Despite greatly improved knowledge of the disease, therapeutic choices are insufficient to prevent some of the disease outcomes, such as development of bronchiectasis, anogenital dysplasia, or invasive cancer. The available therapeutic measures aimed at preventing the risk for infection in WHIM patients are discussed. We critically evaluate the diagnostic criteria of WHIM syndrome, particularly when WHIM syndrome should be suspected in patients with congenital neutropenia and lymphopenia despite the absence of hypogammaglobulinemia and/or warts. Finally, we discuss recent results of trials evaluating plerixafor, a selective antagonist of CXCR4, as a mechanism-oriented strategy for treatment of WHIM patients.

WHIM-09 is the first patient described with WHIM syndrome, an autosomal dominant form of neutropenia related to bone marrow retention of neutrophils. Originally diagnosed incorrectly with autoimmune neutropenia, the patient underwent splenectomy at age 9, but the absolute neutrophil count (ANC) did not rise. Subsequently, she was spontaneously cured by chromothripsis (chromosome shattering), which deleted the disease allele CXCR4 R334X , and 163 other genes, on chromosome 2 in a single hematopoietic stem cell (HSC). Chromothriptic CXCR4 +/o HSCs replaced CXCR4 +/R334X WHIM HSCs, and the ANC rose to a new sustained and benign baseline ~ 2-3-fold above normal that had remained unexplained. Here, we show that splenectomized Cxcr4 +/o mice had sustained and benign neutrophilia, phenocopying neutrophilia in WHIM-09. In addition, WHIM-09's granulocyte-macrophage precursor cells possessed increased granulocyte colony-forming activity ex vivo. Thus, WHIM-09's neutrophilia may be multifactorial, involving neutrophil-extrinsic factors (splenectomy), as well as CXCR4 haploinsufficiency-dependent neutrophil-intrinsic factors (increased myeloid precursor cell differentiation). The strong bone marrow retention signal for neutrophils conferred by the WHIM mutation may have prevented neutrophilia after splenectomy until the mutation was deleted by chromothripsis.

WHIM syndrome is a rare combined primary immunodeficiency disorder caused by autosomal dominant gain-of-function mutations in the chemokine receptor CXCR4. It is the only Mendelian condition known to be caused by mutation of a chemokine or chemokine receptor. As such, it provides a scientific opportunity to understand chemokine-dependent immunoregulation in humans and a medical opportunity to develop mechanism-based treatment and cure strategies.This review covers the clinical features, genetics, immunopathogenesis and clinical management of WHIM syndrome. Clinical trials of targeted therapeutic agents and potential cure strategies are also included.WHIM syndrome may be particularly amenable to mechanism-based therapeutics for three reasons: 1) CXCR4 has been validated as the molecular target in the disease by Mendelian genetics; 2) the biochemical abnormality is excessive CXCR4 signaling; and 3) antagonists selective for CXCR4 have been developed. Plerixafor is FDA-approved for hematopoietic stem cell (HSC) mobilization and has shown preliminary safety and efficacy in phase I clinical trials in WHIM syndrome. Gene editing may represent a viable cure strategy, since chromothriptic deletion of the disease allele in HSCs resulted in clinical cure of a patient and because CXCR4 haploinsufficiency enhances engraftment of transplanted HSCs in mice.

WHIM syndrome is a rare congenital immunodeficiency disease, named after its main clinical manifestations: warts, hypogammaglobulinemia, infections, and myelokathexis, which refers to abnormal accumulation of mature neutrophils in the bone marrow. The disease is primarily caused by C-terminal truncation mutations of the chemokine receptor CXCR4, giving these CXCR4-WHIM mutants a gain of function in response to their ligand CXCL12. Considering the broad functions of CXCR4 in maintaining leukocyte homeostasis, patients are panleukopenic and display altered immune responses, likely as a consequence of impairment in the differentiation and trafficking of leukocytes. Treatment of WHIM patients currently consists of symptom relief, leading to unsatisfactory clinical responses. As an alternative and potentially more effective approach, we tested the potency and efficacy of CXCR4-specific nanobodies on inhibiting CXCR4-WHIM mutants. Nanobodies are therapeutic proteins based on the smallest functional fragments of heavy chain antibodies. They combine the advantages of small-molecule drugs and antibody-based therapeutics due to their relative small size, high stability, and high affinity. We compared the potential of monovalent and bivalent CXCR4-specific nanobodies to inhibit CXCL12-induced CXCR4-WHIM-mediated signaling with the small-molecule clinical candidate AMD3100. The CXCR4-targeting nanobodies displace CXCL12 binding and bind CXCR4-wild type and CXCR4-WHIM (R334X/S338X) mutants and with (sub-) nanomolar affinities. The nanobodies' epitope was mapped to extracellular loop 2 of CXCR4, overlapping with the binding site of CXCL12. Monovalent, and in particular bivalent, nanobodies were more potent than AMD3100 in reducing CXCL12-mediated G protein activation. In addition, CXCR4-WHIM-dependent calcium flux and wound healing of human papillomavirus-immortalized cell lines in response to CXCL12 was effectively inhibited by the nanobodies. Based on these in vitro results, we conclude that CXCR4 nanobodies hold significant potential as alternative therapeutics for CXCR4-associated diseases such as WHIM syndrome.

Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome immunodeficiency is caused by autosomal dominant gain-of-function mutations in chemokine receptor CXCR4. Patient WHIM-09 was spontaneously cured by chromothriptic deletion of 1 copy of 164 genes, including the CXCR4(WHIM) allele, presumably in a single hematopoietic stem cell (HSC) that repopulated HSCs and the myeloid lineage. Testing the specific contribution of CXCR4 hemizygosity to her cure, we previously demonstrated enhanced engraftment of Cxcr4(+/o) HSCs after transplantation in WHIM (Cxcr4(+/w)) model mice, but the potency was not quantitated. We now report graded-dose competitive transplantation experiments using lethally irradiated Cxcr4(+/+) recipients in which mixed BM cells containing approximately 5 Cxcr4(+/o) HSCs and a 100-fold excess of Cxcr4(+/w) HSCs achieved durable 50% Cxcr4(+/o) myeloid and B cell chimerism in blood and approximately 20% Cxcr4(+/o) HSC chimerism in BM. In Cxcr4(+/o)/Cxcr4(+/w) parabiotic mice, we observed 80%–100% Cxcr4(+/o) myeloid and lymphoid chimerism in the blood and 15% Cxcr4(+/o) HSC chimerism in BM from the Cxcr4(+/w) parabiont, which was durable after separation from the Cxcr4(+/o) parabiont. Thus, CXCR4 haploinsufficiency likely significantly contributed to the selective repopulation of HSCs and the myeloid lineage from a single chromothriptic HSC in WHIM-09. Moreover, the results suggest that WHIM allele silencing of patient HSCs is a viable gene therapy strategy.

AMD3100 (plerixafor, Mozobil®) was first identified as an anti-HIV agent specifically active against the T4-lymphotropic HIV strains, as it selectively blocked the CXCR4 receptor. Through interference with the interaction of CXCR4 with its natural ligand, SDF-1 (also named CXCL12), it also mobilized the CD34+stem cells from the bone marrow into the peripheral blood stream. In December 2008, AMD3100 was formally approved by the US FDA for autologous transplantation in patients with Non-Hodgkin's Lymphoma or multiple myeloma. It may be beneficially used in various other malignant diseases as well as hereditary immunological disorders such as WHIM syndrome, and physiopathological processes such as hepatopulmonary syndrome. ispartof: Antivir Chem Chemother vol:27 pages:2040206619829382- ispartof: location:England status: published

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.

WHIM syndrome (warts, hypogammaglobulinemia, infections, and myelokathexis), a primary immunodeficiency disorder involving panleukopenia, is caused by autosomal dominant gain-of-function mutations in CXC chemokine receptor 4 (CXCR4). Myelokathexis is neutropenia caused by neutrophil retention in bone marrow. Patients with WHIM syndrome are often treated with granulocyte colony-stimulating factor (G-CSF), which can increase neutrophil counts but does not affect cytopenias other than neutropenia. In this investigator-initiated, open-label study, three severely affected patients with WHIM syndrome who could not receive G-CSF were treated with low-dose plerixafor, a CXCR4 antagonist, for 19 to 52 months. Myelofibrosis, panleukopenia, anemia, and thrombocytopenia were ameliorated, the wart burden and frequency of infection declined, human papillomavirus-associated oropharyngeal squamous-cell carcinoma stabilized, and quality of life improved markedly. Adverse events were mainly infections attributable to the underlying immunodeficiency. One patient died from complications of elective reconstructive surgery. (Funded by the National Institutes of Health.).

Primary immunodeficiency diseases (PIDs) comprise a genetically heterogeneous group of rare disorders that are caused by genetic defects or developmental defects of the immune system.

Chemokines and chemokine receptors regulate multiple processes such morphogenesis, angiogenesis and immune responses. Among the chemokine receptors, CXCR4 stands out for its pleiotropic roles as well as for its involvement in several pathological conditions, including immune diseases, viral infections and cancer. For these reasons, CXCR4 represents a crucial target in drug development. In this review, we discuss of CXCR4 receptor properties and signaling in health and diseases, focusing on the WHIM syndrome, an inherited immunodeficiency caused by mutations of the CXCR4 gene.

WHIM syndrome is an autosomal dominant immunodeficiency disease caused by mutations affecting the carboxy-terminus of CXCR4. To characterize novel genetic causes of the syndrome, we recruited a pediatric patient with possible WHIM syndrome, performed CXCR4 gene sequencing and compared his clinical phenotype and CXCR4 tail amino acid sequences with other patients with WHIM syndrome carrying CXCR4 (R334X) mutations. We identified and biochemically characterized a heterozygous 5 base pair deletion (nucleotides 986-990) located in the portion of the open reading frame (ORF) of CXCR4 that encodes the carboxy-terminal domain of the receptor. This CXCR4 (L329fs) mutation causes a frame-shift at codon 329 resulting in replacement of the final 24 predicted amino acids of the receptor with 12 missense amino acids. Like previously reported WHIM mutations, this frame-shift mutation CXCR4 (L329fs) decreased receptor downregulation in response to the CXCR4 agonist CXCL12 in patient PBMCs as well as in transfected K562 and HEK 293 cells, but increased calcium flux responses in K562 cells to CXCL12 stimulation. Thus, CXCR4 (L329fs) appears to be a de novo autosomal dominant frame-shift gain-of-function mutation that like other carboxy-terminus mutations causes WHIM syndrome. The same CXCR4 (L329fs) frame-shift variant has been reported to occur in tumor cells from a patient with Waldenström's Macroglobulemia (WM), but is caused by a distinct genetic mechanism: insertion of a single nucleotide in the L329 codon, providing additional evidence that the carboxy-terminus of CXCR4 is a genetic hotspot for mutation.

Given its pleiotropic functions, including its prominent role in inflammation, immune responses and cancer, the C-X-C chemokine receptor type 4 (CXCR4) has gained significant attention in recent years and has become a relevant target in drug development. Although the signaling properties of CXCR4 have been extensively studied, several aspects deserve deeper investigations. Mutations in the C-term tail of the CXCR4 gene cause WHIM syndrome, a rare congenital immunodeficiency associated by chronic leukopenia. Similar mutations have also been recently identified in 30% of patients affected by Waldenstrom’s macroglobulinaemia, a B-cell neoplasia with bone marrow accumulation of malignant cells. An ample body of work has been generated to define the impact of WHIM mutations on CXCR4 signaling properties and evaluate their role on pathogenesis, diagnosis, and response to therapy, although the identity of disease-causing signaling pathways and their relevance for disease development in different genetic variants are still open questions. This review discusses the current knowledge on biochemical properties of CXCR4 mutations to identify their prototypic signaling profile potentially useful to highlighting novel opportunities for therapeutic intervention.

The WHIM syndrome is a rare immunodeficiency characterized by Warts, Hypogammaglobulinemia, recurrent respiratory bacterial Infections, and Myelokathexis. Early studies identified that neutrophils in WHIM patients are retained in the bone marrow; severe peripheral neutropenia and lymphocytopenia were also observed. Infections of the upper respiratory tracts and lesions due to human papillomavirus (HPV) are common. Association with tetralogy of Fallot and Waldenstrom macroglobulinemia was described. The WHIM syndrome is mostly caused by heterozygous mutations on chromosome 2q21 that truncate the C-terminal tail of the chemokine receptor CXCR4, the cognate receptor to chemokine CXCL12. The CXCR4-CXCL12 interaction plays a key role in the recruitment of hematopoietic progenitors into the bone marrow and in the regulation of lymphocyte trafficking within secondary lymphoid organs. Current recommendations for WHIM syndrome include vaccinations and antibiotics to prevent infections, as well as usage of G-CSF and as an immune cell-mobilizing agent to combat myelokathexis. Intravenous immunoglobulin (IVIG) injections can be used to treat hypogammaglobulinemia.

Cysteine-X-cysteine chemokine receptor 4 (CXCR4) is a broadly expressed and multifunctional G protein-coupled chemokine receptor critical for organogenesis, hematopoiesis, and antimicrobial host defense. In the hematopoietic system, the binding of CXCR4 to its cognate chemokine ligand, CXCL12, mediates leukocyte trafficking, distribution, survival, activation, and proliferation. Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is a rare, autosomal dominant, combined immunodeficiency disorder caused by mutations in the C-terminus of CXCR4 that prevent receptor downregulation and therefore result in pathologically increased signaling. The “M„ in the acronym WHIM refers to myelokathexis, the retention of neutrophils in the bone marrow resulting in neutropenia, which explains in part the increased susceptibility to bacterial infection. However, WHIM patients also present with B and T lymphopenia, which may explain the susceptibility to human papillomavirus (HPV), the cause of warts. The impact of WHIM mutations on lymphocytes and adaptive immunity has received less attention than myelokathexis and is the focus of this review.

WHIM syndrome is a rare, autosomal dominant immunodeficiency which is named for the four key manifestations: Warts, Hypogammaglobulinemia, Infections, and Myelokathexis. It results from heterozygous gain-of-function mutations in the chemokine receptor CXCR4 which is widely expressed on leukocytes and has profound influences on immune system homeostasis and organogenesis. New treatments for the disease using drugs to reduce CXCR4 function are excellent examples of precision medicine. Since CXCR4 and its ligand CXCL12 play an important role in a variety of infectious, inflammatory, autoimmune, and malignant diseases, the study of WHIM syndrome provides important insights into both the physiologic and disease roles of these molecules.

For gene therapy of gain-of-function autosomal dominant diseases, either correcting or deleting the disease allele is potentially curative. To test whether there may be an advantage of one approach over the other for WHIM (warts, hypogammaglobulinemia, infections, and myelokathexis) syndrome - a primary immunodeficiency disorder caused by gain-of-function autosomal dominant mutations in chemokine receptor CXCR4 - we performed competitive transplantation experiments using both lethally irradiated WT (Cxcr4+/+) and unconditioned WHIM (Cxcr4+/w) recipient mice. In both models, hematopoietic reconstitution was markedly superior using BM cells from donors hemizygous for Cxcr4 (Cxcr4+/o) compared with BM cells from Cxcr4+/+ donors. Remarkably, only approximately 6% Cxcr4+/o hematopoietic stem cell (HSC) chimerism after transplantation in unconditioned Cxcr4+/w recipient BM supported more than 70% long-term donor myeloid chimerism in blood and corrected myeloid cell deficiency in blood. Donor Cxcr4+/o HSCs differentiated normally and did not undergo exhaustion as late as 465 days after transplantation. Thus, disease allele deletion resulting in Cxcr4 haploinsufficiency was superior to disease allele repair in a mouse model of gene therapy for WHIM syndrome, allowing correction of leukopenia without recipient conditioning.

Although some members of the viral family Papillomaviridae cause benign skin warts (papillomas), many human papillomavirus (HPV) infections are not associated with visible symptoms. For example, most healthy adults chronically shed Gammapapillomavirus (Gamma) virions from apparently healthy skin surfaces. To further explore the diversity of papillomaviruses, we performed viromic surveys on immunodeficient individuals suffering from florid skin warts. Our results nearly double the number of known Gamma HPV types and suggest that WHIM syndrome patients are uniquely susceptible to Gamma HPV-associated skin warts. Preliminary results suggest that treatment with the drug plerixafor may promote resolution of the unusual Gamma HPV skin warts observed in WHIM patients., Several immunodeficiencies are associated with high susceptibility to persistent and progressive human papillomavirus (HPV) infection leading to a wide range of cutaneous and mucosal lesions. However, the HPV types most commonly associated with such clinical manifestations in these patients have not been systematically defined. Here, we used virion enrichment, rolling circle amplification, and deep sequencing to identify circular DNA viruses present in skin swabs and/or wart biopsy samples from 48 patients with rare genetic immunodeficiencies, including patients with warts, hypogammaglobulinemia, infections, myelokathexis (WHIM) syndrome, or epidermodysplasia verruciformis (EV). Their profiles were compared with the profiles of swabs from 14 healthy adults and warts from 6 immunologically normal children. Individual patients were typically infected with multiple HPV types; up to 26 different types were isolated from a single patient (multiple anatomical sites, one time point). Among these, we identified the complete genomes of 83 previously unknown HPV types and 35 incomplete genomes representing possible additional new types. HPV types in the genus Gammapapillomavirus were common in WHIM patients, whereas EV patients mainly shed HPVs from the genus Betapapillomavirus. Preliminary evidence based on three WHIM patients treated with plerixafor, a leukocyte mobilizing agent, suggest that longer-term therapy may correlate with decreased HPV diversity and increased predominance of HPV types associated with childhood skin warts. IMPORTANCE Although some members of the viral family Papillomaviridae cause benign skin warts (papillomas), many human papillomavirus (HPV) infections are not associated with visible symptoms. For example, most healthy adults chronically shed Gammapapillomavirus (Gamma) virions from apparently healthy skin surfaces. To further explore the diversity of papillomaviruses, we performed viromic surveys on immunodeficient individuals suffering from florid skin warts. Our results nearly double the number of known Gamma HPV types and suggest that WHIM syndrome patients are uniquely susceptible to Gamma HPV-associated skin warts. Preliminary results suggest that treatment with the drug plerixafor may promote resolution of the unusual Gamma HPV skin warts observed in WHIM patients.

The CXCL12/CXCR4 signaling axis plays an essential role in the maintenance of hematopoietic stem and progenitor cell (HSPC) homeostasis and constitutes a key pathway through which the niches and HSPCs communicate in the bone marrow (BM). Heterozygous gain-of-function mutations of CXCR4, which engender a truncated receptor and affect its homologous desensitization in response to CXCL12, have been reported in the WHIM Syndrome (WS); a rare immunodeficiency notably characterized by lymphopenia. The mechanisms underpinning this remain obscure. Using a mouse model harboring a naturally occurring WS-linked Cxcr4 gain-of-function mutation, we explored the possibility that the lymphopenia in WS arise from defects at the HSPC level in the BM. We showed that Cxcr4 desensitization is required for proper quiescence/cycling balance of short-term HSCs as well as their differentiation into multipotent progenitors and downstream lymphoid-biased progenitors. Thus, our results suggest that efficient Cxcr4 desensitization is critical for lymphoid differentiation of HSPCs, and its impairment is a key mechanism underpinning the lymphopenia observed in WS mice. The role of Cxcr4 desensitization in regulating such lympho-hematopoiesis process implicated both intrinsic and extrinsic properties, thus raising the question of the impact of a gain-of-Cxcr4-function mutation on BM stroma. Therefore, the main part of my PhD project was dedicated to evaluate using this relevant knock-in model the impact of Cxcr4 desensitization on maintenance of BM mesenchymal elements. We have found unexpectedly that such regulatory mechanism is intrinsically required for regulating quiescence/cycling balance of mesenchymal stem cells (MSCs) and preserving their osteogenic potential through the control of Cxcl12 expression and availability in an autocrine manner. Therefore, these findings support autocrine and paracrine actions of the Cxcl12/Cxcr4 signaling axis within MSCs to regulate osteoblast differentiation while contributing to HSPC niches and hematopoiesis., Le couple CXCL12/CXCR4 joue un rôle essentiel dans le maintien de l’homéostasie des cellules souches et progéniteurs hématopoïétiques (CSPHs) et constitue un axe clé par lequel les niches et les CSPHs communiquent au sein de la moelle osseuse (MO). Des mutations hétérozygotes du gène CXCR4, qui tronquent le domaine C-terminal de la protéine et entraînent un défaut de désensibilisation homologue de CXCR4 et une hypersensibilité à CXCL12, ont été identifiées dans le Syndrome WHIM (SW), une immunodéficience rare caractérisée notamment par une lymphopénie. Les mécanismes sous-jacents de cette anomalie restaient inconnus. Grâce à un modèle murin porteur d’une mutation gain de fonction de Cxcr4 identifiée chez certains patients et phénocopiant la lymphopénie du SW, nous avons exploré la possibilité qu’un défaut de domiciliation, de différenciation ou d’expansion des CSPHs dans la MO soit à l’origine de la lymphopénie circulante. Nous avons mis en évidence que la désensibilisation de Cxcr4 régule la balance quiescence/cycle des CSHs à court terme ainsi que leur différenciation en progéniteurs multipotents et progéniteurs engagés vers le lignage lymphoïde. Nos travaux révèlent donc que la désensibilisation de Cxcr4 est requise à la différenciation lymphoïde des CSPHs et suggèrent que l’absence de ce processus soit à l’origine de la lymphopénie observée chez les souris mutantes et, par extrapolation, chez les patients. Ces altérations lymphoïdes impliquaient à la fois des défauts intrinsèques (CSPHs) et extrinsèques (stroma), ce qui nous a conduit à considérer l’impact de la mutation gain de fonction de Cxcr4 sur le stroma médullaire. Dans ce contexte, l’objectif principal de mon projet de thèse a consisté à investiguer à l’aide du modèle murin du SW le rôle de la désensibilisation de Cxcr4 dans le maintien des composantes mésenchymateuses au sein de la MO. Nos données ont permis de mettre en lumière que la désensibilisation de Cxcr4 est intrinsèquement requise à la régulation de l’équilibre quiescence/cycle des cellules souches mésenchymateuses (CSMs), ainsi qu’à la préservation de leur potentiel ostéogénique en contrôlant l'expression et la biodisponibilité de Cxcl12 de manière autocrine. Par conséquent, nos travaux suggèrent que les actions autocrines et paracrines de l’axe de signalisation Cxcl12/Cxcr4 au sein des CSMs régulent leur différenciation en ostéoblastes tout en contribuant au maintien des niches des CSPHs et au processus d’hématopoïèse.