The chemokine receptor CXCR4 and its functional ligand, CXCL12, are essential regulators of development and homeostasis of hematopoietic and lymphoid organs. Heterozygous truncating mutations in the CXCR4 intracellular tail cause a rare genetic disease known as WHIM syndrome (warts, hypogammaglobulinemia, infections, myelokathexis), whose pathophysiology remains unclear. We report CXCR4 function in 3 patients with WHIM syndrome carrying heterozygous truncating mutations of CXCR4. We show that CXCR4 gene mutations in WHIM patients do not affect cell surface expression of the chemokine receptor and its internalization upon stimulation with CXCL12. Moreover, no significant differences in calcium mobilization in response to CXCL12 are found. However, the chemotactic response of both polymorphonuclear cells and T lymphocytes in response to CXCL12 is increased. IntroductionWHIM syndrome is a rare disease characterized by warts, hypogammaglobulinemia, recurrent respiratory bacterial infections, and myelokathexis, defined as the presence of an increased proportion of mature myeloid cells with degenerative changes in the bone marrow, associated with severe neutropenia in the peripheral blood. [1][2][3] This condition, most often inherited as an autosomal dominant trait, is caused by heterozygous truncating mutations in the C-terminus tail of the chemokine receptor CXCR4. 4 CXCR4 is the only cognate receptor for the CXC-chemokine L12 (CXCL12, or stromal-derived factor-1␣ ). 5 CXCL12 is constitutively produced by stromal and endothelial cells (ECs) ubiquitously; the highest concentrations of CXCL12 are found in bone marrow, spleen red pulp, and lymph node medulla. CXCR4-CXCL12 interaction plays a key role in regulating bone marrow homeostasis [6][7][8][9] and is involved in lymphocyte trafficking. 10,11 Chemotaxis and integrin-mediated adhesion are the main cellular responses to CXCL12 9,12-16 ; in addition, CXCR4 signaling participates in several cellular activation and proliferation processes. 17,18 Both CXCR4-and CXCL12-deficient mice display a lethal phenotype, with severe impairment of myeloid and B-cell generation, reduced proliferation of both triple-negative and doublepositive thymocytes, and developmental defects in cerebellum, heart, and gut vascularization. These abnormalities illustrate that this pair of molecules plays an indispensable role in controlling cell migration and influences (either directly or indirectly) survival/ proliferation of different cell types during embryogenesis. [19][20][21] Mice reconstituted with progenitor cells infected with CXCL12 intrakine (which prevents surface CXCR4 expression) suffer from impaired hematopoiesis that involves both myeloid and lymphoid cell lineages. 22 AMD3100, a pharmacologic CXCR4 antagonist, induces a rapid mobilization of hematopoietic progenitors and mature cells in a dose-dependent manner. 23 In contrast, overexpression of CXCR4 in transgenic T lymphocytes induces their accumulation in the bone marrow and causes a reduction of these cells in peripheral...

IntroductionMendelian predisposition to mycobacterial diseases is characterized by the occurrence of clinically evident infectious episodes sustained by weakly virulent mycobacteria or salmonellae in patients with no other infections. These inherited conditions are caused by germline mutations affecting genes involved in the IFN-␥ and IL-12 signaling pathways. 1,2 However, complete Signal Transducer and Activator of Transcription 1 (STAT1) deficiency is more severe than other deficiencies in IFN-␥ receptor 1 (IFN-␥R1) or IFN-␥ receptor 2 (IFN-␥R2) because STAT1 signaling is common to many transduction pathways and affects not only IFN-␥. [3][4][5] Indeed, STAT1 is also required for the cellular response to many cytokines with antiviral activities, such as IFN-␣/␤/1 or cytokines with immunomodulatory functions on natural killer (NK) and B cells, such as IL-15, IL-21, and IL-27. [6][7][8] After type I IFN stimulation, the receptor is phosphorylated by Janus kinases JAK1 and TYK2, leading to subsequent docking and phosphorylation of both STAT1 and STAT2. Active STAT1/STAT2 heterodimers are released into the cytosol and translocate to the nucleus, where they bind to type I IFN-stimulated response elements (ISREs) in the promoter of target genes. 9,10 Stimulation with IFN-␥ leads to activation of the Janus kinase JAK1 and JAK2, which create a docking site for 2 STAT1 molecules, which are phosphorylated and released into the cytosol as active STAT1 homodimers. The active complex translocates to the nucleus where it binds to the IFN-␥ response region (GRR). 9,11,12 Complete STAT1 deficiency, inherited as an autosomal recessive trait, is characterized by complete lack of STAT1 expression and abolition of STAT1-dependent responses to both IFN-␥ and IFN-␣. These subjects display profound susceptibility to infections with intracellular pathogens and viruses, including life-threatening infections caused by herpes simplex virus, cytomegalovirus (CMV), and Epstein-Barr virus (EBV). 3,4 Partial STAT1 deficiency is inherited as an autosomal recessive or autosomal dominant disease. [13][14][15][16] Patients with heterozygous mutations of STAT1, unlike patients who are homozygous, display an impaired tyrosine phosphorylation or DNA-binding activity in response to IFN-␥ but retain a normal or partial response to IFN-␣ and are not susceptible to viral disease. 13,15,17 We describe here a patient with homozygous STAT1 splicing mutation leading to skipping of exon 3 who developed generalized mycobacterial infections and severe viral disease sustained by CMV. We report that the patient's cells display a complete defect of STAT1 DNA-binding activity after stimulation with IFN-␥ and IFN-␣ and fail to respond at even high doses of these cytokines. These biologic defects are associated with a partial impairment of NK functional activity, which might contribute to the susceptibility of the patient to infections with viral and intracellular pathogens. The online version of this article contains a data supplement.The publication costs of thi...

The treatment of children affected by severe congenital neutropenia (SCN) with G-CSF strongly reduces the risk of sepsis by reversing neutropenia. However, SCN patients who respond to the treatment with the growth factor still have an elevated risk of succumbing to sepsis. Because the disease is usually caused by heterozygous mutations of ELA2, a gene encoding for neutrophil elastase (NE), we have investigated in G-CSF-responder and nonresponder patients affected by SCN the expression of polypeptides that constitute the antimicrobial machinery of these cells. In peripheral blood-derived neutrophils of patients with heterozygous mutations of ELA2 who were treated with G-CSF, NE was nearly absent as detected by immunofluorescence and immunoblotting, suggesting that production of the mutant protein interferes with normal gene expression. This defect was associated with abnormal expression of other granule-associated proteins such as myeloperoxidase, lactoferrin, cathepsin G, and human-neutrophil-peptide.Moreover, in one patient with partial response to G-CSF, we observed an impairment of neutrophil antimicrobial activity against Candida albicans, and, to a lower extent against Escherichia coli. Thereby, we propose that the treatment with G-CSF is not sufficient to correct all of the functional deficiency of neutrophils, and this might account for the consistent risk of infections observed in SCN patients. IntroductionNeutrophils are essential components of the innate immune system because they constitute the first line of defense against bacterial and fungal pathogens. An efficient response against these microorganisms requires that neutrophils carry a fully operational machinery, including proteases, antimicrobial peptides, and reactive oxygen. 1,2 In contrast, a reduction of neutrophil blood counts or a defect in their antimicrobial apparatus exposes the host to threats from many pathogens as observed in chronic granulomatous disease and in other functional defects of phagocytes. [3][4][5][6] Severe congenital neutropenia (SCN) is an uncommon hematologic disorder characterized by reduction of absolute neutrophil counts (ANCs; usually Ͻ 0.2 ϫ 10 9 cells/L), due to maturation arrest of neutrophil precursors in the bone marrow at the promyelocyte stage. If left untreated, the large majority of children affected by SCN dies in the first years of life from invasive infections. 7,8 However, the empiric use of the polypeptide granulocyte colonystimulating factor (G-CSF) for the treatment of SCN has drastically changed the clinical outcome of this condition by increasing absolute neutrophil count values and reducing the episodes of infection in the vast majority of patients. [7][8][9][10] Nonetheless, SCN patients who are receiving G-CSF are at high risk of myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). In particular, a higher risk of MDS/AML is observed in SCN children who display a poor response to the treatment with G-CSF and/or receive large doses of the growth factor (above 8 g/kg per day). In addi...

Key Points• The CXCR4 mutations in WHIM syndrome impair the stability of the T cell-antigen-presenting cell immunologic synapse.WHIM (warts, hypogammaglobulinemia, infections, myelokathexis) syndrome is a rare disease characterized by diverse symptoms indicative of aberrantly functioning immunity. It is caused by mutations in the chemokine receptor CXCR4, which impair its intracellular trafficking, leading to increased responsiveness to chemokine ligand and retention of neutrophils in bone marrow. Yet WHIM symptoms related to adaptive immunity, such as delayed IgG switching and impaired memory B-cell function, remain largely unexplained. We hypothesized that the WHIM-associated mutations in CXCR4 may affect the formation of immunologic synapses between T cells and antigen-presenting cells (APCs). We show that, in the presence of competing external chemokine signals, the stability of T-APC conjugates from patients with WHIM-mutant CXCR4 is disrupted as a result of impaired recruitment of the mutant receptor to the immunologic synapse. Using retrogenic mice that develop WHIM-mutant T cells, we show that WHIM-mutant CXCR4 inhibits the formation of long-lasting T-APC interactions in ex vivo lymph node slice time-lapse microscopy. These findings demonstrate that chemokine receptors can affect T-APC synapse stability and allow us to propose a novel mechanism that contributes to the adaptive immune response defects in WHIM patients. (Blood. 2013;122(5):666-673)