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.

CXCR4 signaling controls dendritic cell location and activation at steady state and in inflammation.

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

Lymphoid differentiation of hematopoietic stem cells requires efficient Cxcr4 desensitization.

The CXCL12/CXCR4 signaling exerts a dominant role in promoting hematopoietic stem and progenitor cell (HSPC) retention and quiescence in bone marrow. Gain-of-function CXCR4 mutations that affect homologous desensitization of the receptor have been reported in the WHIM Syndrome (WS), a rare immunodeficiency characterized by lymphopenia. The mechanisms underpinning this remain obscure. Using a mouse model with a naturally occurring WS-linked gain-of-function Cxcr4 mutation, we explored the possibility that the lymphopenia in WS arises from defects at the HSPC level. We reported that Cxcr4 desensitization is required for quiescence/cycling balance of murine short-term hematopoietic stem cells and their differentiation into multipotent and downstream lymphoid-biased progenitors. Alteration in Cxcr4 desensitization resulted in decrease of circulating HSPCs in five patients with WS. This was also evidenced in WS mice and mirrored by accumulation of HSPCs in the spleen, where we observed enhanced extramedullary hematopoiesis. Therefore, efficient Cxcr4 desensitization is critical for lymphoid differentiation of HSPCs, and its impairment is a key mechanism underpinning the lymphopenia observed in mice and likely in WS patients.

Symptomatic Improvement in Human Papillomavirus-Induced Epithelial Neoplasia by Specific Targeting of the CXCR4 Chemokine Receptor.

Human papillomavirus (HPV) infection is estimated to be the causal agent in 5% of all human cancers and is the leading cause of genital warts, which is the most common sexually transmitted viral disease. Currently, there are no medications to treat HPV infection, and therapeutic strategies primarily target HPV-related cancer rather than viral infection. HPV infection has severe effects on patients who display selective susceptibility to the virus in the context of primary immunodeficiencies, such as the warts, hypogammaglobulinemia, infections, and myelokathexis syndrome, which is caused by dysfunctions of CXCR4, the receptor for the CXCL12 chemokine. In this study we showed in a transgenic mouse model of HPV-induced epidermal neoplasia the beneficial effects of Cxcl12/Cxcr4 pathway blockade with the selective CXCR4 antagonist AMD3100. Daily treatment with AMD3100 for 28 days potently reduced the abnormal ear epidermal thickening in all mice. This effect was associated with reductions in keratinocyte hyperproliferation and immune cell infiltration, both of which are linked to neoplastic progression. Moreover, we observed the abnormal coordinate expression of Cxcl12 and p16INK4a (a surrogate marker of HPV-induced cancers) in dysplastic epidermal keratinocytes, which was inhibited by AMD3100 treatment. These results provide strong evidence for the therapeutic potential of CXCL12/CXCR4 pathway blockade in HPV-induced pathogenesis.

Altered chemotactic response to CXCL12 in patients carrying GATA2 mutations.

GATA2 deficiency-formerly described as MonoMAC syndrome; dendritic cells, monocytes, B cells, and natural killer cell deficiency; familial myelodysplastic syndrome/acute myeloid leukemia; or Emberger syndrome-encompasses a range of hematologic and nonhematologic anomalies, mainly characterized by monocytopenia, B lymphopenia, natural killer cell cytopenia, neutropenia, immunodeficiency, and a high risk of developing acute myeloid leukemia. Herein, we present 7 patients with GATA2 deficiency recruited into the French Severe Chronic Neutropenia Registry, which enrolls patients with all kinds of congenital neutropenia. We performed extended immunophenotyping of their whole blood lymphocyte populations, together with the analysis of their chemotactic responses. Lymphopenia was recorded for B and CD4(+) T cells in 6 patients. Although only 3 patients displayed natural killer cell cytopenia, the CD56(bright) natural killer subpopulation was nearly absent in all 7 patients. Natural killer cells from 6 patients showed decreased CXCL12/CXCR4-dependent chemotaxis, whereas other lymphocytes, and most significantly B lymphocytes, displayed enhanced CXCL12-induced chemotaxis compared with healthy volunteers. Surface expression of CXCR4 was significantly diminished in the patients' natural killer cells, although the total expression of the receptor was found to be equivalent to that of natural killer cells from healthy individual controls. Together, these data reveal that GATA2 deficiency is associated with impaired membrane expression and chemotactic dysfunctions of CXCR4. These dysfunctions may contribute to the physiopathology of this deficiency by affecting the normal distribution of lymphocytes and thus potentially affecting the susceptibility of patients to associated infections.

Dissection of agonistic and blocking effects of CD200 receptor antibodies.

The CD200 receptor (CD200R) is present mainly on myeloid cells and gives inhibitory signals when engaged by its ligand CD200. The interaction is currently of therapeutic interest in cancer and inflammation. However functional effects are complicated by the fact that CD200R is itself polymorphic and also a member of a paired receptor family with four closely related gene products in mice called CD200RLa etc. We show that a second allele of CD200R (termed CD200R(2)) that differs in 7 amino acids also binds CD200 but did not react with the widely used CD200R antibody OX110. Biochemical and functional analysis showed that the CD200/CD200R interaction was blocked by the OX131, mAb that recognises both CD200R(1) and CD200R(2), but not by OX110 mAb. Both mAb can give agonistic inhibitory signals but functional analysis shows OX131 mAb also has the potential to block inhibition by preventing the ligand-receptor interaction and hence gives opposing effects. Although OX131 mAb cross-reacts with the activating receptor CD200RLe, it is specific for CD200R in C57BL/6 whilst OX110 mAb cross-reacts on CD200RLc. The results show the importance of the repertoire of paired receptors in strains or individuals and mAb used with implications for paired receptor analysis and therapeutics.

The B subunit of Escherichia coli heat-labile enterotoxin inhibits Th1 but not Th17 cell responses in established experimental autoimmune uveoretinitis.

PURPOSE: To investigate the efficacy of the B subunit of Escherichia coli heat-labile enterotoxin (EtxB) in the treatment of ocular autoimmune disease. Murine experimental autoimmune uveoretinitis (EAU) is an animal model of autoimmune posterior uveitis initiated by retinal antigen-specific Th1 and Th17 CD4(+) T cells, which activate myeloid cells, inducing retinal damage. EtxB is a potent immune modulator that ameliorates other Th1-mediated autoimmune diseases, enhancing regulatory T-cell activity. METHODS: EAU was induced in B10.RIII mice by immunization with peptide hIRBP(161-180). Disease severity was measured by clinical and histologic assessment, and functional responses of macrophages (Mphis) and T cells were assessed, both in vivo and in cocultures in vitro. EtxB was administered intranasally daily for 4 days, starting either 3 days before or 3 days after EAU induction. RESULTS: Preimmunization treatment with EtxB protected mice from EAU, limiting both the number and the activation status of retinal infiltrating immune cells. Treatment after EAU induction did not alter the disease course, despite suppression of IFN-gamma. Although EtxB treatment of in vitro cocultures of T cells and Mphis increased IL-10 production, EtxB treatment in vivo increased the proportion and number of IL-17-producing CD4(+) cells infiltrating the eye. CONCLUSIONS: EtxB preimmunization protects mice from EAU induction by inhibiting Th1 responses, but the resultant reduction in IFN-gamma responses by EtxB does not effect infiltration or structural damage in established EAU, where Th17 responses predominate. These data highlight the critical importance of the dynamics of T-cell phenotype and infiltration during EAU when considering immunomodulatory therapy.