Christelle Piperoglou

Induction of B7-H6, a ligand for the natural killer cell–activating receptor NKp30, in inflammatory conditions

B7-H6, a member of the B7 family of immunoreceptors, is as a cell-surface ligand for the NKp30-activating receptor expressed on natural killer cells. B7-H6 is not detected in normal human tissues at steady state but is expressed on tumor cells. However, whether B7-H6 can be expressed in other conditions remains unknown. We analyzed here the pathways that lead to the expression of B7-H6 in nontransformed cells. In vitro, B7-H6 was induced at the surface of CD14(+)CD16(+) proinflammatory monocytes and neutrophils upon stimulation by ligands of Toll-like receptors or proinflammatory cytokines such as interleukin-1β and tumor necrosis factor α. In these conditions, a soluble form of B7-H6 (sB7-H6) was also produced by activated monocytes and neutrophils. In vivo, B7-H6 was expressed on circulating proinflammatory CD14(+)CD16(+) monocytes in a group of patients in sepsis conditions, and was linked to an increased mortality. sB7-H6 was selectively detected in the sera of patients with gram-negative sepsis and was associated with membrane vesicles that co-sedimented with the exosomal fraction. These findings reveal that B7-H6 is not only implicated in tumor immunosurveillance but also participates in the inflammatory response in infectious conditions.

Clinical impact of the NKp30/B7-H6 axis in high-risk neuroblastoma patients

Natural killer cell activity and NKp30/B7-H6 interaction are prognostic biomarkers in high-risk neuroblastoma patients. Unleashing the natural killer within Natural killer cells, a part of the innate immune system, can kill cancer cells. Neuroblastoma is a common pediatric cancer that is difficult to treat, especially in older children with metastatic disease. The immune system helps to control the spread of neuroblastoma, and immune-based treatments for this cancer are under active investigation. Now, Semeraro et al. analyzed the role of natural killer cells in neuroblastoma and sought to understand why they are not always equally effective against the tumor. The authors found that the key lies in the predominant isoform of a receptor that natural killer cells use to interact with neuroblastoma cells and that the balance between isoforms of this receptor on a patient’s cells can help predict survival. The immunosurveillance mechanisms governing high-risk neuroblastoma (HR-NB), a major pediatric malignancy, have been elusive. We identify a potential role for natural killer (NK) cells, in particular the interaction between the NK receptor NKp30 and its ligand, B7-H6, in the metastatic progression and survival of HR-NB after myeloablative multimodal chemotherapy and stem cell transplantation. NB cells expressing the NKp30 ligand B7-H6 stimulated NK cells in an NKp30-dependent manner. Serum concentration of soluble B7-H6 correlated with the down-regulation of NKp30, bone marrow metastases, and chemoresistance, and soluble B7-H6 contained in the serum of HR-NB patients inhibited NK cell functions in vitro. The expression of distinct NKp30 isoforms affecting the polarization of NK cell functions correlated with 10-year event-free survival in three independent cohorts of HR-NB in remission from metastases after induction chemotherapy (n = 196, P < 0.001), adding prognostic value to known risk factors such as N-Myc amplification and age >18 months. We conclude that the interaction between NKp30 and B7-H6 may contribute to the fate of NB patients and that both the expression of NKp30 isoforms on circulating NK cells and the concentration of soluble B7-H6 in the serum may be clinically useful as biomarkers for risk stratification.

T Cell Polarization toward TH2/TFH2 and TH17/TFH17 in Patients with IgG4-Related Disease

IgG4-related disease (IgG4-RD) is a fibro-inflammatory disorder involving virtually every organ with a risk of organ dysfunction. Despite recent studies regarding B cell and T cell compartments, the disease’s pathophysiology remains poorly understood. We examined and characterized subsets of circulating lymphocytes in untreated patients with active IgG4-RD. Twenty-eight consecutive patients with biopsy-proven IgG4-RD were included in a prospective, multicentric study. Lymphocytes’ subsets were analyzed by flow cytometry, with analysis of TH1/TH2/TH17, TFH cells, and cytokine release by peripheral blood mononuclear cells. Results were compared to healthy controls and to patients with primary Sjögren’s syndrome. Patients with IgG4-RD showed an increase of circulating T regulatory, TH2, TH17, and CD4+CXCR5+PD1+ TFH cell subsets. Accordingly, increased levels of IL-10 and IL-4 were measured in IgG-RD patients. TFH increase was characterized by the specific expansion of TFH2 (CCR6−CXCR3−), and to a lesser extent of TFH17 (CCR6+CXCR3−) cells. Interestingly, CD4+CXCR5+PD1+ TFH cells normalized under treatment. IgG4-RD is characterized by a shift of circulating T cells toward a TH2/TFH2 and TH17/TFH17 polarization. This immunological imbalance might be implicated in the disease’s pathophysiology. Treatment regimens targeting such T cells warrant further evaluation.

Innate Lymphoid Cells in Cancer.

The world of lymphocytes has recently expanded. A group of cells, innate lymphoid cells (ILC), has been defined. It includes lymphoid cells that have been known for decades, such as natural killer (NK) cells and lymphoid tissue–inducer (LTi) cells. NK cells recognize a vast array of tumor cells, which they help to eliminate through cytotoxicity and the production of cytokines, such as IFNγ. Advances in our understanding of NK-cell biology have led to a growing interest in the clinical manipulation of these cells in cancer. The other ILCs are found mostly in the mucosae and mucosal-associated lymphoid tissues, where they rapidly initiate immune responses to pathogens without the need for specific sensitization. Here, we outline the basic features of ILCs and review the role of ILCs other than NK cells in cancer. Much of the role of these ILCs in cancer remains unknown, but several findings should lead to further efforts to dissect the contribution of different ILC subsets to the promotion, maintenance, or elimination of tumors at various anatomic sites. This will require the development of standardized reagents and protocols for monitoring the presence and function of ILCs in human blood and tissue samples. Cancer Immunol Res; 3(10); 1109–14. ©2015 AACR.

NKp30 isoforms and NKp30 ligands are predictive biomarkers of response to imatinib mesylate in metastatic GIST patients

ABSTRACT Despite effective targeted therapy acting on KIT and PDGFRA tyrosine kinases, gastrointestinal stromal tumors (GIST) escape treatment by acquiring mutations conveying resistance to imatinib mesylate (IM). Following the identification of NKp30-based immunosurveillance of GIST and the off-target effects of IM on NK cell functions, we investigated the predictive value of NKp30 isoforms and NKp30 soluble ligands in blood for the clinical response to IM. The relative expression and the proportions of NKp30 isoforms markedly impacted both event-free and overall survival, in two independent cohorts of metastatic GIST. Phenotypes based on disbalanced NKp30B/NKp30C ratio (ΔBClow) and low expression levels of NKp30A were identified in one third of patients with dismal prognosis across molecular subtypes. This ΔBClow blood phenotype was associated with a pro-inflammatory and immunosuppressive tumor microenvironment. In addition, detectable levels of the NKp30 ligand sB7-H6 predicted a worse prognosis in metastatic GIST. Soluble BAG6, an alternate ligand for NKp30 was associated with low NKp30 transcription and had additional predictive value in GIST patients with high NKp30 expression. Such GIST microenvironments could be rescued by therapy based on rIFN-α and anti-TRAIL mAb which reinstated innate immunity.

Inherited GINS1 deficiency underlies growth retardation along with neutropenia and NK cell deficiency

Inborn errors of DNA repair or replication underlie a variety of clinical phenotypes. We studied 5 patients from 4 kindreds, all of whom displayed intrauterine growth retardation, chronic neutropenia, and NK cell deficiency. Four of the 5 patients also had postnatal growth retardation. The association of neutropenia and NK cell deficiency, which is unusual among primary immunodeficiencies and bone marrow failures, was due to a blockade in the bone marrow and was mildly symptomatic. We discovered compound heterozygous rare mutations in Go-Ichi-Ni-San (GINS) complex subunit 1 (GINS1, also known as PSF1) in the 5 patients. The GINS complex is essential for eukaryotic DNA replication, and homozygous null mutations of GINS component–encoding genes are embryonic lethal in mice. The patients’ fibroblasts displayed impaired GINS complex assembly, basal replication stress, impaired checkpoint signaling, defective cell cycle control, and genomic instability, which was rescued by WT GINS1. The residual levels of GINS1 activity reached 3% to 16% in patients’ cells, depending on their GINS1 genotype, and correlated with the severity of growth retardation and the in vitro cellular phenotype. The levels of GINS1 activity did not influence the immunological phenotype, which was uniform. Autosomal recessive, partial GINS1 deficiency impairs DNA replication and underlies intra-uterine (and postnatal) growth retardation, chronic neutropenia, and NK cell deficiency.

NK cell compartment in the peripheral blood and spleen in adult patients with primary immune thrombocytopenia.

Immune thrombocytopenic purpura (ITP) is a disease characterized by antibody-mediated platelet destruction. The T- and B-cell subsets have been extensively studied in primary ITP, but the NK cell compartment has been less thoroughly explored. We investigated the NK cell receptor repertoire and the functionality of NK cells in the peripheral blood and spleen in patients with primary ITP. An immunophenotypic analysis of peripheral blood lymphocytes from patients revealed that the numbers of CD19+ B lymphocytes, CD4+ and CD8+ T lymphocytes and CD3-CD56+ NK cells were within the normal range. No major alteration to the expression of distinct inhibitory or activating NK cell receptors was observed. The functionality of NK cells, as evaluated by their ability to degranulate in conditions of natural cytotoxicity or antibody-dependent cell cytotoxicity (ADCC), was preserved in these patients. By contrast, these stimuli induced lower levels of IFNγ production by the NK cells of ITP patients than by those of healthy controls. We then compared the splenic NK cell functions of ITP patients with those of cadaveric heart-beating donors (CHBD) as controls. The splenic NK cells of ITP patients tended to be less efficient in natural cytotoxicity conditions and more efficient in ADCC conditions than control splenic NK cells. Finally, we found that infusions of intravenous immunoglobulin led to the inhibition of NK cell activation through the modulation of the interface between target cells and NK cells.

CK2-regulated schwannomin-interacting protein IQCJ-SCHIP-1 association with AnkG contributes to the maintenance of the axon initial segment.

The axon initial segment (AIS) plays a central role in electrogenesis and in the maintenance of neuronal polarity. Its molecular organization is dependent on the scaffolding protein ankyrin (Ank) G and is regulated by kinases. For example, the phosphorylation of voltage‐gated sodium channels by the protein kinase CK2 regulates their interaction with AnkG and, consequently, their accumulation at the AIS. We previously showed that IQ motif containing J‐Schwannomin‐Interacting Protein 1 (IQCJ‐SCHIP‐1), an isoform of the SCHIP‐1, accumulated at the AIS in vivo. Here, we analyzed the molecular mechanisms involved in IQCJ‐SCHIP‐1‐specific axonal location. We showed that IQCJ‐SCHIP‐1 accumulation in the AIS of cultured hippocampal neurons depended on AnkG expression. Pull‐down assays and surface plasmon resonance analysis demonstrated that AnkG binds to CK2‐phosphorylated IQCJ‐SCHIP‐1 but not to the non‐phosphorylated protein. Surface plasmon resonance approaches using IQCJ‐SCHIP‐1, SCHIP‐1a, another SCHIP‐1 isoform, and their C‐terminus tail mutants revealed that a segment including multiple CK2‐phosphorylatable sites was directly involved in the interaction with AnkG. Pharmacological inhibition of CK2 diminished both IQCJ‐SCHIP‐1 and AnkG accumulation in the AIS. Silencing SCHIP‐1 expression reduced AnkG cluster at the AIS. Finally, over‐expression of IQCJ‐SCHIP‐1 decreased AnkG concentration at the AIS, whereas a mutant deleted of the CK2‐regulated AnkG interaction site did not. Our study reveals that CK2‐regulated IQJC‐SCHIP‐1 association with AnkG contributes to AIS maintenance. The axon initial segment (AIS) organization depends on ankyrin (Ank) G and kinases. Here we showed that AnkG binds to CK2‐phosphorylated IQCJ‐SCHIP‐1, in a segment including 12 CK2‐phosphorylatable sites. In cultured neurons, either pharmacological inhibition of CK2 or IQCJ‐SCHIP‐1 silencing reduced AnkG clustering. Overexpressed IQCJ‐SCHIP‐1 decreased AnkG concentration at the AIS whereas a mutant deleted of the CK2‐regulated AnkG interaction site did not. Thus, CK2‐regulated IQJC‐SCHIP‐1 association with AnkG contributes to AIS maintenance.

Corrigendum: Evidence of innate lymphoid cell redundancy in humans.

Frédéric Vély, Vincent Barlogis, Blandine Vallentin, Bénédicte Neven, Christelle Piperoglou, Thibaut Perchet, Maxime Petit, Nadia Yessaad, Fabien Touzot, Julie Bruneau, Nizar Mahlaoui, Nicolas Zucchini, Catherine Farnarier, Gérard Michel, Despina Moshous, Stéphane Blanche, Arnaud Dujardin, Hergen Spits, Jörg H W Distler, Andreas Ramming, Capucine Picard, Rachel Golub, Alain Fischer & Eric Vivier Nat. Immunol. 17, 1291–1299 (2016); published online 12 September 2016; corrected after print 19 October 2016