DOCK11 deficiency in patients with X-linked actinopathy and autoimmunity.

Dedicator of cytokinesis (DOCK) proteins play a central role in actin cytoskeleton regulation. This is highlighted by the DOCK2 and DOCK8 deficiencies leading to actinopathies and immune deficiencies. DOCK8 and DOCK11 activate CDC42, a Rho-guanosine triphosphate hydrolases involved in actin cytoskeleton dynamics, among many cellular functions. The role of DOCK11 in human immune disease has been long suspected but, to the best of our knowledge, has never been described to date. We studied 8 male patients, from 7 unrelated families, with hemizygous DOCK11 missense variants leading to reduced DOCK11 expression. The patients were presenting with early-onset autoimmunity, including cytopenia, systemic lupus erythematosus, skin, and digestive manifestations. Patients' platelets exhibited abnormal ultrastructural morphology and spreading as well as impaired CDC42 activity. In vitro activated T cells and B-lymphoblastoid cell lines from patients exhibited aberrant protrusions and abnormal migration speed in confined channels concomitant with altered actin polymerization during migration. Knock down of DOCK11 recapitulated these abnormal cellular phenotypes in monocytes-derived dendritic cells and primary activated T cells from healthy controls. Lastly, in line with the patients' autoimmune manifestations, we also observed abnormal regulatory T-cell (Treg) phenotype with profoundly reduced FOXP3 and IKZF2 expression. Moreover, we found reduced T-cell proliferation and impaired STAT5B phosphorylation upon interleukin-2 stimulation of the patients' lymphocytes. In conclusion, DOCK11 deficiency is a new X-linked immune-related actinopathy leading to impaired CDC42 activity and STAT5 activation, and is associated with abnormal actin cytoskeleton remodeling as well as Treg phenotype, culminating in immune dysregulation and severe early-onset autoimmunity.

Combined germline and somatic human FADD mutations cause autoimmune lymphoproliferative syndrome.

BACKGROUND: The autoimmune lymphoproliferative syndrome (ALPS) is a noninfectious and nonmalignant lymphoproliferative disease frequently associated with autoimmune cytopenia resulting from defective FAS signaling. We previously described germline monoallelic FAS (TNFRSF6) haploinsufficient mutations associated with somatic events, such as loss of heterozygosity on the second allele of FAS, as a cause of ALPS-FAS. These somatic events were identified by sequencing FAS in DNA from double-negative (DN) T cells, the pathognomonic T-cell subset in ALPS, in which the somatic events accumulated. OBJECTIVE: We sought to identify whether a somatic event affecting the FAS-associated death domain (FADD) gene could be related to the disease onset in 4 unrelated patients with ALPS carrying a germline monoallelic mutation of the FADD protein inherited from a healthy parent. METHODS: We sequenced FADD and performed array-based comparative genomic hybridization using DNA from sorted CD4+ or DN T cells. RESULTS: We found homozygous FADD mutations in the DN T cells from all 4 patients, which resulted from uniparental disomy. FADD deficiency caused by germline heterozygous FADD mutations associated with a somatic loss of heterozygosity was a phenocopy of ALPS-FAS without the more complex symptoms reported in patients with germline biallelic FADD mutations. CONCLUSIONS: The association of germline and somatic events affecting the FADD gene is a new genetic cause of ALPS.

Abnormal biomarkers predict complex FAS or FADD defects missed by exome sequencing.

BACKGROUND: Elevated TCRαß+CD4-CD8- double-negative T cells (DNT) and serum biomarkers help identify FAS mutant patients with autoimmune lymphoproliferative syndrome (ALPS). However, in some patients with clinical features and biomarkers consistent with ALPS, germline or somatic FAS mutations cannot be identified on standard exon sequencing (ALPS-undetermined: ALPS-U). OBJECTIVE: We sought to explore whether complex genetic alterations in the FAS gene escaping standard sequencing or mutations in other FAS pathway-related genes could explain these cases. METHODS: Genetic analysis included whole FAS gene sequencing, copy number variation analysis, and sequencing of FAS cDNA and other FAS pathway-related genes. It was guided by FAS expression analysis on CD57+DNT, which can predict somatic loss of heterozygosity (sLOH). RESULTS: Nine of 16 patients with ALPS-U lacked FAS expression on CD57+DNT predicting heterozygous "loss-of-expression" FAS mutations plus acquired somatic second hits in the FAS gene, enriched in DNT. Indeed, 7 of 9 analyzed patients carried deep intronic mutations or large deletions in the FAS gene combined with sLOH detectable in DNT; 1 patient showed a FAS exon duplication. Three patients had reduced FAS expression, and 2 of them harbored mutations in the FAS promoter, which reduced FAS expression in reporter assays. Three of the 4 ALPS-U patients with normal FAS expression carried heterozygous FADD mutations with sLOH. CONCLUSION: A combination of serum biomarkers and DNT phenotyping is an accurate means to identify patients with ALPS who are missed by routine exome sequencing.

Loss of Function of RIMS2 Causes a Syndromic Congenital Cone-Rod Synaptic Disease with Neurodevelopmental and Pancreatic Involvement.

Congenital cone-rod synaptic disorder (CRSD), also known as incomplete congenital stationary night blindness (iCSNB), is a non-progressive inherited retinal disease (IRD) characterized by night blindness, photophobia, and nystagmus, and distinctive electroretinographic features. Here, we report bi-allelic RIMS2 variants in seven CRSD-affected individuals from four unrelated families. Apart from CRSD, neurodevelopmental disease was observed in all affected individuals, and abnormal glucose homeostasis was observed in the eldest affected individual. RIMS2 regulates synaptic membrane exocytosis. Data mining of human adult bulk and single-cell retinal transcriptional datasets revealed predominant expression in rod photoreceptors, and immunostaining demonstrated RIMS2 localization in the human retinal outer plexiform layer, Purkinje cells, and pancreatic islets. Additionally, nonsense variants were shown to result in truncated RIMS2 and decreased insulin secretion in mammalian cells. The identification of a syndromic stationary congenital IRD has a major impact on the differential diagnosis of syndromic congenital IRD, which has previously been exclusively linked with degenerative IRD.

Compound heterozygous mutations in the kinase domain of IKKα lead to immunodeficiency and immune dysregulation.

IKKα, encoded by CHUK , is crucial in the non-canonical NF-κB pathway and part of the IKK complex activating the canonical pathway alongside IKKß. Absence of IKKα cause fetal encasement syndrome in human, fatal in utero, while an impaired IKKα-NIK interaction was reported in a single patient and cause combined immunodeficiency. Here, we describe compound heterozygous variants in the kinase domain of IKKα in a female patient with hypogammaglobulinemia, recurrent lung infections, and Hay-Wells syndrome-like features. We showed that both variants were loss-of-function. Non-canonical NF-κB activation was profoundly diminished in stromal and immune cells while the canonical pathway was partially impaired. Reintroducing wild-type CHUK restored non-canonical NF-κB activation. The patient had neutralizing autoantibodies against type I IFN, akin to non-canonical NF-κB pathway deficiencies. Thus, this is the first case of bi-allelic CHUK mutations disrupting IKKα kinase function, broadening non-canonical NF-κB defect understanding and suggesting IKKα's role in canonical NF-κB target gene expression in human.

Activating FcγR function depends on endosomal-signaling platforms.

Cell surface receptor internalization can either terminate signaling or activate alternative endosomal signaling pathways. We investigated here whether endosomal signaling is involved in the function of the human receptors for Fc immunoglobulin fragments (FcRs): FcαRI, FcγRIIA, and FcγRI. All these receptors were internalized after their cross-linking with receptor-specific antibodies, but their intracellular trafficking was different. FcαRI was targeted directly to lysosomes, while FcγRIIA and FcγRI were internalized in particular endosomal compartments described by the insulin esponsive minoeptidase (IRAP), where they recruited signaling molecules, such as the active form of the kinase Syk, PLCγ and the adaptor LAT. Destabilization of FcγR endosomal signaling in the absence of IRAP compromised cytokine secretion downstream FcγR activation and macrophage ability to kill tumor cells by antibody-dependent cell-mediated cytotoxicity (ADCC). Our results indicate that FcγR endosomal signaling is required for the FcγR-driven inflammatory reaction and possibly for the therapeutic action of monoclonal antibodies.

NBEAL2 deficiency in humans leads to low CTLA-4 expression in activated conventional T cells.

Loss of NBEAL2 function leads to grey platelet syndrome (GPS), a bleeding disorder characterized by macro-thrombocytopenia and α-granule-deficient platelets. A proportion of patients with GPS develop autoimmunity through an unknown mechanism, which might be related to the proteins NBEAL2 interacts with, specifically in immune cells. Here we show a comprehensive interactome of NBEAL2 in primary T cells, based on mass spectrometry identification of altogether 74 protein association partners. These include LRBA, a member of the same BEACH domain family as NBEAL2, recessive mutations of which cause autoimmunity and lymphocytic infiltration through defective CTLA-4 trafficking. Investigating the potential association between NBEAL2 and CTLA-4 signalling suggested by the mass spectrometry results, we confirm by co-immunoprecipitation that CTLA-4 and NBEAL2 interact with each other. Interestingly, NBEAL2 deficiency leads to low CTLA-4 expression in patient-derived effector T cells, while their regulatory T cells appear unaffected. Knocking-down NBEAL2 in healthy primary T cells recapitulates the low CTLA-4 expression observed in the T cells of GPS patients. Our results thus show that NBEAL2 is involved in the regulation of CTLA-4 expression in conventional T cells and provide a rationale for considering CTLA-4-immunoglobulin therapy in patients with GPS and autoimmune disease.

Compromised mitochondrial quality control triggers lipin1-related rhabdomyolysis.

LPIN1 mutations are responsible for inherited recurrent rhabdomyolysis, a life-threatening condition with no efficient therapeutic intervention. Here, we conduct a bedside-to-bench-and-back investigation to study the pathophysiology of lipin1 deficiency. We find that lipin1-deficient myoblasts exhibit a reduction in phosphatidylinositol-3-phosphate close to autophagosomes and late endosomes that prevents the recruitment of the GTPase Armus, locks Rab7 in the active state, inhibits vesicle clearance by fusion with lysosomes, and alters their positioning and function. Oxidized mitochondrial DNA accumulates in late endosomes, where it activates Toll-like receptor 9 (TLR9) and triggers inflammatory signaling and caspase-dependent myolysis. Hydroxychloroquine blocks TLR9 activation by mitochondrial DNA in vitro and may attenuate flares of rhabdomyolysis in 6 patients treated. We suggest a critical role for defective clearance of oxidized mitochondrial DNA that activates TLR9-restricted inflammation in lipin1-related rhabdomyolysis. Interventions blocking TLR9 activation or inflammation can improve patient care in vivo.

Fgfr3 Is a Positive Regulator of Osteoblast Expansion and Differentiation During Zebrafish Skull Vault Development.

Gain or loss-of-function mutations in fibroblast growth factor receptor 3 (FGFR3) result in cranial vault defects highlighting the protein's role in membranous ossification. Zebrafish express high levels of fgfr3 during skull development; in order to study FGFR3's role in cranial vault development, we generated the first fgfr3 loss-of-function zebrafish (fgfr3lof/lof ). The mutant fish exhibited major changes in the craniofacial skeleton, with a lack of sutures, abnormal frontal and parietal bones, and the presence of ectopic bones. Integrated analyses (in vivo imaging and single-cell RNA sequencing of the osteoblast lineage) of zebrafish fgfr3lof/lof revealed a delay in osteoblast expansion and differentiation, together with changes in the extracellular matrix. These findings demonstrate that fgfr3 is a positive regulator of osteogenesis. We conclude that changes in the extracellular matrix within growing bone might impair cell-cell communication, mineralization, and new osteoblast recruitment. © 2020 American Society for Bone and Mineral Research.

Full-length cytokeratin-19 is released by human tumor cells: a potential role in metastatic progression of breast cancer.

INTRODUCTION: We evaluated whether CK19, one of the main cytoskeleton proteins of epithelial cells, is released as full-length protein from viable tumor cells and whether this property is relevant for metastatic progression in breast cancer patients. METHODS: EPISPOT (EPithelial ImmunoSPOT) assays were performed to analyze the release of full-length CK19 by carcinoma cells of various origins, and the sequence of CK19 was analyzed with mass spectrometry. Additional functional experiments with cycloheximide, Brefeldin A, or vincristine were done to analyze the biology of the CK19-release. CK19-EPISPOT was used to detect disseminated tumor cells in bone marrow (BM) of 45 breast cancer patients who were then followed up over a median of 6 years. RESULTS: CK19 was expressed and released by colorectal (HT-29, HCT116, Caco-2) and breast (MCF-7, SKBR3, and MDA-MB-231) cancer cell lines. The CK19-EPISPOT was more sensitive than the CK19-ELISA. Dual fluorescent EPISPOT with antibodies against different CK19 epitopes showed the release of the full-length CK19, which was confirmed by mass spectrometry. Functional experiments indicated that CK19 release was an active process and not simply the consequence of cell death. CK19-releasing cells (RCs) were detectable in BM of 44% to 70% of breast cancer patients. This incidence and the number of CK19-RCs were correlated to the presence of overt metastases, and patients with CK19-RCs had a reduced survival as compared with patients without these cells (P = 0.025, log-rank test; P = 0.0019, hazard ratio, 4.7; multivariate analysis). CONCLUSIONS: Full-length CK19 is released by viable epithelial tumor cells, and CK19-RCs might constitute a biologically active subset of breast cancer cells with high metastatic properties.

Autoimmune Lymphoproliferative Syndrome-FAS Patients Have an Abnormal Regulatory T Cell (Treg) Phenotype but Display Normal Natural Treg-Suppressive Function on T Cell Proliferation.

Objective: Autoimmune lymphoproliferative syndrome (ALPS) with FAS mutation (ALPS-FAS) is a nonmalignant, noninfectious, lymphoproliferative disease with autoimmunity. Given the central role of natural regulatory T cells (nTregs) in the control of lymphoproliferation and autoimmunity, we assessed nTreg-suppressive function in 16 patients with ALPS-FAS. Results: The proportion of CD25highCD127low Tregs was lower in ALPS-FAS patients than in healthy controls. This subset was correlated with a reduced CD25 expression in CD3+CD4+ T cells from ALPS patients and thus an abnormally low proportion of CD25highFOXP3+ Helios+ T cells. The ALPS patients also displayed a high proportion of naïve Treg (FOXP3lowCD45RA+) and an unusual subpopulation (CD4+CD127lowCD15s+CD45RA+). Despite this abnormal phenotype, the CD25highCD127low Tregs' suppressive function was unaffected. Furthermore, conventional T cells from FAS-mutated patients showed normal levels of sensitivity to Treg suppression. Conclusion: An abnormal Treg phenotype is observed in circulating lymphocytes of ALPS patients. However, these Tregs displayed a normal suppressive function on T effector proliferation in vitro. This is suggesting that lymphoproliferation observed in ALPS patients does not result from Tregs functional defect or T effector cells insensitivity to Tregs suppression.

Lymphadenopathy driven by TCR-Vγ8Vδ1 T-cell expansion in FAS-related autoimmune lymphoproliferative syndrome.

FAS-dependent apoptosis in Vδ1 T cells makes the latter possible culprits for the lymphadenopathy observed in patients with FAS mutations.Rapamycin and methylprednisolone resistance should prompt clinicians to look for Vδ1 T cell proliferation in ALPS-FAS patients.

Modulation of interleukin-7 receptor expression characterizes differentiation of CD8 T cells specific for HIV, EBV and CMV.

OBJECTIVES: To further understand differentiation and homeostasis of CD8 T cells specific for HIV, Epstein-Barr Virus (EBV) and cytomegalovirus (CMV) during HIV infection, we investigated interleukin-7 receptor alpha (IL-7Ralpha) expression on those virus-specific T cells. METHODS: Microarrays and cytometry analyses were performed on peripheral blood mononuclear cells (PBMC), total and tetramer-binding virus-specific CD8 T cells from 66 HIV-infected patients. RESULTS: Microarray analysis revealed reduced levels of IL-7Ralpha and increased levels of perforin with disease progression in total PBMC. This loss of IL-7Ralpha expression was observed on CD8 T cells and was inversely related to perforin expression. The relative expression of both molecules defined three new subsets: IL-7Ralpha(pos)Perforin(neg); IL-7Ralpha(loneg)Perforin(lo); and IL-7Ralpha(loneg)Perforin(hi) corresponding to naive and effector-memory CD8 differentiation, as assessed by CD45RA/CD11a. The IL-7Ralpha expression decreased along the CD8 differentiation pathway defined by CD27 and CD28. In contrast, IL-7Ralpha expression was down-modulated on all the CD8 T cells specific for HIV, EBV and CMV that were almost exclusively IL-7Ralpha(lo/neg)Perforin(lo) and was parallel with the CD27 expression. In addition, this low IL-7Ralpha expression on HIV-specific CD8 T cells was independent of virus load and T-cell activation and remained stable during the first 6 months of antiretroviral therapy despite successful control of HIV replication. CONCLUSION: The relative expression of IL-7Ralpha, perforin reveals new aspects of virus-specific CD8 T cell differentiation, independently of T-cell activation and virus load. This opens new perspectives for understanding homeostasis of those cells and immune-based therapeutic strategies.

Identical strength of the T cell responses against E2, nsP1 and capsid CHIKV proteins in recovered and chronic patients after the epidemics of 2005-2006 in La Reunion Island.

To characterize the immunity developed by patients infected by chikungunya virus (CHIKV), we studied the intensity and specificity of CHIKV-specific T cells mediated responses in chronic and recovered patients at 12 to 24 months post-infection. T cells were challenged in vitro against CHIKV synthetic peptides covering the length of three viral proteins, capsid, E2 and nsP1 proteins as well as all inactivated virus particles. Cytokine production was assessed by ELISPOT and intracellular labeling. T cells producing IFN-γ were detected against CHIKV in 85% patient's cells either by direct ELISPOT assay (69% of patients) or after expansion of memory T cells allowing the detection of both CD4 and CD8 specific-T cells in 16% additional cases. The IFN-γ response was mainly engaged in response to nsP1 or E2 (52% and 46% cases, respectively) but in only 27% cases against the capsid. The anti-E2 response represented half the magnitude of the total CHIKV IFN-γ production and was mainly directed against the C-terminal half part of the protein. Almost all patients had conserved a T cell specific response against CHIKV with a clear hierarchy of T cell responses (CD8 > CD4) engaged against E2 > nsP1 > capsid. More importantly, the intensity of responses was not significantly different between recovered and chronic patients. These findings constitute key elements to a better understanding of patient T cell immunoreactivity against CHIKV and argue against a possible defect of T cell immunoresponse in the chronicity post-CHIKV infection.