IL-10-Dependent Crosstalk between Murine Marginal Zone B Cells, Macrophages, and CD8α+ Dendritic Cells Promotes Listeria monocytogenes Infection.

Type 1 CD8α+ conventional dendritic cells (cDC1s) are required for CD8+ T cell priming but, paradoxically, promote splenic Listeria monocytogenes infection. Using mice with impaired cDC2 function, we ruled out a role for cDC2s in this process and instead discovered an interleukin-10 (IL-10)-dependent cellular crosstalk in the marginal zone (MZ) that promoted bacterial infection. Mice lacking the guanine nucleotide exchange factor DOCK8 or CD19 lost IL-10-producing MZ B cells and were resistant to Listeria. IL-10 increased intracellular Listeria in cDC1s indirectly by reducing inducible nitric oxide synthase expression early after infection and increasing intracellular Listeria in MZ metallophilic macrophages (MMMs). These MMMs trans-infected cDC1s, which, in turn, transported Listeria into the white pulp to prime CD8+ T cells. However, this also facilitated bacterial expansion. Therefore, IL-10-mediated crosstalk between B cells, macrophages, and cDC1s in the MZ promotes both Listeria infection and CD8+ T cell activation.

Hematopoietic cell transplantation for DOCK8 deficiency: Results from a prospective clinical trial.

BACKGROUND: DOCK8 deficiency is a primary immunodeficiency in which allogeneic hematopoietic cell transplantation (HCT) represents the only known cure. We tested the ability of a busulfan-based regimen to achieve reliable engraftment and high levels of donor chimerism with acceptable toxicity in a prospective clinical trial in DOCK8 deficiency. OBJECTIVES: To both evaluate the ability of HCT to reverse the clinical phenotype and to correct the immunologic abnormalities by 1 year post HCT. METHODS: We conducted a prospective HCT trial for recipients with DOCK8 deficiency. Subjects were recruited from October 5, 2010, to December 30, 2022. Donor sources included fully matched related and unrelated donors and haploidentical donors. The reduced toxicity, myeloablative conditioning regimen contained no serotherapy. Graft-versus-host disease (GVHD) prophylaxis included either a calcineurin inhibitor with methotrexate or post-HCT cyclophosphamide (PT/Cy) followed by tacrolimus and mycophenolate mofetil. The trial was later amended to study PT/Cy in all patients. (Pilot Study of Reduced-Intensity Hematopoietic Stem Cell Transplant of DOCK8 [NCT01176006].) RESULTS: Thirty-six subjects, both children and adults (median age 16.4 years), underwent HCT for DOCK8 deficiency. Most patients, 33 of 36 (92%), achieved full (≥98%) donor chimerism in whole blood as early as day +30. With a median potential follow-up of 7.4 years, 29 (80.6%) were alive with no evidence of new DOCK8 deficiency-related complications. PT/Cy was effective in reducing the risk of acute GVHD in patients who had received matched unrelated donor and haploidentical transplants, but it was associated with transient delays in immune-reconstitution and hemorrhagic cystitis. CONCLUSIONS: A busulfan-based HCT regimen using PT/Cy for GVHD prophylaxis and a broad range of donor types and hematopoietic cell sources were well tolerated, leading to the reversal of the clinical immunophenotype.

Regulatory T-cell dysfunction and cutaneous exposure to Staphylococcus aureus underlie eczema in DOCK8 deficiency.

BACKGROUND: Dedicator of cytokinesis 8 (DOCK8)-deficient patients have severe eczema, elevated IgE, and eosinophilia, features of atopic dermatitis (AD). OBJECTIVE: We sought to understand the mechanisms of eczema in DOCK8 deficiency. METHODS: Skin biopsy samples were characterized by histology, immunofluorescence microscopy, and gene expression. Skin barrier function was measured by transepidermal water loss. Allergic skin inflammation was elicited in mice by epicutaneous sensitization with ovalbumin (OVA) or cutaneous application of Staphylococcus aureus. RESULTS: Skin lesions of DOCK8-deficient patients exhibited type 2 inflammation, and the patients' skin was colonized by Saureus, as in AD. Unlike in AD, DOCK8-deficient patients had a reduced FOXP3:CD4 ratio in their skin lesions, and their skin barrier function was intrinsically intact. Dock8-/- mice exhibited reduced numbers of cutaneous T regulatory (Treg) cells and a normal skin barrier. Dock8-/- and mice with an inducible Dock8 deletion in Treg cells exhibited increased allergic skin inflammation after epicutaneous sensitization with OVA. DOCK8 was shown to be important for Treg cell stability at sites of allergic inflammation and for the generation, survival, and suppressive activity of inducible Treg cells. Adoptive transfer of wild-type, but not DOCK8-deficient, OVA-specific, inducible Treg cells suppressed allergic inflammation in OVA-sensitized skin of Dock8-/- mice. These mice developed severe allergic skin inflammation and elevated serum IgE levels after topical exposure to Saureus. Both were attenuated after adoptive transfer of WT but not DOCK8-deficient Treg cells. CONCLUSION: Treg cell dysfunction increases susceptibility to allergic skin inflammation in DOCK8 deficiency and synergizes with cutaneous exposure to Saureus to drive eczema in DOCK8 deficiency.

Reconstitution of norovirus-specific T cell responses following hematopoietic stem cell transplantation in patients with inborn errors of immunity and chronic norovirus infection.

BACKGROUND: Chronic norovirus infection (CNI) causes significant morbidity in immunocompromised patients. No effective prevention or treatment currently exists. METHODS: Two patients with inborn errors of immunity, X- linked severe combined immunodeficiency (X-SCID) and DOCK8 deficiency, were followed longitudinally for clinical course, immune reconstitution, norovirus-specific T cell (NST) response, B cell reconstitution, and norovirus-specific antibody production. Samples were obtained in the peri-hematopoietic stem cell transplant setting (HSCT) before and after CNI clearance. The norovirus strain causing CNI was followed longitudinally for norovirus stool viral loads and sequencing. RESULTS: The noroviruses were identified as GII.4 Sydney[P4 New Orleans] in one patient and GII.17[P17] in the other. An exacerbation of diarrhea post-HSCT in the patient with X-SCID was consistent with norovirus infection but not with graft-vs-host-disease on pathologic samples. Both patients recovered polyfunctional NSTs in the CD4 and CD8 T cell compartments which recognized multiple norovirus structural and non-structural viral antigens. T cell responses were minimal during active CNI but detectable after resolution. Mapping of norovirus-specific T cell responses between the patient with DOCK8 and his matched sibling donor were nearly identical. B cell reconstitution or new endogenous antibody production for IgA or IgG were not observed. CONCLUSION: This report is the first to demonstrate reconstitution of norovirus-specific T cell immunity after HSCT closely temporally aligned with clearance of CNI suggesting that cellular immunity is sufficient for norovirus clearance.

DOCK8 deficiency due to a deep intronic variant in two kindreds with hyper-IgE syndrome.

Dedicator of cytokinesis 8 (DOCK8) deficiency underlies the majority of cases of patients with autosomal recessive form of the hyper-immunoglobulin E syndrome (HIES). Most DOCK8 mutations involve deletions and splice junction mutations that abrogate protein expression. However, a few patients whose presentation is reminiscent of DOCK8 deficiency have no identifiable mutations. Using Whole Exome Sequencing (WES), we identified a deep intronic homozygous DOCK8 variant located in intron 36 (c.4626 + 76 A > G) in two unrelated patients with features of HIES that resulted in an in-frame 75 base pair intronic sequence insertion in DOCK8 cDNA, resulting in a premature stop codon (p.S1542ins6Ter). This variant resulted in variable decrease in DOCK8 expression that was associated with impaired T cell receptor-triggered actin polymerization, decreased IL-6-induced STAT3 phosphorylation, reduced expression of the Th17 cell markers CCR6 and IL-17, and higher frequencies of GATA3+ T cells indicative of Th2 skewing. Our approach extends the reach of WES in identifying disease-related intronic variants. It highlights the role of non-coding mutations in immunodeficiency disorders, including DOCK8 deficiency, and emphasizes the need to explore these mutations in unexplained inborn errors of immunity.

A novel hemizygous CD40L mutation of X-linked hyper IgM syndromes and compound heterozygous DOCK8 mutations of hyper IgE syndromes in two Chinese families.

X-linked hyper-immunoglobulin M (X-HIGM) syndrome and autosomal recessive hyper-immunoglobulin E syndrome (HIES) are rare inborn errors of immunity characterized by recurrent infections due to immune system impairment. In this study, we identified a novel hemizygous CD40 ligand (CD40L) mutation and compound heterozygous dedicator of cytokinesis-8 (DOCK8) mutations in two Han Chinese families with X-HIGM and HIES, respectively. We aimed to investigate the association between their genotypes and phenotypes. Genomic DNA was extracted from peripheral blood samples obtained from the families. Whole exome sequencing and Sanger sequencing were performed to identify and verify pathogenic variants in the two families. Clinical analyses of the probands were also performed. A novel hemizygous mutation of CD40L in exon 2 (c.257delA) was identified in the first proband, resulting in the substitution of glycine with glutamic acid at codon 86 of the protein. This leads to premature termination of translation at downstream codon 9 (p.E86Gfs*9). Sanger sequencing confirmed that the variant was inherited from the mother. The second proband carried two novel compound heterozygous mutations in DOCK8: one at exon 14 (c.1546C > G) inherited from the father, and the other at intron 41 (c.5355 + 6C > T; splicing) inherited from the mother. This study enhances our understanding of the pathogenetic mutation spectrum of CD40L and DOCK8 genes, facilitating the prenatal diagnosis of X-HIGM and HIES and enabling timely treatment of patients.

Clinical, immunological, and genetic description of a Mexican cohort of patients with DOCK8 deficiency.

PURPOSE: We aimed to describe the clinical, immunological, and genetic features of patients with DOCK8 deficiency (DOCK8-Def) in a tertiary care center for children. METHODS: Retrospective chart review of patients' clinical, immunological, and genetic characteristics with DOCK8-Def. Genetic analysis was performed with targeted- or whole-exome sequencing; we also assessed DOCK8 protein expression and a lymphoproliferation assay and analyzed survival by the Kaplan-Meier method. RESULTS: We described 11 patients from 8 unrelated kindreds. The median age at symptoms' onset was 10 months (range 1-54 months). The median follow-up time was 53.4 months (4.8-118.8). All patients presented eczema and recurrent sinopulmonary and cutaneous infections. Besides those symptoms, the most frequent manifestations were bronchiectases (8/11), food allergies (6/11), and severe infections (6/11). Infrequent characteristics were detection of CMV in bronchial lavage, C. parvum-driven sclerosing cholangitis, Takayasu vasculitis, neurological syndromes, pulmonary tuberculosis, and lymphomatoid granulomatosis. CONCLUSION: DOCK8-Def has a broad spectrum of manifestations, including allergy, autoimmunity, inflammation, infection, and cancer. The hallmark of this inborn error of immunity is IEI-associated eczema with eosinophilia and increased IgE. Here, we report six new mutations causing human DOCK8 deficiency and symptoms previously unrecognized to occur in DOCK8-Def. Therefore, an early diagnosis of DOCK8-Def is essential to facilitate an adequate treatment such as HSCT.

DOCK8 Mutation in Patient with Juvenile Idiopathic Arthritis and Sjögren's Syndrome.

This study investigated the association between autoimmunity and immunodeficiency in pediatric patients, focusing on the case of a 15-year-old female diagnosed with juvenile idiopathic arthritis (JIA) and secondary Sjögren's syndrome. The patient presented with a variety of symptoms, including joint pain, bronchial asthma, leukopenia, and skin lesions. Genetic testing revealed a de novo mutation in the DOCK8 gene, associated with DOCK8 deficiency, a condition usually associated with immunodeficiencies. The clinical course, diagnostic pathway, and treatment history are detailed, highlighting the importance of molecular diagnostics in understanding the genetic basis of rheumatic diseases. This case highlights the need to consider innate immune errors in patients with multiple diseases or atypical symptoms of rheumatic diseases. Furthermore, the study highlights the importance of targeted treatment, including genetic counseling, to improve patient outcomes. The observed association between autoimmunity and immune deficiency reinforces the importance of molecular testing in elucidating the causes of previously idiopathic rheumatic diseases, contributing to improved patient care and quality of life.

DOCK8 is essential for neutrophil mediated clearance of cutaneous S. aureus infection.

DOCK8 deficient patients are susceptible to skin infection with Staphylococcus aureus which is normally cleared by neutrophils. We examined the mechanism of this susceptibility in mice. Dock8-/- mice had delayed clearance of S. aureus from skin mechanically injured by tape stripping. The numbers and viability of neutrophils in infected but not in uninfected, tape stripped skin were significantly reduced in Dock8-/- mice compared to WT controls. This is despite comparable numbers of circulating neutrophils, and normal to elevated cutaneous expression of Il17a and IL-17A inducible neutrophil attracting chemokines Cxcl1, Cxcl2 and Cxcl3. DOCK8 deficient neutrophils were significantly more susceptible to cell death upon in vitro exposure to S. aureus and exhibited reduced phagocytosis of S. aureus bioparticles but had a normal respiratory burst. Impaired neutrophil survival in infected skin and defective neutrophil phagocytosis likely underlie the susceptibility to cutaneous S. aureus infection in DOCK8 deficiency.

Treatment of inborn errors of immunity patients with inflammatory bowel disease phenotype by allogeneic stem cell transplantation.

Patients with inborn errors of immunity (IEI) can suffer from treatment-refractory inflammatory bowel disease (IBD) causing failure to thrive and consequences of long-term multiple immunosuppressive treatments. Allogeneic haematopoietic stem cell transplantation (alloHSCT) can serve as a curative treatment option. In this single-centre retrospective cohort study we report on 11 paediatric and young adult IEI patients with IBD and failure to thrive, who had exhausted symptomatic treatment options and received alloHSCT. The cohort included chronic granulomatous disease (CGD), lipopolysaccharide-responsive and beige-like anchor protein (LRBA) deficiency, STAT3 gain-of-function (GOF), Wiskott-Aldrich syndrome (WAS), dedicator of cytokinesis 8 (DOCK8) deficiency and one patient without genetic diagnosis. All patients achieved stable engraftment and immune reconstitution, and gastrointestinal symptoms were resolved after alloHSCT. The overall survival was 11/11 over a median follow-up of 34.7 months. Graft-versus-host disease (GVHD) was limited to grade I-II acute GVHD (n = 5), one case of grade IV acute GVHD and one case of limited chronic GVHD. Since treatment recommendations are limited, this work provides a centre-specific approach to treatment prior to transplant as well as conditioning in IEI patients with severe IBD.

Atypical Localization of Eczema Discriminates DOCK8 or STAT3 Deficiencies from Atopic Dermatitis.

PURPOSE: Autosomal recessive dedicator of cytokinesis 8 (DOCK8-/-) and autosomal dominant signal transducer and activator of transcription 3 (STAT3-/+) deficiencies are inborn errors of immunity (IEI) disorders present with the classic features of eczema and create a dilemma during differentiation from atopic dermatitis (AD). Therefore, an appropriate approach is required for eczema to diagnose DOCK8-/- and STAT3-/+ early. Here, we described a set of clinical and immunological variables, including atypical AD localizations and lymphocyte subsets, to differentiate DOCK8-/- or STAT3-/+ from AD. METHODS: This multicenter study involved 100 patients with DOCK8-/- and STAT3-/+ and moderate/severe AD. We recruited disease manifestations, including detailed localizations of eczema, infections, and allergy. Principle component analysis (PCA) was used to discriminate DOCK8-/- or STAT3-/+ from AD. RESULTS: There were 43 patients with DOCK8-/-, 23 with STAT3-/+, and 34 with AD. Pneumonia, severe infections, mucocutaneous candidiasis, and skin abscesses were commonly observed in DOCK8 and STAT3 deficiencies. Atypical skin involvement with neonatal rash, retro auricular, axillary, sacral, and genital eczema discriminate DOCK8-/- and STAT3-/+ from AD with high specificity ranges between 73.5 and 94.1% and positive predictive index ranges between 55 and 93.1%. Together with using absolute numbers of CD3+, CD4+, and CD8+ T cells, the combined clinical and laboratory features showed perfect differentiation between DOCK8-/- or STAT3-/+ and AD via PCA. CONCLUSIONS: The described features can be easily implemented by physicians providing early diagnosis of DOCK8 and STAT3 deficiencies.

Clinical, immunological and molecular findings of patients with DOCK-8 deficiency from India.

DOCK8 deficiency affects various cell subsets belonging to both the innate and adaptive immune systems. Clinical diagnosis is challenging, as many cases present with severe atopic dermatitis as the only initial manifestation. Though flow cytometry helps in the presumptive diagnosis of DOCK8-deficient patients by evaluating their DOCK8 protein expression, it requires subsequent confirmation by molecular genetic analysis. Currently, haematopoietic stem cell transplantation (HSCT) is the only curative treatment option available for these patients. There is a paucity of data from India on the clinical diversity and molecular spectrum of DOCK8 deficiency. In the present study, we report the clinical, immunological and molecular findings of 17 DOCK8-deficient patients from India diagnosed over the last 5 years.

Oral anaphylaxis to peanut in a mouse model is associated with gut permeability but not with Tlr4 or Dock8 mutations.

BACKGROUND: The etiology of food allergy is poorly understood; mouse models are powerful systems to discover immunologic pathways driving allergic disease. C3H/HeJ mice are a widely used model for the study of peanut allergy because, unlike C57BL/6 or BALB/c mice, they are highly susceptible to oral anaphylaxis. However, the immunologic mechanism of this strain's susceptibility is not known. OBJECTIVE: We aimed to determine the mechanism underlying the unique susceptibility to anaphylaxis in C3H/HeJ mice. We tested the role of deleterious Toll-like receptor 4 (Tlr4) or dedicator of cytokinesis 8 (Dock8) mutations in this strain because both genes have been associated with food allergy. METHODS: We generated C3H/HeJ mice with corrected Dock8 or Tlr4 alleles and sensitized and challenged them with peanut. We then characterized the antibody response to sensitization, anaphylaxis response to both oral and systemic peanut challenge, gut microbiome, and biomarkers of gut permeability. RESULTS: In contrast to C3H/HeJ mice, C57BL/6 mice were resistant to anaphylaxis after oral peanut challenge; however, both strains undergo anaphylaxis with intraperitoneal challenge. Restoring Tlr4 or Dock8 function in C3H/HeJ mice did not protect from anaphylaxis. Instead, we discovered enhanced gut permeability resulting in ingested allergens in the bloodstream in C3H/HeJ mice compared to C57BL/6 mice, which correlated with an increased number of goblet cells in the small intestine. CONCLUSIONS: Our work highlights the potential importance of gut permeability in driving anaphylaxis to ingested food allergens; it also indicates that genetic loci outside of Tlr4 and Dock8 are responsible for the oral anaphylactic susceptibility of C3H/HeJ mice.

Multi-Omics Profiling in PGM3 and STAT3 Deficiencies: A Tale of Two Patients.

Hyper-IgE Syndrome (HIES) is a heterogeneous group of primary immune-deficiency disorders characterized by elevated levels of IgE, eczema, and recurrent skin and lung infections. HIES that is autosomally dominant in the signal transducer and activator of transcription 3 (STAT3), and autosomal recessive mutations in phosphoglucomutase 3 (PGM3) have been reported in humans. An early diagnosis, based on clinical suspicion and immunological assessments, is challenging. Patients' metabolomics, proteomics, and cytokine profiles were compared to DOCK 8-deficient and atopic dermatitis patients. The PGM3 metabolomics profile identified significant dysregulation in hypotaurine, hypoxanthine, uridine, and ribothymidine. The eight proteins involved include bifunctional arginine demethylase and lysyl hydroxylase (JMJD1B), type 1 protein phosphatase inhibitor 4 (PPI 4), and platelet factor 4 which aligned with an increased level of the cytokine GCSF. Patients with STAT3 deficiency, on the other hand, showed significant dysregulation in eight metabolites, including an increase in protocatechuic acid, seven proteins including ceruloplasmin, and a plasma protease C1 inhibitor, in addition to cytokine VEGF being dysregulated. Using multi-omics profiling, we identified the dysregulation of endothelial growth factor (EGFR) and tumor necrosis factor (TNF) signaling pathways in PGM3 and STAT3 patients, respectively. Our findings may serve as a stepping stone for larger prospective HIES clinical cohorts to validate their future use as biomarkers.

Insights into immunity from clinical and basic science studies of DOCK8 immunodeficiency syndrome.

DOCK8 immunodeficiency syndrome (DIDS) is a progressive combined immunodeficiency that can be distinguished from other combined immunodeficiencies or hyperimmunoglobulinemia E syndromes in featuring (a) profound susceptibility to virus infections of the skin, with associated skin cancers, and (b) severe food allergies. The DOCK8 locus has many repetitive sequence elements that predispose to the generation of large germline deletions as well as recombination-mediated somatic DNA repair. Residual DOCK8 protein contributes to the variable disease phenotype. The severe virus infections of the skin, and probably also VZV-associated vasculopathy, reflect an important function of DOCK8, which is normally required to maintain lymphocyte shape integrity as the cells migrate through dense tissues. Loss of DOCK8 also causes immune deficits through other mechanisms including a milder generalized cell survival defect and skewing of T helper cell subsets. Recent work has uncovered the roles for DOCK8 in dendritic cell responses that can also help explain the virus susceptibility, as well as in regulatory T cells that might help explain autoimmunity in a minority of patients. Fortunately, hematopoietic stem cell transplantation cures the eczema and infection susceptibility of DIDS, but not necessarily the other disease manifestations including food allergies.

Hyper-IgE Syndrome due to an Elusive Novel Intronic Homozygous Variant in DOCK8.

Rare, biallelic loss-of-function mutations in DOCK8 result in a combined immune deficiency characterized by severe and recurrent cutaneous infections, eczema, allergies, and susceptibility to malignancy, as well as impaired humoral and cellular immunity and hyper-IgE. The advent of next-generation sequencing technologies has enabled the rapid molecular diagnosis of rare monogenic diseases, including inborn errors of immunity. These advances have resulted in the implementation of gene-guided treatments, such as hematopoietic stem cell transplant for DOCK8 deficiency. However, putative disease-causing variants revealed by next-generation sequencing need rigorous validation to demonstrate pathogenicity. Here, we report the eventual diagnosis of DOCK8 deficiency in a consanguineous family due to a novel homozygous intronic deletion variant that caused aberrant exon splicing and subsequent loss of expression of DOCK8 protein. Remarkably, the causative variant was not initially detected by clinical whole-genome sequencing but was subsequently identified and validated by combining advanced genomic analysis, RNA-seq, and flow cytometry. This case highlights the need to adopt multipronged confirmatory approaches to definitively solve complex genetic cases that result from variants outside protein-coding exons and conventional splice sites.

A set of clinical and laboratory markers differentiates hyper-IgE syndrome from severe atopic dermatitis.

Hyper-IgE syndrome (HIES) patients may share many features observed in severe atopic dermatitis (SAD), making a diagnostic dilemma for physicians. Determining clinical and laboratory markers that distinguish both disorders could provide early diagnosis and treatment. We analyzed patients (DOCK8 deficiency:14, STAT3-HIES:10, SAD:10) with early-onset SAD. Recurrent upper respiratory tract infection and pneumonia were significantly frequent in HIES than SAD patients. Characteristic facial appearance, retained primary teeth, skin abscess, newborn rash, and pneumatocele were more predictable for STAT3-HIES, while mucocutaneous candidiasis and Herpes infection were common in DOCK8 deficiency, which were unusual in SAD group. DOCK8-deficient patients had lower CD3+ and CD4+T cells with a senescent phenotype that unique for this form of HIES. Both DOCK8 deficiency and STAT3-HIES patients exhibited reduced switched memory B cells compared to the SAD patients. These clinical and laboratory markers are helpful to differentiate HIES from SAD patients.

Lineage-Specific Chimerism and Outcome After Hematopoietic Stem Cell Transplantation for DOCK8 Deficiency.

Bi-allelic variants in the dedicator of cytokinesis 8 (DOCK8) gene cause a combined immunodeficiency, characterized by recurrent sinopulmonary and skin infections, food allergies, eczema, eosinophilia, and elevated IgE. Long-term outcome is poor given susceptibility to infections, malignancy, and vascular complications. Allogeneic hematopoietic stem cell transplantation is currently the only curative treatment option and has shown promising outcome. The impact of mixed chimerism on long-term outcome is unclear. We reasoned that reversal of disease phenotype would depend on cell lineage-specific chimerism. DOCK8 variants were confirmed by Sanger and/or exome sequencing and immunoblot and/or intracellular flow cytometry. Donor chimerism was analyzed by XY-fluorescence in situ hybridization or quantitative short tandem repeat PCR. Outcome was assessed by laboratory tests, lymphocyte subsets, intracellular DOCK8 protein flow cytometry, T-cell proliferation analysis, and multiparameter immunoblot allergy screening. We report on nine patients, four of whom with mixed chimerism, with a median follow-up of 78 months after transplantation. Overall, we report successful transplantation with improvement of susceptibility to infections and allergies, and resolution of eczema in all patients. Immunological outcome in patients with mixed chimerism suggests a selective advantage for wild-type donor T-cells but lower donor B-cell chimerism possibly results in a tendency to hypogammaglobulinemia. No increased infectious and allergic complications were associated with mixed chimerism. Aware of the relatively small cohort size, we could not demonstrate a consistent detrimental effect of mixed chimerism on clinical outcomes. We nevertheless advocate aiming for complete donor chimerism in treating DOCK8 deficiency, but recommend reduced toxicity conditioning.

DOCK8 deficiency causes a skewing to type 2 immunity in the gut with expansion of group 2 innate lymphoid cells.

Dedicator of cytokinesis 8 (DOCK8) is a guanine nucleotide exchange factor (GEF) for Cdc42. In humans, homozygous or compound heterozygous deletions in DOCK8 cause a combined immunodeficiency characterized by various allergic diseases including food allergies. Although group 2 innate lymphoid cells (ILC2s) contribute to the development of allergic inflammation by producing interleukin (IL)-5 and IL-13, the role of ILC2s in DOCK8 deficiency has not been fully explored. With the use of cytometry by time-of-flight (CyTOF), we performed high-dimensional phenotyping of intestinal immune cells and found that DOCK8-deficient (Dock8-/-) mice exhibited expansion of ILC2s and other leukocytes associated with type 2 immunity in the small intestine. Moreover, IL-5- and IL-13-producing cells markedly increased in Dock8-/- mice, and the majority of them were lineage-negative cells, most likely ILC2s. Intestinal ILC2s expanded when DOCK8 expression was selectively deleted in hematopoietic cells. Importantly, intestinal ILC2 expansion was also observed in Dock8VAGR mice having mutations in the catalytic center of DOCK8, thereby failing to activate Cdc42. Our findings indicate that DOCK8 is a negative regulator of intestinal ILC2s to inhibit their expansion via Cdc42 activation, and that deletion of DOCK8 causes a skewing to type 2 immunity in the gut.

DOCK8 Serves as a Prognostic Biomarker and Is Related to Immune Infiltration in Patients With HPV Positive Head and Neck Squamous Cell Carcinoma.

PURPOSE: Dedicator of cytokinesis 8 (DOCK8) was reported to have a vital link to immunoregulation. However, the mechanisms by which it drives immune infiltration in cancer remain uncertain. We tried to assess the role of DOCK8 in patients with cancer, especially human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC). METHODS: Data on the expression and survival of DOCK8 in patients with various cancers were analyzed using the Oncomine and TIMER databases. The TIMER database assessed the relationship of DOCK8 with immune infiltration levels and various markers of multiple immune cells. Gene set enrichment analysis revealed tumor-associated biological processes related to DOCK8. ENCODE database was used to explore relevant transcription factors of DOCK8, and a PPI network was constructed using GENEMINIA. The expression and survival role of DOCK8 was confirmed in patients from independent GEO datasets. RESULTS: We determined that DOCK8 expression was upregulated or downregulated in various cancers unlike in healthy tissues. A high expression of DOCK8 was significantly correlated with a favorable prognosis in HPV-positive HNSCC and lung adenocarcinoma (LUAD). Furthermore, multivariate Cox regression analysis revealed that DOCK8 was an independent prognostic factor of HPV-positive HNSCC. Additionally, elevated DOCK8 expression was positively correlated with multiple immune cell infiltration levels and immune marker expression associated with particular immune cell subsets. Also, 14 pathways involved in immune activities and carcinogenesis, 22 potential TFs, and co-expression proteins of DOCK8 indicated DOCK8 to be related to tumor-associated biological processes. Ultimately, we verified that DOCK8 is upregulated and confers a favorable overall survival and progression-free survival status in patients with HPV-positive HNSCC. CONCLUSION: These results elucidate that high expression of DOCK8 indicates a favorable prognosis in patients with HPV-positive HNSCC as well as increased microenvironmental immune infiltration levels. It would provide new insights into the prognosis predicting and clinical regimen decision making in patients with HPV-positive HNSCC.