Integrating circulating T follicular memory cells and autoantibody repertoires for characterization of autoimmune disorders.

Introduction: Autoimmune diseases are heterogeneous and often lack specific or sensitive diagnostic tests. Increased percentages of CD4+CXCR5+PD1+ circulating T follicular helper (cTfh) cells and skewed distributions of cTfh subtypes have been associated with autoimmunity. However, cTfh cell percentages can normalize with immunomodulatory treatment despite persistent disease activity, indicating the need for identifying additional cellular and/or serologic features correlating with autoimmunity. Methods: The cohort included 50 controls and 56 patients with autoimmune cytopenias, gastrointestinal, pulmonary, and/or neurologic autoimmune disease. Flow cytometry was used to measure CD4+CXCR5+ T cell subsets expressing the chemokine receptors CXCR3 and/or CCR6: CXCR3+CCR6- Type 1, CXCR3-CCR6- Type 2, CXCR3+CCR6+ Type 1/17, and CXCR3- CCR6+ Type 17 T cells. IgG and IgA autoantibodies were quantified using a microarray featuring 1616 full-length, conformationally intact protein antigens. The 97.5th percentile in the control cohort defined normal limits for T cell subset percentages and total number (burden) of autoantibodies. Results: This study focused on CD4+CXCR5+ T cells because CXCR5 upregulation occurs after cognate T-B cell interactions characteristic of autoimmune diseases. We refer to these cells as circulating T follicular memory (cTfm) cells to acknowledge the dynamic nature of antigen-experienced CXCR5+ T cells, which encompass progenitors of cTfh or Tfh cells as well as early effector memory T cells that have not yet lost CXCR5. Compared to controls, 57.1% of patients had increased CXCR5+CXCR3+CCR6+ cTfm1/17 and 25% had increased CXCR5+CXCR3-CCR6+ cTfm17 cell percentages. Patients had significantly more diverse IgG and IgA autoantibodies than controls and 44.6% had an increased burden of autoantibodies of either isotype. Unsupervised autoantibody clustering identified three clusters of patients with IgG autoantibody profiles distinct from those of controls, enriched for patients with active autoimmunity and monogenic diseases. An increased percentage of cTfm17 cells was most closely associated with an increased burden of high-titer IgG and IgA autoantibodies. A composite measure integrating increased cTfm1/17, cTfm17, and high-titer IgG and/or IgA autoantibodies had 91.1% sensitivity and 90.9% specificity for identifying patients with autoimmunity. Percentages of cTfm1/17 and cTfm17 percentages and numbers of high-titer autoantibodies in patients receiving immunomodulatory treatment did not differ from those in untreated patients, thus suggesting that measurements of cTfm can complement measurements of other cellular markers affected by treatment. Conclusions: This study highlights two new approaches for assessing autoimmunity: measuring CD4+CXCR5+ cTfm subsets as well as total burden of autoantibodies. Our findings suggest that these approaches are particularly relevant to patients with rare autoimmune disorders for whom target antigens and prognosis are often unknown.

Basophils Play a Protective Role in the Recovery of Skin Barrier Function from Mechanical Injury in Mice.

Physical trauma disrupts skin barrier function. How the skin barrier recovers is not fully understood. We evaluated in mice the mechanism of skin barrier recovery after mechanical injury inflicted by tape stripping. Tape stripping disrupted skin barrier function as evidenced by increased transepidermal water loss. We show that tape stripping induces IL-1-, IL-23-, and TCRγδ+-dependent upregulation of cutaneous Il17a and Il22 expression. We demonstrate that IL-17A and IL-22 induce epidermal hyperplasia, promote neutrophil recruitment, and delay skin barrier function recovery. Neutrophil depletion improved the recovery of skin barrier function and decreased epidermal hyperplasia. Single-cell RNA sequencing and flow cytometry analysis of skin cells revealed basophil infiltration into tape-stripped skin. Basophil depletion upregulated Il17a expression, increased neutrophil infiltration, and delayed skin barrier recovery. Comparative analysis of genes differentially expressed in tape-stripped skin of basophil-depleted mice and Il17a-/- mice indicated that basophils counteract the effects of IL-17A on the expression of epidermal and lipid metabolism genes important for skin barrier integrity. Our results demonstrate that basophils play a protective role by downregulating Il17a expression after mechanical skin injury, thereby counteracting the adverse effect of IL-17A on skin barrier function recovery, and suggest interventions to accelerate this recovery.

Rethinking Immunological Risk: A Retrospective Cohort Study of Severe SARS-Cov-2 Infections in Individuals With Congenital Immunodeficiencies.

BACKGROUND: Debates on the allocation of medical resources during the coronavirus disease 2019 (COVID-19) pandemic revealed the need for a better understanding of immunological risk. Studies highlighted variable clinical outcomes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in individuals with defects in both adaptive and innate immunity, suggesting additional contributions from other factors. Notably, none of these studies controlled for variables linked with social determinants of health. OBJECTIVE: To determine the contributions of determinants of health to risk of hospitalization for SARS-CoV-2 infection among individuals with inborn errors of immunodeficiencies. METHODS: This is a retrospective, single-center cohort study of 166 individuals with inborn errors of immunity, aged 2 months through 69 years, who developed SARS-CoV-2 infections from March 1, 2020, through March 31, 2022. Risks of hospitalization were assessed using a multivariable logistic regression analysis. RESULTS: The risk of SARS-CoV-2-related hospitalization was associated with underrepresented racial and ethnic populations (odds ratio [OR] 4.50; 95% confidence interval [95% CI] 1.57-13.4), a diagnosis of any genetically defined immunodeficiency (OR 3.32; 95% CI 1.24-9.43), obesity (OR 4.24; 95% CI 1.38-13.3), and neurological disease (OR 4.47; 95% CI 1.44-14.3). The COVID-19 vaccination was associated with reduced hospitalization risk (OR 0.52; 95% CI 0.31-0.81). Defects in T cell and innate immune function, immune-mediated organ dysfunction, and social vulnerability were not associated with increased risk of hospitalization after controlling for covariates. CONCLUSIONS: The associations between race, ethnicity, and obesity with increased risk of hospitalization for SARS-CoV-2 infection indicate the importance of variables linked with social determinants of health as immunological risk factors for individuals with inborn errors of immunity.

Clinical utility of measuring CD4+ T follicular cells in patients with immune dysregulation.

Mechanistic studies of autoimmune disorders have identified circulating T follicular helper (cTfh) cells as drivers of autoimmunity. However, the quantification of cTfh cells is not yet used in clinical practice due to the lack of age-stratified normal ranges and the unknown sensitivity and specificity of this test for autoimmunity. We enrolled 238 healthy participants and 130 patients with common and rare disorders of autoimmunity or autoinflammation. Patients with infections, active malignancy, or any history of transplantation were excluded. In 238 healthy controls, median cTfh percentages (range 4.8%-6.2%) were comparable among age groups, sexes, races, and ethnicities, apart from a significantly lower percentages in children less than 1 year of age (median 2.1%, CI: 0.4%-6.8, p < 0.0001). Among 130 patients with over 40 immune regulatory disorders, a cTfh percentage exceeding 12% had 88% sensitivity and 94% specificity for differentiating disorders with adaptive immune cell dysregulation from those with predominantly innate cell defects. This threshold had a sensitivity of 86% and specificity of 100% for active autoimmunity and normalized with effective treatment. cTfh percentages exceeding 12% distinguish autoimmunity from autoinflammation, thereby differentiating two endotypes of immune dysregulation with overlapping symptoms and different therapies.

IL-4 receptor alpha blockade dampens allergic inflammation and upregulates IL-17A expression to promote Saureus clearance in antigen sensitized mouse skin.

BACKGROUND: Skin colonization with Staphylococcus aureus aggravates atopic dermatitis and exaggerates allergic skin inflammation in mice. IL-4 receptor α (IL-4Rα) blockade is beneficial in atopic dermatitis and reduces Saureus skin colonization through unknown mechanisms. The cytokine IL-17A restrains Saureus growth. OBJECTIVES: This study sought to examine the effect of IL-4Rα blockade on Saureus colonization at sites of allergic skin inflammation in mice and determine the mechanism involved. METHODS: BALB/c mice were epicutaneously sensitized with ovalbumin (OVA). Immediately after, PSVue 794-labeled S aureus strain SF8300 or saline was applied and a single dose of anti-IL-4Rα blocking antibody, a mixture of anti-IL-4Rα and anti-IL-17A blocking antibodies, or IgG isotype controls were administered intradermally. Saureus load was assessed 2 days later by in vivo imaging and enumeration of colony forming units. Skin cellular infiltration was examined by flow cytometry and gene expression by quantitative PCR and transcriptome analysis. RESULTS: IL-4Rα blockade decreased allergic skin inflammation in OVA-sensitized skin, as well as in OVA-sensitized and Saureus-exposed skin, evidenced by significantly decreased epidermal thickening and reduced dermal infiltration by eosinophils and mast cells. This was accompanied by increased cutaneous expression of Il17a and IL-17A-driven antimicrobial genes with no change in Il4 and Il13 expression. IL-4Rα blockade significantly decreased Saureus load in OVA-sensitized and S aureus-exposed skin. IL-17A blockade reversed the beneficial effect of IL-4Rα blockade on Saureus clearance and reduced the cutaneous expression of IL-17A-driven antimicrobial genes. CONCLUSIONS: IL-4Rα blockade promotes Saureus clearance from sites of allergic skin inflammation in part by enhancing IL-17A expression.

Clinical utility of measuring CD4 + T follicular cells in patients with immune dysregulation.

Mechanistic studies of autoimmune disorders have identified circulating T follicular helper (cTfh) cells as drivers of autoimmunity. However, the quantification of cTfh cells is not yet used in clinical practice due to the lack of age-stratified normal ranges and the unknown sensitivity and specificity of this test for autoimmunity. We enrolled 238 healthy participants and 130 patients with common and rare disorders of autoimmunity or autoinflammation. Patients with infections, active malignancy, or any history of transplantation were excluded. In 238 healthy controls, median cTfh percentages (range 4.8% - 6.2%) were comparable among age groups, sexes, races, and ethnicities, apart from a significantly lower percentages in children less than 1 year of age (median 2.1%, CI: 0.4% - 6.8, p< 0.0001). Among 130 patients with over 40 immune regulatory disorders, a cTfh percentage exceeding 12% had 88% sensitivity and 94% specificity for differentiating disorders with adaptive immune cell dysregulation from those with predominantly innate cell defects. This threshold had a sensitivity of 86% and specificity of 100% for active autoimmunity and normalized with effective treatment. cTfh percentages exceeding 12% distinguish autoimmunity from autoinflammation, thereby differentiating two endotypes of immune dysregulation with overlapping symptoms and different therapies.

Rethinking immunologic risk: a retrospective cohort study of severe SARS-CoV-2 infections in individuals with congenital immunodeficiencies.

Background: Debates on the allocation of medical resources during the COVID-19 pandemic revealed the need for a better understanding of immunologic risk. Studies highlighted variable clinical outcomes of SARS-CoV-2 infections in individuals with defects in both adaptive and innate immunity, suggesting additional contributions from other factors. Notably, none of these studies controlled for variables linked with social determinants of health. Objective: To determine the contributions of determinants of health to risk of hospitalization for SARS-CoV-2 infection among individuals with inborn errors of immunodeficiencies. Methods: This is a retrospective, single-center cohort study of 166 individuals with inborn errors of immunity, aged two months through 69 years, who developed SARS-CoV-2 infections from March 1, 2020 through March 31, 2022. Risks of hospitalization was assessed using a multivariable logistic regression analysis. Results: The risk of SARS-CoV-2-related hospitalization was associated with underrepresented racial and ethnic populations (odds ratio [OR] 5.29; confidence interval [CI], 1.76-17.0), a diagnosis of any genetically-defined immunodeficiency (OR 4.62; CI, 1.60-14.8), use of B cell depleting therapy within one year of infection (OR 6.1; CI, 1.05-38.5), obesity (OR 3.74; CI, 1.17-12.5), and neurologic disease (OR 5.38; CI, 1.61-17.8). COVID-19 vaccination was associated with reduced hospitalization risk (OR 0.52; CI, 0.31-0.81). Defective T cell function, immune-mediated organ dysfunction, and social vulnerability were not associated with increased risk of hospitalization after controlling for covariates. Conclusions: The associations between race, ethnicity, and obesity with increased risk of hospitalization for SARS-CoV-2 infection indicate the importance of variables linked with social determinants of health as immunologic risk factors for individuals with inborn errors of immunity. Highlights: What is already known about this topic? Outcomes of SARS-CoV-2 infections in individuals with inborn errors of immunity (IEI) are highly variable. Prior studies of patients with IEI have not controlled for race or social vulnerability. What does this article add to our knowledge ? For individuals with IEI, hospitalizations for SARS-CoV-2 were associated with race, ethnicity, obesity, and neurologic disease. Specific types of immunodeficiency, organ dysfunction, and social vulnerability were not associated with increased risk of hospitalization. How does this study impact current management guidelines? Current guidelines for the management of IEIs focus on risk conferred by genetic and cellular mechanisms. This study highlights the importance of considering variables linked with social determinants of health and common comorbidities as immunologic risk factors.

Mechanisms underlying genetic susceptibility to multisystem inflammatory syndrome in children (MIS-C).

BACKGROUND: Multisystem inflammatory syndrome in children (MIS-C) is a pediatric complication of severe acute respiratory syndrome coronavirus 2 infection that is characterized by multiorgan inflammation and frequently by cardiovascular dysfunction. It occurs predominantly in otherwise healthy children. We previously reported haploinsufficiency of suppressor of cytokine signaling 1 (SOCS1), a negative regulator of type I and II interferons, as a genetic risk factor for MIS-C. OBJECTIVES: We aimed to identify additional genetic mechanisms underlying susceptibility to severe acute respiratory syndrome coronavirus 2-associated MIS-C. METHODS: In a single-center, prospective cohort study, whole exome sequencing was performed on patients with MIS-C. The impact of candidate variants was tested by using patients' PBMCs obtained at least 7 months after recovery. RESULTS: We enrolled 18 patients with MIS-C (median age = 8 years; interquartile range = 5-12.25 years), of whom 89% had no conditions other than obesity. In 2 boys with no significant infection history, we identified and validated hemizygous deleterious defects in XIAP, encoding X-linked inhibitor of apoptosis, and CYBB, encoding cytochrome b-245, beta subunit. Including the previously reported SOCS1 haploinsufficiency, a genetic diagnosis was identified in 3 of 18 patients (17%). In contrast to patients with mild COVID-19, patients with defects in SOCS1, XIAP, or CYBB exhibit an inflammatory immune cell transcriptome with enrichment of differentially expressed genes in pathways downstream of IL-18, oncostatin M, and nuclear factor κB, even after recovery. CONCLUSIONS: Although inflammatory disorders are rare in the general population, our cohort of patients with MIS-C was enriched for monogenic susceptibility to inflammation. Our results support the use of next-generation sequencing in previously healthy children who develop MIS-C.

Multisystem inflammation and susceptibility to viral infections in human ZNFX1 deficiency.

BACKGROUND: Recognition of viral nucleic acids is one of the primary triggers for a type I interferon-mediated antiviral immune response. Inborn errors of type I interferon immunity can be associated with increased inflammation and/or increased susceptibility to viral infections as a result of dysbalanced interferon production. NFX1-type zinc finger-containing 1 (ZNFX1) is an interferon-stimulated double-stranded RNA sensor that restricts the replication of RNA viruses in mice. The role of ZNFX1 in the human immune response is not known. OBJECTIVE: We studied 15 patients from 8 families with an autosomal recessive immunodeficiency characterized by severe infections by both RNA and DNA viruses and virally triggered inflammatory episodes with hemophagocytic lymphohistiocytosis-like disease, early-onset seizures, and renal and lung disease. METHODS: Whole exome sequencing was performed on 13 patients from 8 families. We investigated the transcriptome, posttranscriptional regulation of interferon-stimulated genes (ISGs) and predisposition to viral infections in primary cells from patients and controls stimulated with synthetic double-stranded nucleic acids. RESULTS: Deleterious homozygous and compound heterozygous ZNFX1 variants were identified in all 13 patients. Stimulation of patient-derived primary cells with synthetic double-stranded nucleic acids was associated with a deregulated pattern of expression of ISGs and alterations in the half-life of the mRNA of ISGs and also associated with poorer clearance of viral infections by monocytes. CONCLUSION: ZNFX1 is an important regulator of the response to double-stranded nucleic acids stimuli following viral infections. ZNFX1 deficiency predisposes to severe viral infections and a multisystem inflammatory disease.

Hypomorphic variants in AK2 reveal the contribution of mitochondrial function to B-cell activation.

BACKGROUND: The gene AK2 encodes the phosphotransferase adenylate kinase 2 (AK2). Human variants in AK2 cause reticular dysgenesis, a severe combined immunodeficiency with agranulocytosis, lymphopenia, and sensorineural deafness that requires hematopoietic stem cell transplantation for survival. OBJECTIVE: We investigated the mechanisms underlying recurrent sinopulmonary infections and hypogammaglobulinemia in 15 patients, ranging from 3 to 34 years of age, from 9 kindreds. Only 2 patients, both of whom had mildly impaired T-cell proliferation, each had a single clinically significant opportunistic infection. METHODS: Patient cells were studied with next-generation DNA sequencing, tandem mass spectrometry, and assays of lymphocyte and mitochondrial function. RESULTS: We identified 2 different homozygous variants in AK2. AK2G100S and AK2A182D permit residual protein expression, enzymatic activity, and normal numbers of neutrophils and lymphocytes. All but 1 patient had intact hearing. The patients' B cells had severely impaired proliferation and in vitro immunoglobulin secretion. With activation, the patients' B cells exhibited defective mitochondrial respiration and impaired regulation of mitochondrial membrane potential and quality. Although activated T cells from the patients with opportunistic infections demonstrated impaired mitochondrial function, the mitochondrial quality in T cells was preserved. Consistent with the capacity of activated T cells to utilize nonmitochondrial metabolism, these findings revealed a less strict cellular dependence of T-cell function on AK2 activity. Chemical inhibition of ATP synthesis in control T and B cells similarly demonstrated the greater dependency of B cells on mitochondrial function. CONCLUSIONS: Our patients demonstrate the in vivo sequelae of the cell-specific requirements for the functions of AK2 and mitochondria, particularly in B-cell activation and antibody production.