Patients with STAT1 Gain-of-function Mutations Display Increased Apoptosis which is Reversed by the JAK Inhibitor Ruxolitinib.

INTRODUCTION: The signal transducer and activator of transcription (STAT1) gain-of-function (GOF) syndrome accounts for most cases of chronic mucocutaneous candidiasis but is characterized by a broader clinical phenotype that may include bacterial, viral, or invasive fungal infections, autoimmunity, autoinflammatory manifestations, vascular complications, or malignancies. The severity of lymphopenia may vary and influence the infectious morbidity. METHODS: In our cohort of seven STAT1-GOF patients, we investigated the mechanisms that may determine T lymphopenia, we characterized the interferon gene signature (IGS) and analyzed the effect of ruxolitinib in reverting the immune dysregulation. RESULTS: STAT1-GOF patients exhibited increased T lymphocyte apoptosis that was significantly augmented in both resting conditions and following stimulation with mitogens and IFNα, as evaluated by flow cytometry by Annexin V/ Propidium iodide assay. The JAK inhibitor ruxolitinib significantly reduced the IFNα-induced hyperphosphorylation of STAT1 and reverted the stimulation-induced T-cell apoptosis, in vitro. In two adult STAT1-GOF patients, the JAKinib treatment ameliorated chronic mucocutaneous candidiasis and lymphopenia. Most STAT1-GOF patients, particularly those who had autoimmunity, presented increased IGS that significantly decreased in the two patients during ruxolitinib treatment. CONCLUSION: In STAT1-GOF patients, T lymphocyte apoptosis is increased, and T lymphopenia may determine higher risk of severe infections. The JAKinib target therapy should be evaluated to treat severe chronic candidiasis and lymphopenia, and to downregulate the IFNs in patients with autoinflammatory or autoimmune manifestations.

Sustained post-rituximab B-cell depletion is common in ANCA-associated vasculitis and is affected by sex and renal function.

OBJECTIVE: Despite the increasing use of rituximab in anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), it remains unclear what the optimal dosing is, especially for maintenance of remission. A deeper understanding of post-rituximab B-cell repopulation patterns may aid better-tailored treatment. METHODS: This is a monocentric, retrospective study including ANCA-positive AAV patients receiving a single course of rituximab induction. CD19+ B cells were longitudinally monitored with flow cytometry. B-cell repopulation was defined as CD19+ >10 cells/µL. RESULTS: Seventy-one patients were included, the majority with microscopic polyangiitis (75%), myeloperoxidase-ANCA positivity (75%) and with renal involvement (79%). During a median follow-up of 54 months since the first rituximab infusion, 44 patients (62%) repopulated B cells, with a median time to repopulation of 39 months (range 7-102). Patients experiencing B-cell depletion lasting longer than the overall median time to repopulation (39 months) exhibited a lower risk of flare and higher risk of serious infection. In multivariate Cox regression, higher estimated glomerular filtration rate (eGFR) [hazard ratio (HR) 1.84, 95% confidence interval (CI) 1.13-2.98 per 30 mL/min/1.73 m2 eGFR] and female sex (HR 2.70, 95% CI 1.37-5.31) were independent predictors of increased rate of B-cell repopulation. CONCLUSION: A subset of AAV patients develop sustained post-rituximab B-cell depletion, which associates with reduced risk of flare and increased risk of serious infection in the long term. Preserved renal function and female sex are associated with faster B-cell repopulation. These observations further highlight the need to personalize immunosuppression to improve clinical outcomes.

A distinct human cell type expressing MHCII and RORγt with dual characteristics of dendritic cells and type 3 innate lymphoid cells.

Recent studies have characterized various mouse antigen-presenting cells (APCs) expressing the lymphoid-lineage transcription factor RORγt (Retinoid-related orphan receptor gamma t), which exhibit distinct phenotypic features and are implicated in the induction of peripheral regulatory T cells (Tregs) and immune tolerance to microbiota and self-antigens. These APCs encompass Janus cells and Thetis cell subsets, some of which express the AutoImmune REgulator (AIRE). RORγt+ MHCII+ type 3 innate lymphoid cells (ILC3) have also been implicated in the instruction of microbiota-specific Tregs. While RORγt+ APCs have been actively investigated in mice, the identity and function of these cell subsets in humans remain elusive. Herein, we identify a rare subset of RORγt+ cells with dendritic cell (DC) features through integrated single-cell RNA sequencing and single-cell ATAC sequencing. These cells, which we term RORγt+ DC-like cells (R-DC-like), exhibit DC morphology, express the MHC class II machinery, and are distinct from all previously reported DC and ILC3 subsets, but share transcriptional and epigenetic similarities with DC2 and ILC3. We have developed procedures to isolate and expand them in vitro, enabling their functional characterization. R-DC-like cells proliferate in vitro, continue to express RORγt, and differentiate into CD1c+ DC2-like cells. They stimulate the proliferation of allogeneic T cells. The identification of human R-DC-like cells with proliferative potential and plasticity toward CD1c+ DC2-like cells will prompt further investigation into their impact on immune homeostasis, inflammation, and autoimmunity.

Clinical and transcriptomic characteristics of a novel SMARCD2 mutation that disrupts neutrophil maturation and function.

We report a novel case of SMARCD2 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily D, member 2) mutation successfully treated with hematopoietic stem cell transplantation. The female patient presented delayed cord separation, chronic diarrhea, skin abscesses, skeletal dysmorphisms, and neutropenia with specific granule deficiency. Analysis of the transcriptomic profile of peripheral blood sorted mature and immature SMARCD2 neutrophils showed defective maturation process that associated with altered expression of genes related to specific, azurophilic, and gelatinase granules, such as LTF, CRISP3, PTX3, and CHI3L1. These abnormalities account for the prevalence of immature neutrophils in the peripheral blood, impaired function, and deregulated inflammatory responses.

Rituximab Monotherapy Is Effective as First-Line Treatment for Granulomatous Lymphocytic Interstitial Lung Disease (GLILD) in CVID Patients.

Granulomatous lymphocytic interstitial lung disease (GLILD) represents a fatal immune dysregulatory complication in common variable immunodeficiency (CVID). Evidence-based diagnostic guidelines are lacking, and GLILD treatment consists in immunosuppressive drugs; nonetheless, therapeutical strategies are heterogeneous and essentially based on experts' opinions and data from small case series or case reports.We aimed to evaluate the efficacy and safety of first-line Rituximab monotherapy for CVID-related GLILD, by assessing symptoms and quality of life alterations, immunological parameters, pulmonary function tests, and lung computed tomography.All six GLILD patients received Rituximab infusions as a first-line treatment. Rituximab was administered at 375 mg/m2 monthly for six infusions followed by maintenance every 3 months; none of the patients experienced severe adverse events. Symptom burden and quality of life significantly improved in treated patients compared to a control group of CVID patients without GLILD. Rituximab treatment indirectly caused a trend toward reduced T-cell activation and exhaustion markers sCD25 and sTIM-3. Lung function improved in treated patients, with statistically significant increases in TLC and DLCO. Lung CT scan findings expressed by means of Baumann scoring system displayed a reduction in the entire cohort.In conclusion, first-line monotherapy with Rituximab displayed high efficacy in disease remission in all treated patients, with improvement of symptoms and amelioration of quality of life, as well as restoration of PFTs and lung CT scan findings.

Antibody Deficiency in Patients with Biallelic KARS1 Mutations.

Biallelic KARS1 mutations cause KARS-related diseases, a rare syndromic condition encompassing central and peripheral nervous system impairment, heart and liver disease, and deafness. KARS1 encodes the t-RNA synthase of lysine, an aminoacyl-tRNA synthetase, involved in different physiological mechanisms (such as angiogenesis, post-translational modifications, translation initiation, autophagy and mitochondrial function). Although patients with immune-hematological abnormalities have been individually described, results have not been collectively discussed and functional studies investigating how KARS1 mutations affect B cells have not been performed. Here, we describe one patient with severe developmental delay, sensoneurinal deafness, acute disseminated encephalomyelitis, hypogammaglobulinemia and recurrent infections. Pathogenic biallelic KARS1 variants (Phe291Val/ Pro499Leu) were associated with impaired B cell metabolism (decreased mitochondrial numbers and activity). All published cases of KARS-related diseases were identified. The corresponding authors and researchers involved in the diagnosis of inborn errors of immunity or genetic syndromes were contacted to obtain up-to-date clinical and immunological information. Seventeen patients with KARS-related diseases were identified. Recurrent/severe infections (9/17) and B cell abnormalities (either B cell lymphopenia [3/9], hypogammaglobulinemia [either IgG, IgA or IgM; 6/15] or impaired vaccine responses [4/7]) were frequently reported. Immunoglobulin replacement therapy was given in five patients. Full immunological assessment is warranted in these patients, who may require detailed investigation and specific supportive treatment.

Longitudinal Characterization of Immune Response in a Cohort of Children Hospitalized with Multisystem Inflammatory Syndrome.

BACKGROUND: Multisystem Inflammatory Syndrome in Children (MIS-C) is a severe complication of SARS-CoV-2 infection caused by hyperactivation of the immune system. METHODS: this is a retrospective analysis of clinical data, biochemical parameters, and immune cell subsets in 40 MIS-C patients from hospital admission to outpatient long-term follow-up. RESULTS: MIS-C patients had elevated inflammatory markers, associated with T- and NK-cell lymphopenia, a profound depletion of dendritic cells, and altered monocyte phenotype at disease onset, while the subacute phase of the disease was characterized by a significant increase in T- and B-cell counts and a rapid decline in activated T cells and terminally differentiated B cells. Most of the immunological parameters returned to values close to the normal range during the remission phase (20-60 days after hospital admission). Nevertheless, we observed a significantly reduced ratio between recently generated and more differentiated CD8+ T- and B-cell subsets, which partially settled at longer-term follow-up determinations. CONCLUSIONS: The characterization of lymphocyte distribution in different phases of MIS-C may help to understand the course of diseases that are associated with dysregulated immune responses and to calibrate prompt and targeted treatments.

CARD11 dominant negative mutation leads to altered human Natural Killer cell homeostasis.

Dominant negative mutations in CARD11 have been reported in patients with immune dysregulation, severe atopic features, and variable T cell alterations. Data on Natural killer (NK) cells from affected patients are lacking. We report on a 12-year-old boy with severe atopic dermatitis, food induced anaphylaxis and hypogammaglobulinemia harbouring a novel de novo heterozygous variant c.169G > A; p.Glu57Lys in CARD11. The dominant negative effect of this mutation was confirmed on both CD4+ and CD8+. CTLA4+Foxp3+CD4+ Tregs were severely reduced. Patient's NK cells showed reduced expression of NKp46, NKG2D and CD69. Patient's CD56bright NK cells showed in vitro impaired production of IFN-γ. Steady state pS6 levels on patient's NK cells were increased and remained elevated upon IL2 + IL12 + IL18 overnight stimulation. Overall, the effect of CARD11 mutation on mTORC1 differs between T and NK cells. These findings may explain the increased susceptibility to viral infections and the reduced immune surveillance in affected patients.

Reduced G protein signaling despite impaired internalization and ß-arrestin recruitment in patients carrying a CXCR4Leu317fsX3 mutation causing WHIM syndrome.

WHIM syndrome is an inherited immune disorder caused by an autosomal dominant heterozygous mutation in CXCR4. The disease is characterized by neutropenia/leukopenia (secondary to retention of mature neutrophils in bone marrow), recurrent bacterial infections, treatment-refractory warts, and hypogammaglobulinemia. All mutations reported in WHIM patients lead to the truncations in the C-terminal domain of CXCR4, R334X being the most frequent. This defect prevents receptor internalization and enhances both calcium mobilization and ERK phosphorylation, resulting in increased chemotaxis in response to the unique ligand CXCL12. Here, we describe 3 patients presenting neutropenia and myelokathexis, but normal lymphocyte count and immunoglobulin levels, carrying what we believe to be a novel Leu317fsX3 mutation in CXCR4, leading to a complete truncation of its intracellular tail. The analysis of the L317fsX3 mutation in cells derived from patients and in vitro cellular models reveals unique signaling features in comparison with R334X mutation. The L317fsX3 mutation impairs CXCR4 downregulation and ß-arrestin recruitment in response to CXCL12 and reduces other signaling events - including ERK1/2 phosphorylation, calcium mobilization, and chemotaxis - all processes that are typically enhanced in cells carrying the R334X mutation. Our findings suggest that, overall, the L317fsX3 mutation may be causative of a form of WHIM syndrome not associated with an augmented CXCR4 response to CXCL12.

Lack of a prompt normalization of immunological parameters is associated with long-term care and poor prognosis in COVID-19 affected patients receiving convalescent plasma: a single center experience.

OBJECTIVES: Being COVID-19 convalescent plasma (CCP) a therapeutic option that can have a potential impact on the normalization of immunological parameters of COVID-19 affected patients, a detailed analysis of post-infusion immunological changes was conducted in CCP treated patients, aiming to identify possible predictive hallmarks of disease prognosis. METHODS: This prospective observational study describes a cohort of 28 patients who received CCP shortly after being hospitalized for COVID-19 and diagnosed for Acute Respiratory Distress Syndrome. All patients were subjected to a detailed flow cytometry based evaluation of immunological markers at baseline and on days +3 and +7 after transfusion. RESULTS: At baseline almost all patients suffered from lymphopenia (25/28 on T-cells and 16/28 on B-cells) coupled with neutrophil-lymphocyte ratio exceeding normal values (26/28). Lymphocyte subsets were generally characterized by increased percentages of CD19+CD20-CD38hiCD27+ plasmablasts and reduction of CD4+CD45RA+CCR7+CD31+ recent thymic emigrants, while monocytes presented a limited expression of CD4 and HLA-DR molecules. Amelioration of immunological parameters began to be evident from day +3 and became more significant at day +7 post-CCP transfusion in 18 patients who recovered within 30 days from hospitalization. Conversely, baseline immunological characteristics generally persisted in ten critical patients who eventually progressed to death (6) or long-term care (4). CONCLUSIONS: This study demonstrates that proper immunophenotyping panels can be potentially useful for monitoring CCP treated patients from the first days after infusion in order to presume higher risk of medical complications.

Timely Leukapheresis May Interfere with the "Fitness" of Lymphocytes Collected for CAR-T Treatment in High Risk DLBCL Patients.

The development of chimeric antigen receptor (CAR)-T cell therapy has revolutionized the treatment of hematological diseases. However, approximately 60% of patients relapse after CAR-T cell therapy, and no clear cause for this failure has been identified. The objective of the Bio-CAR-T BS study (ClinicalTrials.gov: NCT05366569) is to improve our understanding of the lymphocyte harvest to maximize the quality of the CAR-T cell product. Of the 14 patients enrolled, 11 were diagnosed with DLBCL, 2 with PMBCL, and 1 with ALL. Five of 11 DLBCL patients met the criteria for "pre-emptive" Lymphocytes-apheresis (being at high risk of second relapse), and 6 were included in the standard-of-care Lymphocytes-apheresis group. Previous autologous stem cell transplantation (ASCT) and age were significantly different between the two groups. At the time of Lymphocyte-apheresis, patients in the "pre-emptive" group had more "fit" lymphocytes (higher CD4+/CD8+ ratio; higher naïve T cells levels) compared with standard group, probably due to the impact of ASCT. At the same time, also being older than 60 years results in a more "exhausted" lymphocyte profile. Overall, "pre-emptive" Ly-apheresis in DLBCL patients at high risk of relapse appears to be feasible and may allow the timely collection of "fit" lymphocytes for CAR-T cell manufacturing.

Abnormal B-Cell Maturation and Increased Transitional B Cells in CBL Syndrome.

CBL syndrome is a Noonan-like RASopathy with heterogeneous clinical phenotype and predisposition to juvenile myelomonocytic leukemia (JMML). Here we describe two patients with identical germline CBL mutation and clinical and immune-hematological overlapping features with autoimmune lymphoproliferative syndrome (ALPS) and B-cell expansion with NF-κB and T-cell anergy (BENTA) syndrome. Increased immature/transitional B cells can be depicted in CBL syndrome, ALPS, and BENTA. Nonetheless, our patients here described showed peculiar B-cell phenotype due to increased immature/transitional CD34+ B cells. This feature differentiates CBL syndrome from BENTA, pointing toward an abnormal proliferation of B-cell early precursors.

Lymphocyte alterations in patients with Common Variable Immunodeficiency (CVID) and autoimmune manifestations.

INTRODUCTION: Autoimmunity is a common feature in CVID patients. To date the mechanisms leading to the development of such complications are not fully elucidated. MATERIALS AND METHODS: Data from 122 CVID patients subdivided in three groups based on the absence of autoimmunity (n-AI) or the presence of hematologic autoimmune phenomena (Cy-AI) or non-hematologic autoimmune phenomena (n-Cy-AI) were evaluated. RESULTS: We identified a total of 128 autoimmune manifestations in 55/122 patients (45.1%). 30/122 (24.6%) patients presented hematologic autoimmune phenomena while 29/122 (23.8%) presented gastrointestinal autoimmune involvement. Immune thrombocytopenia was the most common manifestation (27/122; 22.1%), followed by autoimmune hemolytic anemia (18/122; 14.8%) and autoimmune enteropathy (17/122; 13.9%). Cy-AI patients displayed higher CD4+ effector memory and terminally differentiated CD8+ cells with lower percentages of naïve and recent thymic emigrants (RTEs) CD4+ cells and a significant expansion of the CD19hiCD21low population. CONCLUSIONS: CVID patients developing autoimmune cytopenias display characteristic immune phenotypic features.

Identical EP300 variant leading to Rubinstein-Taybi syndrome with different clinical and immunologic phenotype.

The Rubinstein-Taybi syndrome (RSTS) is a rare developmental disorder characterized by craniofacial dysmorphisms, broad thumbs and toes, intellectual disability, growth deficiency, and recurrent infections. Mutations in the cyclic adenosine monophosphate response element-binding protein (CREB)-binding protein (CREBBP) or in the E1A-associated protein p300 (EP300) genes have been demonstrated in 55% (RSTS1) and up to 8% of the patients (RSTS2), respectively. Dysfunction of immune response has been reported in a subgroup of individuals with RSTS. Here we characterize two patients carrying the same EP300 variant and distinctive RSTS features (including congenital heart abnormalities, short stature, feeding problems, and gastroesophageal reflux). Whole exome sequencing did not support a dual molecular diagnosis hypothesis. Nonetheless, patients showed distinct clinical manifestations and immunological features. The most severe phenotype was associated with reduced T-cell production and diversity. This latter feature was confirmed in a control group of four RSTS patients.

SARS-CoV-2 infection in dialysis and kidney transplant patients: immunological and serological response.

BACKGROUND: Dialysis and kidney transplant patients with moderate-severe COVID-19 have a high mortality rate, around 30%, that is similar in the two populations, despite differences in their baseline characteristics. In these groups, the immunology of the disease has been poorly explored. METHODS: Thirty-two patients on dialysis or with kidney transplant and SARS-CoV-2 infection requiring hospitalization (COV group) were included in our study. Lymphocyte subsets, dendritic cell (DC) counts and monocyte activation were studied. SARS-CoV-2 anti-spike/anti-nucleocapsid were monitored, and baseline cytokines and chemokines were measured in 10 patients. RESULTS: The COV group, compared to healthy subjects and uninfected dialysis/kidney transplant controls, showed lower numbers of CD4 + and CD8 + T cells, Natural-Killer (NK), B cells, plasmacytoid and myeloid DCs, while the proportion of terminally differentiated B-cells was increased. IL6, IL10, IFN-α and chemokines involved in monocyte and neutrophil recruitment were higher in the COV group, compared to uninfected dialysis/kidney transplant controls. Patients with severe disease had lower CD4 + , CD8 + and B-cell counts and lower monocyte HLA-DR expression. Of note, when comparing dialysis and kidney transplant patients with COVID-19, the latter group presented lower NK and pDC counts and monocyte HLA-DR expression. Up to 60 days after symptom onset, kidney transplant recipients showed lower levels of anti-spike antibodies compared to dialysis patients. CONCLUSIONS: During SARS-CoV-2 infection, dialysis and kidney transplant patients manifest immunophenotype abnormalities; these are similar in the two groups, however kidney transplant recipients show more profound alterations of the innate immune system and lower anti-spike antibody response.

Immunological Evaluation of Patients Affected with Jacobsen Syndrome Reveals Profound Not Age-Related Lymphocyte Alterations.

PURPOSE: Jacobsen syndrome (JS) is a rare form of genetic disorder that was recently classified as a syndromic immunodeficiency. Available detailed immunological data from JS patients are limited. METHODS: Clinical and immunological presentation of twelve pediatric patients with JS by means of revision of clinical records, flow cytometry, real-time PCR, and lymphocyte functional testing were collected. RESULTS: Recurrent infections were registered in 6/12 patients (50%), while bleeding episodes in 2/12 (16.7%). White blood cell and absolute lymphocyte counts were reduced in 8/12 (66.7%) and 7/12 (58.3%) patients, respectively. Absolute numbers of CD3+ and CD4+ T cells were reduced in 8/12 (66.7%) and 7/12 (58.3%), respectively. Of note, recent thymic emigrants (RTE) were reduced in all tested patients (9/9), with T-cell receptor excision circle analysis (TRECs) showing a similar trend in 8/9 patients; naïve CD4+ T cells were low only in 5/11 patients (45.4%). Interestingly, B-cell counts, IgM memory B cells, and IgM serum levels were reduced in 10/12 (83.3%) patients. Natural killer (NK) cell counts were mostly normal but the percentages of CD16+CD56low/- cells were expanded in 7/7 patients tested. The observed immunological alterations did not correlate with patients' age. Finally, responses to proliferative stimuli were normal at presentation for all patients, although they may deteriorate over time. CONCLUSIONS: Our data suggest that patients affected with JS may display important numeric and maturational alterations in the T-, B-, and NK-cell compartments. These findings suggest that JS patients should be regularly monitored from an immunological point of view.

Case Report: Analysis of Inflammatory Cytokines IL-6, CCL2/MCP1, CCL5/RANTES, CXCL9/MIG, and CXCL10/IP10 in a Cystic Fibrosis Patient Cohort During the First Wave of the COVID-19 Pandemic.

Since the beginning of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, data registered in the European countries revealed increasing cases of infection in cystic fibrosis (CF) patients. In the course of this pandemic, we enrolled 17 CF patients for a study evaluating inflammatory markers. One of them developed COVID-19, giving us the possibility to analyze inflammatory markers in the acute phase as compared to levels detected before and after the infectious episode and to levels measured in the other CF patients enrolled to the study who did not experience COVID-19 and 23 patients referred to our center for SARS-CoV-2 infection.

Case Report: Hypomorphic Function and Somatic Reversion in DOCK8 Deficiency in One Patient With Two Novel Variants and Sclerosing Cholangitis.

DOCK8 deficiency is a combined immunodeficiency due to biallelic variants in dedicator of cytokinesis 8 (DOCK8) gene. The disease has a wide clinical spectrum encompassing recurrent infections (candidiasis, viral and bacterial infections), virally driven malignancies and immune dysregulatory features, including autoimmune (cytopenia and vasculitis) as well as allergic disorders (eczema, asthma, and food allergy). Hypomorphic function and somatic reversion of DOCK8 has been reported to result in incomplete phenotype without IgE overproduction. Here we describe a case of DOCK8 deficiency in a 8-year-old Caucasian girl. The patient's disease was initially classified as autoimmune thrombocytopenia, which then evolved toward a combined immunodeficiency phenotype with recurrent infections, persistent EBV infection and lymphoproliferation. Two novel variants (one deletion and one premature stop codon) were characterized, resulting in markedly reduced, but not absent, DOCK8 expression. Somatic reversion of the DOCK8 deletion was identified in T cells. Hypomorphic function and somatic reversion were associated with restricted T cell repertoire, decreased STAT5 phosphorylation and impaired immune synapse functioning in T cells. Although the patient presented with incomplete phenotype (absence of markedly increase IgE and eosinophil count), sclerosing cholangitis was incidentally detected, thus indicating that hypomorphic function and somatic reversion of DOCK8 may delay disease progression but do not necessarily prevent from severe complications.