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Case history: We present the case of a 31-year-old Hispanic male with history of recurrent bronchiectasis, invasive aspergillosis, and severe persistent asthma, who is now status post lung transplant for end-stage lung disease. He initially presented at 7 years of age with diarrhea, failure to thrive, and nearly absent immunoglobulin levels (IgG < 33 mg/dL, IgA < 7 mg/dL, IgM = 11 mg/dL, IgE = 4 IU/dL) necessitating IVIG treatment. Small intestinal biopsy showed villous atrophy consistent with autoimmune enteropathy. Sweat chloride was reported as indeterminate (44 me/dL). Initial WBC, platelet, and T- and NK-cell counts were within normal range, and B-cell count and percentage were borderline low. Most recently, he was found to have increased immature B-cell count (CD21low), decreased memory B-cells, and poor pneumococcal vaccine antibody response. Patient has been hospitalized numerous times with increasingly severe bronchiectasis, pneumonitis, and COVID-19 infections twice despite vaccination, leading to respiratory failure and lung transplantation. Family history is negative for immune deficiency and lung diseases. Discussion(s): Of these 3 VUSs (see the table), the one in IRF2BP2 has the most pathogenic potential due to its autosomal dominant inheritance, its location in a conserved domain (Ring), and previous case reports of pathogenic variants at the same or adjacent alleles 1-3. Baxter et al reported a de novo truncating mutation in IRF2BP2 at codon 536 (c.1606CinsTTT), which is similar to our patient's mutation. This patient was noted to have an IPEX-like presentation, with chronic diarrhea, hypogammaglobulinemia, and recurrent infections. Variant Functional Prediction Score for our variant predicts a potentially high damage effect. There are 2 other case reports of heterozygous mutations in loci adjacent to this allele;one (c.1652G>A)2 with a similar clinical phenotype to our patient and the other (C.625-665 del)3 with primarily inflammatory features and few infections. Impact: This case highlights a variant in IRF2BP2 associated with severe hypogammaglobulinemia, recurrent pulmonary infections, and autoimmune enteropathy. [Table presented]Copyright © 2023 Elsevier Inc.
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Introduction: COVID-19 related encephalitis has been reported in pediatric patients;however, there are no reports in patients with inborn errors of immunity (IEI). Activated PI3K Delta Syndrome (APDS) is a disease of immune dysregulation with immunodeficiency, autoimmunity, and abnormal lymphoproliferation resulting from autosomal dominant gain-offunction variants in PIK3CD or PIK3R1 genes. We investigate a family with APDS, one mother and three children, one of whom developed COVID-19 related encephalitis. Method(s): Patients were consented to an IRB-approved protocol at our institution. Medical records and detailed immunophenotyping were reviewed. Family members were sequenced for IEI with a targeted gene panel. Result(s): The index case is a 10-year-old female with a known pathogenic variant in PIK3CD (c.3061 G > A, p.Glu1021Lys), who contracted SARS-COV-2 despite one COVID-19 vaccination in the series. Her disease course included COVID-related encephalitis with cerebellitis and compression of the pons, resulting in lasting truncal ataxia and cerebellar mutism. At that time, the patient was not on immunoglobulin replacement therapy (IgRT), but was receiving Sirolimus. Besides the index case, 3 family members (2 brothers, 1 mother) also share the same PIK3CD variant with variable clinical and immunological phenotypes. All children exhibited high transitional B-cells, consistent with developmental block to follicular B cell stage. Increased non-class switched IgM+ memory B cells and skewing towards CD21lo B cell subset, which is considered autoreactive-like, was observed in all patients. Of note, the patient had low plasmablasts, but normal immunoglobulins. Of her family members, only one was receiving both sirolimus and IgRT. Conclusion(s): We describe a rare case of COVID-19-related encephalitis in a patient with inborn error of immunity while not on IgRT. This may indicate infection susceptibility because of a lack of sufficient immunity to SARS-CoV-2, unlike the rest of her family with the same PIK3CD variant.Copyright © 2023 Elsevier Inc.
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Introduction: STAT1 gain-of-function (GOF) disease is associated with chronic mucocutaneous candidiasis (CMC) and a broad spectrum of infectious, inflammatory, and vascular manifestations. The Janus Kinase inhibitor ruxolitinib has been used successfully for CMC and autoimmune phenomena. We describe a case of warm autoimmune hemolytic anemia (WAIHA) in a patient with STAT1 GOF disease after initiating ruxolitinib. Case report: A 36-year-old man with STAT1 c.850G>A (p.Glu284Lys) mutation presented with CMC as well as recurrent viral and bacterial infections, lymphadenopathy, enteritis, nodular regenerative hyperplasia (NRH) and splenomegaly. Immune workup confirmed a combined immunodeficiency with hypogammaglobulinemia and T-cell lymphopenia. Ruxolitinib was initiated at 5 mg twice daily (due to pre-existing thrombocytopenia) with up titration over 3 months to 20 mg twice daily. He improved with weight gain, increased energy, resolution of chronic anemia, and improved lymphadenopathy and splenomegaly on imaging. Serum CXCL9 only minimally decreased from 4660 pg/ml to 3990 pg/ml. Soon after reaching ruxolitinib 20 mg twice daily, he developed JC viremia, prompting dose reduction to 15 mg BID. Within two weeks, he developed a non-COVID upper respiratory tract infection followed by fatigue, shortness of breath with ambulation, and dark urine. Emergency evaluation revealed warm antibody positive hemolytic anemia with a hemoglobin of 5 g/dL, and worsened thrombocytopenia. He was treated with blood transfusions, pulse steroids, and high-dose IVIG with stabilization but continued hemolysis. Due to the JC viremia, there was concern to give rituximab with increased PML risk. Bone marrow showed trilineage hematopoiesis, a mild increase in megakaryocytes and RBC precursors, and a loss of B-cell progenitors with retention of mature B cells. His B and T lymphocyte numbers had increased since prior to ruxolitinib, with a predominance of Tfh1-cells (58.7% of total Tfh-cells). He was started on sirolimus with a slow taper of prednisone with continued stable hemoglobin and platelets, and resolution of hemolysis after 3 months. Conclusion(s): To our knowledge, this is the first case of a STAT1 GOF patient developing WAIHA while receiving ruxolitinib therapy. Treatment choices were complicated by the risks of PML. Sirolimus combined with ruxolitinib allowed wean of corticosteroid and subsequent resolution of hemolysis.Copyright © 2023 Elsevier Inc.
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Antibody deficiencies constitute the majority of primary immunodeficiencies in adults. These patients have a well-established increased risk of bacterial infections but there is a lack of knowledge regarding the relative risks upon contracting COVID-19. In this monocentric study the disease course of COVID-19 in 1 patient with Good's syndrome and in 13 patients with common variable immunodeficiency (CVID) is described. The severity of disease ranged from very mild to severe. Several patients required hospitalization and immunomodulatory treatment but all survived. Although viral infections are not a typical feature of humoral immunodeficiencies we recommend that vigilance is increased in the management of patients with Good's syndrome and CVID during the COVID-19 pandemic.Copyright © 2021
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Introduction: Despite the growing number of studies on COVID-19, interpreting the data and extracting meaning from these data is challenging. Method(s): We used a multivariate patient similarity network (PSN) approach to analyse ~85 immune (cellular and humoral) and ~70 clinical features in 250 prospectively recruited patients 4-8 weeks after a SARS-CoV-2 diagnostic PCR. Result(s): Our multivariate analysis identified a minimal immune signature (CD69+CD4+ T-lymphocytes, CTLA-4+ Tlymphocytes and immature B-lymphocytes) that was strongly associated with disease severity and manifested even 4-8 weeks after COVID-19. Visualisation of clinical and laboratory parameters in patient clusters detected by PSN showed that a history of severe COVID-19 was associated with less frequent anosmia/ageusia and higher levels of IgM and IgG 4-8 weeks after COVID-19 compared with mild disease. Conclusion(s): Ours study reveals the relationship between immune profiles and severity of COVID-19, showing that more severe COVID-19 is typical lower proportion on immature B cells and cytotoxic T- lymphocytes. Our study demonstrates the benefits of implementing multivariate data-mining approaches for interpreting complex datasets, not only relating to COVID-19.
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Objectives. The peripheral lymphocyte compartment of patients with primary Sjogren's syndrome (pSS) differs strongly from healthy individuals. Whether this altered lymphocyte composition also abnormally changes during immune reactions, especially in the context of novel mRNA-vaccines, is unknown. Methods. Peripheral blood samples from 26 pSS patients were compared to 6 healthy controls before Coronavirus-2 (CoV-2) vaccination (BNT162b2, ChAdOx1, mRNA-1273) and 7 days after secondary vaccination. Spike. 1 (S1)-receptor binding domain (RBD)-neutralizing IgG antibodies were measured in serum samples. Within peripheral blood mononuclear cells (PBMC), lymphocytes were characterized using spectral flow cytometry and B and T cell subpopulations were phenotypically analyzed. Results. Immunization induced CoV-2 specific serum antibodies in all pSS and healthy participants. When analyzing pSS and healthy individuals together, frequencies of circulating IgG+ RBD-binding antibody-secreting cells (ASC) and anti-CoV-2 serum titers correlated (r=0.42, p=0.022). Previously described alterations of peripheral B cells in pSS patients (like reduced memory B cells, increased naive and transitional B cells and higher maturity of ASCs) remained stable during vaccination. Also the subset distribution of CD4+ and CD8+ T cells mainly stayed unchanged. However, CD4+CXCR5-PD-1+ T cells phenotypically mimicking peripheral helper TPH cells increased in pSS patients comparing pre- and post-vaccination (p=0.020), while circulating CD4+CXCR5+PD-1+ follicular helper TFH cells declined (p=0.024). Conclusions. An immune reaction induced by vaccination with the novel mRNA technology yields adequate antibody production and vaccine specific lymphocytes in pSS patients and controls. However, no major changes within the typical composition of lymphocyte subpopulations of pSS patients were observed despite small changes in TPH and TFH subsets.
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Background: Infectious complications are a major cause of morbidity and mortality in Chronic Lymphocytic Leukaemia (CLL). Therapeutic approaches that deplete CLL cells also affect normal B-cells. Optimal treatment would result in eradication of CLL cells and recovery of normal immune function. FLAIR (ISRCTN01844152) is a phase III trial for previously untreated CLL comparing ibrutinib plus rituximab (IR) with fludarabine, cyclophosphamide and rituximab (FCR) and subsequently amended to also compare ibrutinib plus venetoclax (I+V) and ibrutinib alone (I) with FCR. Measurable residual disease (MRD) and normal B-cell levels were assessed at multiple timepoints. Aims: To assess the depletion of normal B-cells during treatment and recovery after end of treatment. Methods: Participants aged under 75 years with <20% TP53-deleted cells were initially randomised to FCR or IR and subsequently to FCR, IR, I+V or I with the IR arm closed after randomisation of 771 participants to FCR/IR. FCR was given for 6 cycles, while treatment in the IR, I and I+V arms continued for up to 6 years except in participants attaining <0.01% MRD who continued treatment for the time taken to achieved MRD <0.01% and then stopped if MRD remained <0.01%. Month (M) 24 was earliest permitted stopping point. MRD flow cytometry was performed according to ERIC guidelines (panel: CD19/5/20/43/79/81+ROR1, acquisition of 0.5-2.2 million cells, BD Biosciences Lyric). Additional analysis of normal B-cell subsets was performed in a cohort of >500 patients (panel: CD19 to identify B-cells, CD20/5/79b+ROR1 and CD3 to exclude CLL & contaminating cells, with CD27/ 38/IgD/IgM to characterise normal B-cell subsets using a Coulter Cytoflex LX). Results: Normal B-cells were undetectable during FCR treatment and only rarely detectable until 12 months after last FCR cycle. Circulating normal B-cells were reduced in number or undetectable in participants receiving ibrutinibcontaining regimens with greater depletion in the I+V and IR arms relative to I monotherapy. B-progenitors persist through FCR treatment but were depleted during I, I+R or I+V treatment. Normal B-cell levels at 24 and 36 months after randomisation, with time off-treatment if applicable, are shown in Figure 1. In the ibrutinib-containing arms (IR, I, and I+V), there was a trend towards fewer COVID-associated SAE at any time point for participants with detectable B-cells at 24M (4/181, 2.2%) compared to those with no detectable B-cells (14/344, 4.1%) and COVID-associated SAEs were not observed in FCR-treated participants who had recovered any level of normal B-cells by 24M (0/215). However, the data on COVID infections are limited and there was no apparent association between normal B-cell levels at 24M with the proportion of participants experiencing an infectious SAE overall. Assessment of normal B-cell subsets during ibrutinib-based treatment demonstrated a mix of naïve and memory B-cells. Serological response to COVID infection/vaccination in this cohort is currently being performed. Participants stopping I+V treatment at 24-30 months post-randomisation due to MRD eradication showed rapid recovery of normal naive B-cells within 6-12 months after end of treatment in the vast majority (>95%) of evaluable cases. Summary/Conclusion: Normal circulating B-cells are depleted during treatment with rituximab but can persist at a low level during I, IR or I+V treatment. Most patients in remission after treatment with FCR or I+V show recovery of normal B-cells at 12 months of stopping treatment.
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Background: Vaccination is considered efficient in controlling infections incl. SARS-CoV-2. Prior studies showed that patients receiving rituximab (RTX) with low B cell counts are at increased infectious risk (1) and risk of inadequate vaccination responses (2, 3). Thus, the ability to further defne and predict vaccination responses in these patients may guide their optimal protection. Objectives: To assess predictive biomarkers of vaccination responses upon SARS-CoV-2 vaccination in RTX treated patients. Methods: B cell characteristics before vaccination were evaluated to predict responses in 15 patients with autoimmune infammatory rheumatic diseases receiving RTX. 11 patients with rheumatoid arthritis on other therapies (RA), 11 kidney transplant recipients (KTR) and 15 healthy volunteers (HC) served as controls. A multidimensional analysis of B cell subsets and a correlation matrix were performed to identify predictive biomarkers. Results: Signifcant differences regarding absolute B cell counts and specifc subset distribution pattern between the groups were validated at baseline. Here, the majority of B cells from vaccination responders of the RTX group (RTX IgG+) comprised naïve and transitional B cells, whereas vaccination non-responders (RTX IgG-) carried preferentially plasmablasts and double negative (CD27-IgD-) B cells (Figure 1). Moreover, there was a positive correlation between neutralizing antibodies and absolute B cell numbers with B cells expressing HLA-DR and CXCR5 (involved in antigen presentation and germinal center formation) as well as an inverse correlation with CD95 expression and CD21low expression (marker for activation and exhaustion) on B cells. Conclusion: Substantial repopulation of naïve B cells upon RTX therapy appears to be essential for an adequate vaccination response requiring germinal center formation. In contrast, expression of exhaustion markers (CD21low, CXCR5-, CD95+) indicate negative predictors of vaccination responses. These results may guide optimized vaccination strategies in RTX treated patients clearly requiring antigen-inexperienced B cells for appropriate protection.
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Background: Oncohematological patients may have a lower immune response against SARS-CoV-2, both to natural infection and to vaccines. Most studies have focused on the analysis of the humoral response, which means that the information available on the cellular response against SARS-CoV-2 in these patients is limited. Current recommendations include vaccination against SARS-CoV-2 in patients undergoing autologous hematopoietic stem cell transplantation (AHSTC), regardless of whether they have been previously exposed to the virus. These recommendations are based on previous studies with other vaccines. Therefore, it is necessary to analyze the immune response that is developed in these patients in order to make specific recommendations for COVID-19 vaccination. Objective: To study the humoral and cellular immune response before and after AHSTC in patients with oncohematological neoplasms who were exposed to SARS-CoV-2 before the transplantation. Materials & methods: Nine patients with previous exposure to SARS-CoV-2 who underwent AHSTC (Table 1) and 8 healthy donors who recovered from mild COVID-19 were recruited from Hospital Ramón y Cajal and Primary Healthcare Center Pedro Laín Entralgo (Madrid, Spain), respectively. Specific direct cellular cytotoxicity (DCC) of PBMCs from these patients against Vero E6 cells infected with pseudotyped SARS-CoV-2 was determined. The activation of caspase-3 in Vero cells was measured after 1 hour of co-culture with PBMCs, in which cytotoxic cell populations were analyzed by flow cytometry. Antibody-dependent cellular cytotoxicity (ADCC) was analyzed by quantifying the binding of Annexin V to rituximab-coated Raji cells as targets of PBMCs. Results: 1) 66% of AHSTC patients did not develop detectable levels of IgGs against SARS-CoV-2 (Fig. 1). In 33% of these patients with detectable IgG, the titers decreased after AHSTC, as well as their neutralizing capacity (Fig. 1B and C). 2) AHSTC patients showed increased levels of immature B cells (9.5-fold;p=0.0586) and plasmablasts (28.8-fold), in comparison with healthy donors who had mild COVID-19, while naive and resting memory B cells decreased 1.7- and 6.9-fold, respectively. 3) Specific DCC against SARS-CoV-2-infected cells increased 1.5-fold in comparison with healthy donors (Fig. 2A). Cytotoxic populations with NK phenotypes (CD3-CD56+CD16+), NKT (CD3+CD56+CD16+), and CD8+ T cells (CD3+CD8+TCRγδ+) increased 1.9- (p=0.0311), 1.9- (p=0.0592), and 1.6-fold, respectively (Fig. 2B). ADCC increased 2.1-fold in PBMCs from AHSTC patients in comparison with healthy donors (p = 0.0592). Conclusions: Our data show for the first time how the humoral and cellular immune response against the natural infection by SARS-Cov-2 may be modified in patients who were subsequently subjected to AHSTC. Although the humoral response may be reduced after AHSTC, the specific cellular response showed an increased cytotoxic activity. These results could be extrapolated to patients who were vaccinated against COVID-19 prior to AHSTC. Therefore, this information could be useful to define the recommendations for COVID-19 vaccination after AHSTC. [Formula presented] Disclosures: Garcia-Gutiérrez: Pfizer: Research Funding;Incyte: Consultancy;Novartis: Consultancy;Bristol-Myers Squibb: Consultancy.
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Background: Within seconds of antigen-encounter, B-cell receptor (BCR) signaling induces dramatic changes of cell membrane lipid composition, including >40-fold increases of local PIP3-concentrations within lipid rafts. While several structural elements, including pleckstrin homology (PH) domains have been identified as PIP3-binding proteins, the underlying mechanisms that amplify BCR-signaling to assemble large signaling complexes within lipid rafts within 15 to 30 seconds, remained elusive. To understand the mechanistic and biophysical requirements for PIP3 accumulation during normal B-cell activation and acute oncogenic transformation, we identified PIP3-interacting proteins by cell-surface proteomic analyses. Results: In addition to proteins known to bind PIP3 with their PH-domains, we identified the short 133 aa protein IFITM3 (interferon-inducible transmembrane protein 3) as a top-ranking PIP3 scaffold. This was unexpected because IFITM3 was previously identified as endosomal protein that blocks viral infection by stiffening endosomal membranes to firmly contain viral cargo. Previous studies revealed that polymorphisms that lead to the expression of truncated IFITM3 are associated with increased susceptibility to viral infections, including SARS-CoV2. Among known cell membrane lipids, PIP3 has the highest negative charge. Instead of a PH-domain, IFITM3 laterally sequestered PIP3 through electrostatic interactions with two basic lysine residues (K83 and K104) located at the membrane-solution interface. Together with three other basic lysine and arginine residues K83 and K104 form a conserved intracellular loop (CIL), which enable IFITM3 to efficiently capture two PIP3 molecules. Bivalent PIP3-binding of the IFITM3-CIL enables a crosslinking mechanism that results in dramatic amplification of B-cell activation signals and clustering of large signaling complexes within lipid rafts. In normal resting B-cells, Ifitm3 was minimally expressed and mainly localized in endosomes. However, B-cell activation and oncogenic kinases induced phosphorylation at IFITM3-Y20, resulting in translocation of IFITM3 from endosomes and massive accumulation at the cell surface. Ifitm3ˉ /ˉ naïve B-cells developed at normal numbers, however, activation by antigen encounter was compromised. In Ifitm3ˉ /ˉ B-cells, lipid rafts were depleted of PIP3, resulting in defective expression of >60 lipid raft-associated surface receptors and impaired PI3K-signaling. Ifitm3ˉ /ˉ B-cells were unable to undergo affinity maturation and di not contribute to germinal center formation upon immunization. Analyses of gene expression and clinical outcome data from patients in six clinical cohorts for pediatric and adult B-ALL, mantle cell lymphoma, CLL and DLBCL, we consistently identified IFITM3 as a top-ranking predictor of poor clinical outcome. Inducible activation of BCR-ABL1 and NRAS G12D rapidly induced development of B-ALL but failed to transform and initiate B-ALL from Ifitm3ˉ /ˉ B-cell precursors. Conversely, the phospho-mimetic IFITM3-Y20E mutation, mimicking phosphorylation of the IFITM3 N-terminus at Y20 induced constitutive membrane localization of IFITM3, spontaneous aggregation of large oncogenic signaling complexes and readily initiated transformation in a genetic model of pre-malignant B-cells. Conclusions: We conclude that phosphorylation of IFITM3 upon B-cell activation induces a dynamic switch from antiviral effector functions in endosomes to oncogenic signal-amplification at the cell-surface. IFITM3-dependent amplification of PI3K-signaling is critical to enable rapid expansion of activated B-cells. In addition, multiple oncogenes depend on IFITM3 to assemble PIP3-dependent signaling complexes and amplify PI3K-signaling for malignant transformation and initiation of B-lymphoid leukemia and lymphoma. [Formula presented] Disclosures: Weinstock: SecuraBio: Consultancy;ASELL: Consultancy;Bantam: Consultancy;Abcuro: Research Funding;Verastem: Research Funding;Daiichi Sankyo: Consultancy, Research Funding;AstraZeneca: Consultanc ;Travera: Other: Founder/Equity;Ajax: Other: Founder/Equity.