B-cell Lymphopenia Research Articles

Intrinsic functional defects in B cells of patients with NFKB2 mutations.

Mutations in the human nuclear factor-κB2 gene (NFKB2) are associated with common variable immunodeficiency (CVID) or combined immunodeficiency diseases (CID), characterized by B-cell lymphopenia, hypogammaglobulinemia, and T cell dysfunction. This study investigated whether B cells with NFKB2 mutations exhibit intrinsic impairments in activation, class-switch recombination, and differentiation. We analyzed five patients from four unrelated families with CVID, each carrying a heterozygous NFKB2 mutation: P1 (C.2595_2614del, p.A867Gfs*12), P2 (C.2597G>A, p.S866N), P3 (C.2540dupT, p.R848Efs*38), and P4 and P5 (C.2570_2571insCAGCACA, p.A860Qfs*28). The patients with frameshift mutations (P1, P3, P4, and P5) exhibited truncated proteins detectable in their peripheral blood mononuclear cells, while P2 had a missense mutation. All identified mutations disrupted the processing of p100 into the active p52 form, resulting in NF-κB2 loss-of-function and IκBδ gain-of-function. Clinically, P1, P2, and P3 exhibited B-cell lymphopenia, and all five patients presented with hypogammaglobulinemia. Notably, P2 exhibited a markedly low B-cell count, associated with increased proportions of memory B and IgD-CD27- double negative B cells. In vitro experiments with naïve B cells from P1 and P4 demonstrated decreased survival, impaired activation, and reduced differentiation into CD27+IgD- cells and plasmablasts, while class switch recombination was unaffected. These findings reveal novel B cell-intrinsic functional defects in patients with NFKB2 mutations.

Biallelic PI4KA Mutations Disrupt B-Cell Metabolism and Cause B-Cell Lymphopenia and Hypogammaglobulinemia

Biallelic PI4KA Mutations Disrupt B-Cell Metabolism and Cause B-Cell Lymphopenia and Hypogammaglobulinemia

Noninfectious Complications in B-Lymphopenic Common Variable Immunodeficiency.

Common variable immunodeficiency (CVID) is considered the most symptomatic type of inborn errors of immunity in humans. Along with infectious complications, which have numerous consequences, noninfectious complications are a major challenge among CVID patients. All CVID patients registered in the national database were included in this retrospective cohort study. Patients were divided into 2 groups based on the presence of B-cell lymphopenia. Demographic characteristics, laboratory findings, noninfectious organ involvement, autoimmunity, and lymphoproliferative diseases were evaluated. Among 387 enrolled patients, 66.4% were diagnosed with noninfectious complications and 33.6% with isolated infectious presentations. Enteropathy, autoimmunity, and lymphoproliferative disorders were reported in 35.1%, 24.3%, and 21.4% of patients, respectively. Some complications, including autoimmunity and hepatosplenomegaly, were reported to be significantly more frequent among patients with B-cell lymphopenia. As for organ involvement, the dermatologic, endocrine, and musculoskeletal systems were predominantly affected in CVID patients with B-cell lymphopenia. Among autoimmune manifestations, the frequency of rheumatologic, hematologic, and gastrointestinal autoimmunity was reported to be higher than that of other types of autoimmunity not associated with B cell-lymphopenia. Furthermore, hematological cancers, particularly lymphoma, were the most common type of malignancy. The mortality rate was 24.5%, and respiratory failure and malignancies were the most common causes of death, with no significant differences between the 2 groups. Considering that some of the noninfectious complications might be associated with B-cell lymphopenia, regular patient monitoring and follow-up with proper medication (in addition to immunoglobulin replacement therapy) are highly recommended to prevent sequelae and increase patient quality of life.

Blood RNA-sequencing across the continuum of ANA-positive autoimmunity reveals insights into initiating immunopathology

ObjectiveMechanisms underpinning clinical evolution to systemic lupus erythematosus (SLE) from preceding antinuclear antibodies (ANA) positivity are poorly understood. This study aimed to understand blood immune cell transcriptional signatures associated with...

Lessons from prospective longitudinal follow-up of a French APECED cohort.

APECED syndrome is a rare disease caused by biallelic mutations of the AIRE gene, usually presenting with the triad "hypoparathyroidism-adrenal failure-chronic mucocutaneous candidiasis (CMC)" and non-endocrine manifestations. The aim of this study was to determine the molecular profile of the AIRE gene, the prevalence of rare manifestations and to characterize immunological disturbances in a French cohort. A national, multicenter prospective observational study to collect genetic, clinical, biological and immunological data (NCT03751683). 25 patients (23 families) were enrolled. Eleven distinct AIRE variants were identified, two of which were not previously reported: an intronic variant, c.653-70G > A, and a c.1066del (p.Arg356GlyfsX22) variant (exon 9). The most common was the Finnish variant c.769C > T (16 alleles), followed by the variant c.967_979del13 (15 alleles), which seemed associated with a less severe phenotype. 17/25 patients were homozygote. The median number of clinical manifestations was seven; 19/25 patients presented with the hypoparathyroidism-adrenal failure-CMC triad, 8/13 showed pulmonary involvement, 20/25 had ectodermal dystrophy, 8/25 had malabsorption, and 6/23 had asplenia. Fifteen out of 19 patients had NK cell lymphopenia with an increase in CD4+ and CD8+ T lymphocytes and an age-dependent alteration of B lymphocyte homeostasis compared with matched controls (p < 0.001), related to the severity of the disease. All tested sera (n = 18) were positive for anti-interferon-α, 15/18 for anti-interleukin-22 antibodies, and 13/18 for anti-interleukin-17F antibodies, without clear phenotypic correlation other than with CMC. This first prospective cohort showed a high AIRE genotype variability, with two new gene variants. The prevalence of potentially life-threatening non-endocrine manifestations, was higher with systematic screening. These manifestations could, along with age-dependent B-cell lymphopenia, contribute to disease severity. Systematic screening for all the manifestations of the syndrome would allow earlier diagnosis, supporting vaccination, and targeted therapeutic approaches.

B-cells in pulmonary arterial hypertension: friend, foe or bystander?

There is an unmet need for new therapeutic strategies that target alternative pathways to improve the prognosis of patients with pulmonary arterial hypertension (PAH). As immunity has been involved in the development and progression of vascular lesions in PAH, we review the potential contribution of B-cells in its pathogenesis and evaluate the relevance of B-cell-targeted therapies. Circulating B-cell homeostasis is altered in PAH patients, with total B-cell lymphopenia, abnormal subset distribution (expansion of naïve and antibody-secreting cells, reduction of memory B-cells) and chronic activation. B-cells are recruited to the lungs through local chemokine secretion, and activated by several mechanisms: 1) interaction with lung vascular autoantigens through cognate B-cell receptors; 2) costimulatory signals provided by T follicular helper cells (interleukin (IL)-21), type 2 T helper cells and mast cells (IL-4, IL-6 and IL-13); and 3) increased survival signals provided by B-cell activating factor pathways. This activity results in the formation of germinal centres within perivascular tertiary lymphoid organs and in the local production of pathogenic autoantibodies that target the pulmonary vasculature and vascular stabilisation factors (including angiotensin-II/endothelin-1 receptors and bone morphogenetic protein receptors). B-cells also mediate their effects through enhanced production of pro-inflammatory cytokines, reduced anti-inflammatory properties by regulatory B-cells, immunoglobulin (Ig)G-induced complement activation, and IgE-induced mast cell activation. Precision-medicine approaches targeting B-cell immunity are a promising direction for select PAH conditions, as suggested by the efficacy of anti-CD20 therapy in experimental models and a trial of rituximab in systemic sclerosis-associated PAH.

MYSM1 attenuates DNA damage signals triggered by physiologic and genotoxic DNA breaks

Patients with deleterious variants in MYSM1 have an immune deficiency characterized by B-cell lymphopenia, hypogammaglobulinemia, and increased radiosensitivity. MYSM1 is a histone deubiquitinase with established activity in regulating gene expression. MYSM1 also localizes to sites of DNA injury but its function in cellular responses to DNA breaks has not been elucidated. This study sought to determine the activity of MYSM1 in regulating DNA damage responses (DDRs) to DNA double-stranded breaks (DSBs) generated during immunoglobulin receptor gene (Ig) recombination and by ionizing radiation. MYSM1-deficient pre- and non-B cells were used to determine the role of MYSM1 in DSB generation, DSB repair, and termination of DDRs. Genetic testing in a newborn with abnormal screen for severe combined immune deficiency, T-cell lymphopenia, and near absence of B cells identified a novel splice variant in MYSM1 that results in nearly absent protein expression. Radiosensitivity testing in patient's peripheral blood lymphocytes showed constitutive γH2AX, a marker of DNA damage, in B cells in the absence of irradiation, suggesting a role for MYSM1 in response to DSBs generated during Ig recombination. Suppression of MYSM1 in pre-B cells did not alter generation or repair of Ig DSBs. Rather, loss of MYSM1 resulted in persistent DNA damage foci and prolonged DDR signaling. Loss of MYSM1 also led to protracted DDRs in U2OS cells with irradiation induced DSBs. MYSM1 regulates termination of DNA damage responses but does not function in DNA break generation and repair.

Progressive Multifocal Leukoencephalopathy in Systemic Lupus Erythematosus: A Consequence of Patient-Intrinsic or -Extrinsic Factors?

Progressive multifocal leukoencephalopathy (PML) is a severe demyelinating disease of the central nervous system (CNS) caused by reactivation of the polyomavirus JC (JCV) typically in immunocompromised individuals. The risk of PML among rheumatic diseases may be higher for systemic lupus erythematosus (SLE), without, however, a clear association with the type and intensity of background therapy. We present the development and outcome of PML in a 32-year-old female lupus patient under mild immunosuppressive treatment, yet with marked B-cell lymphopenia in the peripheral blood and bone marrow (<1% of total lymphocytes). Despite treatment with the immune checkpoint inhibitor pembrolizumab, the patient showed progressive neurological and brain imaging deterioration and eventually died 15 months after PML diagnosis. To unveil possible underlying genetic liabilities, whole exome sequencing was performed which identified deleterious variants in GATA2 and CDH7 genes, which both have been linked to defective T- and/or B-lymphocyte production. These findings reiterate the possible role of disease-/patient-intrinsic factors, rather than that of drug-induced immunosuppression, in driving immune dysregulation and susceptibility to PML in certain patients with SLE.

MECOM Deficiency: from Bone Marrow Failure to Impaired B-Cell Development.

MECOM deficiency is a recently identified inborn error of immunity and inherited bone marrow failure syndrome caused by haploinsufficiency of the hematopoietic transcription factor MECOM. It is unique among inherited bone marrow failure syndromes, many of which present during later childhood or adolescence, because of the early age of onset and severity of the pancytopenia, emphasizing the importance and gene dose dependency of MECOM during hematopoiesis. B-cell lymphopenia and hypogammaglobulinemia have been described in a subset of patients with MECOM deficiency. While the mechanisms underlying the B-cell deficiency are currently unknown, recent work has provided mechanistic insights into the function of MECOM in hematopoietic stem cell (HSC) maintenance. MECOM binds to regulatory enhancers that control the expression of a network of genes essential for HSC maintenance and self-renewal. Heterozygous mutations, as seen in MECOM-deficient bone marrow failure, lead to dysregulated MECOM network expression. Extra-hematopoietic manifestations of MECOM deficiency, including renal and cardiac anomalies, radioulnar synostosis, clinodactyly, and hearing loss, have been reported. Individuals with specific genotypes have some of the systemic manifestations with isolated mild thrombocytopenia or without hematologic abnormalities, highlighting the tissue specificity of mutations in some MECOM domains. Those infants with MECOM-associated bone marrow failure require HSC transplantation for survival. Here, we review the expanding cohort of patient phenotypes and accompanying genotypes resulting in MECOM deficiency, and the proposed mechanisms underlying MECOM regulation of human HSC maintenance and B-cell development.

B cell depletion in infants after intra uterine exposure to immunomodulating chemotherapy with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP): A case series and review of the literature

The immunomodulating chemotherapeutic drugs rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) have the ablity to pass the placenta during pregnancy and might affect the development of the immune system of exposed infants. In particular rituximab causes a transient and almost complete depletion of CD20 expressing B cells and can remain detectable in the infant several months after birth.In this case series we report on the clinical and immunological outcomes of 3 infants exposed to R-CHOP during pregnancy because of maternal B-cell lymphoma and review other cases that have been published. We show that R-CHOP in pregnancy has a profound effect on the immune system in the first year of life, including B-cell lymphopenia, hypogammaglobinemia, neutropenia and decreased response to immunization. Immune monitoring of exposed infants is warranted.

Abstract A51: Divergent effects of MDS predisposing SAMD9L point mutations, in humans, mice, and cellular models

Abstract We previously showed that germline SAMD9 and SAMD9L (SAMD9/9L) disorders are the most common predisposition to childhood myelodysplastic syndromes with bone marrow failure (BMF). Heterozygous mutations in both genes are growth inhibitory and undergo negative selection by acquiring compensatory rescue events in blood. Here, we describe the first report of SAMD9L genetic rescue occurring during embryonal development and resulting in germline triple-allelic mosaicism (1 wildtype (WT) and 2 mutant alleles), where only one mutant allele was transmitted to each of the two diseased children. In a family of 2 affected siblings with hypocellular BMF, the Index case carried germline heterozygous SAMD9L V1512M mutation and her brother was heterozygous for V1512L mutation. The clinical presentation for Index with V1512M was more severe compared to her brother with V1512L mutation. Genetic assessment of the parents showed the asymptomatic mother to be a triple-allelic mosaic, carrying WT SAMD9L allele at ~50% frequency in all tissues, while both V1512M and V1512L mutant alleles “competed” in their allelic distribution depending on the tissue origin. Single-cell DNA sequencing on her peripheral blood (PB) revealed 3 independent diploid clones: V1512M in 14%, V1512L in 68%, and WT (due to revertant UPD7q) in 18% of cells. Because her parents were SAMD9L WT, one of the mutations likely arose de novo and underwent failed embryonic rescue attempt leading to a second mutation. Towards studying the effect on hematopoiesis in vitro, we knocked in V1512M and V1512L mutations in inducible pluripotent stem cells (iPSC). For in vivo functional validation, we created constitutive mouse models with ortholog mutations (V1507M and V1507L). Mutant hematopoietic progenitor cells from iPSC had severely compromised proliferative capacity and yielded fewer erythroid and myeloid cells. This effect was more severe in V1512M vs. V1512L mutants. The divergent mutational phenotypes were also replicated in our mouse models: two-thirds (13/19) of the heterozygous V1507M (V1507Mhet) pups died before 4 weeks of age. In contrast, V1507Lhet mice had overall survival equal to WT mice, while one-third of homozygous V1507L (V1507Lhom) mice showed decreased survival post 19 weeks of age. At baseline, V1507Mhet mice showed severe growth retardation with multisystemic issues, which were absent in the V1507L mutant models. PB cytopenia (anemia, B-cell lymphopenia) though observed in both V1507Mhet and V1507Lhom mice, the degree of severity was high in the V1507Mhet than in the V1507Lhom model. Meanwhile, V1507Lhet mice had blood counts similar to WT. In summary, V1512M compared to V1512L mutation resulted in a more severe phenotype in all analyzed model systems. This observation showed how different mutational permutations of the same amino acid can exert divergent phenotypic effects in SAMD9L BMF disorder. Therefore, together with our genetic analysis, we could postulate that the V1512L mutation arose as a failed rescue attempt of V1512M during embryogenesis. Citation Format: Sushree S Sahoo, Charnise Goodings, Shondra M. Pruett-Miller, Maria A Lillo, Lei Han, Baranda Hansen, Ti-Cheng Chang, Tim Lammens, Mattias Hofmans, Marta Derecka, Barbara De Moerloose, Marcin W Wlodarski. Divergent effects of MDS predisposing SAMD9L point mutations, in humans, mice, and cellular models [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A51.

Diagnostic significance of TREC and KRE Cevaluation in case of primary immunodeficiencies

IntroductionTREC and KREC are products of V(D)J recombination of Tand B-lymphocyte receptors in thymus and bone marrow, respectively. Low levels of TREC/KREC in peripheral blood are connected to T- and/or B-cell lymphopenia Materials and methodsWe have studied DNA of healthy children (n = 98) aged 0.0 (0–15.0) years, patients with primary immunodeficiency (n = 84) (SCID (n = 13), AT (n = 15), NBS (n = 17), X-linked agammaglobulinemia (n = 15), diseases of immune dysregulation (n = 14), Wicksott-Aldrich syndrome (n = 10)) aged 7.0 (0.1–15.0) years by multiplex RQ-PCR.The analysis of diagnostic significance has been performed by tracing of characteristic ROC curves and defining the area under the AUC curve. ResultsThe AUC(TREC) and KREC for the SCID diagnostics has been equal to 1.00 ± 0.0 (p < 0.0001) with a diagnostic sensitivity and specificity of 100%.The AUC(TREC) for the X-linked agammaglobulemia diagnosis has made 0.62 ± 0.07 (p = 0.16) with diagnostic sensitivity and specificity of 14% and 93.6%, respectively. The AUC(KREC) has been 1.00 ± 0.0 (p < 0.0001) with diagnostic sensitivity and specificity of 100%.AUC(TREC) for patients with syndromes of chromosomal instability has been equal to 0.99 ± 0.007 (p < 0.0001) with diagnostic sensitivity of 93.4% and specificity of – 97.4%. The AUC(KREC) has been 0.98 ±0.015 (p < 0.0001)) with diagnostic sensitivity of 93.5% and specificity of 98.7%.In case of diseases of immune dysregulation, AUC(TREC) has been equal to 0.99 ± 0.007 (p < 0.0001) with diagnostic sensitivity of 93.3% and specificity of 96.3%. The AUC(KREC) has been 0.87 ± 0.06 (p < 0.0001) with diagnostic sensitivity of 86.6% and specificity of 94.6%.The AUC(TREC) for Wiskott-Aldrich syndrome diagnostics using analysis has made 0.63 ± 0.1 (p = 0.16), with sensitivity and specificity of 30% and 78.1%, respectively. AUC(KREC) has been 0.61 ± 0.09 (p = 0.23) with sensitivity of 20.0% and specificity of 92.35%. ConclusionEvaluation of TREC/KREC is diagnostically significant during the primary diagnosis of diseases, associated with severe combined immune deficiency, agammaglobulinemia, syndromes of chromosomal instability, immune dysregulation. Evaluation of TREC/KREC has low information content and, thus, has no diagnostic significance for Wiskott-Aldrich syndrome.

Persistent Immunity against SARS-CoV-2 in Individuals with Oncohematological Diseases Who Underwent Autologous or Allogeneic Stem Cell Transplantation after Vaccination

The high morbimortality due to SARS-CoV-2 infection in oncohematological diseases (OHD) and hematopoietic stem cell transplant (HSCT) recipients in the pre-vaccine era has made vaccination a priority in this group. After HSCT, the immune responses against common vaccines such as tetanus, varicella, rubella, and polio may be lost. However, the loss of immunity developed by COVID-19 vaccination after HSCT has not been completely defined. In this study, both humoral and cellular immunity against SARS-CoV-2 were analyzed in 29 individuals with OHD who were vaccinated before receiving allogeneic (n = 11) or autologous (n = 18) HSCT. All participants had low but protective levels of neutralizing IgGs against SARS-CoV-2 after HSCT despite B-cell lymphopenia and immaturity. Although antibody-dependent cellular cytotoxicity was impaired, direct cellular cytotoxicity was similar to healthy donors in participants with autologous-HSCT, in contrast to individuals with allogeneic-HSCT, which severely deteriorated. No significant changes were observed in the immune response before and after HSCT. During follow-up, all reported post-HSCT SARS-CoV-2 infections were mild. This data emphasizes that COVID-19 vaccination is effective, necessary, and safe for individuals with OHD and also supports the persistence of some degree of immune protection after HSCT, at least in the short term, when patients cannot yet be revaccinated.

Hematopoietic and Immunological Assessment in Fanconi Anemia after Ex Vivo Lentiviral FANCA Gene Therapy with RP-L102

Introduction: Fanconi anemia (FA) is the most frequent cause of genetic bone marrow failure (BMF) resulting from inherited defects in the FA pathway, one of several essential deoxyribonucleic acid (DNA) repair mechanisms. Although allogeneic hematopoietic stem cell transplantation (alloHSCT) offers hematolymphoid cure, it confers significant morbidity and mortality and is thereby indicated only after development of severe BMF or dysplasia. Ex vivo hematopoietic stem and progenitor cell (HSPC)-based gene therapy (GT) offers an alternative strategy for earlier and potentially safer intervention with the goal of preventing BMF and the need for alloHSCT. Core assessments of safety and efficacy are conducted as part of ongoing GT clinical trials for FA-type A patients who have received ex vivo lentiviral FANCA GT. In these additional exploratory research studies we seek to better understand how GT may affect HSPC behavior, mutational acquisition and immune function. Methods: To deepen our understanding of the effects of ex vivo lentiviral FANCA GT on FA biology, we collected serial bone marrow (BM) and peripheral blood (PB) samples from FA-Type A patients who were enrolled at Stanford University on the Phase I and II RP-L102-0418 and RP-L102-0319 clinical trials. We initiated the assessment of Phase I subjects at 18 months post treatment and the assessment of Phase II subjects at study enrollment. BM was assessed to characterize HSPC distribution by flow cytometry, proliferative capacity by colony-forming count (CFC) assays, transcriptomics by single-cell RNA sequencing (scRNASeq) and mutation acquisition by targeted next generation sequencing (NGS). Immune system composition and function were assessed using PB flow cytometry, immunoglobulin levels and T cell proliferative assays. Results: Of the 8 enrolled patients, 5 subjects met eligibility criteria and were treated with RP-L102 GT. As of April 2022, 4 remain on study, now 18 to 36 months post-treatment. Of these 4 subjects, 3 have demonstrated increasing vector copy number and MMC-resistance. At time of enrollment, HSCs were markedly reduced in these patients compared to healthy controls, even in patients with no or mild cytopenia, and to-date have remained decreased post-GT. Functional HSPC proliferation capacity, as assessed by CFCs, was reduced in all patients at screening but showed evidence of improvement or stabilization in 3 of the 4 post-GT patients, as did BM total cellularity and CD34+ counts. scRNASeq showed reduced proportions of HSCs pre- and post-GT relative to healthy controls. Expression of ribosome biogenesis genes, an indicator of cancer risk, was downregulated post-GT. Targeted bulk-NGS revealed that none of the patients evaluated on these studies had mutations known to be associated with myeloid malignancies pre- or post-GT treatment. Detailed immune monitoring revealed that 3 patients had B-cell lymphopenia at time of enrollment. Additionally, 1 patient showed an increase in B-cell numbers post-GT. IgG levels were normal in all 8 screened patients pre-GT and remained stable post-GT. Interestingly, among 7 vaccinated patients evaluated at enrollment, 3 did not have protective IgG levels to Hepatitis B, 1 did not have protective IgG levels to Varicella Zoster Virus (VZV), and 3 showed equivocal levels of anti-VZV IgG. Conclusions: Although our results are based on a small cohort and are interim within ongoing clinical trials, they indicate the need for more thorough assessment of BM and immune characterization in FA patients beyond reliance on blood counts. Importantly, the paucity of HSCs in FA patients even prior to the development of cytopenias supports the need for early intervention for HSC preservation which may be possible with GT upon diagnosis. Additionally, these studies show that RP-L102 GT appears to be a safe approach for hematopoietic preservation in FA without acquisition of detectable mutations in HSPCs to-date. Moreover, downregulation of ribosome biogenesis genes can potentially lead to decreased leukemogenesis which could decrease cancer risk post-GT. Finally, we show laboratory evidence of baseline immune dysfunction in FA patients mainly consisting of B-cell lymphopenia and poor response to certain vaccines. Given this, post-GT re-vaccination of patients may be clinically indicated and would enable assessment of functional immune recovery potentially associated with the GT treatment.

Circulating B-Cell Precursor Cells As Potential Diagnostic and Therapeutic Biomarker in Patients with WHIM Syndrome

Background Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is a combined immunodeficiency caused in most known cases by gain-of-function mutations in the CXCR4 gene. The wide spectrum of multisystem clinical presentations, often with incomplete penetrance and expressivity, can pose a diagnostic challenge. WHIM syndrome has historically been a clinicopathologic diagnosis, with myelokathexis being the pathognomonic feature of WHIM. As genetic testing is becoming readily available, sequencing of the CXCR4 gene is often used to confirm a clinical suspicion of WHIM syndrome by finding genetic variants. There is a need for additional easily accessible and noninvasive biomarkers for diagnosing WHIM syndrome. We hypothesize that B-cell compartment might also show pathognomonic features of WHIM syndrome. Patients and Methods We analyzed peripheral blood B-cell subsets in 13 patients diagnosed with WHIM syndrome from 8 families with pathogenic CXCR4 mutations using 9-multicolor flow cytometry after informed consent approval. Samples from 2 patients who received plerixafor were collected at baseline before and after treatment. The duration of treatment was approximately 6 months. Results Using multicolor flow cytometry, we analyzed circulating B-cell subsets (CD10,CD19,CD21,CD27,CD38,IgA,IgD,IgG and IgM) in 13 patients (pediatric, n=7; adult, n=6) with WHIM syndrome (9 patients with p.Arg334Stop mutation, 2 patients with p.Val320Glufs*23 mutation, 1 patient with p.Ser338Stop mutation, and 1 patient with p.Ser346Stop mutation). We observed severe peripheral B-cell lymphopenia, accompanied by the presence of a consistent and specific scarce CD19+CD10+CD38+IgM-IgD-CD21-CD27- B-cell subpopulation. A B-cell subpopulation with an equivalent marker expression pattern was not found in healthy controls (n=50) or in non-WHIM disease controls (n=20). The rare B-cell population found in WHIM syndrome was further characterized as belonging mostly to the pre-B-cell stage revealed by intracellular expression of IgM, CD179a (VpreB chain), and CD179b (lambda 5). The presence of these early B-cell precursors was independent of age and was stable over time in patients with WHIM syndrome. The number of these peripheral B-cell precursors found specifically in patients with WHIM syndrome was greatly reduced when the patients were treated with granulocyte colony-stimulating factor (G-CSF) or plerixafor; counts of the precursor cells returned to baseline after the patients discontinued plerixafor treatment. Conclusions Taken together, we identify a unique CD19+CD10+CD38+IgM-IgD-CD21-CD27- B-cell subpopulation in patients with WHIM syndrome that might be used as a noninvasive biomarker to help identify patients with this rare primary combined immunodeficiency. In addition, these circulating B-cell precursors may be used to monitor response to therapy in patients with WHIM syndrome. The observed impact of G-CSF on the B-cell precursor population may be attributed to downregulation of CXCL12/CXCR4, which influences myelokathexis and impairs egression of the precursor population from the bone marrow but needs further experimental validation.

THROMBOCYTOPENIA, HYPOGAMMAGLOBULINEMIA, AND POOR VACCINE RESPONSE IN A PATIENT WITH TAKENOUCHI-KOSAKI SYNDROME

<h3>Introduction</h3> Takenouchi-Kosaki Syndrome (TKS) is a rare congenital developmental disorder characterized by thrombocytopenia, facial abnormalities, and varied multisystemic anomalies including immunodeficiency. Heterozygous deficiency of CDC42, an intracellular signaling GTPase, has been found in all TKS patients described to date. The immunological phenotypes of TKS patients are heterogeneous: recurrent infections have been noted in some patient series but with variable penetrance. Herein we describe a case of TKS causing hypogammaglobulinemia, B-cell lymphopenia, and poor vaccine response. <h3>Case Description</h3> A fifteen-year-old girl with history of developmental delay, multiple congenital defects, and lymphedema was referred to the immunology clinic for evaluation of recurrent ear infections that began at 3 months of age requiring tympanostomy tube placement at 18 months. Her physical exam was notable for abnormal facies, scoliosis, strabismus, abnormal thumbs, and unilateral lymphedema. Her laboratory evaluation revealed macrothrombocytopenia, mild leukopenia with low T-cells and markedly low B-cells by flow cytometry, and notably low IgA (11.7, RR 69 – 309) and IgG (423, RR 613 – 1295). She had a poor response to a full course of PCV7 with PPSV23 booster (9 of 23 > 1.3). Whole exome sequencing identified a heterozygous p.Y64C mutation in CDC42 consistent with TKS. <h3>Discussion</h3> The Y64C mutation in CDC42 is a TKS Group I lesion causing ineffective intracellular signaling, resulting in <i>in vitro</i> defects including abnormal cellular proliferation and disruption of cell polarity. Actin remodeling disorders such as Wiskott-Aldrich Syndrome and TKS should be considered in patients with immunodeficiency and congenital thrombocytopenia.

DON'T SWEAT IT: A CASE OF PID-LUNG DISEASE MISDIAGNOSED AS CYSTIC FIBROSIS

<h3>Introduction</h3> Chronic lung disease in primary immunodeficiency is common and contributes to significant morbidity and mortality. Bronchiectasis has a broad differential which includes Cystic Fibrosis and several other disease processes. In patients with primary immunodeficiency, this inflammatory destruction of airway walls is a complication of recurrent pneumonia and immune dysregulation. <h3>Case Description</h3> A 16-year-old boy from Bangladesh with history of Cystic Fibrosis (CF) was referred by pulmonology for management of known hypogammaglobulinemia. Patient came to the United States for treatment of CF-associated severe lung disease. His course in Bangladesh had been complicated by multiple episodes of sinopulmonary infections and failure to thrive secondary to severe diarrhea and pancreatic insufficiency. He had been initially diagnosed with CF in 2019 in Thailand with positive sweat chloride test with additional finding of hypogammaglobulinemia. Chest CT at our institution showed severe bronchiectasis with multifocal consolidations and ground glass opacities consistent with CF. Our immunological evaluation revealed agammaglobulinemia with IgG <109, IgA <2, IgM <5, as well as B-cell and T-cell lymphopenia. Pulmonology then performed repeat sweat chloride testing and genetics which were both negative for CF. GI evaluation revealed evidence of colitis with no pancreatic insufficiency. Patient's history could all be explained by primary immunodeficiency (PID). PID gene panel was unrevealing and whole exome sequencing is pending. Following his diagnosis, patient was managed with IVIG, prophylactic antibiotics, and enteral feeds with great improvement. <h3>Discussion</h3> This case illustrates the importance of early recognition of primary immunodeficiency, especially in resource-poor areas. Hypogammaglobulinemia should be on the differential for childhood-onset bronchiectasis.

RECURRENT FUNGAL INFECTIONS FROM BIRTH TO ADULTHOOD: UNKNOWN DIAGNOSIS UNTIL GENETIC TESTING FOR STAT-1 VARIANTS

<h3>Introduction</h3> Signal transducer and activator of transcription 1 (STAT-1) is a transcription factor that plays key roles in cell viability and pathogen responses. Mutations in STAT-1 result in differing degrees of susceptibility to viral, fungal and bacterial infections, vascular alterations, failure to thrive, and autoimmunity. We report a case of STAT-1 variant presenting with a life-long history of recurrent fungal infections where the diagnosis required genetic testing. <h3>Case Description</h3> A 33-year-old man presented for an evaluation of recurrent fungal infections. He experienced oral thrush, tinea pedis, tinea corporis, and onychomycosis since infancy. He was hospitalized twice for Candida esophagitis requiring IV antifungals. He required treatments with oral antifungals every few months, as he was refractory to topical treatments. He had no bacterial or viral infections, and otherwise unremarkable past medical history. He had thorough workups through multiple specialists with no diagnosis made as his immunologic laboratory studies were normal. However, his 4-year-old son was diagnosed with chronic mucocutaneous candidiasis (CMCC) and had genetic testing showing a STAT-1 variant (c.1159A>G (p.Thr387Ala)). The patient then underwent genetic testing which revealed the same pathogenic STAT-1 variant. Further evaluation showed normal T-cell mitogen studies, total IgG/A/M/E, IgG subclass levels, and vaccine responses. Flow cytometry showed mild B-cell lymphopenia and normal T/NK cells. <h3>Discussion</h3> Patients with pathogenic STAT-1 variants can present with variable infection histories and normal immunologic testing. This case highlights the importance of genetic testing for patients presenting with CMCC regardless of normal immunologic testing to identify a specific diagnosis and defects associated with CMCC.

PROFOUND LYMPHOPENIA AND HYPOGAMMAGLOBULINEMIA ASSOCIATED WITH ROTAVIRUS VACCINATION

<h3>Introduction</h3> Rotavirus is the most common cause of severe infectious gastroenteritis in infants. The live vaccine, while effective in immunocompetent patients, has been associated with life-threatening infection in infants with Severe Combined Immunodeficiency (SCID). We present a case of lymphopenia and hypogammaglobulinemia following rotavirus vaccination in a previously immunocompetent patient. <h3>Case Description</h3> A 3-month-old, ex-full-term Latinx female, presented with worsening diarrhea beginning one-week post rotavirus vaccination. She was meeting age-appropriate milestones, had a negative newborn screen (NBS), including SCID screen, and no pertinent family history. She was admitted for un-resolving diarrhea, electrolyte abnormalities, lymphopenia and hypogammaglobulinemia. Stool rotavirus PCR was positive 8 weeks after vaccination. Total IgG and IgM were low. The presence of a thymus was confirmed on ultrasound. EGD and flexible sigmoidoscopy were grossly normal. She received palivizumab, IVIG, and bactrim and fluconazole prophylaxis. Diarrhea resolved one week after hospitalization. Repeat NBS at 4 months of age demonstrated TRECs of 28/ul. Absolute CD45RA and CD45RO were low. ADA and PNP enzyme, whole exome genome sequencing, chromosomal microarray, and plasma amino acid profile were normal. At 8 months of age, she had mild B-cell lymphopenia with decreased absolute counts and normal percentage. Her leukopenia, neutropenia, and T-cell lymphopenia resolved. <h3>Discussion</h3> The temporal relationship of vaccine administration with onset of clinical findings, the persistently positive stool rotavirus PCR, and the absence of other clear etiology suggests rotavirus vaccine as the inciting factor for this patient's presentation. This case highlights a potentially rare subset of immunocompetent patients who could contract rotavirus infection from the rotavirus vaccine.

Inflammatory profile of convalescent plasma to treat COVID: Impact of amotosalen/UVA pathogen reduction technology.

Blood products in therapeutic transfusion are now commonly acknowledged to contain biologically active constituents during the processes of preparation. In the midst of a worldwide COVID-19 pandemic, preliminary evidence suggests that convalescent plasma may lessen the severity of COVID-19 if administered early in the disease, particularly in patients with profound B-cell lymphopenia and prolonged COVID-19 symptoms. This study examined the influence of photochemical Pathogen Reduction Treatment (PRT) using amotosalen‐HCl and UVA light in comparison with untreated control convalescent plasma (n= 72 – paired samples) - cFFP, regarding soluble inflammatory factors: sCD40L, IFN-alpha, IFN-beta, IFN-gamma, IL-1 beta, IL-6, IL-8, IL-10, IL-18, TNF-alpha and ex-vivo inflammatory bioactivity on endothelial cells. We didn’t observe significant modulation of the majority of inflammatory soluble factors (8 of 10 molecules tested) pre- or post-PRT. We noted that IL-8 concentrations were significantly decreased in cFFP with PRT, whereas the IL-18 concentration was increased by PRT. In contrast, endothelial cell release of IL-6 was similar whether cFFP was pre-treated with or without PRT. Expression of CD54 and CD31 in the presence of cFFP were similar to control levels, and both were significant decreased in when cFFP had been pre-treated by PRT. It will be interesting to continue investigations of IL-18 and IL-8, and the physiopathological effect of PRT- treated convalescent plasma and in clinical trials. But overall, it appears that cFFP post-PRT were not excessively pro-inflammatory. Further research, including a careful clinical evaluation of CCP-treated patients, will be required to thoroughly define the clinical relevance of these findings.