The recombination-activating genes (RAG) 1 and 2 are indispensable for diversifying the primary B cell receptor repertoire and pruning self-reactive clones via receptor editing in the bone marrow; however, the impact of RAG1/RAG2 on peripheral tolerance is unknown. Partial RAG deficiency (pRD) manifesting with late-onset immune dysregulation represents an ‘experiment of nature’ to explore this conundrum. By studying B cell development and subset-specific repertoires in pRD, we demonstrate that reduced RAG activity impinges on peripheral tolerance through the generation of a restricted primary B cell repertoire, persistent antigenic stimulation and an inflammatory milieu with elevated B cell-activating factor. This unique environment gradually provokes profound B cell dysregulation with widespread activation, remarkable extrafollicular maturation and persistence, expansion and somatic diversification of self-reactive clones. Through the model of pRD, we reveal a RAG-dependent ‘domino effect’ that impacts stringency of tolerance and B cell fate in the periphery.

Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome (WS) is a combined immunodeficiency caused by gain-of-function mutations in the C-X-C chemokine receptor type 4 (CXCR4) gene. We characterize a unique international cohort of 66 patients, including 57 (86%) cases previously unreported, with variable clinical phenotypes. Of 17 distinct CXCR4 genetic variants within our cohort, 11 were novel pathogenic variants affecting 15 individuals (23%). All variants affect the same CXCR4 region and impair CXCR4 internalization resulting in hyperactive signaling. The median age of diagnosis in our cohort (5.5 years) indicates WHIM syndrome can commonly present in childhood, although some patients are not diagnosed until adulthood. The prevalence and mean age of recognition and/or onset of clinical manifestations within our cohort were infections 88%/1.6 years, neutropenia 98%/3.8 years, lymphopenia 88%/5.0 years, and warts 40%/12.1 years. However, we report greater prevalence and variety of autoimmune complications of WHIM syndrome (21.2%) than reported previously. Patients with versus without family history of WHIM syndrome were diagnosed earlier (22%, average age 1.3 years versus 78%, average age 5 years, respectively). Patients with a family history of WHIM syndrome also received earlier treatment, experienced less hospitalization, and had less end-organ damage. This observation reinforces previous reports that early treatment for WHIM syndrome improves outcomes. Only one patient died; death was attributed to complications of hematopoietic stem cell transplantation. The variable expressivity of WHIM syndrome in pediatric patients delays their diagnosis and therapy. Early-onset bacterial infections with severe neutropenia and/or lymphopenia should prompt genetic testing for WHIM syndrome, even in the absence of warts.

BackgroundPrimary immunodeficiency is common among patients with autoimmune cytopenia.ObjectiveThe purpose of this study is to retrospectively identify key clinical features and biomarkers of primary immunodeficiency (PID) in pediatric patients with autoimmune cytopenias (AIC) so as to facilitate early diagnosis and targeted therapy.MethodsElectronic medical records at a pediatric tertiary care center were reviewed. We selected 154 patients with both AIC and PID (n=17), or AIC alone (n=137) for inclusion in two cohorts. Immunoglobulin levels, vaccine titers, lymphocyte subsets (T, B and NK cells), autoantibodies, clinical characteristics, and response to treatment were recorded.ResultsClinical features associated with AIC-PID included splenomegaly, short stature, and recurrent or chronic infections. PID patients were more likely to have autoimmune hemolytic anemia (AIHA) or Evans syndrome than AIC-only patients. The AIC-PID group was also distinguished by low T cells (CD3 and CD8), low immunoglobulins (IgG and IgA), and higher prevalence of autoantibodies to red blood cells, platelets or neutrophils. AIC diagnosis preceded PID diagnosis by 3 years on average, except among those with partial DiGeorge syndrome. AIC-PID patients were more likely to fail first-line treatment.ConclusionsAIC patients, especially those with Evans syndrome or AIHA, should be evaluated for PID. Lymphocyte subsets and immune globulins serve as a rapid screen for underlying PID. Early detection of patients with comorbid PID and AIC may improve treatment outcomes. Prospective studies are needed to confirm the diagnostic clues identified and to guide targeted therapy.

In the era of newborn screening (NBS) for severe combined immunodeficiency (SCID) and the possibility of gene therapy (GT), it is important to link SCID phenotype to the underlying genetic disease. In western countries, X-linked interleukin 2 receptor gamma chain (IL2RG) and adenosine deaminase (ADA) deficiency SCID are two of the most common types of SCID and can be treated by GT. As a challenge, both IL2RG and ADA genes are highly polymorphic and a gene–based diagnosis may be difficult if the variant is of unknown significance or if it is located in non-coding areas of the genes that are not routinely evaluated with exon-based genetic testing (e.g., introns, promoters, and the 5′and 3′ untranslated regions). Therefore, it is important to extend evaluation to non-coding areas of a SCID gene if the exon-based sequencing is inconclusive and there is strong suspicion that a variant in that gene is the cause for disease. Functional studies are often required in these cases to confirm a pathogenic variant. We present here two unique examples of X-linked SCID with variable immune phenotypes, where IL2R gamma chain expression was detected and no pathogenic variant was identified on initial genetic testing. Pathogenic IL2RG variants were subsequently confirmed by functional assay of gamma chain signaling and maternal X-inactivation studies. We propose that such tests can facilitate confirmation of suspected cases of X-linked SCID in newborns when initial genetic testing is inconclusive. Early identification of pathogenic IL2RG variants is especially important to ensure eligibility for gene therapy.

Autoimmunity is becoming an increasingly recognized complication in patients with primary immunodeficiencies (PIDs), including a variety of combined immune deficiencies such as Recombination Activating Gene (RAG) defects. The approach to treating autoimmunity in PID patients is complex, requiring a balance between immunosuppression and susceptibility to infection. Inflammatory arthritis is a feature of immune dysregulation in many PIDs, and the optimal treatment may differ from first line therapies that usually consist of disease-modifying anti rheumatic drugs (DMARDs). An example of mechanism-based therapy of arthritis in PID uses blockade of IL-6 signaling with tocilizumab for patients with STAT 3 gain-of-function (GOF) mutation and augmented IL-6 pathway. Herein, we describe two PID cases with arthritis who were found to have defects in RAG. One patient with refractory inflammatory arthritis experienced remarkable improvement in symptoms with tocilizumab therapy. Arthritis can be a clinical feature of immune dysregulation in RAG deficiency, and tocilizumab therapy has been suggested to have utility in treatment of arthritis in RAG deficiency.

BackgroundPatients with inborn errors of immunity (IEI) have increased risk of developing cancers secondary to impaired anti-tumor immunity. Treatment of patients with IEI and cancer is challenging as chemotherapy can exacerbate infectious susceptibility. However, the literature on optimal cancer treatment in the setting of IEI is sparse.ObjectivesWe present a patient with specific antibody deficiency with normal immunoglobins (SADNI), immune dysregulation (ID), and stage III ovarian carcinoma as an example of the need to modify conventional treatment in the context of malignancy, IEI, and ongoing infections.MethodsThis is a retrospective chart review of the patient’s clinical manifestations, laboratory evaluation and treatment course.ResultsOur patient is a female with SADNI and ID diagnosed with stage III ovarian carcinoma at 60 years of age. Her ID accounted for antinuclear antibody positive (ANA+) mixed connective tissue diseases, polyarthralgia, autoimmune neutropenia, asthma, autoimmune thyroiditis, and Celiac disease. Due to the lack of precedent in the literature, her treatment was modified with continuous input from infectious disease, allergy/immunology and oncology specialist using a multidisciplinary approach.The patient completed debulking surgery and 6 cycles of chemotherapy. The dosing for immunoglobulin replacement therapy was increased for prophylaxis. Chemotherapy doses were lowered for all cycles preemptively for IEI. The therapy included carboplatin, paclitaxel, bevacizumab, and pegfilgrastim. The patient completed six-months of maintenance medication involving bevacizumab.Her treatment course was complicated by Mycobacterium avium-complex (MAC) infection, elevated bilirubin and liver enzymes attributed to excessive immunoglobulin replacement therapy, and urinary tract infection (UTI) and incontinence.Cancer genetic analysis revealed no targetable markers and primary immunodeficiency gene panel of 407 genes by Invitae was unrevealing. Lab tests revealed no evidence of Epstein-Barr Virus (EBV) infection. Post-chemotherapy imaging revealed no evidence of cancer for 1 year and 4 months, but the disease relapsed subsequently. The patient’s lung scarring requires vigilance.ConclusionsOur patient with ovarian cancer and IEI required modified treatment and prevention of complications. In cases of IEI, optimal chemotherapy should be titrated to minimize immunosuppression yet treat cancer aggressively while decreasing the risk of infection with prophylactic antibiotics and prolonged post-treatment surveillance, including pulmonary evaluation.

Objective: The purpose of this study is to increase awareness and improve diagnosis of primary immune deficiency (PID) in the heterogenous group of patients with autoimmune cytopenia (AIC) by identifying clinical characteristics and laboratory biomarkers that distinguish PID patients from patients with AIC alone. This is especially relevant in genetically-defined PIDs which may be resistant to conventional therapy and mechanism-based treatment approach is required. Evans syndrome, defined by multi-lineage autoantibodies (two or more positive anti-platelet, anti-neutrophil, or direct Coombs test) is a known risk factor for PID; therefore we also investigate the incidence of PID in a broad cohort of AIC patients with single and multi-lineage cytopenias. Methods: Patients with autoimmune cytopenias (autoimmune hemolytic anemia (AIHA), immune thrombocytopenia (ITP), or autoimmune neutropenia (AIN)) were referred to our Immune Dysregulation clinical team and prospectively enrolled in the study period of 2016-2019. Detailed immune phenotyping (IgG, IgA, IgM, lymphocyte subsets, vaccine titers, lymphocyte proliferation to mitogens/antigens), serum lipopolysaccharide (LPS) and autoantibodies were measured and/or collected by chart review and genetic testing for PID was pursued. Results: From 2016 to 2019, 78 patients were enrolled to our study. The patients were predominantly children (ages 1-82 years, average age 16.82 years). Of 78 patients with AIC, 48 (61%) were diagnosed with underlying PID based on immune phenotyping and/or genetic testing. Of 39 patients with genetic testing to date, 19 (49%) have been diagnosed with genetically-defined PID (pathogenic variants in CTLA-4, NFKB1, ALPS-related genes, WAS, POLE-1, PI3K, CYBB, or 22q11 [partial DiGeorge syndrome]); the remainder were classified as combined immune deficiency or common variable immune deficiency based on immune phenotyping; an additional two patients were classified as ALPS based on clinical history/flow cytometry/family history of ALPS. Lymphocyte subsets (CD4+ T, CD8+ T, CD19+ B, CD56+ NK) and immune globulins (IgG, IgA, IgM) tended to be lower in AIC-PID patients vs AIC-only (p<0.05). Evans syndrome was more commonly found in AIC-PID patients (13/31, 42%) compared to AIC-only (4/42, 10%). LPS was significantly elevated in the serum of AIC patients compared to healthy controls (mean 719 vs 87 pg/mL, p<0.001). Excluding partial DiGeorge syndrome patients (average LPS 222pg/mL), LPS levels were significantly higher in genetically-defined untreated PID patients (average 1463 pg/mL) vs. other PID (average 444 pg/mL) (p=0.02) or patients with AIC alone (average 667 pg/mL) (p=0.03). Studies are ongoing on specific subsets that are linked to immune dysregulation (T-regulatory cells, double negative T cells, T follicular helper cells). Conclusions: An unexpectedly high fraction of patients with AIC were identified with underlying PID in our Immune Dysregulation program. Routine basic immune evaluation with immunoglobulin levels and lymphocyte subsets expedited diagnosis of PID. Genetic evaluation distinguished a group of patients with AIC-PID and highly elevated LPS level, reflecting high bacterial load, and this may distinguish them from the rest of patients with AIC. The source of bacterial LPS can be multifactorial and is yet to be determined. Our studies continue focusing on biomarkers that can be applied to the heterogenous group of patients with AIC. This will allow early detection and timely initiation of targeted therapies. Disclosures No relevant conflicts of interest to declare.

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