Krisztian Csomos

Broad-spectrum antibodies against self-antigens and cytokines in RAG deficiency

Patients with mutations of the recombination-activating genes (RAG) present with diverse clinical phenotypes, including severe combined immune deficiency (SCID), autoimmunity, and inflammation. However, the incidence and extent of immune dysregulation in RAG-dependent immunodeficiency have not been studied in detail. Here, we have demonstrated that patients with hypomorphic RAG mutations, especially those with delayed-onset combined immune deficiency and granulomatous/autoimmune manifestations (CID-G/AI), produce a broad spectrum of autoantibodies. Neutralizing anti-IFN-α or anti-IFN-ω antibodies were present at detectable levels in patients with CID-G/AI who had a history of severe viral infections. As this autoantibody profile is not observed in a wide range of other primary immunodeficiencies, we hypothesized that recurrent or chronic viral infections may precipitate or aggravate immune dysregulation in RAG-deficient hosts. We repeatedly challenged Rag1S723C/S723C mice, which serve as a model of leaky SCID, with agonists of the virus-recognizing receptors TLR3/MDA5, TLR7/-8, and TLR9 and found that this treatment elicits autoantibody production. Altogether, our data demonstrate that immune dysregulation is an integral aspect of RAG-associated immunodeficiency and indicate that environmental triggers may modulate the phenotypic expression of autoimmune manifestations.

Identification of Patients with RAG Mutations Previously Diagnosed with Common Variable Immunodeficiency Disorders

PurposeCombined immunodeficiency (CID) presents a unique challenge to clinicians. Two patients presented with the prior clinical diagnosis of common variable immunodeficiency (CVID) disorder marked by an early age of presentation, opportunistic infections, and persistent lymphopenia. Due to the presence of atypical clinical features, next generation sequencing was applied documenting RAG deficiency in both patients.MethodsTwo different genetic analysis techniques were applied in these patients including whole exome sequencing in one patient and the use of a gene panel designed to target genes known to cause primary immunodeficiency disorders (PIDD) in a second patient. Sanger dideoxy sequencing was used to confirm RAG1 mutations in both patients.ResultsTwo young adults with a history of recurrent bacterial sinopulmonary infections, viral infections, and autoimmune disease as well as progressive hypogammaglobulinemia, abnormal antibody responses, lymphopenia and a prior diagnosis of CVID disorder were evaluated. Compound heterozygous mutations in RAG1 (1) c256_257delAA, p86VfsX32 and (2) c1835A>G, pH612R were documented in one patient. Compound heterozygous mutations in RAG1 (1) c.1566G>T, p.W522C and (2) c.2689C>T, p. R897X) were documented in a second patient post-mortem following a fatal opportunistic infection.ConclusionAstute clinical judgment in the evaluation of patients with PIDD is necessary. Atypical clinical findings such as early onset, granulomatous disease, or opportunistic infections should support the consideration of atypical forms of late onset CID secondary to RAG deficiency. Next generation sequencing approaches provide powerful tools in the investigation of these patients and may expedite definitive treatments.

Ligase-4 Deficiency Causes Distinctive Immune Abnormalities in Asymptomatic Individuals

PurposeDNA Ligase 4 (LIG4) is a key factor in the non-homologous end-joining (NHEJ) DNA double-strand break repair pathway needed for V(D)J recombination and the generation of the T cell receptor and immunoglobulin molecules. Defects in LIG4 result in a variable syndrome of growth retardation, pancytopenia, combined immunodeficiency, cellular radiosensitivity, and developmental delay.MethodsWe diagnosed a patient with LIG4 syndrome by radiosensitivity testing on peripheral blood cells, and established that two of her four healthy siblings carried the same compound heterozygous LIG4 mutations. An extensive analysis of the immune phenotype, cellular radiosensitivity, telomere length, and T and B cell antigen receptor repertoire was performed in all siblings.ResultsIn the three genotypically affected individuals, variable severities of radiosensitivity, alterations of T and B cell counts with an increased percentage of memory cells, and hypogammaglobulinemia, were noticed. Analysis of T and B cell antigen receptor repertoires demonstrated increased usage of alternative microhomology-mediated end-joining (MHMEJ) repair, leading to diminished N nucleotide addition and shorter CDR3 length. However, overall repertoire diversity was preserved.ConclusionsWe demonstrate that LIG4 syndrome presents with high clinical variability even within the same family, and that distinctive immunologic abnormalities may be observed also in yet asymptomatic individuals.

Prevalence and clinical challenges among adults with primary immunodeficiency and recombination-activating gene deficiency

To the Editor: Recombination-activating gene (RAG) deficiency has an estimated disease incidence of 1:181,000, including severe combined immunodeficiency (SCID) at a rate of 1:330,000. Complete or hypomorphic variants of SCID secondary to low recombinase activity (<5%) present early with severe infections and/or clinical signs of systemic inflammation, such as severe dermatitis, colitis, or both. Hypomorphic RAG1/2 mutations with more preserved residual V(D)J recombination activity (5% to 30%) result in a distinct phenotype of combined immunodeficiency with granuloma, autoimmunity, or both. Beyond combined immunodeficiency, RAG deficiency has been found in patients with predominantly primary antibody deficiencies and naive CD4 T-cell lymphopenia in most cases. Currently, there is no published systematic evaluation for the presence of an underlying RAG deficiency in patients with primary antibody deficiencies. There is great variability among diagnostic modalities for evaluation and treatment for inflammatory lung disease in case reports of RAG deficiency with no standardized guidelines. Clinical features and lung disease for patients with late presentation of RAG deficiency have not been studied extensively. In addition, no studies have examined the prevalence of RAG deficiency in cohorts of adults with primary immunodeficiency (PID). Here we describe a cohort of 15 patients with late presentation of RAG deficiency. We also estimate the prevalence of RAG deficiency in adults with PID after genetic analysis in 2 separate large cohorts of patients with PID. We have analyzed the canonical regions of RAG1 and RAG2 in a total of 692 patients with PID from 2 separate cohorts, one from the United Kingdom (UK) and one from Austria (Vienna). The UK cohort is part of the National Institute for Health Research BioResource–Rare Diseases PID study, as previously described (Tuijnenburg et al). In the National Institute for Health Research BioResource–Rare Diseases PID cohort of 558 patients (299 adults) and the Vienna cohort of 134 patients (106 adults), we report a total of 5 newly identified cases of RAG deficiency. For details, see the Methods section and Tables E1 to E3 in this article’s Online Repository at www.jacionline.org. Based on these findings, we estimate that the prevalence of RAG deficiency in adults with PID ranges from 1% to 1.9%. For all adult patients with PID currently registered with the UK Primary Immunodeficiency Network database (3294 patients older than age 18 years), we expect to find an additional 32.9 to 62.6 cases of RAG deficiency. Gene variants are shown in Fig 1, A. Cohort demographics are discussed in the Methods section in this article’s Online Repository. Functional characterization of novel RAG variants is discussed in the Methods section in this article’s Online Repository. The activity of mutant RAG1 and RAG2 proteins normally required for catalyzingV(D)J recombination events are shown in Table E2. In addition to the method previously described, we also used a

Reduced numbers of circulating group 2 innate lymphoid cells in patients with common variable immunodeficiency

Recent studies identified an emerging role of group 2 and 3 innate lymphoid cells (ILCs) as key players in the generation of T‐dependent and T‐independent antibody production. In this retrospective case‐control study, CD117+ ILCs (including the majority of ILC2 and ILC3) were reduced in patients with common variable immunodeficiency (CVID). The reduction in CD117+ ILCs was distinctive to CVID and could not be observed in patients with X‐linked agammaglobulinemia. Patients with a more pronounced reduction in CD117+ ILC numbers showed significantly lower numbers of peripheral MZ‐like B cells and an increased prevalence of chronic, non‐infectious enteropathy. Subsequent phenotyping of ILC subsets in CVID revealed that the reduction in CD117+ ILC numbers is due to a reduction in ILC2 numbers. In vitro expansion of CVID ILC2 in response to IL‐2, IL‐7, IL‐25 and IL‐33 was impaired. Furthermore, upregulation of MHCII and IL‐2RA in response to IL‐2, IL‐7, IL‐25 and IL‐33 was impaired in CVID ILC2. Thus, our results indicate a dysregulation of ILC subsets with a reduction in ILC2 numbers in CVID, however, further studies are needed to explore whether ILC abnormalities are a primary finding or secondary to disease complications encountered in CVID.

Adult-Onset Myopathy in a Patient with Hypomorphic RAG2 Mutations and Combined Immune Deficiency

To the Editor: Severe loss of function mutations in recombination activating genes (RAG1 and RAG2), required for the initial phase of V(D)J recombination in T cell and B cell receptors, are associated with a T-B-NK+ form of severe combined immunodeficiency (SCID) [1]. However, hypomorphic mutations in the same RAG genes can lead to atypical (or Bleaky^) forms of SCID, which may present with a broad spectrum of phenotypes [1–5]. These include Omenn syndrome, which presents in infancy, and is characterized by erythroderma, eosinophilia, elevated IgE level, lymphadenopathy, and hepatosplenomegaly. Often milder and with later onset, a range of immune dysregulation syndromes can be seen. These can be associated with autoantibodies against self-antigens and cytokines [1, 6, 7], as well as defects in Tcell and B cell tolerance [1]. Some patients present with a combined immunodeficiency phenotype with granulomatous disease and autoimmunity, including autoimmune cytopenias, vitiligo, myasthenia gravis, and psoriasis [1–3, 6]. Patientswith hypomorphicmutations in SCID-associated genes may appear to have common variable immune deficiency (CVID). Of note, a sizeable proportion of patients with CVID present with autoimmunity (approximately 30%) and/or granulomatous disease (approximately 10%) [8]. While there can be significant overlap in laboratory and clinical manifestation of CVID and hypomorphic RAG mutations, features such as opportunistic infections, significant T and B cell defects, and the presence of autoantibodies, including those targeting cytokines and causing autoimmunity, should prompt investigation for an underlying monogenic defect, which could include hypomorphic RAG deficiency.We found two cases ofRAGmutation in the literature that were uncovered in patients previously diagnosed with CVIDwho developed opportunistic infections as well as autoimmunity (vitiligo and autoimmune cytopenias) [1, 9]) as well as two cases of RAGmutation in patients with selective antibody deficiency with impaired production of antibody against bacterial polysaccharide [10].

Congenital Immunodeficiency Diseases: Crossroad of Infection, Autoimmunity, and Hyperinflammation

Primary immunodeficiency disorders (PIDs) are genetically predetermined conditions of innate or adaptive immune system dysregulation. Susceptibility toward infection has been a long-standing, often diagnosis-requiring, feature. More recently, autoimmunity as a central pathology in PIDs has been described. Our growing ability to link unique genetic defects with downstream mechanisms that drive hyperinflammation and loss of self-tolerance is extremely powerful. Ultimately, successful management of the PID patient requires not only prevention of infection, but early detection and treatment of autoimmune complications through the use of mechanism-based therapies, which will directly address the immune dysregulation at play.

266. G-Force Loading of Virus Vectors into Vesicles for Enhanced Gene Therapy Vehicles

Adeno-associated virus (AAV) vectors have shown remarkable efficiency in a number of preclinical models of disease in several organs including the eye, brain, muscle, heart, and liver. A major limitation to long term transduction, especially via the systemic route, is pre-existing immunity (humoral and cell-mediated) as well as the high vector load required to achieve sufficient levels of gene expression in the desired organ. Natural nanoparticles released from all cells, called extracellular vesicles (EVs), may have utility in creating better AAV vectors for human gene therapy. We have previously shown that harvesting AAV vectors from the media of 293T producer cells contains AAV vectors endogenously associated with EVs (called ev-AAV, aka vexosomes). We have gone on to demonstrate that 293T-derived ev-AAV can evade neutralizing anti-AAV antibodies and greatly increase transduction in mice. An alternative strategy would be to load purified AAV vectors into separately purified EVs. This would have the benefit of using EVs from a variety of cellular sources with desired biological activity and also to use donor-derived autologous EVs. Here we demonstrate a simple method for loading EVs with AAV vectors using high speed centrifugation forces.Iodixanol density gradient-purified AAV was mixed with conditioned media containing EVs from 293T cells and also primary human peripheral blood mononuclear cells (PBMCs) and then ultracentrifuged to co-pellet EVs and AAV. The pellet was resuspended in media and used in transduction assays and anti-AAV antibody neutralization assays.Strikingly we found that this process bestowed greatly enhanced transduction of cells in culture (36-fold) as well as resistance to neutralizing anti-AAV antibodies. To demonstrate that EVs were essential to this enhancement, we mixed AAV with plain media (no EVs) or depleted conditioned media of EVs before addition of AAV using either Triton-X-100 EV lysis or ultracentrifugation. In all cases there was a large decrease in the transduction efficiency and antibody evasion compared to conditioned EV media mixed with AAV.Together these data suggest that g-force loading of AAV into EVs represents a promising method to increase performance of the vector. We will continue to optimize the protocol and test the gene delivery functions in vivo.