Irit Tirosh

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.

Characterization of T and B cell repertoire diversity in patients with RAG deficiency

Differences in B and T cell repertoires in patients with RAG deficiency associate with clinical severity. Taking SCID genetics to the clinic Mutations that lead to deficiencies in the recombination-activating genes RAG1 and RAG2 result in a spectrum of immunodeficiencies ranging from loss of T and/or B cell repertoire diversity to a complete lack of T and B cells—severe combined immunodeficiency (SCID). Here, Lee et al. perform next-generation B and T cell repertoire sequencing on 12 patients with RAG mutations who have immunodeficiencies of varying severity. They found that the level of repertoire skewing was associated with the severity of disease and that specific repertoire deficiencies were associated with particular phenotypes. These data support a genotype-phenotype connection for primary immunodeficiencies. Recombination-activating genes 1 and 2 (RAG1 and RAG2) play a critical role in T and B cell development by initiating the recombination process that controls the expression of T cell receptor (TCR) and immunoglobulin genes. Mutations in the RAG1 and RAG2 genes in humans cause a broad spectrum of phenotypes, including severe combined immunodeficiency (SCID) with lack of T and B cells, Omenn syndrome, leaky SCID, and combined immunodeficiency with granulomas or autoimmunity (CID-G/AI). Using next-generation sequencing, we analyzed the TCR and B cell receptor (BCR) repertoire in 12 patients with RAG mutations presenting with Omenn syndrome (n = 5), leaky SCID (n = 3), or CID-G/AI (n = 4). Restriction of repertoire diversity skewed usage of variable (V), diversity (D), and joining (J) segment genes, and abnormalities of CDR3 length distribution were progressively more prominent in patients with a more severe phenotype. Skewed usage of V, D, and J segment genes was present also within unique sequences, indicating a primary restriction of repertoire. Patients with Omenn syndrome had a high proportion of class-switched immunoglobulin heavy chain transcripts and increased somatic hypermutation rate, suggesting in vivo activation of these B cells. These data provide a framework to better understand the phenotypic heterogeneity of RAG deficiency.

Late Onset Hypomorphic RAG2 Deficiency Presentation with Fatal Vaccine-Strain VZV Infection

PurposeHypomorphic mutations in RAG1 and RAG2 are associated with significant clinical heterogeneity and symptoms of immunodeficiency or autoimmunity may be late in appearance. As a result, immunosuppressive medications may be introduced that can have life-threatening consequences. We describe a previously healthy 13-month-old girl presenting with rash and autoimmune hemolytic anemia, while highlighting the importance of vigilance and consideration of an underlying severe immunodeficiency disease prior to instituting immunosuppressive therapy.MethodsGiven clinical deterioration of the patient and a temporal association with recently administered vaccinations, virus genotyping was carried out via 4 real-time Forster Resonance Energy Transfer PCR protocols targeting vaccine-associated single nucleotide polymorphisms. Genomic DNA was extracted from whole blood and analyzed via the next-generation sequencing method of sequencing-by-synthesis. Immune function studies included immunophenotyping of peripheral blood lymphocytes, mitogen-induced proliferation and TLR ligand-induced production of TNFα. Analysis of recombination activity of wild-type and mutant RAG2 constructs was performed.ResultsVirus genotyping revealed vaccine-strain VZV, mumps, and rubella. Next-generation sequencing identified heterozygosity for RAG2 R73H and P180H mutations. Profound lymphopenia was associated with intense corticosteroid therapy, with some recovery after steroid reduction. Residual, albeit low, RAG2 protein activity was demonstrated.ConclusionsBecause of the association of RAG deficiency with late-onset presentation and autoimmunity, live virus vaccination and immunosuppressive therapies are often initiated and can result in negative consequences. Here, hypomorphic RAG2 mutations were linked to disseminated vaccine-strain virus infections following institution of corticosteroid therapy for autoimmune hemolytic anemia.

Recombination activity of human recombination-activating gene 2 (RAG2) mutations and correlation with clinical phenotype

Background: Mutations in recombination‐activating gene (RAG) 1 and RAG2 are associated with a broad range of clinical and immunologic phenotypes in human subjects. Objective: Using a flow cytometry–based assay, we aimed to measure the recombinase activity of naturally occurring RAG2 mutant proteins and to correlate our results with the severity of the clinical and immunologic phenotype. Methods: Abelson virus–transformed Rag2−/− pro‐B cells engineered to contain an inverted green fluorescent protein (GFP) cassette flanked by recombination signal sequences were transduced with retroviruses encoding either wild‐type or 41 naturally occurring RAG2 variants. Bicistronic vectors were used to introduce compound heterozygous RAG2 variants. The percentage of GFP‐expressing cells was evaluated by using flow cytometry, and high‐throughput sequencing was used to analyze rearrangements at the endogenous immunoglobulin heavy chain (Igh) locus. Results: The RAG2 variants showed a wide range of recombination activity. Mutations associated with severe combined immunodeficiency and Omenn syndrome had significantly lower activity than those detected in patients with less severe clinical presentations. Four variants (P253R, F386L, N474S, and M502V) previously thought to be pathogenic were found to have wild‐type levels of activity. Use of bicistronic vectors permitted us to assess more carefully the effect of compound heterozygous mutations, with good correlation between GFP expression and the number and diversity of Igh rearrangements. Conclusions: Our data support genotype‐phenotype correlation in the setting of RAG2 deficiency. The assay described can be used to define the possible disease‐causing role of novel RAG2 variants and might help predict the severity of the clinical phenotype. Graphical abstract Figure. No caption available.