A hypomorphic R229Q Rag2 mouse mutant recapitulates human Omenn syndrome

Abstract

Rag enzymes are the main players in V(D)J recombination, the process responsible for rearrangement of TCR and Ig genes. Hypomorphic Rag mutations in humans, which maintain partial V(D)J activity, cause a peculiar SCID associated with autoimmune-like manifestations, Omenn syndrome (OS). Although a deficient ability to sustain thymopoiesis and to produce a diverse T and B cell repertoire explains the increased susceptibility to severe infections, the molecular and cellular mechanisms underlying the spectrum of clinical and immunological features of OS remain poorly defined. In order to better define the molecular and cellular pathophysiology of OS, we generated a knockin murine model carrying the Rag2 R229Q mutation previously described in several patients with OS and leaky forms of SCID. These Rag2(R229Q/R229Q) mice showed oligoclonal T cells, absence of circulating B cells, and peripheral eosinophilia. In addition, activated T cells infiltrated gut and skin, causing diarrhea, alopecia, and, in some cases, severe erythrodermia. These findings were associated with reduced thymic expression of Aire and markedly reduced numbers of naturally occurring Tregs and NKT lymphocytes. In conclusion, Rag2(R229Q/R229Q) mice mimicked most symptoms of human OS; our findings support the notion that impaired immune tolerance and defective immune regulation are involved in the pathophysiology of OS.

Figure 1. Generation of Rag2^R229Q/R229Q ES cells and mice.

(A) A targeting vector was designed to replace endogenous Rag2 with the gene carrying the R229Q substitution and an NheI restriction site. GFP was fused in frame with the Rag2 neo cassette flanked by loxP sites (arrows), which was cloned downstream of the Rag2 UTR (black box). Gray boxes denote the 3′ probe used for genomic screening. (B) Southern blot analysis using the 3′ probe on EcoRV-digested genomic DNAs. Lane 1, 129Sv wild-type ES cells; lane 2, Rag2^+/R229QneoΔ recombinant ES cells; lane 3, heterozygous Rag2^+/R229QneoΔ mouse after neo excision due to Cre cross; lane 4, 129Sv Rag2^+/+ mouse; lane 5, Rag2^+/R229QneoΔ mouse. (C) Chromatogram showing nucleotide substitutions, denoted by underline: the first 2 changes obtained the NheI restriction site, the last 2 caused the R to Q amino acid change (bold).

Figure 2. Histological analysis of Rag2^R229Q/R229Q mice.

(A) Phenotypic aspect of a 3-month-old Rag2^R229Q/R229Q mouse, showing severe alopecia and skin erythrodermia. (B) Skin biopsy revealed marked dermal inflammation (top) composed of CD3⁺ lymphocytes (bottom left) and containing numerous eosinophils (bottom right). Original magnification, ×10 (top); ×20 (bottom). (C) Similarly, the gut showed dense inflammatory infiltration (left), mainly composed of CD3⁺ cells (right). Original magnification, ×20. (D and E) Comparison between thymic tissue from age-matched 6-week-old Rag2^+/+ and Rag2^R229Q/R229Q mice: the normal corticomedullary differentiation observed in Rag2^+/+ thymus (D), as highlighted by the anti–cytokeratin 5 immunostaining (D, inset), was absent in the Rag2^R229Q/R229Q mouse (E and inset). Original magnification, ×4 (D and E). (F) B220 immunostaining showed B follicles (b) and paracortical areas (p) in Rag2^+/+ mice (left) in contrast to the abnormal architecture and severe depletion of B cells observed in Rag2^R229Q/R229Q mice (right). Original magnification, ×4. (G) Nodal parenchyma from the Rag2^R229Q/R229Q (left) shows admixture of histiocytes, large activated lymphoid cells (arrow), and eosinophils (arrowhead); most lymphoid cells were CD3⁺ lymphocytes (top right) that expressed the activation antigen CD30 (bottom right). Original magnification, ×20 (left); ×40 (right).

Figure 3. Impaired T cell development and aberrant peripheral T cell activation in Rag2^R229Q/R229Q mice.

(A) Top left: Fluorescence-activated cell sorting analysis of thymocytes from the indicated mice stained with CD4 and CD8 antibodies. Top right: CD4^–CD8^– cells were gated and analyzed for CD44 and CD25 expression. Bottom left: TCR-β and CD69 expression in electronically gated CD4⁺8⁺ cells. The percentage of TCR-β^brightCD69⁺ cells is indicated. Bottom right: Overlay of TCR-β surface expression in the indicated thymocyte subsets (gray histograms, Rag2^+/+; open histograms, Rag2^R229Q/R229Q). (B) Left: Percentage of cells during different T cell differentiation stages in the thymus. Right: Percentage of differentiating DN cells. Results are the mean and SD of 5 Rag2^+/+ and 10 Rag2^R229Q/R229Q mice. P values were determined by unpaired Student’s t test.

Figure 4. Peripheral T cells and T cell proliferation.

(A) Top: Fluorescence-activated cell sorting analysis of lymph node cells stained with CD4 and CD8 antibodies. Bottom: CD4⁺ and CD8⁺ cells from the indicated mice were analyzed for CD44 and CD62L distribution. (B) Proliferation of Rag2^+/+ and Rag2^R229Q/R229Q CD90⁺ cells isolated from splenocytes, plated at 2 × 10⁵ cells per well, and stimulated as indicated. Proliferation was assessed by ³H-thymidine incorporation in triplicate wells. Plotted values are cpm for each individual mouse. Bars represent the median for each group.

Figure 5. Immunoscope analysis of TCR repertoire.

(A) Quantitative TCRVβ repertoire determined by real-time PCR analysis on T cells from the 2 thymi and 4 spleens from Rag2^R229Q/R229Q mice (designated as 1–4) and from 2 representative Rag2^+/+ mice (designated as 1 and 2). (B) Representative immunoscope profiles of TCRVβ analysis in the thymi and spleens of the mice as in A. The x axes represent CDR3 length, and y axes represent arbitrary fluorescence intensity of the runoff products.

Figure 6. Impaired B cell development in Rag2^R229Q/R229Q mice.

(A) Top: Relative representation and SD of B220^low and B220^high cells in the bone marrow of the indicated mice (n = 6). Bottom: Dot-plot analysis of bone marrow cells from the indicated mice labeled with B220 and CD2 antibodies and IgD and IgM expression of electronically gated B220⁺ bone marrow cells. (B) Top: Relative representation and SD of B220⁺ cells in the spleens of the indicated mice (n = 6). Bottom: Dot-plot analysis of B220⁺ splenocytes labeled with CD21 and CD23 antibodies or IgD and IgM antibodies. P values were determined by unpaired Student’s t test. Numbers indicate the percentage of cells in the indicated gates.

Figure 7. Serum concentration of Igs.

Sera from Rag2^+/+ and Rag2^R229Q/R229Q mice were collected and their IgG1, IgG2, IgG3, IgA, and IgM were evaluated by Bioplex analysis, while ELISA assay was performed to measure IgE production. Bars represent the median for each group. P values were determined by Mann-Whitney test.

Figure 8. AIRE expression analysis and cellular infiltration in target organs.

(A) Amplification of Aire cDNAs obtained from Rag2^+/+, Rag2 ^R229Q/R229Q, and Rag2^–/– thymic mRNAs. As an internal control, Gapdh was used. (B–E) Inflammatory infiltration in the lungs and livers of Rag2^+/+ and Rag2^R229Q/R229Q mice. Compared with the Rag2^+/+ mouse (B), histological analysis of a representative 4-month-old Rag2^R229Q/R229Q mouse revealed dense peribronchiolar and perivascular inflammatory infiltration in the lung (C) composed mainly of CD3⁺ lymphocytes (C, inset). Similarly, in contrast to the control mouse (D), dense inflammatory infiltration composed of CD3⁺ T cells was found in liver portal tracts (E and inset). Original magnification, ×10 (B–E); ×40 (insets).

Figure 9. Analysis of nTregs in Rag2^R229Q/R229Q mice.

(A) Fluorescence-activated cell sorting analysis of thymus and spleen cells stained with CD25 and Foxp3 antibodies electronically gated on CD4⁺ cells. Numbers indicate the percentages of total live cells. (B) Histograms show percentage of CD25^highFoxp3⁺ cells in thymi, spleens, and lymph nodes of Rag2^+/+ and Rag2^R229Q/R229Q mice. P values were determined by unpaired Student’s t test.