A Spontaneous RAG1 Nonsense Mutation Unveils Naturally Occurring N-Terminal Truncated RAG1 Isoforms

RAG1^NX mice have lymphocyte developmental blocks at Ag receptor rearrangement steps.

Thymi from 4- to 5-wk-old WT, NX, and Het littermates were analyzed by flow cytometry. (A) CD4 versus CD8 frequencies and (B) total cell counts per thymus. Gating: live, singlets, dump⁻ (B220, CD11b, CD11c, Gr1, NK1.1, Ter119), TCRγδ⁻. (C) DN1-4 frequencies and (D) total cell counts per thymus. Gating: CD4⁻, CD8⁻, TCRβ^lo. Early B cell development was analyzed in the bone marrow. (E and F) Frequencies of early B cell progenitors of B220⁺CD93⁺IgM⁻ cells were quantified. Pregating: live, singlets, dump⁻ (TCRβ, NK1.1, Ter119, CD11c, Gr1). Data combined from at least three independent experiments. Bars indicate mean ± SEM. Statistics: one-way ANOVA with Tukey honest significant difference posttest. **p < 0.01, ****p < 0.0001. n.s., p > 0.05.

RAG1^NX thymocytes have reduced TCRVβ recombination and an altered Vβ TCR repertoire.

Sorted DN3 thymocytes from WT and NX mice were assayed by qPCR for frequencies of respective Vβ to DβJβ1 rearrangements (A) and Vβ to DβJβ2 rearrangements (B), four mice per genotype. A number sign (#) denotes not detected. Gating: live, singlets, dump⁻ (B220, CD8, CD4, CD11b, CD11c, Gr1, NK1.1, Ter119), CD4⁻, CD8⁻, CD25⁺, CD44⁻. Vβ repertoire was assayed by flow cytometry of (C) DP thymocytes and (D) SP thymocytes. Representative gating in Supplemental Fig. 1A, 1B. (E) The ratio of Vβ usage by DP versus SP thymocytes was calculated. Data combined from two independent experiments. Error bars indicate SEM. Statistics: multiple t tests with Holm-Sidak correction. *p < 0.05, **p < 0.01, ***p < 0.0001, ****p < 0.0001. n.s., p > 0.05.

RAG1^NX mice have reduced numbers of mature B and T cells and increased frequencies of Tregs and T memory cells.

(A) Numbers of leukocytes were enumerated from spleens of 4- to 5-wk-old RAG1^WT (WT), RAG1^NX (NX), or heterozygous (Het) littermates by flow cytometry. Relative frequencies and numbers of peripheral Tregs (B), memory CD4⁺ T cells (C), and memory CD8⁺ T cells (D) in the spleen were quantified. Data combined from at least three independent experiments. Bars indicate mean ± SEM. Statistics: one-way ANOVA with Tukey honest significant difference posttest. Gating: live, singlets, B cells (CD19⁺TCRβ⁻), T cells (TCRβ⁺, CD19⁻, CD4⁺, or CD8⁺), NK cells (TCRβ⁻, CD19⁻, NK1.1⁺), dendritic cells (TCRβ⁻, CD19⁻, NK1.1⁻, CD11c⁺), neutrophils (TCRβ⁻, CD19⁻, NK1.1⁻, CD11c⁻, CD11b⁺, Ly6G⁺), inflammatory monocytes (TCRβ⁻, CD19⁻, NK1.1⁻, CD11c⁻, CD11b⁺, Ly6G⁻, Ly6C^hi). T_CM, central memory; T_EM, effector memory. **p < 0.01, ****p < 0.0001. n.s., p > 0.05.

RAG1^NX mice develop increased age-associated B cells and ANA.

(A–D) T cells from 4- to 5-wk-old WT and NX mice were stimulated ex vivo with PMA and ionomycin, and cytokine expression was measured by flow cytometry. Statistics: one-way ANOVA with Tukey honest significant difference posttest. Combined data from two independent experiments. (E) Twenty- to twenty-two–month-old WT and NX mice were compared for peripheral leukocyte populations. Gating: neutrophils (CD3⁻, Ly6G⁺), B cells (CD3⁻, Ly6G⁻, CD19⁺), T cells (CD3⁺, CD19⁻, Ly6G⁻, CD4⁺, or CD8⁺), eosinophils (CD3⁻, CD19⁻, Ly6G⁻, Siglec-F⁺), macrophages (CD3⁻, CD19⁻, Ly6G⁻, Siglec-F⁻, F4/80⁺), monocytes (CD3⁻, CD19⁻, Ly6G⁻, Siglec-F⁻, F4/80⁻, CD11b⁺). (F) Frequency of ABCs of total B cells was quantified in 20- to 22-mo-old female WT and NX mice. Statistics: Mann–Whitney U test. (G) ANA staining intensity on Hep-2a cells was assayed for in young (4- to 5-wk-old) and old (20- to 22-mo-old) WT and NX mice. Bars indicate mean ± SEM. Statistics: one-way nonparametric ANOVA with Tukey honest significant difference posttest. See representative images in Supplemental Fig. 2. *p < 0.05, ***p < 0.0001, ****p < 0.0001.