BN and LEW rats exhibit different proportions and numbers of natural Foxp3⁺ T_reg cells. (A) Representative flow cytometry profiles of Foxp3⁺ T cells in CD4⁺-gated lymphocytes in PBMCs, LNs, and spleen from LEW and BN rats. The values on each cytometry profile represent the mean percentages of Foxp3⁺ cells in the CD4⁺ T cell population (mean ± SEM; n = 4). Data are representative of four independent experiments. (B) Absolute numbers (mean ± SEM) of Foxp3⁺CD4⁺ cells in the LNs and spleen from BN (n = 6) and LEW (n = 10) rats. (C) Representative flow cytometry profiles of Foxp3 expression in DP CD4⁺CD8⁺ and SP CD4⁺CD8⁻ thymocytes from naive BN and LEW rats. The value on each profile represents the percentages of Foxp3⁺ thymocytes (mean ± SEM; n = 6). (D) Absolute numbers of Foxp3⁺ cells in DP CD4⁺CD8⁺ and SP CD4⁺ thymocytes from BN (n = 6) and LEW (n = 6) rats. Bars represent the means ± SEM. Data are representative of three independent experiments. (E) Percentages of Foxp3⁺ cells among CD4⁺ T cells of donor origin in PBMCs, LNs, and spleen originating from (LEW × BN) F1 recipients of bone marrow from BN or LEW rats 14 wk after engraftment. Bars represent the mean ± SEM (n = 6 rats per group). Data are representative of two independent experiments. **, P < 0.01.

Biochemical analyses of VAV1 in HEK293 cells transfected with VAV1 variants reveal that VAV1-W63 is constitutively active. (A and B) Analysis of VAV1 phosphorylation in HEK293 transfected with different human VAV1 plasmids. VAV1 was immunoprecipitated and immunoblotted with anti-phosphotyrosine 4G10 mAb (A) or anti–phospho-VAV1 (Y174) antibody (B). After stripping, membranes were probed with anti-VAV1 antibody. IP, immunoprecipitation. (C) Analysis of GEF activity of VAV1 using pull-down with Rac1G15A agarose beads on serum-starved HEK293 cells transfected with different human VAV1 plasmids. (D and E) Analysis of Rac1 or RhoA activities in HEK293 cells transfected with different VAV1 variants using pull-down assay to detect relative amounts of Rac1-GTP (D) or RhoA-GTP (E). Total Rac1 and RhoA levels were used as loading controls. In each case, results show one blot of a representative experiment and a graph representing the mean ± SEM of three to four independent experiments. WB, Western blot. (F) Confocal analysis of HEK293 cells transfected with different VAV1 variants. Actin was visualized with phalloidin–Alexa Fluor 488. Representative images of three independent experiments are shown. *, P < 0.05; **, P < 0.01; ns, not significant.

Fort1, a 117-kb locus of chromosome 9, controls the proportion of CD4⁺Foxp3⁺ T_reg cell and Vav1 protein expression. (A) Percentages of Foxp3⁺ cells among blood CD4⁺ T cells from the BN parental strain and a series of BN.LEWc9 congenic lines and sublines. Each point represents the value found in individual rats (n = 4–9). (B) Genetic maps of congenic lines. Microsatellite markers are indicated on the left. Black, white, and gray segments indicate chromosomal regions of BN, LEW, or undetermined origin, respectively. On the right is shown the physical map of the Fort1 117-kb region constructed using information from the Celera Genomics genome assembly available in the NCBI Rat Genome Resource, which contains four genes, C3, Gpr108, Trip10, and Vav1. (C) BN (closed) or Bf (open) CD25⁺ and CD25⁻ FACS sorted T cells were cultured for 3 d with allogeneic APCs. Proliferation was assessed with an 18-h [³H]thymidine pulse added after 48 h of culture. Data represent mean ± SEM. (D) Suppressor activity of CD4⁺CD25⁺ T_reg cells was measured by T cell proliferation assay. T_reg cells from BN or Bf were co-cultured with effector T cells (CD4⁺CD45RC^high from F1 rats) at various ratios of T_reg cell/T_eff cell. The proliferation level of responder cells alone was assigned a value of 100%. Results are representative of two independent experiments. (E) Western blot analysis of Vav1 protein expression in thymic and LN CD4⁺ T cell lysates from LEW, BN, and Bf rats. (F) Western blot analysis of VAV1 protein in HEK293 cells transfected with plasmids encoding human wild-type VAV1 (VAV1-R63), mutated VAV1 (VAV1-W63), or oncogenic VAV1 (VAV1-ΔCH). (E and F) β-Actin was used as a loading control. In each case, results show one blot of a representative experiment and a graph representing the mean ± SEM of three to six independent experiments. **, P < 0.01; ns, not significant.

In primary CD4 T cells, Vav1 R63W polymorphism affects Vav1 phosphorylation, Rac1/RhoA GTPases activities, and Ca²⁺ flux under TCR stimulation. (A and B) Analysis of Vav1 phosphorylation by immunoblot with anti-phosphotyrosine 4G10 mAb (A) or anti–phospho-Vav1(Y174) antibody (B) in BN (Vav1-W63) and Bf (Vav1-R63) LN CD4⁺ T cells stimulated (stim) or not (unstim) with anti-TCR mAb. Lysates were probed with anti-Vav1Ab. IP, immunoprecipitation. (C and D) Analysis of Rac1 or RhoA activation in serum-starved BN and Bf CD4⁺ T cells stimulated or not with anti-TCR mAb using pull-down assay to detect the relative amounts of Rac1-GTP (C) or RhoA-GTP (D). Total Rac1 and RhoA levels were used as loading controls. In each case, results show one blot of a representative experiment and a graph representing the mean ± SEM of three to five independent experiments. WB, Western blot. (E) Confocal microscopy analyses of CD4⁺ T cells of BN and Bf rats that were stimulated or not with anti-TCR mAb. Cells were staining for TCR (red) and Vav1 (green) using the appropriate second reagents. Line scan analysis indicates the distribution of the TCR and Vav1 along the white lines. Representative images of three independent experiments are shown. (F) Analysis of Ca²⁺ influx in purified CD4⁺ T cells from LEW, BN, and Bf LNs (left) or thymus (middle) loaded with Indo-1 and incubated with an anti-TCR mAb. Analyses were first performed in the absence of rabbit anti–mouse (RAM) immunoglobulin cross-linker to measure the baseline Ca²⁺ level in unstimulated CD4⁺ T cells. Flow was halted for the addition of 50 µg/ml RAM (first arrow). The increase in intracellular Ca²⁺ flux was then recorded in real time over 200 s. Cell flow was again halted for the addition of ionomycin (Iono; second arrow) to measure the maximal signal from the Indo-1 indicator. Data are representative of three independent experiments involving a total of five or six rats per strain. These data are expressed in the right panel as percentages of the variation of maximum Ca²⁺ flux observed in the Bf congenic subline compared with the BN (left) and LEW (right) parental strains.