Lymphocyte development in SIT-deficient mice. Bone marrow cells (A), lymph nodes cells (B), peritoneal cavity cells (C), thymocytes (D), double-negative thymocytes (E), and splenocytes (F) from 5- to 8-week-old mice were stained with MAbs for CD43, B220, IgM, CD4, CD8, TCR-β, CD44, CD25, γδTCR, IgD, and CD5 and analyzed by flow cytometry. Numbers represent the percentages of cells that fall into the indicated boxes or quadrant areas of total gated living cells.

Enhanced positive selection in the absence of SIT. (A) Thymocytes from H-Y female mice were stained with CD4 and CD8 and assessed for the expression of CD5 and HSA on gated DP and CD8 SP cells. The histograms represent overlaid profiles of cells from a representative SIT^+/− (shaded) and SIT^−/− (thick lines) mouse. (B) Enhanced ERK1/2 activation in SIT^−/− H-Y thymocytes. Single-cell suspensions from thymi were immediately fixed and stained for CD4, CD8, and phospho-ERK1/2. Numbers indicate MFI from one representative experiment out of four. Dotted lines indicate the levels of staining with the secondary antibody alone. (C) Representative dot plots of the expression of the clonotypic transgenic TCRα chain (T3.70) and transgenic TCRβ (F23.1) (upper panels) or CD4 and CD8 (lower panels) in lymph node cells. Numbers indicate the percentages of cells in each subset.

SIT is dispensable for negative selection. (A) Flow cytometric analysis of CD4/CD8 profiles of the T3.70⁺ population in H-Y TCR transgenic male mice. (B) DP thymocyte deletion in response to TCR cross-linking. Thymocytes were cultured in 24-well plates coated with anti-CD3 or anti-CD3 plus anti-CD28 or in the presence of ConA for 24 h, and their viability was determined. One of two independent experiments is shown, each using three pairs of mice. Black and open bars represent wild-type and knockout mice, respectively.

Altered peripheral T-cell function in SIT^−/− mice. (A) Enhanced T-cell proliferation. Purified splenic T cells were stimulated in 96-well plates coated with anti-CD3 antibody or PMA and ionomycin (P+I), pulsed with [³H]thymidine, and processed for standard scintillation counting. Results from seven wild-type and nine knock-out mice are shown as mean counts per minute (cpm). One of two independent experiments is shown. *, P = 0.01; **, P = 0.007. (B) Augmented cytokine production by anti-CD3-stimulated SIT^−/− T cells. T cells were purified and stimulated with 0.3 μg/ml anti-CD3 or PMA and ionomycin (P+I). Results from 8 wild-type and 10 knockout mice are shown. IL-2, interleukin-2; IFN-γ, gamma interferon; TNF-α, tumor necrosis factor alpha. *, P = 0.02; **, P < 0.005. (C) Increased incidence of EAE in the absence of SIT. Mice were examined daily for signs of disease and graded on a scale of increasing severity from 0 to 5 as follows: 0, no signs; 0.5, partial tail weakness; 1, limp tail or slight slowing of righting from supine position; 1.5, limp tail and slight slowing of righting; 2, partial hind-limb weakness or marked slowing of righting; 2.5, dragging of hind limb(s) without complete paralysis; 3, complete paralysis of at least one hind limb; 3.5, hind-limb paralysis and slight weakness of forelimbs; 4, severe forelimb weakness; 5, moribund or dead.

Expression of SIT in mouse lymphocytes and generation of SIT-deficient mice. (A) Lysates prepared from freshly isolated thymocytes (T), splenocytes (S), purified splenic T cells (CD3⁺), or purified splenic B cells (B220⁺) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted with an anti-SIT MAb. To demonstrate equal amounts of protein loading, the blot was stripped and reprobed with an anti-Erk polyclonal antibody. (B) Partial restriction map of the SIT locus and the targeting cassette. Filled boxes represent exons. Restriction sites are indicated as follows: N, NotI; B, BamHI; M, MluI; P, PstI; X, XbaI. Abbreviations: NEO, neomycin resistance cassette; TK, thymidine kinase. (C) PCR analysis of SIT wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mutant alleles. (D) Anti-SIT Western blot analysis showing absence of SIT protein in SIT^−/− mice. Postnuclear lysates were prepared from thymocytes, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and blotted with an anti-SIT MAb. The blot was successively stripped and reprobed with an anti-ERK1/2 antibody as a loading control.

Enhanced expression of CD5 and CD69 on SIT^−/− DP thymocytes. Ungated (total) or CD4⁺ CD8⁺-gated (DP) thymocytes were assessed for surface expression of CD5 and CD69. The histograms represent profiles of cells from a representative SIT^+/+ (shaded) and SIT^−/− (thick lines) mouse. All data are representative of a minimum of 10 individual experiments.

The absence of SIT partially converts positive selection to negative selection. Flow cytometric analysis of positive selection in thymus is shown. Thymocytes from H-Y (A) and P14 (B) TCR SIT^−/− or SIT^+/− transgenic mice were analyzed for transgenic TCR expression by staining with T3.70, a clonotypic antibody specific for H-Y TCRα chain, or V_α2, the P14 TCRα chain. The dot plots show CD4 and CD8 profiles of cells gated on TCR^high. Numbers indicate percentages of cells in each subset. One representative animal for each group is shown. The graphs show the mean cellularity of TCR transgenic thymocyte subsets from seven SIT^+/− and seven SIT^−/− H-Y⁺ tg mice and three SIT^+/− and three SIT^−/− P14⁺ tg mice.

γδ T-cell populations in SIT^−/− mice. (A) Proportion of CD3⁺ γδ TCR⁺ lymph node cells expressing CD44, CD45RB, and CD62L. Ten mice for each genotype were included in the groups. ***, P = 0.0003; **, P = 0.0038; *, P = 0.0352. (B) γδ T-cell distribution in thymi from adult or newborn mice. Black bars and open boxes represent wt and SIT-deficient mice, respectively.