Generation of Tgfb1^{flox-ex 6} mice. a: A Cre-LoxP based conditional gene targeting scheme depicting the relevant piece of Tgfb1 genomic locus, the Tgfb1^{flox-ex 6} targeting vector and the Tgfb1^{flox-ex 6} targeted and deleted locus. PCR primers and southern blot probes are indicated. E, EcoRV; St, StuI; A, ApaI; S, SacI; As, AscI; Sg, SgfI. b: Southern blot screening. StuI cuts both outside and inside and EcoRV cuts only outside at both ends of the targeting vector. AscI and SgfI are only present in the LoxP. Probe A is an outside side probe. Only two (clone #1 and #17) of the five originally targeted clones are shown. StuI, ApaI, and AscI/EcoRV blots confirm the presence of a targeted floxed (exon 6, neo) allele. Positive clones were identified by 3.0 kb (StuI blot), 8.9 kb (ApaI blot), and 10.1 kb (AscI/EcoRV blot). A positive targeted floxed (exon 6, neo) clone (#17) was treated with Cre recombinase and the floxed-neo was deleted in these ES cells. c: Long PCR shows the presence of an expected 8.9-kb band before the Cre-excision and a 7.8-kb band after the Cre-mediated excision in #17 clone, indicating a successful removal of floxed-neo from the targeted floxed (exon 6) allele in ES cells. d: ApaI blot confirms the presence of Tgfb1^{flox-ex 6} allele in germline chimera. Wild type (+/+) mice and targeted floxed (exon 6, neo) ES cells are used as controls. e: PCR genotyping of wild type, Tgfb1 floxed heterozygous (+/flox (exon 6)) and Tgfb1 floxed homozygous (flox-ex 6/flox-ex 6) mice. f: PCR amplification indicating partial but specific Cre-mediated excision in thymus of Tgfb1^{flox-ex 6/flox-ex 6}/LckCre Tg mice. PCR primer p3 is a hybrid primer with sequences from both the Tgfb1 and LoxP and detects specifically Tgfb1^{flox-ex 6} allele but not the wild-type allele under conditions described in the Materials and Methods. There is no floxed deleted allele (1.23 kb) in control mice which is seen in Tgfb1^{flox-ex 6/flox-ex 6}/LckCre Tg mice. Note that no deletion product(s) at the Tgfb1 locus in liver, heart, lung, colon and brain tissue of Tgfb1^{flox-ex 6/flox-ex 6}/LckCre nTg or Tgfb1^+/+/LckCre Tg mice was found (not shown), indicating that Cre-mediated excision occurred only in the thymus of Tgfb1^{flox-ex 6/flox-ex 6}/LckCre Tg mice. g: Q-RT-PCR analysis showing significantly decreased Tgfb1 expression (*P = 3.5747e-3) in T cells from Tgfb1^{flox-ex 6/flox-ex 6}/LckCre Tg mice as compared to the control non-transgenic mice. Each control value is normalized to 1.0.

T cell-specific deletion of Tgfb1 results in increased T cell activation in Tgfb1^{flox-ex 6/flox-ex 6}/LckCre Tg mice. Splenocytes from 5-month-old control and Tgfb1^{flox-ex 6/flox-ex 6}/LckCre Tg mice are analyzed for CD4, CD8, CD44 and CD62L by flow cytometry. Expression of CD44 and CD62L is analyzed on CD4⁺ or CD8⁺ T cells by gating on these subsets. The percent of cells positive for each marker is shown in the quadrants. a: FACS analyses showing an increased CD44 and reduced CD62L on CD4⁺ cells in Tgfb1^{flox-ex 6/flox-ex 6}/LckCre Tg mice. b: CD44 and CD62L expression on CD8⁺ T cells. CD44 expression is increased, whereas CD62L expression is reduced on CD8⁺ T cells in Tgfb1^{flox-ex 6/flox-ex 6}/LckCre Tg mice.

T cell-specific deletion of Tgfb1 results in increased organ inflammation and Th1-effector cytokine production. a–f: Hematoxylin and eosin staining of serial sections of lung (a–c) and salivary gland (d–f) from 5-month-old Tgfb1^{f-ex 6/f-ex 6} control (a, d), Tgfb1^{flox-ex 6/flox-ex 6}/LckCre Tg (b, e) and Tgfb1^{f-ex 6/−} LckCre Tg (c, f) mice. These histopathology data are representative results of at least three mice per genotype analyzed. All photomicrographs are taken at equal magnification. Tgfb1^{flox-ex 6/flox-ex 6}/LckCre Tg mice exhibit moderate inflammation in both the lungs (arrows in b) and the salivary gland (arrows in e) which becomes severe in both organs (arrows in c, f) in Tgfb1^{flox-ex 6/−}/LckCre Tg mice. g: INF-γ production. h: IL-2 production. Splenic T cells (1.5 × 10⁶/ml) from 2-month-old control and Tgfb1^{flox-ex 6/flox-ex 6}/LckCre Tg mice are stimulated for 72 h with T cell receptor-dependent (plate-bound anti-CD3 mAb (5 μg/ml) and Con A (1 μg/ml)) and receptor-independent (PMA (25 ng/ml) plus ionomycin (250 ng/ml)) mitogens. The cytokines in the culture supernatants are assayed using ELISA as described in the Materials and Methods section. The mitogen-stimulated T cells of Tgfb1^{flox-ex 6/flox-ex 6}/LckCre Tg mice exhibit significant increase in the levels (mean ±SD) of IFNγ (G) (*P < 0.0001 for anti-CD3, *P < 0.0001 for Con A, *P = 0.0017 for PMA+ionomycin; and IL-2 (H) (*P < 0.0001 for anti-CD3, *P < 0.0001 for Con A, *P < 0.0001 (PMA+ionomycin).

T cell-specific deletion of Tgfb1 affects T cell homeostasis in Tgfb1^{flox-ex 6/flox-ex 6}/LckCre Tg mice. Splenocytes are prepared from 5-month-old control and Tgfb1^{flox-ex 6/flox-ex 6}/LckCre Tg mice and immunostained for surface expression of CD4, CD8, and CD3 and analyzed by flow cytometry. a: CD3 expression and percent of CD3⁺ splenocytes. Both the mean fluorescence intensity (MFI) and the percent of CD3⁺-T cells are decreased in Tgfb1^{flox-ex 6/flox-ex 6}/LckCre Tg mice. b: Analysis of CD4⁺ and CD8⁺ cells. c: CD3⁺-gated splenocytes indicating distribution of CD4⁺ and CD8⁺ T cells. The % of cells positive for each marker is shown in the quadrants in (a, b). The % of CD4⁺ T cells is reduced and percent of CD8⁺ T cells is increased in both non-gated and CD3-gated splenocytes. d: CD8 expression and percent of CD8⁺ splenocytes. The percent of CD8⁺-T cells is increased but their MFI is slightly reduced in Tgfb1^{flox-ex 6/flox-ex 6}/LckCre Tg mice.

Analysis of body weight and thymus and spleen weight and number of thymocytes and splenocytes in Tgfb1^{flox-ex 6/flox-ex 6}/LckCre Tg mice. a: Body weight at 5 month. b: Thymic weight at 2 month. c: Spleen weight at 2 month. d: Number of thymocytes at 1 month. e: Number of splenocytes at 5 months. Tgfb1^{flox-ex 6/flox-ex 6}/LckCre Tg mice exhibit significant reduction in body weight (n = 4, *P = 8.9415e-4) and thymic (n = 3, *P = 0.0166) and splenic weight (n = 4 for control and n = 5 for Tgfb1^{flox-ex 6/-}/LckCre Tg, *P = 0.0165), and number of total thymocytes (n = 3, *P = 2.4698e-3) and splenocytes (n = 3, *P = 0.0236).