Impaired suppressive function of BN^m Treg is involved in the development of intestinal lesions.

<p>(A) Absolute numbers of Foxp3+ CD4+ T cells in thymus, spleen and mLN from BN (n = 7) and BN^m (n = 13) rats. Data are representative of three independent experiments. (B) Suppressive activity of thymic CD25^bright CD4+ SP cells (top panel) and peripheral CD25^bright CD4+ T cells (bottom panel) from BN or BN^m rats was assessed in co-culture experiments with CFSE-labeled naive LEW CD4 T cells as effector cells. Proliferation was assessed by CFSE dilution (percentages indicate the proportion of CFSE^low cells). Data are representative of three independent experiments. (C, D) Disease frequency (C) and duodenum microscopic scores (D) in 12 week-old BN^m rats injected with PBS (white column or symbols; n = 21) or with 4.10⁶ BN CD25^bright CD4+ T cells (grey columns or symbols; n = 12) at 4 weeks of age. (E) Cytokine protein expression in duodenum from control BN^m rats with microscopic intestinal scores (white columns; n = 8) and from BN^m rats transferred with CD25^bright CD4+ T cells (grey columns; n = 12).</p

BN^m rats exhibit a T cell autonomous lymphopenia restricted to CD4 T cells.

<p>(A) Percentage of TCRαβ positive cells in PBMC from BN (n = 15, black bars), BN^m (n = 9, white bars) and (BN×BN^m) F1 (n = 5, grey bars) rats. (B) CD4 and CD8 expression in BN and BN^m spleen T cells; numbers indicate the cell percentages in the outlined area. (C) Absolute numbers of BN (n = 4) and BNm (n = 9) T cells, CD4+ and CD8+ T cells, and B cells in spleen. Data in B and C are representative of five independent experiments. (D) CD4 and CD8 expression in BN and BN^m thymocytes; numbers indicate cell percentages in the outlined area. (E) Absolute numbers of BN (n = 5) and BN^m (n = 5) in thymocyte subsets (DN: double negative; DP: double positive; SP: simple positive). Data in D and E are representative of six independent experiments. (F) Absolute numbers of lymphocytes in spleen of lethally irradiated (LEW×BN) F1 rats reconstituted with BN (n = 6) or BN^m (n = 8) T cell-depleted bone marrow cells. Data are representative of three independent experiments. (G) Absolute numbers of donor CD4 SP and CD8 SP cells in thymus of sub-lethally irradiated (LEW×BN) F1 rats 15 days after intrathymic injection of DN thymocytes from BN (n = 4) and BN^m (n = 4) rats. (BN: black columns, BN^m: white columns).</p

BN^m rats develop inflammatory bowel disease.

<p>(A) Small intestine and colon from 12 week-old BN and BN^m rats. (B, C) Length of small intestine and colon (B) and thickness of the duodenum, jejunum, ileum and colon (C) of BN (n = 10) and BN^m rats with macroscopic signs of intestinal lesions (n = 7). (D) Hematoxylin-eosin staining of duodenum, jejunum and colon from 8 to 10 week-old BN and BN^m rats. Images are representative of microscopic lesions observed in all affected BN^m rats (original magnification: 100 X). Stars indicate granulomas; arrows point to infiltration and double head arrows indicate the thickness of the intestinal wall. (E) CD68, CD3 and B220 immunoperoxydase staining on sections of jejunum from 8 week-old BN^m rats. Positive staining results in a brown reaction product. HE: hematoxylin-eosin staining showing the presence of polymorphonuclear leucocytes (original magnification: 400 X). (F) Myeloperoxidase (MPO) activity in the duodenum, jejunum, ileum and colon tissue samples from BN (n = 30) and BN^m rats with macroscopic lesions (n = 21). (G) Cytokine and chemokine protein expression in duodenum from BN (n = 7) and BN^m rats with macroscopic lesions (n = 7). (H) Relative mRNA expression of IL-2 and IL-17A in duodenum from BN (n = 4) and BN^m rats exhibiting intestinal macroscopic lesions (n = 7). (BN: black columns, BN^m: white columns).</p

BN^m rats carry a disrupted <i>Themis</i> gene.

<p>(A) Genome scan for loci controlling the percentage of CD4 T cells in the blood of 44 (BN^m×DA)×BN^m rats. Horizontal lines represent genome-wide significance thresholds of 5% (significant) and 0.1% (highly significant) as determined by permutation tests. (B) Percentages of CD4 T cells in 44 (BN^m×DA)×BN^m backcross rats classified according to their genotypes at the microsatellite marker of chromosome 1 nearest to the QTL. In each group, horizontal bars represent the mean values. nn: homozygous BN (n = 23); nd: heterozygous BN-DA (n = 21). (C) Fine mapping of the BN^m mutation in 28 (BN^m×DA) F2 or (BN^m×DA)×BN^m backcross rats, among which 16 showed normal proportions of CD4 T lymphocytes (Unaffected) and 12 showed CD4 T cell lymphopenia (Affected). The position of each microsatellite marker on chromosome 1 is indicated in megabases (Mb). White: homozygous BN^m; black: heterozygous BN^m/DA. Rec: number of rats characterized by a given recombination. The physical map of the critical 1.5 Mb interval containing 6 genes is shown underneath. (D) Relative mRNA expression of the 6 genes in BN^m (n = 4) and BN (n = 4) thymocytes. Data are representative of two independent experiments. (E) Electrophoregram, nucleic acid sequences and corresponding amino acids of the BN and BN^m Themis gene in the region surrounding the 4 nucleotide insertion corresponding to the BN^m mutation (boxed). (F) Immunoblot analysis of Themis and β-actin in thymocytes from BN, BN^m, LEW and DA rats, C57BL/6 mice and in human PBMC using anti-Themis antibodies specific either for the mouse C-terminal (left panel) or the human N-terminal (right panel) portion of the protein.</p