Volume 119, Issue 4, Pages 983-995 (October 2000) Limited CD4 T-cell diversity associated with colitis in T-cell receptor α mutant mice requires a T helper 2 environment  Atsushi Mizoguchi, Emiko Mizoguchi, Lawrence J. Saubermann, Koichi Higaki, Richard S. Blumberg, Atul K. Bhan  Gastroenterology  Volume 119, Issue 4, Pages 983-995 (October 2000) DOI: 10.1053/gast.2000.18153 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 (A) Percentage of Vβ (Vβ1-20) expression in the colonic LP cells of TCRα−/− mice (6 months of age) without (top panel) and with (lower panel) colitis. ■, Vβ8.2 expression. In TCRα−/− mice without colitis, the Vβ usage in the colon is different from mouse to mouse. In contrast, the Vβ usage is more restricted to Vβ8.2 in the diseased colon. (B) Vβ8.1 and Vβ8.2 expressions in the colonic LP cells are compared in TCRα−/− mice (6 months of age) with and without colitis. A dot represents the result of an individual mouse. (C) CDR3 length display of Vβ8.2 in the colonic LP cells of TCRα−/− mice with and without colitis. The amplified Vβ8.2 products were resolved on 6% sequence gel and visualized by silver staining. The arrow represents a CDR3 β length of 6 amino acids. (D) Restriction ratio (see Materials and Methods) of the CDR3 region of Vβ8.2 in the colonic LP of C57BL/6 and TCRα−/− (6 months old) mice with and without colitis. A more restricted distribution pattern of CDR3 is found in the colonic LP of TCRα−/− mice with colitis than in C57BL/6 or TCRα−/− mice without colitis. Gastroenterology 2000 119, 983-995DOI: (10.1053/gast.2000.18153) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Amino acid sequence of CDR3β in the dominant Vβ8.2 clones obtained from the colonic LP of TCRα−/− mice (6 months old) with and without colitis and wild-type C57BL/6 mice (6 months old). The CDR3 region is in bold, and the second position of CDR3 region isunderlined. The disease severity is graded as normal (0), mild (1), moderate (2), and severe (3) colitis. In the mice indicated by the asterisk the cecal contents were analyzed and no known pathogenic organism was detectable. D, aspartic acid; E, glutamic acid; G, glycine; R, arginine; A, alanine; T, threonine; V, valine; S, serine; P, proline; Q, glutamine; N, asparagine; L, leucine; H, histidine; I, isoleucine; K, lysine; F, phenylalanine; W, tryptophan; Y, tyrosine. Gastroenterology 2000 119, 983-995DOI: (10.1053/gast.2000.18153) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 The frequency (%) of amino acid (D, aspartic acid; E, glutamic acid; G, glycine; R, arginine; A, alanine; T, threonine; V, valine; S, serine; P, proline; Q, glutamine; N, asparagine; L, leucine) usage in the second position of the CDR3 region in Vβ8.2 clones from colonic LP of TCRα−/− mice (6 months of age) with (■) and without (▩) colitis and C57BL/6 mice (2). The results were obtained from 203 clones of TCRα−/− mice with colitis, 130 clones of TCRα−/− mice without colitis, and 51 clones of C57BL/6 mice. Gastroenterology 2000 119, 983-995DOI: (10.1053/gast.2000.18153) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 (A) Frequency of TCRβ+ and TCRδ+ T cells in the colonic LP of TCRα−/− mice (6-month-old) with or without colitis maintained under specific pathogen–free conditions or germfree (germ-reduced) conditions (GF). TCRα−/− mice maintained in germfree conditions did not develop colitis. Colonic LP cells were incubated with FITC–anti-TCRδ (GL3) and PE–anti-TCRβ (H57-597) and then subjected to flow-cytometric analysis. Percentages of cells in the quadrants are indicated in the corners. (B) The expression of Cβ and Vβ8.2 (BV8S2) in the colonic LP of appendectomized and sham-operated TCRα−/− mice was detected by reverse-transcription PCR/Southern blot analysis. TCRα−/− mice underwent appendectomy (resection of cecal patch) or sham operation at 3 weeks of age and were killed at 6 months of age. (C) Amino acid sequence of CDR3 usage of Vβ8.2 in the colonic LP cells from these appendectomized TCRα−/− mice. There is no obvious restriction of the CDR3 repertoire of Vβ8.2 in the colon of appendectomized mice. Gastroenterology 2000 119, 983-995DOI: (10.1053/gast.2000.18153) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 (A) Proliferation of TCRα−β+ T cells under in vitro conditions. Enriched TCRα−β+ T cells from the diseased colons of the pooled TCRα−/− mice (3–8 mice, 6 months old) were cultured for 4 days in media alone or in the presence of activating reagents (plate-coated anti-TCRβ or concanavalin A) or epithelial cells/APCs with or without Th1 (rIL-2, rIL-12, anti–IL-4 mAb) or Th2 (rIL-2, rIL-4, anti–IL-12 mAb) conditions. After pulse with [3H]thymidine for 16 hours, [3H]thymidine incorporation was measured. Each experiment was performed 3 times, yielding similar results. (B) Purified TCRα−β+ T cells from the diseased colons of the pooled TCRα−/− mice (3–8 mice, 6 months old) were cultured in media alone or under Th1 (rIL-2, rIL-12, anti–IL-4 mAb) or Th2 (rIL-2, rIL-4, anti–IL-12 mAb) conditions or in the presence of epithelial cells/APCs with or without Th1 or Th2 conditions. After 14 days of culture, viable lymphocytes were counted by the trypan blue dye exclusion test. The survival ratio (percentage) was calculated as the number of total viable lymphocytes after 14 days of culture divided by the number of lymphocytes seeded when cultures were initiated. Each experiment was performed 4 times with similar results. Gastroenterology 2000 119, 983-995DOI: (10.1053/gast.2000.18153) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 Enriched TCRα−β+ T cells pooled from the colons of young TCRα−/− mice (12 weeks old) without colitis were cultured with epithelial Ags/APCs in Th1 (rIL-2, rIL-12, anti–IL-4 mAb) or Th2 (rIL-2, rIL-4, anti–IL-12 mAb) conditions. (A) Cβ expression was detectable in the freshly isolated cells (Fresh) and those cultured for 14 days with epithelial cells (Ags)/APCs in the presence of Th2 (Th2) conditions, but was not detectable in the cells cultured with epithelial cells (Ags)/APCs in Th1 (Th1) conditions. (B) Expression of the selected Vβ in the freshly isolated cells (top panel) and those cultured for 14 days with epithelial cells (Ags)/APCs under Th2 conditions (lower panel). (C and D) Results from 2 separate experiments. cDNA from cells freshly isolated (Fresh) and those cultured for 7 days with epithelial Ags/APCs under Th1 (Th1) or Th2 (Th2) conditions was amplified using Vβ8.2- and Cβ-specific primers. After separation on 6% sequence gels, DNA from the dominant bands (C) and D) was eluted and sequenced. The clones identical to the ones detected in vivo in the diseased colon of TCRα−/− mice (#9 and #12 in Figure 1) are underlined. (E and F) Lymphocytes freshly isolated (Fresh) or cultured for 4 days with epithelial cells (Ags)/APCs in the presence of Th1 (Th1) or Th2 (Th2) conditions were intraperitoneally transferred into RAG-1−/− mice. These recipient mice were killed 6 weeks after cell transfer. (E) H&E-stained section of the colon showing mild colitis in RAG-1−/− mice reconstituted with cells cultured under Th2 conditions. (F) The proliferation index (number of BrdU+ cells/mm length of colon) of colonic epithelial cells in the recipient mice (3–5 mice) was assessed by detection of the BrdU-incorporated cells. Gastroenterology 2000 119, 983-995DOI: (10.1053/gast.2000.18153) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 7 (A) CDR3 length display of Vβ8.2 in the colonic LP of IL-4−/− × TCRα−/− mice (top panel) and TCRα−/− mice (lower panel). The arrowhead indicates 6–amino acid length of CDR3β. (B) Restriction ratio (see Materials and Methods) of the CDR3 region of Vβ8.2 in the colonic LP of IL-4−/− × TCRα−/− mice (6 months old) and TCRα−/− (6 months old) mice. (C) Amino acid sequence of CDR3β in Vβ8.2 clones from IL-4−/− × TCRα−/− mice (6 months old). No obvious restricted pattern of Vβ8.2 clones can be seen. Gastroenterology 2000 119, 983-995DOI: (10.1053/gast.2000.18153) Copyright © 2000 American Gastroenterological Association Terms and Conditions