Exacerbated colitis associated with elevated levels of activated CD4+ T cells in TCRα chain transgenic mice  Immo Prinz, Uwe Klemm, Stefan H.E. Kaufmann, Ulrich Steinhoff  Gastroenterology  Volume 126, Issue 1, Pages 170-181 (January 2004) DOI: 10.1053/j.gastro.2003.10.062

Figure 1 Expression of transgenic TCRα chains in TCRα−/− mice. (A) RT-PCR showing TCRα 7.2 chain expression in lymphocytes derived from thymus and spleen of TCRα−/− Tg α7.2 mice. Nonreverse-transcribed RNA was used as negative, β-actin as positive control. (B) Dot plots show TCRβ and TCR Vα8 expression profiles of PBL of TCRα−/− and TCRα−/− Tg α8.2 mice. The percentage of T-cell subsets is shown in each dot plot quadrant. (C) Impaired T-cell maturation in TCRα−/− and TCRα−/− Tg α7.2 mice. T-cell differentiation was analyzed by staining thymocytes from 6-week-old mice for CD4 and CD8. Data are representative of 3 independent experiments. Gastroenterology 2004 126, 170-181DOI: (10.1053/j.gastro.2003.10.062)

Figure 2 Increased frequencies of CD4+ TCRα−β+ cells in TCRα−/− Tg α7.2 mice. Peripheral blood lymphocytes (PBL) from TCRα+, TCRα−/−, TCRα−/− Tg α7.2, and TCRα−/− Tg α7.2 mice at 4–6 weeks of age were stained for CD4, CD8, CD62L, and TCRβ chain surface expression. (A) Comparison of the frequencies of TCRβ positive cells in PBL of the indicated mice. (B) Dot plots show TCRβ and CD4 expression profiles of PBL of the indicated mice. The percentage of T-cell subsets is shown in each dot plot quadrant. (C) Dot plots showing CD4 and CD62L expression profiles of PBL of the same mice. FACS profiles are representative of at least 5 independent experiments. Gastroenterology 2004 126, 170-181DOI: (10.1053/j.gastro.2003.10.062)

Figure 3 Analysis of activation markers and cytokine profile in peripheral CD4+ TCRα−β+ cells. Cells were prepared from mesenteric lymph nodes (MLN) and spleens of 6-week-old TCRα+, TCRα−/−, and TCRα−/− Tg α7.2 littermates and stained for expression of the cell surface activation markers CD44, CD62L, and CD69 plus CD4. (A) Cells were gated on CD4; dot plots show CD44 and CD62L activation marker profiles. The percentage of T-cell subsets is shown in each dot plot quadrant. (B) Cells were gated on CD4 expression, histograms showing expression of CD69 activation marker. Similar results were obtained from 3 independent experiments. (C) Relative expression of the indicated genes in CD4+ T cells isolated from MLN of TCRα−/− and TCRα−/− Tg α7.2 mice compared with control littermates. After stimulation by PMA/ionomycin for 3 hours, cDNA was prepared and analyzed by semiquantitative real-time PCR analysis. Data are shown as fold differences to control normalized to β-actin expression. Gastroenterology 2004 126, 170-181DOI: (10.1053/j.gastro.2003.10.062)

Figure 4 Severe and accelerated course of IBD in TCRα−/− Tg α7.2 mice. TCRα+, TCRα−/−, and TCRα−/− Tg α7.2 mice from heterozygous breedings were monitored for 4 months. At an age of 8 weeks, TCRα−/− Tg α7.2 mice began to develop severe IBD characterized by chronic diarrhea and progressive wasting syndrome. TCRα+ and TCRα−/− littermates remained disease free during the period of observation. Data represent 8 mice per group from 2 independent experiments. Gastroenterology 2004 126, 170-181DOI: (10.1053/j.gastro.2003.10.062)

Figure 5 Histologic analysis of IBD in control, TCRα−/− Tg α7.2, and TCRα−/− mice. Histology of the colon from 10-week-old TCRα+ and TCRα−/− Tg α7.2 mice and from elder TCRα−/− mice with IBD. Hematoxylin-eosin staining of transverse and longitudinal sections of TCRα+ (A and B) and TCRα−/− Tg α7.2 mice (C and D) littermates. Compared with control animals (A and B), typical signs of pathology in TCRα−/− Tg α7.2 mice (C and D) are elongated crypts, loss of goblet cells (large white cells), and massive cell infiltrates in the lamina propria. For comparison, immune cell infiltration in a 5-month-old (E) and severe pathology in a 10-month-old TCRα−/− mouse (F). (Original magnifications ×200; bars = 100 μm.) Gastroenterology 2004 126, 170-181DOI: (10.1053/j.gastro.2003.10.062)

Figure 6 Analysis of T-cell infiltrates in TCRα−/− Tg α7.2 mice. Frozen sections of the colon from 10-week-old mice with IBD were immunohistochemically stained with anti-CD4 mAb (A and B), anti-TCR-γδ mAb (C and D), or anti-IL-4 (F). (E) Overview hematoxylin-eosin staining of the granulomatous foci at lower magnification (Original magnification ×50, bar = 500 μm). Focal granulomatous accumulation of CD4+ T cells (A and B) and scattered distribution of γδ T cells (C and D) are shown at low (Original magnification ×100, bars = 200 μm) and high (Original magnification ×400, bars = 50 μm) magnification. (C and D) Arrows indicate crypt abscesses. Gastroenterology 2004 126, 170-181DOI: (10.1053/j.gastro.2003.10.062)

Figure 7 Analysis of T-cell infiltrates after adoptive transfer. Histology of the colon from TCRβ−/− mice 9 weeks after adoptive transfer of (A) PBS, or 2.5 × 105 FACS-sorted CD4+ TCRβ+ cells from MLN of (B) control, (C) TCRα−/−, and (D) TCRα−/− Tg α7.2 mice. Frozen sections were immunohistochemically stained with anti-CD4 mAb. (Original magnifications ×100 for A and B and ×200 for C.) Gastroenterology 2004 126, 170-181DOI: (10.1053/j.gastro.2003.10.062)

Figure 8 Prolonged half-life of TCRβ chains in TCRα−/− Tg α7.2 mice. (A) Direct association and stabilization of TCRβ chains by TCR α7.2. Thymocytes from TCRα+, TCRα−/−, and TCRα−/− Tg α7.2 mice were radiolabeled with [35S]methionine for 5 minutes and then chased in the presence of excess unlabeled methionine for the indicated times. Lysates were immunoprecipitated with anti-TCRβ mAb and analyzed by reducing SDS-PAGE. (B) Extended half-life of TCRβ chains in TCRα−/− Tg α7.2 mice. Nonlinear regression plots of labeled TCRβ chains and of coimmunoprecipitated TCRα7.2 as quantified by densitometry. This experiment was repeated twice with similar results. Gastroenterology 2004 126, 170-181DOI: (10.1053/j.gastro.2003.10.062)

Figure 9 Analysis of thymocytes and their susceptibility to apoptosis. (A) Total thymocyte counts in TCRα+ (□), TCRα−/− (▩), and TCRα−/− Tg α7.2 (■) mice. (B) Analysis of apoptotic Annexin V positive thymocytes directly after isolation (0 hours) or after 24-hour incubation in serum-free medium. Representative results of 3 independent experiments with 3 mice per group. Gastroenterology 2004 126, 170-181DOI: (10.1053/j.gastro.2003.10.062)