K^b−/−D^b−/−CIITA^−/− mice show signs of intestinal inflammation and insulitis. (a) K^b−/−D^b−/−CIITA^−/− mice showed swollen intestines and enlarged mesenteric lymph nodes (green arrow). (middle, inset) Rectal prolapse is shown. (b) Small intestine cross sections. H&E staining shows moderate to severe inflammation in K^b−/−D^b−/−CIITA^−/− mice, which was absent in control mice. Bar, 800 μm. (c) H&E-stained pancreatic sections show insulitis in K^b−/−D^b−/−CIITA^−/− mice but not in control mice. Bar, 800 μm.

Increased numbers of CD8⁺ T cells in secondary lymphoid organs of K^b−/−D^b−/−CIITA^−/− mice. (a) Spleen cells from the indicated mouse strains were stained for anti-CD4 and anti-CD8 antibodies. Relative percentages of CD4 and CD8 cells are analyzed on the basis of forward/side scatter–based lymphocyte gating. (b) Total number of spleen cells was counted using a hemocytometer (Bright-Line, Hausser Scientific). Different subsets of T cells are calculated from the percentage multiplied by the total number of cells. Horizontal lines represent means. (c) Spleen cells were stained with anti-CD4, anti-DX5, and anti-TCRαβ antibodies. Most CD4⁺ T cells in K^b−/−D^b−/−CIITA^−/− mice are NKT cells.

CD8⁺ T cells from K^b−/−D^b−/−CIITA^−/− mice induce intestinal inflammation upon adoptive transfer. (a) Sorted CD8⁺ T cells from K^b−/−D^b−/−CIITA^−/− (♦) or OT-1 TCR Tg (▪) mice were adoptively transferred to RAG^−/−pfn^−/− mice. Recipients of CD8⁺ T cells from K^b−/−D^b−/−CIITA^−/− mice showed progressive weight loss. Error bars represent means ± SEM. (b) Intestinal (top) and pancreatic (bottom) cross sections revealed ongoing inflammation in mice that received cells from K^b−/−D^b−/−CIITA^−/− mice. Bar, 600 μm. (c) Depletion of CD8⁺ T cells rescues K^b−/−D^b−/−CIITA^−/− mice from colonic inflammation. 8-wk-old mice were depleted of CD8⁺ T cells by injecting anti-CD8 antibodies. Every 2 wk a group of mice was killed, and the thickness of the colon was measured. The CD8 T cell–depleted (▪) and control (♦) groups are shown. Error bars represent means ± SEM. (d) Histological analysis of cross sections of intestine (top) and pancreata (bottom) show the reduction of inflammation in CD8⁺ T cell–depleted animals. Bar, 600 μm.

Expression of distinct MHC molecules on splenocytes. (a) Splenocytes from the indicated mouse strains were stained with 28-8-6 and 25-9-17 antibodies. K^b−/−D^b−/−CIITA^−/− and β₂m^−/−CIITA^−/− mice did not express MHC class Ia or MHC class II molecules. (b) Expression of Qa-1^b, Qa-2, and H2-M3 on splenocytes as revealed by staining with 6A8.6F10.1A, 1-1-2, and H2-M3.130 antibodies, respectively. Black lines represent isotype control and red lines show expression of class I–like molecules. (c) MHC class II–restricted CD4⁺ T cells or MHC class Ia–restricted CD8⁺ T cells cannot survive in K^b−/−D^b−/−CIITA^−/− (TKO) mice. TCR Tg CD4⁺ T cells (OT-II) or CD8⁺ T cells (OT-I) were purified, CFSE stained, and adoptively transferred to RAG-2^−/− or K^b−/−D^b−/−CIITA^−/− mice. 1 wk later, spleens of these mice were evaluated for the presence of CFSE-labeled T cells.

Characteristics of CD8⁺ T cells in K^b−/−D^b−/−CIITA^−/− mice. (a) Cells from spleen (top) and thymus (bottom) of C57BL/6 (black line) and K^b−/−D^b−/−CIITA^−/− (red line) mice were stained with anti-CD8 antibodies and antibodies directed against various activation markers as indicated. CD8⁺ T cells in K^b−/−D^b−/−CIITA^−/− mice are activated in both spleen and thymus. (b) Staining with anti-CD4 and anti-CD8 antibodies shows no abnormal expansion of single-positive cells in thymus. (c) Spleen cells from wild-type, K^b−/−D^b−/−, and K^b−/−D^b−/−CIITA^−/− mice were stained with anti-CD8α, anti-TCRβ, and anti-NKG2A/B/C antibodies. The cells shown are gated on CD8α⁺ cells. (d) Spleen cells or thymocytes from wild-type or K^b−/−D^b−/−CIITA^−/− mice that were fed BrdU for 9 d were stained with anti-CD4, anti-CD8α, and anti-BrdU. Incorporation of BrdU in CD8⁺ T cells was determined by gating on CD4⁻CD8⁺ cells. The numbers in a–d represent the percentages of cells in the total displayed populations. (e) Purified CD8⁺ T cells from wild-type, K^b−/−D^b−/−, and K^b−/−D^b−/−CIITA^−/− mice were activated overnight on plate-bound anti-CD3 and anti-CD28 antibodies in the absence (B) and presence of IL-2 (C) and compared with unstimulated cells (A). Cytokines in the supernatants were measured by the Luminex multiplex cytokine assay. Error bars represent means ± SEM.