IL-22 +/+ or IL-22 −/− CD4+ CD45RB^high CD25− NK1.1− T cells (5×10⁵) were transferred i.p. into IL-22−/− RAG1−/− double knockout mice.
(A) Mice were massed weekly and percent change from week 0 was calculated. Each dot represents one mouse, bar indicates mean, crosses represent dead mice or mice that reached 30% mass loss and were euthanized according to protocol. For statistics, dead mice were assigned mass loss of −30%. * = p value of <0.05 by an unpaired two-tailed Student’s t test.
(B) Histology of H&E stained sections from the ascending colons of the indicated mice, 3 weeks post-transfer. Shown are representative sections from one mouse of 3–4 mice/group. Bottom image is a higher magnification of the top row image.
(C) At four weeks post-transfer, cytokine mRNA expression was assessed in the colons of the mice by real-time RT-PCR. Bars represent the mean±SD expression of the cytokine gene to HPRT using the ΔΔC_T method. ND=not detected. Experiment was performed two times with similar results.
(D) Total numbers of CD4+ TCRβ+ cells in the inflamed colons as determined by FACS. Mean±SD.

NK1.1+ TCRβ− CD27^low (CD27low) or NK1.1+ TCRβ− CD27^high (CD27high) NK cells were sorted from the spleen and lymph nodes of C57BL/6 mice. Cells were stimulated in vitro with IL-15 and the indicated cytokines. 18 hrs post-stimulation, IL-22 and IFNγ mRNA and protein expression was analyzed by (A) real-time RT-PCR or (B) ELISA of the supernatants, respectively. Experiment was performed three times with similar results.

(A) IL-22 is secreted from the colon during DSS-mediated colitis. C57BL/6 mice were given 3% DSS ad libitum in their drinking water, or remained untreated, and three days later the mice were euthanized and their colons were excised. Colons were cultured for three days as described in the Experimental Procedures and IL-22 was detected in the supernatant by ELISA. Bars represent mean±SD of 7 mice/group.
(B) IL-22 +/+ or IL-22−/− mice were given 3% DSS ad libitum in their drinking water for seven days. Mice were massed daily and percent mass change from day 0 was calculated. Mean±SD. * indicates p value <0.05. 8–10 mice/group.
(C) Survival of IL-22+/+ or IL-22−/− mice in panel B after receiving 3% DSS in their drinking water.

(A) C57BL/6 CD4+ CD45RB^high CD25− NK1.1− T cells (5×10⁵) were transferred i.p. to RAG1−/− mice and five weeks post-transfer, when clinical signs of IBD were evident, cytokine mRNA in the colon was assessed by real-time RT-PCR. As a control, cytokine in the colons of RAG1−/− that did not receive T cells was also assessed. Bars represent the mean±SD expression of the cytokine gene to HPRT using the ΔΔC_T method. Experiment was performed two times with similar results. 5–7 mice/group. ND=not detected.
(B) C57BL/6 CD4+ CD45RB^high CD25− NK1.1− T cells (5×10⁵) were transferred i.p. to RAG1−/− mice and at different weeks post-transfer (0, 1, 2, 3 and 4 weeks) cytokine mRNA expressed in the colon was semi-quantitated by real-time RT-PCR. Mean±SD; dashed line indicates limit of detection. 4 mice/group.
(C) From the colon samples in (A), a 1 cm section of the ascending colons of the above mice were cultured ex vivo for three days. Secreted IFNγ (left) or IL-22 (right) from the supernatants were quantiated by ELISA, mean±SD of 5–7 mice/group. ND=not detected, dashed line indicates limit of detection.

IL-22 +/+ or IL-22 −/−CD4+ CD45RB^high CD25− NK1.1− T cells (5×10⁵) were transferred i.p. into RAG1−/− mice.
(A) 6 weeks post-transfer expression of cytokines mRNA in the ascending colons of the mice was assessed by real-time RT-PCR. The cytokine gene message was compared to HPRT using the ΔΔC_T method. Each dot represents one mouse, bar indicates the mean. Experiment was performed three times with similar results.
(B) Excised colon sections from either RAG−/− mice that did not receive transferred cells or RAG1−/− mice that received IL-22−/− T cells 6 weeks prior, were cultured for three days ex vivo and then cytokine levels in the supernatant were quantitated by ELISA. Bar indicates mean±SD; dashed line indicates limit of detection.
(C) Mice from (A) were massed bi-weekly and percent mass change from day 0 was calculated (top graph). At the same time, IL-22 +/+ or IL-22 −/−CD4+ CD45RB^high CD25− NK1.1− T cells (5×10⁵) were transferred i.p. into IL-22−/− RAG1−/− mice and their body mass was also monitored (bottom graph). Points represent the mean percent change in body mass.

(A) NK-attracting chemokines are upregulated in the colon during IBD. Two weeks post-transfer of IL-22 +/+ CD4+ CD45RB^high CD25− NK1.1− T cells (5×10⁵) into RAG1−/− mice, or control RAG1−/− mice that did not receive cells, mRNA levels of the chemokines Cxcl9, Cxcl10, and Cxcl11, were examined in the ascending colon by real-time RT-PCR. mean±SD.
(B) IL-22−/− CD4+ CD45RB^high CD25− NK1.1− T cells (5×10⁵) cells were transferred into RAG1−/− mice and 45 days post-transfer, when mice exhibited clinical disease, spleens and colons were harvested and FACS stained for CD4 and NK1.1. Numbers indicate the mean±SD percentage of CD4+ NK1.1− (top gate) or CD4− NK1.1+ (right gate) cells out of total lymphocytes isolated from the colon. 5 mice/group.
(C) Total numbers of CD4 (CD4+ TCRβ+) and NK (NK1.1+ TCRβ−) cells in the colons of mice presented in B as determined by FACS. mean±SD. B6=C57BL/6 mouse, ND=not detected
(D) IL-22+/+ or IL-22−/− CD4+ CD45RB^high CD25− NK1.1− T cells were transferred into RAG2−/−γc−/− mice (5×10⁵ cells; i.p.). 42 days post-transfer cytokine induction in the ascending colon was semi-quantitated by real-time RT-PCR, as well as in untransferred RAG2−/−γc−/− mice or C57BL/6 (B6) mice. Each dot represents one mouse; bar indicates mean; ND = not detected.

RAG1−/− mice were given 3% DSS ad libitum in their drinking water for four days. On the third day, mice were either i.p. injected with (A) 250 μg of anti-NK1.1 depleting Ab or control IgG2a or (B) 500 μg anti-asialo GM1 Ab or untreated. Twenty-four hours later, mice were euthanized, and colons were excised. Colons were cultured for three days as described in the Experimental Procedures and IL-22 or IL-6 were detected in the supernatant by ELISA. Each dot represents one colon segment, bars represent mean of 4–5 mice/group; 4 segments per mouse. * indicates p<0.05. Experiment was performed twice with similar results.