iIEL NK receptor expression upon DSS-induced colitis in Ly49E WT versus Ly49E KO mice.

<p>A) Colon iIEL subpopulation frequencies (mean ±SD; n = 5 on day 0, n = 3 on day 7 and n = 4 on day 11, where each value is derived from a pool of 3 mice). TCRαβ CD4, TCRαβ CD8αβ and TCRαβ CD8αα iIEL subpopulation frequencies are shown as a percentage of the total TCRαβ iIELs. TCRγδ DN and TCRγδ CD8αα iIEL subpopulation frequencies are shown as a percentage of the total TCRγδ iIEL. B) Colon iIEL NK receptor and activation marker expression on TCRαβ CD8αα iIELs and TCRγδ CD8αα iIELs (mean ±SD; n = 5 on day 0, n = 3 on day 7 and n = 4 on day 11, where each value represents the mean of a pool of 3 mice). C) Colon iIEL NK receptor expression on TCRαβ CD8αα iIELs and TCRγδ CD8αα iIELs on day 7 following induction of DSS-induced colitis. Numbers indicate the percentage of cells expressing the indicated Ly49 receptors, and are each representative of 5 mice. Representative data are shown on the left. Mean fluorence intensity (MFI) values for each receptor are shown on the right.</p

iIEL NKG2D expression upon DSS-induced colitis in Ly49E WT and Ly49E KO mice.

<p>Colitis was induced in Ly49E WT and Ly49E KO littermate mice by administration of DSS in drinking water for 7 days. A) NKG2D expression on TCRαβ CD8αα iIELs. B) NKG2D expression on TCRγδ CD8αα iIELs. N = 2 for control mice, n = 3 for DSS-treated mice.</p

Clinical symptoms of DSS-induced colitis in Ly49E WT versus Ly49E KO mice.

<p>Colitis was induced in Ly49E WT and Ly49E KO mice by administration of DSS in drinking water for 7 days. Thereafter, mice received normal drinking water. Ly49E WT and Ly49E KO mice were scored and compared at the indicated days. A) Relative weight loss (mean ±SD; n = 71). The reference weight was taken as the weight on day 0, at the start of the experiment. B) Disease activity index (mean ±SD; n = 71). C) Colon histological score (mean ±SD; n = 6 on day 7, n = 5 on day 9 and n = 10 on day 11). D) Representative hematoxylin/eosin-stained paraffin sections of the distal colon.</p

iIEL NK receptor expression in the small intestine and colon of Ly49E WT versus Ly49E KO mice.

<p>A) NK receptor expression on TCRαβ CD8αα iIELs and TCRγδ CD8αα iIELs in the small intestine of Ly49E WT versus Ly49E KO mice. B) NK receptor expression on TCRαβ CD8αα iIELs and TCRγδ CD8αα iIELs in the colon of Ly49E WT versus Ly49E KO mice. C) Co-expression of Ly49 receptors on the iIEL surface of TCRαβ CD8αα iIELs and TCRγδ CD8αα iIELs in the small intestine of Ly49E WT versus Ly49E KO mice. Numbers indicate the percentage of cells expressing or co-expressing the indicated Ly49 receptors, and are representative of 3 mice. D) Co-expression of Ly49 receptors on the iIEL surface of TCRαβ CD8αα iIELs and TCRγδ CD8αα iIELs in the colon of Ly49E WT versus Ly49E KO mice. Numbers indicate the percentage of cells expressing or co-expressing the specific Ly49 receptors, and are representative of 3 mice. iIEL NK receptor expression is presented as the mean ±SD (n = 5, small intestine; n = 5, colon, where each value represents the mean of a pool of 3 mice).</p

TNF^ΔARE-induced ileitis on an Ly49E WT versus Ly49E KO background.

<p>A) Weight progression of TNF^ΔARE/WT Ly49E^WT/WT and TNF^ΔARE/WT Ly49E^KO/KO mice as compared to TNF^WT/WT Ly49E WT and Ly49E KO mice (mean ±SD; n = 30 (TNF^ΔARE/WT Ly49E^WT/WT), n = 22 (TNF^ΔARE/WT Ly49E^KO/KO), n = 31 (TNF^WT/WT Ly49E WT) and n = 21 (TNF^WT/WT Ly49E KO)). Weight was monitored weekly from the age of genotyping (5 weeks of age) up to 14 weeks of age. B) Small intestinal iIEL subpopulation frequencies in TNF^ΔARE/WT Ly49E^WT/WT and TNF^ΔARE/WT Ly49E^KO/KO mice as compared to TNF^WT/WT Ly49E WT and Ly49E KO mice (mean ±SD; n = 6). TCRαβ CD4, TCRαβ CD8αβ and TCRαβ CD8αα iIEL subpopulation frequencies are shown as a percentage of the total TCRαβ iIELs. TCRγδ DN and TCRγδ CD8αα iIEL subpopulation frequencies are shown as a percentage of the total TCRγδ iIEL. C) CD69 expression on small intestinal TCRαβ CD8αα iIELs and TCRγδ CD8αα iIELs of TNF^ΔARE/WT Ly49E^WT/WT and TNF^ΔARE/WT Ly49E^KO/KO mice as compared to TNF^WT/WT Ly49E WT and Ly49E KO mice (mean ±SD; n = 6).</p

Analysis of spleen and brain lymphocytes of cerebral malaria developing <i>P. berghei</i> ANKA-infected WT vs Ly49E KO mice.

<p>Freshly isolated spleen (A–C) or brain (D–F) NK (CD3⁻NK1.1⁺), NKT (CD3^dimNK1.1⁺), CD4⁺ T (CD3⁺CD4⁺), CD8⁺ T (CD3⁺CD8⁺) and γδ T cells (CD3⁺TCRγδ⁺) from non-infected WT and Ly49E KO mice or cerebral malaria developing <i>P. berghei</i> ANKA-infected WT and Ly49E KO mice (as indicated in the figure) were analyzed by flow cytometry. The percentage of these lymphocyte populations are shown in A and D. CXCR3 and CD69 expression was analyzed within each lymphocyte population (B and E). NK receptor expression was determined in NK and NKT cells (C and F). Data represent the results of 2 independent experiments (WT, n = 8; KO, n = 11) and are shown as mean ± SEM. Statistical analysis was performed using the 2-tailed Mann Whitney test. * represent a significant difference with p<0.05.</p

Role of Ly49E expression in lymphocyte phenotype and cytokine production in early <i>P. berghei</i> ANKA infection.

<p>Liver lymphocytes (A–C) and spleen cells (D–E) were isolated from non-infected, or from 5 d and 7 d <i>P. berghei</i> ANKA-infected WT and Ly49E KO mice, as indicated in the figure. Freshly isolated liver lymphocytes (A) and spleen cells (D) were analyzed for their subpopulation composition by gating on NK cells (liver: CD3⁻NK1.1⁺DX5⁻ and CD3⁻NK1.1⁺DX5⁺; spleen: CD3⁻NK1.1⁺), NKT cells (CD3^dimNK1.1⁺) and T cells (CD3⁺NK1.1⁻). The absolute cell number was calculated based on the total viable cell number obtained after isolation and the representative percentages of each cell population. CD69, IFN-γ and TNF-α expression by liver (B) and spleen (E) NK, NKT and T cells was determined. (A,B,D,E) Data are represented as mean ± SEM (n = 5). Statistical analysis was performed using the 2-tailed Mann Whitney test. * indicates a significant difference with p<0.05 and ** a significant difference with p<0.01. (C) Overlay histograms show unstained, and CD69-, IFN-γ- and TNF-α-stained DX5⁻ liver NK cells from non-infected and day 5 <i>P. berghei</i> ANKA-infected WT and Ly49E KO mice, as indicated. The histograms of one representative mouse of each group is shown. The mean ± SEM of 5 different mice is shown for <i>P. berghei</i> ANKA-infected (above the gate bar) and for non-infected mice (below the gate bar).</p

Cerebral malaria development in <i>P. berghei</i> ANKA-infected WT vs Ly49E KO mice.

<p><i>P. berghei</i> ANKA-infected WT and Ly49E KO mice were monitored daily for cerebral malaria development. (A) The incidence of cerebral malaria development in 5 separate experiments. The results are presented as (number of animals with cerebral malaria)/(total number of animals studied). The last line represents the total number of mice affected with cerebral malaria during these 5 experiments. (B) The cumulative incidence and kinetics of cerebral malaria development in the 5 pooled experiments (WT, n = 86; Ly49E KO, n = 80). Cerebral malaria development results were analyzed by Log-rank (Kaplan-Meier). There was no significant difference in WT vs Ly49E KO mice.</p

iIEL subpopulation frequencies in the small intestine and colon of Ly49E WT versus Ly49E KO mice.

<p>A) iIEL subpopulation frequencies in the small intestine of Ly49E WT versus Ly49E KO mice. TCRαβ and TCRγδ iIEL population frequencies are shown as a percentage of the total iIELs present in the small intestine and colon of Ly49E WT versus Ly49E KO mice. TCRαβ CD4, TCRαβ CD8αβ and TCRαβ CD8αα iIEL subpopulation frequencies are shown as a percentage of the total TCRαβ iIELs. TCRγδ DN and TCRγδ CD8αα iIEL subpopulation frequencies are shown as a percentage of the total TCRγδ iIELs. B) iIEL subpopulation frequencies in the colon of Ly49E WT versus Ly49E KO mice. Representation of iIEL subpopulation frequencies is the same as for Fig. 1A. iIEL subpopulation frequencies are shown as mean values (n = 5, small intestine; n = 5, colon, where each value is derived from a pool of 3 mice).</p

TNBS-induced colitis in Ly49E WT versus Ly49E KO mice.

<p>Colitis was induced in Ly49E WT and Ly49E KO mice by intra-rectal administration of TNBS. Mice were analysed at the indicated days. A) Survival (n = 16). B) Relative weight loss (mean ±SD; n = 16). The reference weight was taken as the weight on day 0, at the start of the experiment. C) Colon histological score (mean ±SD; n = 5). D) Representative hematoxylin/eosin- stained paraffin sections of the distal colon.</p