Mst1 regulates integrin-dependent thymocyte trafficking and antigen recognition in the thymus

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Mst1 regulates integrin-dependent thymocyte trafficking and antigen recognition in the thymus Yoshihiro Ueda, Koko Katagiri, Takashi Tomiyama, Kaneki Yasuda, Katsuyoshi Habiro, Tomoya Katakai, Susumu Ikehara, Mitsuru Matsumoto, and Tatsuo Kinashi Supplementary Information Supplementary Figures S1 S6

Supplementary Figure S1. Thymocyte subsets used in the experiments. FACS profiles of thymocyte before (left) and after (right) sorting are shown. To avoid the effect of anti-cd4 antibody in MHC class II-dependent Ag recognition within RIP-mOVA thymic tissues, OTII cells are negatively sorted by anti-cd8 antibody. (a) total DP and CD4 SP cells, (b) CD69 + DP cells, (c) CD69 + and CD69 CD4SP cells, (d) OTII CD4 SP cells, (e)cd69 + and CD69 OTII CD4 SP cells. The numbers in the graph indicate the percentage of population in the gates.

Supplementary Figure S2. Mst1 deficiency causes impaired migration of CD69 + and CD69 SP thymocytes within thymic tissues. (a) The average migration speed of Mst1 +/+ and Mst1 / CD69 + DP within the cortex (Mst1 +/+, n=116; Mst1 /, n=94), (b) CD69 + SP cells (Mst1 +/+, n=224; Mst1 /, n=197), and (c) CD69 SP cells (Mst1 +/+, n=328; Mst1 /, n=85) within the medulla are shown. Bars indicate the median.

Supplementary Figure S3. The expression of CCR7 and LFA-1 by SP thymocyte subsets in Mst1 / mice. The expression levels of CCR7 (upper panels), CD18 (middle panels) and CD11a (lower panels) in CD4 + SP cells and CD8 + SP cells from Mst1 +/+ and Mst1 / mice. The blue and red lines represent wild-type and Mst1 / thymocytes, respectively. Dotted lines represent the staining intensity with Fc or the isotype control, whereas the solid lines represent the staining intensity with CCR7-Fc, anti-cd11a and anti-cd18 antibodies.

Supplementary Figure S4. Detection of apoptosis of OT-II cells in RIP-mOVA thymic slices 20 hr after the culture. CD69 + (upper panel) or CD69 (lower panel) OT-II.CD45.1 SP cells were loaded onto CD45.2 + C57BL/6 thymic slices with/without OVA323-339-peptides (OVAp) or onto RIP-mOVA thymic slices. (a) CD45.1 expression, (b) Vα2 and Vβ5 expression in CD45.1 cells (c) Vβ5 expression or (d) Annexin-V and PI binding in Vα2 + Vβ5 + cells by CD69 + or CD69 OT-II.CD45.1 SP cells are shown. The numbers in the graph indicate the percentage of population in the gates.

Supplementary Figure S5. Foxo1 and Foxo3 expression in thymocytes and splenic T cells. (a) Expression of Foxo1, Mst1 and α-tubulin in CD4SP thymocytes or splenic CD4 T cells from 2-3 month old Mst1 +/+ and Mst1 -/- mice. The numbers indicate approximate molecular size of Foxo, Foxo3, Mst1, and α-tubulin. Relative intensities of Foxo1 to α-tubulin (the average ± range, n = 2) are also shown in the bar graphs (low panels). (b) Expression of Foxo3, Mst1 and α-tubulin in whole thymocytes from 2-3 month old Mst1 +/+ and Mst1 -/- mice. Relative intensity of Foxo3 to α-tubulin (the average ± range, n = 2) are also shown in the bar graphs (low panels, n = 2).

Supplementary Figure S6. Leukocyte infiltration in one-year-old Mst1-deficient mice. (a) H&E staining of tissue sections of the kidney and pancreas from one-year-old Mst1 f/f, CAG-Cre;Mst1 f/f, lck-cre;mst1 f/f, or mb-1-cre;mst1 f/f mice (scale bar 400 µm). (b) Cells infiltrating the liver were examined by immunostaining with anti-cd3, anti-b220, anti-cd11b and anti-igκ antibodies (scale bar 200 µm). (c) T cell specific and B cell specific Mst1 deletions were confirmed by immunoblotting.

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