SUPPORTING INFORMATIONS
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1 SUPPORTING INFORMATIONS Mice MT/ret RetCD3ε KO α-cd25 treated MT/ret Age 1 month 3 mnths 6 months 1 month 3 months 6 months 1 month 3 months 6 months 2/87 Survival 87/87 incidence of 17/87 1 ary tumor (19.5) incidence of cut met 1 (2.3) incidence of distant met 2 N.D incidence of 6/87 vitiligo (6.9) 87/87 78/87 (89.7) 52/87 (59.8) 0/30 (0.0) 31/87 (35.6) 42/46 (91.3) 42/42 34/42 (80.9) 4/41 (9.7) 16/42 (38.1) 42/42 8/42 (19.0) 3/42 (7.1) N.D 9/42 (21.4) 39/42 (92.8) 35/39 (89.7) 22/39 (56.4) 3/21 (14.3) 30/39 (76.9) 18/21 (85.7) 18/18 16/18 4/18 (22.2) 16/18 40/40 15/40 (37.5) 2/40 (5.0) 40/40 33/40 (82.5) 9/40 (22.5) 0/12 (0.0) 31/40 (77.5) Table S1: Diagnosis of MT/ret, RetCD3ε KO and anti-cd25 treated MT/ret mice. Survival and prevalence of primary tumors, cutaneous metastases 1, distant metastases 2 and vitiligo in the indicated mice as a function of mouse age. Results are expressed as the number of mice with the indicated symptoms over the total number of analyzed mice. Numbers in brackets represent the percentage of analyzed mice with the indicated symptoms. N.D: Not determined. N.D 24/40 (60.0) 28/28 25/28 (89.3) 11/28 (39.3) 0/28 (0.0) 24/28 (85.7)
2 Mice MT/ret RetCD3ε KO Treatment None αcd8 αnk1.1 αgr1 αly6g CCL2 αil-10r NAC None αgr1 52/87 Survival 87/87 incidence of 78/87 1 ary tumor (89.7) incidence of cut met 1 (59.8) incidence of 0/30 distant (0.0) met 2 incidence of vitiligo 31/87 (35.6) 19/23 (82.6) 19/19 13/19 (68.4) 10/19 (52.6) 7/19 (36.8) 13/14 (92.9) 12/13 (93.3) 8/13 (61.5) 2/13 (15.4) 5/13 (38.5) 6/23 (26.1) 6/6 6/6 6/6 1/6 (16.7) 9/9 8/9 2/9 (22.2) 0/9 (0.0) 8/9 18/18 15/18 (83.3) 5/18 (27.7) 2/18 (11.1) 16/18 20/21 (95.2) 16/20 (80.0) 5/20 (25.0) 0/20 (0.0) 16/20 (80.0) 12/15 (80.0) 12/12 8/12 (66.7) 9/12 (75.0) 3/12 (25.0) 39/42 (92.8) 35/39 (89.7) 22/39 (56.4) 3/21 (14.3) 30/39 (76.9) 9/26 (34.6) 8/9 7/9 (77.7) 7/9 (77.7) 3/9 (33.3) CD4 T cells (C57BL/6) 9/11 (81.8) 9/9 8/9 3/9 (33.3) 2/9 (22.2) CD4 T cells (MT/ret) 16/17 (94.1) 16/16 13/16 (81.2) 4/16 (25.0) 2/16 (12.5) Table S2: Diagnosis of untreated and treated 3-month old MT/ret and RetCD3ε KO mice. Survival and prevalence of primary tumors, cutaneous metastases 1, distant metastases 2 and vitiligo in the indicated mice. Results are expressed as the number of mice with the indicated symptoms over the total number of analyzed mice. Numbers in brackets represent the percentage of analyzed mice with the indicated symptoms.
3 Figure S1: Diagnosis of MT/ret mice. Diagnosis of MT/ret and RetCD3εKO mice was performed from weaning and the development of primary tumors, facial or dorsal cutaneous metastases and vitiligo was recorded weekly. Representative pictures (upper part) and H&E staining (Lower part) of a primary tumor (A), cutaneous (B) and distant (C) metastases. The occurrence of vitiligo was monitored and recorded by the appearance of depigmented areas on (D) the tail, ears, snout and legs, and (E) confirmed at the time of the sacrifice by observation of the dorsal and ventral skin. Mice were classified as without vitiligo or with weak, moderate or extensive vitiligo accordingly.
4 Figure S2: Identification of lymphoid and myeloid cell populations in spleen by flow cytometry. Representative dot plots showing (A) common gating strategy, identification of (B) lymphoid populations and (C) myeloid populations. Dot plots are shown for splenocytes of a 12-week old Ctrl mouse. PE-conjugated anti-nk1.1 (PK136), anti TcRγδ (GL3), PerCPconjugated anti-cd4 (RM4-5), PerCP-Cy5.5 anti-nk1.1 (PK136), anti-cd45 (30F11), APCconjugated anti-cd45 (30F11), FITC-conjugated anti-cd8 (53.6.7), anti-cd19 (1D3), anti- Ly-6G (1A8), PE-Cy7-conjugated anti CD11c (HL3), streptavidin, APC-H7-conjugated anti- CD8 (53-6.7), Pacific Blue conjugated anti-cd4 (RM4-5), biotinylated anti-ly-6c (AL-21), anti-tcrβ (H57-597) antibodies were obtained from BD Biosciences. APC-conjugated anti- CD11b (M1/70) antibody was obtained from ebioscience. Pacific Blue-conjugated streptavidin was obtained from Invitrogen.
5 Figure S3: Identification of lymphoid and myeloid cell populations in cutaneous metastases by flow cytometry. Representative dot plots showing (A) common gating strategy, identification of (B) lymphoid populations and (C) myeloid populations. Dot plots are shown for a cutaneous metastasis cell suspension of a 24-week old MT/ret mouse. Antibodies were the same as in Fig. S2.
6 Figure S4: Proportion of hematopoietic cells in secondary lymphoid organs and tumors. A CD45 staining was systematically performed on cell suspensions of spleen, cervical lymph nodes and cutaneous metastases to assess the tumor invasion of secondary lymphoid organs and the immune infiltration of tumors. PerCP-Cy5.5 and APC-conjugated anti-cd45 (30F11) antibodies were obtained from BD Biosciences. The graph shows the proportion of CD45- expressing cells in the spleen, cervical lymph nodes and cutaneous metastases from 6-month old Ctrl or MT/ret mice with (V) or without (NV) vitiligo.
7 Figure S5: Accumulation of regulatory CD4 + T cells in the spleen and cervical lymph nodes of non-vitiligo MT/ret mice. Absolute numbers of Tregs (CD4 + CD8 - TcRβ + Foxp3 + cells) in cutaneous metastases of 6-month old MT/ret mice developing or not vitiligo and in the spleen and cervical lymph nodes of Ctrl mice and MT/ret mice. For each studied mouse, numbers were calculated as follows: Absolute numbers of cells per indicated tissue (Spleen, cervical lymph nodes or cutaneous metastases) % CD45 + cells in this tissue % CD4 + CD8 - TcRβ + among CD45 + cells % Foxp3 + among CD4 + cells.
8 Figure S6: Percentage of Tregs in MT/ret mice according to their symptoms. Proportion of Foxp3 + cells within CD45 + CD4 + CD8 - TcRβ + cells from the spleen, cervical (Cerv), axillary (Ax) and mesenteric (Mes) lymph nodes of 6-month old Ctrl and MT/ret mice without vitiligo.
9 Figure S7: Effect of CD8 + T-cell or NK-cell depletion on the development of vitiligo and tumor spread at cutaneous level in MT/ret mice. Incidence of vitiligo and cutaneous metastases (Cut met) in MT/ret mice treated with (A) anti-cd8 Ab (n=19) or (B) anti-nk1.1 Ab (n=13).
10 Figure S8: The proportion of circulating granulocytes and Ly-6C high monocytes transiently increases after CCL2 injection. Proportion of Ly-6C high monocytes (Left panel) and granulocytes (Right panel) in the blood of Ctrl mice 0, 2, 4, 6, 24, 48 and 72 hours after one intra-peritoneal injection of 400ng of CCL2.
11 Figure S9: Ly-6C high monocytes/dcs inhibit melanoma-cell proliferation in vitro in part through a ROS dependent mechanism. Melan-ret melanoma cells ( cells) were seeded into wells of 96X E-Plates XCELLigence System (Roche Diagnostics). When cells were in their exponential phase of growth (24 h later), Ly6-C high monocytes/dcs or Ly-6G + granulocytes ( cells) purified from the spleen of C57BL/6 mice were added and tumorcell proliferation was dynamically monitored every 30 min over a 5 hour period based on cell impedance. (A) Graph showing the normalized cell index (nci) values obtained by the RT- CES system for one out of 6 representative experiments. (B) Percentage of inhibition of Melan-ret-cell proliferation 5 hours after the addition of myeloid cells = ((nci (Melan-ret cells) - nci (Melan-ret cells+ly6-c high or Ly6-G + cells)) / ((nci (Melan-ret cells) -1)) x100. (C) Different numbers of Ly6-C high monocytes/dcs ( , , , ) were added corresponding respectively to Ly6-C high /Melan-ret-cell ratios of 4/1, 2/1, 1/1 and 0.5/1. (D) Inhibition of the effect of Ly6-C high monocytes/dcs in presence of NAC (1mM) or anti-tnfα Ab (20µg/ml). Both NAC or anti-tnfα Ab were added at the same time as Ly6-C high monocytes/dcs.
12 Figure S10: Characterization of immune cells infiltrating the skin of 6-week old MT/ret mice with or without vitiligo. (A) Characterization of immune cells infiltrating the skin of 6- week old Ctrl or MT/ret mice with or without vitiligo. The different immune populations were defined as shown in Fig. S2 and Fig. S3. Proportions are shown within CD45 + cells. (B) Quantification of different immune-cell populations infiltrating the skin of 6-week old Ctrl or MT/ret mice with or without vitiligo.
13 Figure S11: IL-10 production by CD4 + T cells. Cell suspensions from the spleen, peripheral lymph nodes (plns), mesenteric lymph nodes (mlns) and the skin of Foxp3-GFP mice were incubated for 4 hours at 37 C in PBS 1X, 10% SVF containing 0.5µg/ml PMA, 0.5µg/ml ionomycin, and 10µg/ml BrefeldinA (all from Sigma). Cells were then stained for surface markers, fixed in 2% paraformaldehyde in PBS, and permeabilized with 0.5% saponin, followed by labeling with PE anti-il-10 Ab (clone JES5-16E3 from BD Bioscience). Percentages are expressed within CD4 + T cells expressing or not Foxp3.
SUPPLEMENTARY INFORMATION
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Supporting Information
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