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Landes Bioscience www.landesbioscience.com Supplemental Material to: Martinet et al. High endothelial venules (HEVs) in human melanoma lesions: Major gateways for tumor-infiltrating lymphocytes OncoImmunology 2012; 1(6) http://dx.doi.org/10.4161/onci.20492 http://www.landesbioscience.com/journals/oncoimmunology/ article/20492/

Supplementary Table S1. Spearman correlation matrix between HEVs and immune parameters HEV number HEV/mm2 CD3 level HEV/mm 2 0.8840* CD8 level CD3 level 0.7050* 0.7089* CD8 level 0.6631* 0.6672* 0.9540* CD20 level 0.6128* 0.5625* 0.7075* 0.7199*

Supplementary Table S2. Characteristics of melanomas (n=225) according to CD3 + tumorinfiltrating lymphocyte density CD3 Grade 1 Grade 2 Grade 3 HEV /mm2 P < 0.001 Median 0.0 2.4 5.4 (Range) (0.0: 17.5) (0.0: 18.7) (0.0: 27.3) Type SSM 42 (44.2) 23 (43.4) 33 (50.8) p < 0.001 Autre 17 (17.9) 12 (22.6) 14 (21.5) Acral-lentiginous 24 (25.3) 2 ( 3.8) 1 ( 1.5) Dubreuilh 12 (12.6) 16 (30.2) 17 (26.2) Missing 4 2 1 Breslow thickness p < 0.02 <=1mm 43 (47.8) 36 (70.6) 37 (57.8) 1.01-2 mm 16 (17.8) 3 ( 5.9) 15 (23.4) >2.0mm 31 (34.4) 12 (23.5) 12 (18.8) Missing 9 4 2 Ulceration p = 0.58 No 84 (88.4) 47 (92.2) 55 (85.9) Yes 11 (11.6) 4 ( 7.8) 9 (14.1) Missing 4 4 2 Regression p < 0.001 No 86 (89.6) 38 (74.5) 42 (65.6) Yes 10 (10.4) 13 (25.5) 22 (34.4) Missing 3 4 2 Clark Level p < 0.03 I-II 30 (31.6) 22 (43.1) 24 (36.4) III 20 (21.1) 17 (33.3) 23 (34.8) IV-V 45 (47.4) 12 (23.5) 19 (28.8) Missing 4 4 0

Supplementary Table S3. Characteristics of melanomas (n=225) according to CD8 + tumorinfiltrating lymphocyte density CD8 Grade 1 Grade 2 Grade 3 HEV /mm2 p < 0.001 Median 0.0 2.8 5.4 (Range) (0.0: 17.5) (0.0: 13.4) (0.0: 27.3) Type p < 0.001 SSM 40 (43.0) 24 (54.5) 27 (49.1) Others 18 (19.4) 6 (13.6) 14 (25.5) Acral-lentiginous 21 (22.6) 1 ( 2.3) 0 ( 0.0) Dubreuilh 14 (15.1) 13 (29.5) 14 (25.5) Missing 4 2 1 Breslow thickness p = 0.17 <=1mm 49 (55.1) 29 (69.0) 31 (57.4) 1.01-2 mm 12 (13.5) 7 (16.7) 12 (22.2) >2. mm 28 (31.5) 6 (14.3) 11 (20.4) Missing 8 4 2 Ulceration p = 0.20 No 84 (90.3) 40 (95.2) 45 (83.3) Yes 9 ( 9.7) 2 ( 4.8) 9 (16.7) Missing 4 4 2 Regression p < 0.005 No 82 (87.2) 29 (69.0) 35 (64.8) Yes 12 (12.8) 13 (31.0) 19 (35.2) Missing 3 4 2 Clark Level p < 0.03 I-II 35 (37.2) 18 (41.9) 21 (37.5) III 19 (20.2) 18 (41.9) 16 (28.6) IV-V 40 (42.6) 7 (16.3) 19 (33.9) Missing 3 3 0

Supplementary Table S4. Characteristics of melanomas (n=225) according to CD20 + tumorinfiltrating lymphocyte density CD20 Grade 1 Grade 2 Grade 3 HEV /mm2 p < 0.001 Median 0.1 5.2 7.9 (Range) (0.0: 17.5) (0.0: 18.9) (0.2: 27.3) Type p = 0.09 SSM 70 (47.3) 18 (42.9) 11 (47.8) Others 26 (17.6) 11 (26.2) 5 (21.7) Acral-lentiginous 25 (16.9) 1 ( 2.4) 1 ( 4.3) Dubreuilh 27 (18.2) 12 (28.6) 6 (26.1) Missing 6 1 0 Breslow Thickness p = 0.86 <=1mm 84 (58.7) 20 (51.3) 12 (52.2) 1.01-2 mm 22 (15.4) 8 (20.5) 4 (17.4) 2.01-4 mm 37 (25.9) 11 (28.2) 7 (30.4) Missing 11 4 0 Ulceration p = 0.58 Non 132 (89.2) 35 (89.7) 19 (82.6) Oui 16 (10.8) 4 (10.3) 4 (17.4) Missing 6 4 0 Regression p < 0.05 Non 123 (82.6) 29 (74.4) 14 (60.9) Oui 26 (17.4) 10 (25.6) 9 (39.1) Missing 5 4 0 Clark level p = 0.72 I-II 54 (36.7) 16 (38.1) 6 (26.1) III 42 (28.6) 9 (21.4) 8 (34.8) IV-V 51 (34.7) 17 (40.5) 9 (39.1) Missing 7 1 0

Supplementary Table S5 : List of antibodies used in immunofluorescence and immunohistochemistry Antigen Antibody Dilution Species Retrieval method Tissue fixative MECA-79 MECA-79 (BD) 1/2 Rat IgM CD3 SP7 (Abcam) Rabbit IgG CD3 F7.2.38 (Dako) R,F CD8 C8/144B (Dako) 95 C TRS PH high (Dako) F, D CD20 L26 (Beckman coulter) CD31 CD45RO Ab28364 (Abcam) UCHL1 (Dako) Rabbit IgG R,F vwb vwb (Dako) Rabbit IgG R, F DARC Fy6 (BD) R, F ICAM1 1H4 (Abcam) R, F G72 G152 G72 G152 1/10 1/10 Mouse IgM Mouse IgM R R HECA-452 CD45RA HECA-452 (BD) (Beckman coulter) Rat IgM Foxp3 236A/E7 (Abcam) 95 C TRS PH high (Dako) R, F DC-LAMP Fascin Melan-A 1010 E 1.01(Dendritics) 55K2 (Dako) A103 (Dako) Rat IgG 95 C TRS PH high (Dako) 95 C TRS PH high (Dako) F, D R: RCL-2, F: Formalin, D: Dubosc. The HEV-specific antibodies G72 and G152 were kindly provided by Dr Reiji Kannagi (Aichi Cancer Center, Nagoya, Japan).

Supplementary Figure S1 Figure S1. Absence of HEV blood vessels in normal skin. Representative pictures of normal skin tissue sections stained with MECA-79 antibody demonstrating the absence of HEV blood vessels in non-pathologic tissues distant from primary melanoma site.

Supplementary Figure S2 A HEV number Grade of infiltration : CD3 CD8 CD20 *** *** *** *** *** 1 2 3 1 2 3 1 2 3 B C % of samples er of a samples Numb melanoma CD3 CD8 CD20 0 HEV - HEV + HEV - HEV + HEV - HEV + CD3 CD8 CD20 1 0.5 Grade of infiltration : Grade 1 Grade 2 Grade 3 HEV - HEV + Grade of infiltration : 1 2 3 1 2 3 1 2 3 Figure S2. Tumor HEVs predict lymphocyte infiltration in melanoma. (A) Graphs showing the absolute numbers of HEVs in melanoma according to the density of CD3 +,CD8 + and CD20 + TILs. (B) Graphs showing the percentage of melanoma with grade 1 (blue bars), grade 2 (green bars) and grade 3 (red bars) levels of CD3+, CD8+ or CD20+ TILs according to the presence or absence of tumor HEVs. (C) Graphs showing the number of melanoma tumors with grade 1, grade 2 and grade 3 density of CD3+, CD8+ or CD20+ TILs according to the presence (red bars) or absence of tumor HEVs (blue bars). *** p <0.001; Mann Whitney test.

Supplementary Figure S3 A /mm2 Spearman r = 0.86 p<0.001 CD3 + cells B 1 2 3 CD3 Grade DC-LAMP cluste er Spearman r = 0.92 p<0.001001 1 2 3 DC-LAMP grade Figure S3 Graphs sho ing the correlation bet een optical grading and a tomatic cell Figure S3. Graphs showing the correlation between optical grading and automatic cell count for CD3 + (A) and DC-LAMP + tumor infiltrating cells (B).

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