ONLINE SUPPLEMENT MATERIAL. CD70 limits atherosclerosis and promotes macrophage function.

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ONLINE SUPPLEMENT MATERIAL CD7 limits atherosclerosis and promotes macrophage function. Holger Winkels* 1,2, Svenja Meiler* 1,2, Esther Smeets* 2, Dirk Lievens 1, David Engel 3, Charlotte Spitz 1, Christina Bürger 1, Petteri Rinne 1, Linda Beckers 2, Angelika Dandl 1, Sigrid Reim 1, Maiwand Ahmadsei 1, Jan Van den Bossche 2, Lesca M. Holdt 7, Remco T.A. Megens 1,3, Martin M. Schmitt 1, Menno de Winther 1,2, Eric A. Biessen 3,, Jannie Borst 4, Alexander Faussner 1, Christian Weber 1,3,6, Esther Lutgens #1,2, Norbert Gerdes #1,2 *, # These authors contributed equally. 1. Institute for Cardiovascular Prevention (IPEK), LMU Munich, Munich, Germany 2. Department of Medical Biochemistry, Academic Medical Center (AMC), University of Amsterdam, Amsterdam, The Netherlands 3. Cardiovascular Research Institute Maastricht (CARIM), University of Maastricht, The Netherlands 4. Division of Immunology, The Netherlands Cancer Institute, Amsterdam, The Netherlands.. Institute for Molecular Cardiovascular Research (IMCAR), RWTH Aachen, Germany 6. DZHK (German Centre for Cardiovascular Research), partner site Munich Heart Alliance, Munich, Germany 7. Department of Laboratory Medicine, LMU Munich, Munich, Germany Short title: Winkels, CD7 in atherosclerosis 1

A B αβt cells γδt cells myeloid cells Macrophages CD7 expression [MFI] 3 2 1 1 C 3 CD7 D 2 3 αβt cells γδt cells myeloid cells Macrophages CD7 expression [MFI] 2 1 CD7 expression [MFI] 2 1 nonmyeloid cells CD11b Ly6G CD11b Ly6G hi Ly6C CD11b Ly6G lo Ly6C CD11b Ly6G nonmyeloid cells CD11b Ly6G CD11b Ly6G hi Ly6C CD11b Ly6G lo Ly6C CD11b Ly6G Supplement Figure I CD7 is expressed on myeloid cells and macrophages. (A) Representative histograms displaying CD7 expression determined by flow cytometry on splenic leukocyte subsets of a 28week old hyperlipidemic mouse. (BC) Quantification of CD7 expression of leukocyte subsets in the (B) spleen, (C) blood, and (D) bone marrow of hyperlipidemic mice (age=28 weeks; n=3). Myeloid cells are all CD11b cells of living leukocytes. Macrophages are CD11b F4/8 cells of living leukocytes. MFI, mean fluorescence intensity.

[% of F4/8 ] 6 4 3 2 1 4 3 2 1 CD64 Cd7 / Cd7 / Supplement Figure II CD7deficiency does not alter M, M1, and M2 abundance in untreated BMDM cultures. Bone marrowderived macrophages (BMDMs) from Cd7 / or Cd7 / mice were analyzed by flow cytometry for CD64 and CD31 as M1 and M2 marker, respectively. Data is presented as mean±sem (n=3). CD64 CD31 CD31

IL12p7 [pg/ml] 1 8 6 4 2 IL6 [pg/ml] 2 2 1 1 TNFα [pg/ml] 2 1 1 Cd7 / Cd7 / n.d. n.d. M M1 M2 M M1 M2 M M1 M2 Supplement Figure III CD7deficiency does change IL12p7, IL6, and TNFa secretion by untreated or polarized macrophages. The supernatant of bone marrowderived macrophages (BMDMs) from Cd7 / or Cd7 / mice left untreated or polarized with LPS or IL4 to generate M1 or M2 macrophages respectively, was assayed by ELISA for concentrations of IL12p7, IL6, and TNFα, n.d. abbreviates not detectable. Data is presented as mean±sem (n=3).

Collagen [SR area % of plaque area] A B C 1 1 Cd7 / Cd7 / Smooth muscle cells [ASMA area % of plaque area 1 1 Cd7 / Cd7 / D E F CD4 [cells/mm²] 2 2 1 1 Cd7 / Cd7 / G Cd7 / Cd7 / CD4 DAPI H CD4 DAPI ICAM area [µm²]/ Plaque endothelial length [µm] 3 2 1 Cd7 / Cd7 / VCAM area [µm²]/ Plaque endothelial length [µm] 4 3 2 1 Cd7 / Cd7 / Collagen [SR area % of plaque area] 4 3 2 1 Smooth muscle cells [ASMA area % of plaque area] 2 1 1 Cd7 / Cd7 / Cd7 / Cd7 / Cd7 / Cd7 / CD4 [cells/mm²] 3 2 1 Supplement Figure IV Lack of CD7 does not change collagen, smooth muscle cell, or CD4 cell content or adhesion molecule expression. (AE) Irradiated mice were reconstituted with Cd7 / or Cd7 / bone marrow (n=114 (: Cd7 / ), n=121 (: Cd7 / )). (A) Percentage of sirius red positive stained area in lesions of the ascending aorta. (BE) Immunofluorescent stainings were analyzed for (B) alpha smooth muscle actin, (C) CD4, representative photomicrographs (right) are displayed (Scale bar = 1 µm), (D) ICAM1, and (E) VCAM1 in crosssections of the ascending aorta. (D,E) ICAM1 and VCAM1 area was quantified on the lesions endothelial area and further correlated to endothelial length. (FH) Quantifications of stainings in crosssections of the ascending aorta of 18week old Cd7 / or Cd7 / mice (n=121). (F) Percentage of sirius red positive stained area in aortic lesions. Immunofluorescent stainings were analyzed for (G) alpha smooth muscle actin and (H) CD4 in crosssections of the ascending aorta.

A CD11b Ly6G [% of CD4 ] 2 2 1 1 Cd7 / Cd7 / Ly6C hi [% of CD11b Ly6G ] 4 3 2 1 Ly6C lo [% of CD11b Ly6G ] 8 6 4 2 D CD11b Ly6G [% of CD4 ] 1 8 6 4 2 Ly6C hi [% of CD11b Ly6G ] 3 2 1 Ly6C lo [% of CD11b Ly6G ] 1 8 6 4 2 G CD11b Ly6G [% of CD4 ] 1 1 Ly6C hi [% of CD11b Ly6G ] 8 6 4 2 Ly6C lo [% of CD11b Ly6G ] 6 4 2 B Annexin V LD [% of Ly6C hi ] 4 3 2 1 22.% 69.3% Cd7 / 1.86% 24.9% 6.27% 66.% Cd7 / 3.2%.7% E Annexin V LD [% of Ly6C hi ] 4 3 2 1 Cd7 / Cd7 / 16.2% 8.66% 17.1% 7.9% 4.19% 73.2% 7.92% 1.74% H Annexin V LD [% of Ly6C hi ] 2 1 1 Cd7 / Cd7 / 6.28%.11% 1.6% 84.7% 3.96% 82.3%.4% 2.11% C Annexin V LD [% of Ly6C lo ] 2 1 1 Cd7 / Cd7 / 9.67%.43% 1.9% 89.6%.26% 86.9%.64% 1.4% F Annexin Annexin V LD [% of Ly6C lo ] 3 2 1 Cd7 / Cd7 / 16.9% 8.69% 14.7% 73.7%.62% 74.7% 9.82%.74% I Annexin V LD [% of Ly6C lo ] 2 2 1 1 Cd7 / Cd7 / 16.4% 6.47% 13.4% 76.7%.46% 82.2% 3.72%.66% LD Supplement Figure V CD7 deficiency does not influence abundance and apoptosis of total monocytes and Ly6C hi and Ly6C lo subsets. Flow cytometric analysis of (AC) blood, (DF) splenic, and (GI) bone marrow suspensions for distribution of total monocytes, Ly6C hi monocytes, and Ly6C lo monocytes from 18 weekold male Cd7 / or Cd7 /. Cell viability assayed by AnnexinV and Live/Dead for (B,C) blood, (E,F) splenic, and (H,I) and bone marrow Ly6C hi and Ly6C lo monocytes, respectively. Representative flow cytometric plots are displayed for each genotype and monocyte subset. Data is presented as mean±sem.

A C 1 8 6 4 2 MFI Ki67 [of Treg] Foxp3 Treg [% of CD4 ] 1 1 Cd7 / Cd7 / B MFI Ki67 [of Treg] Foxp3 Treg [% of CD4 ] 2 2 1 1 2 2 1 1 Cd7 / * ** Cd7 / Normalized to mode D MFI BCL2 [of Treg] 6 4 3 2 1 Cd7 / Cd7 / 8 MFI BCL2 [of Treg] 6 4 2 Cd7 / * Cd7 / Normalized to mode Ki67 E Foxp3 [cells/mm²] 1 8 6 4 2 Cd7 / Cd7 / Cd7 / Cd7 / Cd7 / Cd7 / Foxp3 DAPI BCL2 Cd7 / Cd7 / Foxp3 DAPI F 2 1 1 Cd7 / * Cd7 / Supplement Figure VI CD7 deficiency reduces splenic but not aortic Treg abundance. (AE) Irradiated mice were reconstituted with Cd7 / or Cd7 / bone marrow. Flow cytometric analysis of (A) aortic and (B) splenic suspensions for Foxp3 Treg. (C) Ki67 expression of aortic (left) and splenic (middle) Treg. Representative flow cytometric histograms Ki67 expression on splenic Treg (right). (D) BCL2 expression of aortic (left) and splenic (middle) Treg. Representative flow cytometric histograms depicting BCL2 expression on splenic Treg (right). (E) Immunofluorescent stainings for Foxp3 in crosssections of the ascending aorta. Quantifications (left) and representative photomicrographs (right) are displayed. Arrows indicate positive stained cells; Scale bar = 1 µm. (F) Flow cytometric analysis of splenic suspensions of 18 weekold, male Cd7 / or Cd7 / mice for Foxp3 Treg. (n=71). Data is presented as mean±sem. *p <., **p <.1. Foxp3 Treg [% of CD4 ]

Cholesterol [mm] 1 1 Cd7 / Cd7 / HDL LDL VLDL Supplement Figure VII CD7 deficiency does not affect plasma lipoprotein cholesterol distribution. Cholesterol distribution in different lipoprotein fractions separated by ultracentrifugation. Plasma from mice reconstituted with Cd7 / or Cd7 / bone marrow (n=4/group) was analyzed. HDL, highdensity lipoprotein; VLDL, very lowdensity lipoprotein LDL, lowdensity lipoprotein. Data is presented as mean±sem. Total

Supplement Table I. Distribution of T cell subsets in compound mutant mice. Organ Blood Lymph nodes Spleen Mouse strain Cd7 / Cd7 / Cd7 / Cd7 / Cd7 / Cd7 / Cell subset Parental Gate n Mean SD n Mean SD p n Mean SD n Mean SD p n Mean SD n Mean SD p CD3 of CD4 7 16.49 ±.2 17 16.21 ± 4.47 4.7 ± 6.82 17 48.61 ±.6 7 23.33 ± 4.91 17 19.22 ± 4.2.6 CD4 of CD3 7 4.24 ± 9.76 17 48.34 ±.8 2.16 ± 2.87 17 49.44 ±.23 7 7.84 ± 4.64 17 7.3 ± 4.79 activated CD4 (CD44 CD62L ) not activated CD4 (CD44 CD62L ) of CD4 7 3.9 ± 1. 17 43.7 ± 19.88 2.18 ± 4.37 17 22.3 ±.81 7 3.6 ± 12.7 17 34.43 ± 11. of CD4 7 64.74 ± 1.43 17 6.23 ± 19.89 79.4 ± 4.62 17 77.3 ±.81 7 64.31 ± 12.9 17 6.43 ± 11. Treg (CD4 Foxp3 ) of CD4 1 7.78 ± 1.93 1 6.27 ± 1.2. 1 14.1 ± 1.6 1 11.86 ± 2.1 ** 1 13.97 ± 1.6 1 12.1 ± 1.83 * CD8 of CD3 7 46. ± 8.2 17 43.3 ±.64 42.14 ± 2.86 17 41.9 ± 4.44 7 3.74 ± 4. 17 33.12 ± 6.21 central memory CD8 (CD44 CD62L ) effector memory CD8 (CD44 CD62L ) naive CD8 (CD44 CD62L ) of CD8 7 2.34 ± 7.4 17 22.16 ± 4.87 18.3 ± 6.87 17 2.7 ± 6.32 7 23.39 ±.6 17 23.91 ± 6.92 of CD8 7 8.86 ±.34 17 8.66 ± 4.86.9 ± 3.3 17 2.78 ±.92 *** 7.46 ± 2.28 17 4.64 ± 3.77 of CD8 7 8.64 ± 14.67 17 7.73 ± 12.27 61.28 ± 1.19 17 66.41 ± 7.6 7 66.6 ± 6.26 17 66.1 ± 1.3 Mean ± SD. Statistical significance was calculated for groups pairwise by 2tailed t test. *p <., **p <.1, **p <.1, ***p <.1

Supplement Table II. Distribution of T cells subsets in organs of bone marrow chimeric mice. Organ Lymph node Spleen Bone marrow transplanted into recipient Cd7 / Cd7 / Cd7 / Cd7 / Cell subset Parental Gate n Mean SD n Mean SD p n Mean SD n Mean SD p CD3 of CD4 8 49.6 ± 1.11 8 38.66 ± 8.9 * 8 27.61 ±.22 8 24. ± 3.38 n.s. CD4 of CD3 8 7.71 ± 2.63 8 4.44 ± 2.27 * 8 62.16 ± 3.8 8 9.84 ± 4.1 n.s. activated CD4 (CD44 CD62L ) not activated CD4 (CD44 CD62L ) of CD4 8 27.6 ± 3.16 8 29.94 ± 3.19 n.s. 8 39.61 ± 4.2 8 38.3 ± 3. n.s. of CD4 8 64.91 ± 4.99 8 6.93 ± 4.73 n.s. 8.4 ±.16 8.8 ± 4.22 n.s. Treg (CD4 Foxp3 ) of CD4 7 1. ± 2.37 8 13.6 ± 1.6 n.s. 7 19.71 ± 1. 8 18.4 ± 1.7 * CD8 of CD3 8 33.93 ± 3.1 8 33.99 ± 3.1 n.s. 8 27.4 ± 2. 8 27.48 ± 1.68 n.s. central memory CD8 (CD44 CD62L ) effector memory CD8 (CD44 CD62L ) naive CD8 (CD44 CD62L ) of CD8 8 2.24 ±.76 8 23.8 ± 4.7 n.s. 8 32.43 ±.68 8 28.84 ±.1 n.s. of CD8 8 16.6 ± 4.38 8 1.7 ± 2.44 n.s. 8 12.89 ± 3.88 8 8.48 ± 2.26 * of CD8 8 4.2 ± 7.27 8 4.1 ± 3.63 n.s. 8 49.88 ± 9.9 8 7.13 ± 7.71 n.s. Mean ± SD. Statistical significance was calculated for groups pairwise by 2tailed t test. *p <.

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