Supplementary Table 1: Antibodies used in this study Antigen Clone Conjugate B220 RA3-6B2 APC, Cy5PE, PerCPCy5.5 CD19 1D3 FITC, Biotin, APC, PE, PerCPCy5.5 CD81 Eat-2 PE, Biotin TCRγδ eBioGL3 APC NK1.1 PK316 PE CD3 145-2C11 FITC, PE, APC CD4 H129.19/RM4-5 FITC, PE, PerCPCy5.5 CD8 53-6.7 FITC, APC Gr-1 RB6-8C5 PE, PerCPCy5.5, CD11b Mac-1, M1/70 FITC,PE, APC,PerCPCy5.5 CD11c HL3 APC, PE CD80 16-10A1 FITC,APC CD86 GL1 CD69 H1.2F3 FoxP3 FJK16s CD49b DX5 APC, FITC, PE F4/80 BM8 CD25 PC61.5 EpCAM G8.8 GITR DTA-1 FITC CD278 7E.17G9 CD134 OX-86 CD152 UC10-4F10-11 CD279 RMP1-30 CD137 1AH2

4T1 E0771 LLC Isotype control CD81 Supplementary Figure 1: CD81 expression on tumor cell lines Lewis lung carcinoma (LLC) and 4T1 breast cancer cells express CD81; Breast cancer cells (E0771) do not express CD81.

WT KO Supplementary Figure 2: Analysis of purified CD4+CD25+ Tregs for FoxP3 expression WT or CD81KO Tregs from tumor bearing animals were isolated using MACS militenyi kit. Briefly, CD4+ cells were negativly enriched followed by CD25+ positive selection. Equal numbers of CD3+CD4+CD25+FoxP3+ Tregs were used for suppression assays and for adoptive transfer.

Supplementary Figure 3: Tregs from LLC and E0771 tumor bearing CD81KO C57BL6 mice are less suppressive Purified splenic WT or CD81 KO Tregs from (A) LLC-luc or (B) E0771 tumor-bearing mice were co-cultured at the indicated Treg:T effector cell ratios. CFSE labeled naive CD4+ T cells were stimulated to devide by anti-CD3/CD28 coated beads. The proliferation of CD4+ T cells after 5 days of culture (% CFSE) was calculated with Flowjo software. A B Treg:Teffector Treg:Teffector

WT Teff KO Teff A B Media CD3/CD28 WT Tregs HT Tregs KO Tregs 2:1 1:1 1:2 Treg:CD4Teff Supplementary Figure 4: Naive BALB/c WT or CD81 KO T CD4+ cells are equally suppressed by WT, HT or CD81 KO Tregs Tregs were purified from naive WT, HT or CD81KO spleens and co-cultured at the indicated Treg:T effector cell ratios. VTD labeled naive WT (A) or CD81KO (B) T cells were stimulated to devide by anti-CD3/CD28 coated beads. CD4+ T cell proliferation was evaluated after 5 days of co-culture and shown as histograms.

A B WT MDSC KO Huh-7 WB: anti Arg1 WB: anti actin 35 KDa 44 KDa Supplementary Figure 5: Analysis of the indicated proteins (A) and mRNA (B) expression in purified WT or CD81KO MDSCs MDSCs (CD11b+Gr1+) from blood of day 20 4T1-bearing WT or CD81KO mice were purified. (A) Purified MDSCs were lysed, electrophoresed, blotted and probed for Arg1 protein expression by western blot. Lysate of a hepatocytes cell line, Huh-7, was used as a positive control and actin is shown as a loading control. (B) Expression of the indicated gene expressed by the purified MDSCs. The extracted mRNA was examined by quantitative real time RT-PCR analysis. CT values from each indivual gene is shown as relative expression.

Gated on MDSCs (Gr1+CD11b+) Gated on lymphocytes B WT basal WT TBHP HT basal HT TBHP KO basal KO TBHP Supplementary Figure 6: Analysis of ROS levels measured in MDSCs and Lymphocytes in WT, HT or CD81KO C57BL/6 mice. Splenocytes harvested from naive WT, HT or CD81KO mice were resuspended in RPMI 1640 media. Cells were left untreated (basal) or incubated with 200 µM of tert-butyl hydroperoxide (TBHP) for 1 hr at 37oC to induce ROS production. 500 nM of CellROX® was added to all samples to detect ROS production. ROS production was analyzed by flow cytometry gated on (A) MDSCs (CD11b+Gr1+) and on (B) lymphocytes.

WT KO IC Supplementary Figure 7: WT and CD81KO Tregs express similar levels of the indicated markers Purified WT or CD81KO Tregs from 4T1-tumor bearing mice (day 20) were stained with GITR, ICOS, CD134, CD279, CD137 or CD152 antibodies and analyzed by flow cytometry.