1. 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

2. 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.

3. 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.

4. 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

5. 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.

6. 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.

7. 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.

8. 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.