Supplemental figure legends

Figure S1: Presence of CD4+CD25+Foxp3+ T cells before and after the ex vivo expansion

Freshly isolated (D 0) or ex vivo expanded (D 6) splenocytes harvested from MMC-primed

FVBN202 mice. Red cells were lyzed by using ACK lysis buffer and splenocytes were subjected to three-color staining to determine proportion of Tregs in CD4+ T cells. Data represent three independent experiments.

Figure S2. T cell responses to MMC in the presence or absence of MDSC

A) Freshly isolated and ex vivo expanded T cells were cultured in the presence or absence of irradiated MMC cells for 24 hs and subjected to three color staining to determine the expression of

GrB. Data represent three independent experiments. B) Frequency of MDSC in the blood of

FVBN202 mice before and 7 days after the challenge with MMC cells. C) T cells were harvested from MMC-primed FVBN202 mice and expanded with alternating common gamma-chain cytokines. Briefly, splenocytes (106 cells/mL) were stimulated in complete medium containing 15%

FBS with bryostatin-1 (5 nM) and ionomycin (1 M) along with 80 U/mL of IL-2 (Peprotech) for

16 hs. Cells were washed three times and cultured at 106 cells/mL in complete medium with

40 U/mL IL-2 and media was changed every other day for a total of 6 days. Cells expanded with alternating gamma-chain cytokines were cultured on day 1 with 10 ng/mL IL-7 and 10 ng/mL IL-15

(Peprotech). On day two, 40 U/mL of IL-2 was added. Medium was changed on days 3 and 5, each time culturing with 10 ng/mL each of IL-7 and IL-15 and injections were done on day 7. Production of IFN- by the expanded T cells was determined in the presence or absence of MMC or

MMC+MDSC. Data represent two independent experiments. Figure S3. Expanded T cells do not increase the expression of CD69 or GrB following stimulation with MMC cells in vitro, and include activated NK T cells and NK cells

Ex vivo expanded T cells were cultured in the presence or absence of irradiated MMC or

MMC+MDSC. Expression of Prf was determined in gated neu positive MMC cells (A). Expression of CD69 (B) or GrB (C) was determined in gated CD8+ and CD4+ T cells. Data represent three independent experiments. D) Freshly isolated (D 0) or ex vivo expanded (D 6) splenocytes were gated on CD3+CD49b+ NK T cells or CD3-CD49b+ NK cells and expression of CD25 was determined. Data represent three independent experiments.

Figure S4. Analysis of the ex vivo expanded T cells derived from FVBN202 donor mice after local radiation therapy of the tumor

A) Viability of freshly isolated (D 0) and ex vivo expanded (D 6) T cells was determined by flow cytometry analysis of gated CD8+ or CD4+ T cells. Expression of CD127 (B) and CD122 (C) were determined on gated CD8+ or CD4+ T cells after a 6-day expansion. Data represent 3 independent experiments. D) Proportion of CD4-CD8- non-T cells and CD4+ or CD8+ T cells was determined by flow cytometry analysis of the ex vivo expanded T cells derived from donor mice after radaition therapy of the tumors. Freshly isolated and ex vivo expanded cells were cultured in the presence or absence of irradiated MMC cells and expression of CD69 (E) or GrB (F) was determined. Data represent three independent experiments. Figure S1

Gated CD4+ T cells

D 0 D 6

0.2% 2.4% Foxp3

7.5% 54.9%

CD25 Figure S2

A B Freshly isolated T cells Day 0 Day 7 Gated CD8+ T cells Gated CD4+ T cells medium MMC medium + MMC medium + MMC 100 75 98.12% 96.40% 98.55% 96.84% 50 4.24% 17% Gr1

CD8 25 CD4

% GrB+ T cells 0 CD8 CD4 CD11B C Ex vivo expanded T cells Gated CD8+ T cells Gated CD4+ T cells medium MMC 2500 medium + MMC medium + MMC 100 2000 93.34% 94.35% 75 1500 84.10% 90.88% 50 CD8 CD4 25 1000 % GrB+ T cells

0 IFN-g (pg/ml) 500 GrB CD8 CD4 0

Expanded T cells Figure S3

B C

Gated CD8+ T cells Gated CD8+ T cells

medium + MMC + MMC+ MDSC medium + MMC + MMC+ MDSC A 81.06% 84.41% 82.32% 84.1% 90.88% 92.66% Gated neu+ MMC CD8

0.39% 0.76%

Gated CD4+ T cells Gated CD4+ T cells

medium + MMC + MMC+ MDSC medium + MMC + MMC+ MDSC IFN- Prf 14.85% 18.87% 28.05% 93.34% 94.35% 92.38% CD4

CD69 GrB

D

Gated CD3+CD49b+ Gated CD3-CD49b+

0.8% 17.7% D 0 D 0

CD49B 84.9% CD49b 33% D 6 D 6

CD25 CD25 A

CD8 S4 Figure B

CD8 16.4% D 0

% Annexin V+ T cells CD8+ Tcells 100 20 40 60 80 CD8+ Tcells D 0 0 1.5% % CD127+ T cells 10 0 2 4 6 8 D 6 27.8% D CD4 CD8 D 6 AnnexinV 1.7% D CD4 CD8 D 0 CD4 CD127 D 0 CD4 D 0 30.0% CD4+ Tcells CD4+ Tcells D 0 D 6 1% D 6 D 6 20.5% D 6 0.8%

CD8 C D 0 11.2% CD8+ Tcells

% CD122+ T cells D 20 40 60

CD8 0 19.28% 21.17% D 6 P=0.032 46.9% D CD4 CD8 CD122 D 0 CD4 25.81% D 0 CD4+ Tcells 3.2% D 6 D 6 8.7% E CD8 CD8 F CD8 CD8 eim+MMC medium eim+MMC medium eim+MMC medium eim+MMC medium 57.62% 13.35% Gated CD8+ Tcells CD8+ Gated Gated CD8+ Tcells CD8+ Gated 95.19% Gated CD8+ Tcells CD8+ Gated Gated CD8+ Tcells CD8+ Gated 93.03% Ex vivo Freshly isolated T cells isolated Freshly Freshly isolated T cells isolated Freshly Ex vivo 28.03% 65.14% 53%88.87% 95.36% 92.97% expanded T cells expanded T cells CD69 GrB CD4 CD4 CD4 CD4 eim+MMC medium medium medium medium Gated CD4+ Tcells CD4+ Gated 88.50% 16.34% Gated CD4+ Tcells CD4+ Gated Gated CD4+ Tcells CD4+ Gated 61%38.95% 16.17% Gated CD4+ Tcells CD4+ Gated 96.47% + MMC + MMC + MMC 96.48% 19.43%