CD44 PD-L1 FSC Human/Mouse Stromal Markers FSC DAPI FSC SSC FSC-W FSC-H SSC-W SSC-H Live cells Tumor cells CD44 - CD44 + Supplementary Figure S1. Gating.

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CD44 PD-L1 FSC Human/Mouse Stromal Markers FSC DAPI FSC SSC FSC-W FSC-H SSC-W SSC-H Live cells Tumor cells CD44 - CD44 + Supplementary Figure S1. Gating strategy for isolating tumor cells from primary SCCHN tumors and xenografts. Tumor samples were mechanically and enzymatically digested into single cell suspensions and labeled with an antibody cocktail for flow cytometry. Dot plots illustrating the gating strategy for a representative tumor sample are shown. Dead cells were excluded with DAPI, while human stromal cells were excluded with antibodies against CD2, CD3, CD18, CD31, CD45, CD64 and TE-7 in primary SCCHN tumors. For patient-derived xenografts, mouse stromal cells were excluded with antibodies against H-2K b and CD45. Tumor cells were then profiled for expression of surface markers such as CD44 and PD-L1.

A B C EGFR CD44 CD47 CD44 Gal-9 Supplementary Figure S2. Characterization of CD44 - and CD44 + SCCHN cells. Representative dot plots of CD44 against markers: A, EGFR (n=7), B, CD47 (n=6), and C, Gal-9 (n=5) with corresponding graphs showing their MFI in matched pairs of CD44 - (●) and CD44 + ( ▲ ) gated tumor cells (*P<0.05, **P<0.01, paired t-tests), and CD44 + /CD44 - fold change of MFI values, mean ± SEM. D, relative expression of genes associated with EMT in pairs of sorted CD44 + cells vs CD44 - cells (n=5-8), mean ± SEM. D

CD44 CD271 ABC Supplementary Figure S3. CD44 + CD271 - and CD44 + CD271 + cells express comparable levels of PD-L1 in human SCCHN tumors. A, representative dot plot of CD44 and CD271 expression on a SCCHN sample. B, PD-L1 MFI of matched sets of CD44 - (●), CD44 + CD271 - ( ▲ ) and CD44 + CD271 + ( ■ ) gated tumor cells (n=7, *P<0.05, paired t-tests). C, CD44 + /CD44 - fold change of PD-L1 MFI for CD44 + CD271 - and CD44 + CD271 + cells (n=7), mean ± SEM.

Supplementary Figure S4. Anti-PD-1 blockade partially restores IFNγ secretion by TILs against CD44 + SCCHN cells. CD44 - and CD44 + SCCHN cells were co-cultured with autologous CD8 + TILs with 10μg/ml of control mAb, or 10μg/ml of anti-PD-1. Graph shows the IFNγ concentrations from a single representative co-culture experiment (n=4,*P<0.05, unpaired t-tests), mean ± SD.

WB: p-STAT3 WB: Total-STAT3 CD44 + CD44 - CD44 + CD44 - CD44 + CD44 - SCCHN1SCCHN2SCCHN3 Supplementary Figure S5. Full-length Western blots corresponding to truncated blots shown in Fig 3. Dotted-line boxes highlight the regions of the Western blots shown in Fig 3.

B C A HLA-ABC CD44 IFN γ R1 IFNγ Unt Supplementary Figure S6. CD44 + SCCHN cells have higher IFNγR1 expression. A, representative dot plots of CD44 and IFNγR1 expression on a tumor sample with or without 50U/ml IFNγ treatment for 16h. Graph shows the MFI of IFNγR1 on untreated (●) and 50U/ml IFNγ treated (○) CD44 - and CD44 + gated tumor cells (n=5, *P<0.05, paired t-tests). B, representative dot plots of CD44 and HLA-ABC expression on a tumor sample. Graphs show the MFI of HLA-ABC on CD44 - (●) and CD44 + ( ▲ ) gated tumor cells (n=6, *P<0.05, paired t-tests), and the mean ± SEM of CD44 + /CD44 ‑ fold change of HLA- ABC MFI (n=6). C, HLA-ABC MFI of CD44 - and CD44 + gated tumor cells left untreated (●) or treated with 50U/ml IFNγ for 16h (○) (n=6).