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This file contains Supplementary Figure 1. Supplementary Figure 1 shows the staining pattern of tumoral PD-L1 staining.

This file contains Supplementary Figure 2. Supplementary Figure 2 shows tumoral PD-L1 staining in association with PD-1, CD8, and CD4 TILs.

This file contains Supplementary Figures 3-9. Supplementary Figure 3. Representative multi-color immunofluorescence staining shows that PD-1+CD4+ T cells demonstrate greater PD-1 expression than PD1+CD8+ T cells. Supplementary Figure 4. Representative multi-color immunofluorescence staining of a PD-L1 negative tumor shows that PD-1+CD4+ T cells demonstrate greater PD-1 expression than PD1+CD8+ T cells. Supplementary Figure 5. CD4+ T cells show markedly more specific PD-L1 localization in OTSCC tumors compared to CD8+ T cells. Supplementary Figure 6. Representative multi-color immunofluorescence staining of an OTSCC tumor shows that PD-1+CD4+ T cells demonstrate greater PD-1 expression than PD1+CD8+ T cells. Supplementary Figure 7. Higher magnification multi-color immunofluorescence staining of a representative OTSCC tumor stained with PD-L1, PD-1, CD4, and CD8. Supplementary Figure 8. Diffuse tumoral PD-L1 staining shows low infiltration of CD4+ and CD8+ T cells. Supplementary Figure 9. Representative multi-color immunofluorescence staining of FoxP3, PD1, and Ki67 in OTSCC.

This file contains Supplementary Table 1. Supplementary Table 1 shows the immunohistochemical staining for PD-L1, PD-1, CD4, and CD8 in the samples analyzed in the manuscript.

This file contains Supplementary Table 2. Supplementary Table 2 shows that the proliferative capacity of PD-1 expressing CD8, CD4, and FoxP3+ TILs are decreased compared to non-PD-1 expressing TILs in OTSCC.

This file contains the figure legends for supplementary figures 1-9. Supplementary Figure 1. Staining pattern of tumoral PD-L1 staining. Supplementary Figure 2. Tumoral PD-L1 staining in association with PD-1, CD8, and CD4 TILs. Supplementary Figure 3. Representative multi-color immunofluorescence staining shows that PD-1+CD4+ T cells demonstrate greater PD-1 expression than PD1+CD8+ T cells. Supplementary Figure 4. Representative multi-color immunofluorescence staining of a PD-L1 negative tumor shows that PD-1+CD4+ T cells demonstrate greater PD-1 expression than PD1+CD8+ T cells. Supplementary Figure 5. CD4+ T cells show markedly more specific PD-L1 localization in OTSCC tumors compared to CD8+ T cells. Supplementary Figure 6. Representative multi-color immunofluorescence staining of an OTSCC tumor shows that PD-1+CD4+ T cells demonstrate greater PD-1 expression than PD1+CD8+ T cells. Supplementary Figure 7. Higher magnification multi-color immunofluorescence staining of a representative OTSCC tumor stained with PD-L1, PD-1, CD4, and CD8. Supplementary Figure 8. Diffuse tumoral PD-L1 staining shows low infiltration of CD4+ and CD8+ T cells. Supplementary Figure 9. Representative multi-color immunofluorescence staining of FoxP3, PD1, and Ki67 in OTSCC.

Oral tongue squamous cell carcinoma (OTSCC) is the most common oral cavity tumor. In this study, we examined the basis for the activity of programmed cell death protein (PD-1)-based immune checkpoint therapy that is being explored widely in head and neck cancers. Using multispectral imaging, we systematically investigated the OTSCC tumor microenvironment (TME) by evaluating the frequency of PD-1 expression in CD8+, CD4+, and FoxP3+ tumor-infiltrating lymphocytes (TIL). We also defined the cellular sources of PD-1 ligand (PD-L1) to evaluate the utility of PD-1:PD-L1 blocking antibody therapy in this patient population. PD-L1 was expressed in 79% of the OTSCC specimens examined within the TME. Expression of PD-L1 was associated with moderate to high levels of CD4+ and CD8+ TILs. We found that CD4+ TILs were present in equal or greater frequencies than CD8+ TILs in 94% of OTSCC and that CD4+FOXP3neg TILs were colocalized with PD-1/PD-L1/CD68 more frequently than CD8+ TILs. Both CD4+PD1+ and CD8+PD1+ TILs were anergic in the setting of PD-L1 expression. Overall, our results highlight the importance of CD4+ TILs as pivotal regulators of PD-L1 levels and in determining the responsiveness of OTSCC to PD1-based immune checkpoint therapy. Cancer Res; 77(22); 6365–74. ©2017 AACR.

This file contains Supplementary Table 3. Supplementary Table 3 shows the clinicopathological characteristics of the patient cohort.

Oral tongue squamous cell carcinoma (OTSCC) is the most common oral cavity tumor. In this study, we examined the basis for the activity of PD-1-based immune checkpoint therapy that is being explored widely in head and neck cancers. Using multispectral imaging, we systematically investigated the OTSCC tumor microenvironment (TME) by evaluating the frequency of PD-1 expression in CD8+, CD4+ and FoxP3+ tumor-infiltrating lymphocytes (TIL). We also defined the cellular sources of PD-L1 to evaluate the utility of PD-1:PD-L1 blocking antibody therapy in this patient population. PD-L1 was expressed in 79% of the OTSCC specimens examined within the TME. Expression of PD-L1 was associated with moderate to high levels of CD4+ and CD8+ TIL. We found that CD4+ TIL were present in equal or greater frequencies than CD8+ TIL in 94% of OTSCC, and that CD4+ FOXP3neg TIL were co-localized with PD-1/PD-L1/CD68 more frequently than CD8+ TIL. However, both CD4+PD1+ and CD8+PD1+ TIL were anergic in the setting of PD-L1 expression. Overall, our results highlight the importance of CD4+ TIL as pivotal regulators of PD-L1 levels and in determining the responsiveness of OTSCC to PD1-based immune checkpoint therapy.