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Figure S10. CD8+ T cell phenotype microenvironment heatmaps. Flow cytometry assessment of CD8+ T cell phenotype changes at day 23 for all treatment groups, and further assessed at day 33 and 37 for CPR treatment and compared to tumor size-matched control mice. Data is represented as a z-score of the phenotypic marker’s median fluorescence intensity (MFI) among CD8+ T cells represented as z-scores. (A) Tumor and (B) tdLN CD8+ T cell phenotypic marker expression between all treatment groups at day 23 of treatment (N = 2, n = 7–13 per group). (C) tdLN CD8+ T cell phenotypic marker expression at days 23, 33, and 37 of CPR treatment progression compared to size-matched control tumors (N = 2; n = 7–13 per group). (PDF 3118 kb)

Figure S7. Tumor immune microenvironment data time course. Flow cytometry assessment of tumor was performed at day 23, day 33, and day 37 for the CPR treatment group and major immune cell subset percentages (among CD45+ cells) are shown. (A) Percentage of CD4+ and CD8+ T cell subsets. (B) Percentage E7 tetramer+ CD8+ T cells. (C) Percentage of Tregs. (D) Percentage of major myeloid subsets. (For A-D, Tukey’s multiple comparison test; N = 2; 8–13 per group). (E) Aggregate flow cytometry scatter plots of CD8+ T cells showing E7 tetramer staining (N = 1, representative of 2; n = 4 aggregate samples per group). **p

Figure S6. Tumor immune microenvironment data at day 23. Flow cytometry assessment of tumor was performed at day 23 for all treatment groups and major immune cell subset percentages (among CD45+ cells) are shown. (A) Percentage of CD4+ and CD8+ T cell subsets. (B) Percentage E7 tetramer+ CD8+ T cells. (C) Percentage of Tregs. (D) Percentage of major myeloid subsets. (For A-D, Tukey’s multiple comparison test; N = 2; 8–13 per group). (E) Aggregate flow cytometry scatter plots of CD8+ T cells showing E7 tetramer staining (N = 1, representative of 2; n = 4 aggregate samples per group). *p

Figure S5. CPR increases intratumoral M1-like macrophages. Aggregate flow cytometry scatterplots showing MHCII and iNOS expression among tumor-dwelling macrophages at day 23 of treatment (percentages show mean +/− SD; N = 1 representative of 2; n = 4 aggregate samples per group). (PDF 1299 kb)

Figure S4. CTX/L-NIL improves anti-tumor effect of αPD-1/αCTLA-4 and radiation in the B16 syngeneic melanoma tumor model. Subcutaneous established B16-F0 melanoma tumors (day 4 post tumor cell injection) were treated with αPD-1/αCTLA-4 and radiation alone, or combined with CTX/L-NIL immunomodulation (CPR regimen), mice were euthanized when tumors reached 225 mm2. (A) Average tumor area statistically compared at time of first control mouse euthanization (Tukey’s multiple comparison test; N = 1 representative of 2; n = 7–8 per group). (B) Kaplan Meier survival curves with comparison between treatment groups (Log-rank test; N = 2; n = 10–11 per group). *p

Figure S3. CPR regimen induces minimal weight loss and no gross treatment related toxicities. (A) Normalized weight for treated mice over the course of treatment, normalized to mouse weight 1 week after tumor cell inoculation (N = 1 representative of 2; n = 5–9). (B) Image of mouse treated with full CPR regimen approximately 100 days after tumor clearance with white fur visible in location of tumor clearance. (PDF 1600 kb)

Figure S13: Multiplex raw cellular densities. Raw cellular densities from multiplex analysis used for generation of Fig. 4b and c. For all samples N = 1 and n = 15 images per group (3 mice per group with 5 images assessed per tumor). (PDF 1479 kb)

Figure S7. Tumor immune microenvironment data time course. Flow cytometry assessment of tumor was performed at day 23, day 33, and day 37 for the CPR treatment group and major immune cell subset percentages (among CD45+ cells) are shown. (A) Percentage of CD4+ and CD8+ T cell subsets. (B) Percentage E7 tetramer+ CD8+ T cells. (C) Percentage of Tregs. (D) Percentage of major myeloid subsets. (For A-D, Tukey’s multiple comparison test; N = 2; 8–13 per group). (E) Aggregate flow cytometry scatter plots of CD8+ T cells showing E7 tetramer staining (N = 1, representative of 2; n = 4 aggregate samples per group). **p

Figure S11. Myeloid flow cytometry gating strategy. Flow cytometry strategy used for gating myeloid subsets, with labeled cell type above the respective gate. (PDF 2002 kb)

Figure S6. Tumor immune microenvironment data at day 23. Flow cytometry assessment of tumor was performed at day 23 for all treatment groups and major immune cell subset percentages (among CD45+ cells) are shown. (A) Percentage of CD4+ and CD8+ T cell subsets. (B) Percentage E7 tetramer+ CD8+ T cells. (C) Percentage of Tregs. (D) Percentage of major myeloid subsets. (For A-D, Tukey’s multiple comparison test; N = 2; 8–13 per group). (E) Aggregate flow cytometry scatter plots of CD8+ T cells showing E7 tetramer staining (N = 1, representative of 2; n = 4 aggregate samples per group). *p

Figure S1. Immune checkpoint inhibitors alone provide minimal treatment benefit or microenvironment improvement. (A-C) Subcutaneous established mEER tumors (day 17–18 post tumor cell injection) were treated with 6 total doses of αPD-1 (250 μg/dose) and/or αCTLA-4 (100 μg/dose) according to the schedule in (Fig. 1b), mice were euthanized when tumors reached 225 mm2. (A) Kaplan Meier survival curves with comparison between treatment groups (Log-rank test; N = 1 representative of 2; n = 5–12). (B) Flow cytometry immune profiling of treated tumors at 1 week of treatment (day 23) shown as z-scores for major lymphoid and myeloid subtypes between groups (N = 2; n = 9–12 per group). (C) Percentage of major lymphoid subsets (among CD45+ cells) in tdLN at day 23 of treatment for various treatment groups (Tukey’s multiple comparison test; N = 2; n = 9–12 per group). *p

Figure S9. tdLN immune microenvironment data time course. Flow cytometry assessment of tdLN was performed at day 23, day 33, and day 37 for the CPR treatment group and major immune cell subset percentages (among CD45+ cells) are shown. (A) Percentage of CD4+ and CD8+ T cell subsets. (B) Percentage E7 tetramer+ CD8+ T cells. (C) Percentage of Tregs. (D) Percentage of major myeloid subsets. (For A-D, Tukey’s multiple comparison test; N = 2; 7–13 per group). (E) Aggregate flow cytometry scatter plots of CD8+ T cells showing E7 tetramer staining (N = 1, representative of 2; n = 4 aggregate samples per group). *p

Figure S8. tdLN immune microenvironment data at day 23. Flow cytometry assessment of tdLN was performed at day 23 for all treatment groups and major immune cell subset percentages (among CD45+ cells) are shown. (A) Percentage of CD4+ and CD8+ T cell subsets. (B) Percentage E7 tetramer+ CD8+ T cells. (C) Percentage of Tregs. (D) Percentage of major myeloid subsets. (For A-D, Tukey’s multiple comparison test; N = 2; 7–13 per group). (E) Aggregate flow cytometry scatter plots of CD8+ T cells showing E7 tetramer staining (N = 1, representative of 2; n = 4 aggregate samples per group). *p

Figure S1. Immune checkpoint inhibitors alone provide minimal treatment benefit or microenvironment improvement. (A-C) Subcutaneous established mEER tumors (day 17–18 post tumor cell injection) were treated with 6 total doses of αPD-1 (250 μg/dose) and/or αCTLA-4 (100 μg/dose) according to the schedule in (Fig. 1b), mice were euthanized when tumors reached 225 mm2. (A) Kaplan Meier survival curves with comparison between treatment groups (Log-rank test; N = 1 representative of 2; n = 5–12). (B) Flow cytometry immune profiling of treated tumors at 1 week of treatment (day 23) shown as z-scores for major lymphoid and myeloid subtypes between groups (N = 2; n = 9–12 per group). (C) Percentage of major lymphoid subsets (among CD45+ cells) in tdLN at day 23 of treatment for various treatment groups (Tukey’s multiple comparison test; N = 2; n = 9–12 per group). *p

Figure S10. CD8+ T cell phenotype microenvironment heatmaps. Flow cytometry assessment of CD8+ T cell phenotype changes at day 23 for all treatment groups, and further assessed at day 33 and 37 for CPR treatment and compared to tumor size-matched control mice. Data is represented as a z-score of the phenotypic marker’s median fluorescence intensity (MFI) among CD8+ T cells represented as z-scores. (A) Tumor and (B) tdLN CD8+ T cell phenotypic marker expression between all treatment groups at day 23 of treatment (N = 2, n = 7–13 per group). (C) tdLN CD8+ T cell phenotypic marker expression at days 23, 33, and 37 of CPR treatment progression compared to size-matched control tumors (N = 2; n = 7–13 per group). (PDF 3118 kb)

Table S1. Flow cytometry immune microenvironment staining panels. (DOCX 19 kb)

Table S1. Flow cytometry immune microenvironment staining panels. (DOCX 19 kb)

Figure S8. tdLN immune microenvironment data at day 23. Flow cytometry assessment of tdLN was performed at day 23 for all treatment groups and major immune cell subset percentages (among CD45+ cells) are shown. (A) Percentage of CD4+ and CD8+ T cell subsets. (B) Percentage E7 tetramer+ CD8+ T cells. (C) Percentage of Tregs. (D) Percentage of major myeloid subsets. (For A-D, Tukey’s multiple comparison test; N = 2; 7–13 per group). (E) Aggregate flow cytometry scatter plots of CD8+ T cells showing E7 tetramer staining (N = 1, representative of 2; n = 4 aggregate samples per group). *p

Figure S4. CTX/L-NIL improves anti-tumor effect of αPD-1/αCTLA-4 and radiation in the B16 syngeneic melanoma tumor model. Subcutaneous established B16-F0 melanoma tumors (day 4 post tumor cell injection) were treated with αPD-1/αCTLA-4 and radiation alone, or combined with CTX/L-NIL immunomodulation (CPR regimen), mice were euthanized when tumors reached 225 mm2. (A) Average tumor area statistically compared at time of first control mouse euthanization (Tukey’s multiple comparison test; N = 1 representative of 2; n = 7–8 per group). (B) Kaplan Meier survival curves with comparison between treatment groups (Log-rank test; N = 2; n = 10–11 per group). *p

Figure S5. CPR increases intratumoral M1-like macrophages. Aggregate flow cytometry scatterplots showing MHCII and iNOS expression among tumor-dwelling macrophages at day 23 of treatment (percentages show mean +/− SD; N = 1 representative of 2; n = 4 aggregate samples per group). (PDF 1299 kb)

Figure S13: Multiplex raw cellular densities. Raw cellular densities from multiplex analysis used for generation of Fig. 4b and c. For all samples N = 1 and n = 15 images per group (3 mice per group with 5 images assessed per tumor). (PDF 1479 kb)

Background Chemoradiotherapy (CRT) remains one of the most common cancer treatment modalities, and recent data suggest that CRT is maximally effective when there is generation of an anti-tumoral immune response. However, CRT has also been shown to promote immunosuppressive mechanisms which must be blocked or reversed to maximize its immune stimulating effects. Methods Therefore, using a preclinical model of human papillomavirus (HPV)-associated head and neck squamous cell carcinoma (HNSCC), we developed a clinically relevant therapy combining CRT and two existing immunomodulatory drugs: cyclophosphamide (CTX) and the small molecule inducible nitric oxide synthase (iNOS) inhibitor L-n6-(1-iminoethyl)-lysine (L-NIL). In this model, we treated the syngeneic HPV-HNSCC mEER tumor-bearing mice with fractionated (10 fractions of 3 Gy) tumor-directed radiation and weekly cisplatin administration. We compared the immune responses induced by CRT and those induced by combinatory treatment (CRT + CTX/L-NIL) with flow cytometry, quantitative multiplex immunofluorescence and by profiling immune-related gene expression changes. Results We show that combination treatment favorably remodels the tumor myeloid immune microenvironment including an increase in anti-tumor immune cell types (inflammatory monocytes and M1-like macrophages) and a decrease in immunosuppressive granulocytic myeloid-derived suppressor cells (MDSCs). Intratumoral T cell infiltration and tumor antigen specificity of T cells were also improved, including a 31.8-fold increase in the CD8+ T cell/ regulatory T cell ratio and a significant increase in tumor antigen-specific CD8+ T cells compared to CRT alone. CTX/LNIL immunomodulation was also shown to significantly improve CRT efficacy, leading to rejection of 21% established tumors in a CD8-dependent manner. Conclusions Overall, these data show that modulation of the tumor immune microenvironment with CTX/L-NIL enhances susceptibility of treatment-refractory tumors to CRT. The combination of tumor immune microenvironment modulation with CRT constitutes a translationally relevant approach to enhance CRT efficacy through enhanced immune activation.