Cell Reports, Volume 25

Supplemental Information

Magnitude of Therapeutic STING Activation

Determines CD8+ T Cell-Mediated Anti-tumor Immunity

Kelsey E. Sivick, Anthony L. Desbien, Laura Hix Glickman, Gabrielle L. Reiner, LeticiaCorrales, Natalie H. Surh, Thomas E. Hudson, Uyen T. Vu, Brian J. Francica, TamaraBanda, George E. Katibah, David B. Kanne, Justin J. Leong, Ken Metchette, Jacob R.Bruml, Chudi O. Ndubaku, Jeffrey M. McKenna, Yan Feng, Lianxing Zheng, Steven L.Bender, Charles Y. Cho, Meredith L. Leong, Andrea van Elsas, Thomas W. DubenskyJr., and Sarah M. McWhirter

Figure S1. IT STING Activation Can Be Modulated to Induce Local versus Systemic Immune Activation. Related to Figure 1. (A) Mice bearing a single flank CT26m tumor received one IT injection of vehicle alone or 0.5, 1, 5, 10, 50, 100, or 500 µg S100. Six h post injection, serum cytokines were measured. (B) Mice bearing a single flank B16.SIY tumor received one IT injection of vehicle alone or 0.5, 2, 10, 100, or 200 µg S100. Seven days post injection, PBMCs from each mouse were assessed for the frequency of CD8+H-2Kb-SIY tetramer+ (SIY+) cells by flow cytometry. (C) Mice bearing dual flank CT26m tumors received one IT injection (in the right flank tumor) with vehicle alone or 10, 100, or 500 µg S100. One h post injection, both tumors were dissociated and incubated for an additional four h in the presence of Golgi inhibitors for staining and interrogation by flow cytometry. Shown are TNFα positive frequencies of CD4+ and CD8+ T cells, NK1.1+ NK cells, CD11b+Ly6G+ neutrophils, CD11b+Ly6C+MHC Class II+ monocytes, and CD11b+CD11c+Ly6C-MHC Class II+ macrophages in the injected and distal tumor. (D) Mice bearing a single flank CT26m tumor received one IT injection of vehicle alone or 10 µg S100. Three days post injection, TDLN cells from the injected and distal sides were stained and interrogated by flow cytometry. Shown are histograms depicting CD11c, CD25, Granzyme B (GrzB), Ki-67, and Tbet expression on CD8+AH1+ T cells. (E) Mice bearing a single flank CT26m tumor received one IT injection of vehicle alone or 10 or 500 µg S100. One d post injection, TDLN cells from the injected side were processed, stained, and interrogated by flow cytometry (top) or mounted and stained for CC3 by IHC (bottom). Shown are the Dead positive frequencies on single cells with CD45+CC3+ or CD45-CC3+ positive frequencies on Live Single cells. Bar=1 mm. (F) Mice bearing dual flank CT26m tumors received one IT injection of vehicle alone or 1, 10, 100, or 500 µg S100. Seven days post injection, PBMCs from each mouse were assessed for the frequency of AH1+ cells by flow cytometry. Data are representative of at least two independent experiments with an n=4-8 animals/group. Bars and error represent the mean ± SEM and each symbol represents an individual animal. ns, not significant; N, Naïve; V, vehicle; CC3, cleaved caspase 3. **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure S2. CD8+ T cells Are Necessary and Sufficient for Anti-tumor Immunity Elicited by Immunogenic Doses of S100. Related to Figure 2. (A) Mice bearing dual flank CT26m tumors were injected the intraperitoneal (IP) route with 100 µg anti-CD8 or isotype control on days 6, 8,10,14 and 17 along with IT injection of vehicle alone or 10 µg S100 on day 8. On day 15, PBMCs from each mouse were assessed for CD8+ T cell depletion by flow cytometry (using anti-CD8β antibody recognizing a distinct epitope from the anti-CD8α depleting antibody). Shown are the frequency (left), total number (right), and representative dot plots (bottom) of CD8+ T cells. (B) Mice bearing dual flank CT26m tumors were injected the IP route with 100 µg αCD8 or isotype control on days 4, 8,10,14 and 17 with one IT injection (in the right flank tumor) of vehicle alone or indicated doses of S100. Shown is the percent decrease in tumor growth at day 21 of individual animals compared to the mean of the corresponding PBS group (either isotype or αCD8 treated). (C) Mice bearing single or dual flank CT26m tumors received one (1x) or three (3x) IT injections of 50, 100, 500 µg S100. Along with naïve controls, mice that cured their primary tumors were implanted with 1x106 CT26m tumor cells. Shown is the mean rechallenge tumor growth on d 7. Data are representative of at least two independent experiments with an n=8 animals/group. Bars/lines and error represent the mean ± SEM and each symbol represents an individual animal. Unless indicated otherwise, significance shown above each bar is compared to Naïve control. ns, not significant; SF, single flank; DF, dual flank. **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure S3. Role of Hematopoietic versus Non-hematopoietic Cells in Anti-tumor and Cytokine Responses. Related to Figure 3. (A) Bone marrow cells from wild type (WT) C57BL/6 or goldenticket (Gt) mice lacking functional STING were transferred into irradiated WT or Gt recipient mice, resulting in four sets of bone marrow chimeric mice. At 13 weeks post adoptive transfer, PBMCs from each mouse were assessed for chimerism by flow cytometry. Shown are the frequency of CD45.1 (45.1, gray bars) and CD45.2 (45.2, open bars) positive cells of the single cell, CD11b+, CD4+, and CD8+ cell compartments. Data are representative of two independent experiments with an n=20 animals/group. Bars and error represent the mean ± SEM. (B) Parental B16.SIY cells and four B16.SIY STING-/- clones were stimulated for 2h with vehicle alone or 100 µg S100 before processing for qPCR. Shown is the IFNβ mRNA expression relative to GAPDH. Equal cell numbers of the four B16.SIY STING-/- clones were pooled and expanded prior to implantation. Data are representative of at least two independent experiments. Bars and error represent the mean ± SEM of technical replicates.

Figure S4. Type I IFN, but Not TNFα, is Required for Optimal Anti-tumor Immune Responses in an Immunogenic Setting. Related to Figure 4. (A) Mice bearing a single B16.SIY flank tumor were injected the IP route with 200 μg of αTNFα or isotype control on days 4 and 7 with one IT injection of vehicle alone or 10 µg S100 on d 8. Six h post IT injection, serum cytokines were measured. (B) Mice bearing a single B16.SIY STING-/- flank tumor were injected the IP route with 200 μg of αIFNAR or isotype control on days 7 and 12 with one IT injection of vehicle alone or 2 µg S100 on day 8. On day 15, spleens were dissociated and left unstained (open histogram) or stained with a fluorescently-labeled version of the IFNAR neutralizing antibody clone (filled histograms). Shown are splenocytes from WT mice receiving PBS IP injection (black histogram), WT mice receiving αIFNAR IP injection (gray histogram), and uninjected IFNAR-KO mice (purple histogram). (C) Mice bearing a single B16.SIY STING-/- flank tumor were injected the IP route with 200 μg of αIFNAR or isotype control on day 7 with one IT injection of vehicle alone or 2 µg S100 on d 8. Six h post IT injection, serum cytokines were measured. Bars and error represent the mean ± SEM and each symbol represents an individual animal. Data are representative of two independent experiments with an n=8 animals/group with the exception of panel B showing representative animals. ns, not significant. ***p < 0.001, ****p < 0.0001.

Figure S5. S100 and αPD-1 Synergize to Control Distal Tumors. Related to Figure 5. CT26m dual flank tumors were injected the IP route with 200 μg of αPD-1 or isotype control on day 10 and twice weekly thereafter with one IT injection (in the right flank tumor) of vehicle alone or 10 µg S100 on day 10. 12 days post injection, spleens were dissociated and either left untreated (not shown) or stimulated with AH1 peptide in the presence of Golgi inhibitors for staining and interrogation by flow cytometry. Shown are the AH1 and IFNγ+TNFα+ positive frequencies on bulk CD8+ T cells with CD11c positive frequency on AH1+ T cells. Bars and error represent the mean ± SEM and each symbol represents an individual animal. Data are representative of at least two independent experiments with an n=8-10 animals/group. ns, not significant. *p < 0.05, **p < 0.01, ***p < 0.001.