B Vehicle 1V270 (35 μg) 1V270 (100 μg) Days post tumor implantation. Vehicle 100μg 1V270 biweekly 100μg 1V270 daily
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1 Supplemental Figure 1 A 1 1V7 (8 μg) 1 1V7 (16 μg) B V7 (35 μg) 1V7 (1 μg) C Biweekly Days Implant SCC7 cells Daily Implant SCC7 cells 1V7 i.t. treatment 1V7 i.t. treatment Days D μg 1V7 biweekly 1μg 1V7 daily Supplemental Figure 1. Dose and schedule optimization for i.t. administration of 1V7. (A and B) Dose optimization studies. C3H mice (n=6-9/group) were implanted with 1 5 SCC7 cells. Mice were i.t. treated with 8, 16, 35, or 1 μg/injection of 1V7 starting when tumors reached -4 mm diameter, approximately day 8, daily for 5 days. (C and D) Schedule optimization studies. (C) Experimental protocol to determine optimal schedule of i.t. treatment with 1V7. C3H mice (n=14-15/group) were implanted with 1 5 SCC7 cells in both flanks. Intratumoral 1V7 treatment was given daily on days 8, 9, 1, 11, 1, or biweekly (on day 8, 11, 14, 17, and ). (1% DMSO) or isotype antibody administered as a control. (D) Tumor volume from mice treated daily (blue solid line) or biweekly (green solid line) with 1V7 or vehicle (black dashed line). Data presented are mean ± SEM P<.5, P<.1, and P<.1 (two-way repeated measures ANOVA with Bonferroni post hoc test). Data shown are representative of two independent experiments showing similar results.
2 Supplemental Figure A Days Implant MOC1 cells α-pd-1 mab 1V7 i.t. treatment B 5 Injected site 1V7+αPD-1 C Uninjected site 5 1V7+αPD Supplemental Figure. The combination therapy inhibits MOC1 tumor progression at both injected and uninjected sites. 1 6 MOC1 cells were s.c. implanted in both flanks of C57BL/6 mice (n=5/group). Mice were treated with 1V7 (1 μg/injection) and anti-pd-1 antibody (5 μg/injection).(a) Treatment schedule. (B) Tumor volume at injected sites. (C) Tumor volume at uninjected sites. Data presented are ± SEM P<.5 and P<.1 (two-way ANOVA with Bonferroni post hoc test)
3 Supplemental Figure 3 A CD45 CD CD11b CD11b+ 95. F4/8 F4/ CD6 1V7+α-PD-1 CD CD FSC FSC B D %F4/8 + CD11b + F4/8 + TAMs in CD45 + cells Uninjected site CD45+ Injected site M1/M ratio 6 4 Day 13 CD11b+.8 Uninjected site 1V7 αpd-1 1V7+αPD-1 1V7 αpd-1 1V7+αPD-1 Day 13 M1/M ratio C %F4/8 + CD11b + F4/8 + TAMs in CD45 + cells 6 4 F4/ FSC Injected site Day 1 CD6+.8 FSC CD6-1. Day 1 1V7 αpd-1 1V7+αPD-1 1V7 αpd-1 1V7+αPD-1 Uninjected site E CD45 CD CD11b 1V7 αpd-1 1V7+αPD-1 CD F4/8 1V7 αpd-1 F4/ V7+αPD-1 CD6 1V7+αPD-1 Q Q Q 15.9 Q FSC CD45+.1 CD11+.1 F4/8+.1 Class II Q1.61 Q Q Q3.5 FSC Supplemental Figure. CD11b + F4/8 + macrophages are large populations of tumor filtrating immune cells. (A-C) SCC7 bearing mice were treated as described in Figure 1A. Tumors (injected and uninjected sites) were harvested on days 13 and 1 and TAMs were analyzed by flow cytometry. Single cell suspensions were stained for CD45, CD11b, and F4/8 to identify TAMs. Expression of CD6 was used to identify M macrophages. (A) Representative flow cytometric plots of tumor infiltrating M1 (CD6 ) and M (CD6 + ) macrophages. The plots staining with isotype antibodies are shown on the lower panel. (B and C) The percentage of macrophage in the gated CD45 + cell population in the tumors harvested on days 13 (B) and 1 (C) was calculated and plotted. Each dot represents a tumor from an individual mouse. The horizontal and vertical bars indicate the mean ± SEM. Data shown are representative of two independent experiments showing similar results. P<.5 (Kruskal-Wallis test with Dunn s post hoc test). (D) The ratios of M1 to M (M1/M) were calculated as [% M1 (CD6 ) population in CD45 + CD11b + F4/8 + ]/ [% M (CD6 + ) population in CD45 + CD11b + F4/8 + ]. There were no significant differences in M1/M ratios among groups at uninjected sites (Kruskal-Wallis test with Dunn s post hoc test). (E) Representative flow cytometric plots of CD6 + Class II and CD6 Class II + populations. M1- and M- like macrophages were identified as CD45 + CD11b + F4/8 + CD6 Class II + and CD45 + CD11b + F4/8 + CD6 + Class II populations, respectively.
4 Supplemental Figure 4 A Lymph node B Tumor %OVA-positive cells in F48 + macrophages V7+αPD-1 1V7+αPD-1 Day 13 Day 1 %OVA-positive cells in F48 + TAMs V7+αPD-1 1V7+αPD-1 Day 13 Day 1 Supplemental Figure 4. Antigen associated macrophages were increased in the dlns and tumors on days 13 and 1. SCC7-tumor bearing mice were treated with 1V7 and anti-pd-1 antibody as described in Figure 1A and antigen uptake was evaluated on days 13 and 1 (n=4/group). OVA conjugated with Alexa Fluor 488 was i.t. injected one day before the analysis. Single cell suspensions were obtained from tumors at injected sites and draining lymph nodes (dlns), and stained for CD45, CD11b and F4/8. OVA + cells in the gated macrophage population (CD45 + CD11b + F4/8 + ) in the dlns and tumors were analyzed by FACS. (A) %OVA + cells in CD45 + CD11b + F4/8 + macrophages. (B) %OVA + cells in CD45 + CD11b + F4/8 + TAMs. Data presented are means ± SEM. P<.1 (two-tailed Welch s t test).
5 Supplemental Figure 5 TLR7 TLR9 PD-L1 Relative expression normalized to GAPDH 1..5 Relative expression normalized to GAPDH 1..5 Relative expression normalized to GAPDH RAW SCC7 MEER. RAW SCC7 MEER. RAW SCC7 MEER Supplemental Figure 5. TLR7, TLR9 and PD-L1 expression in SCC7 and MEER cells. SCC7 and MEER cells were cultured in vitro, harvested, and RNA was isolated. Quantitative RT-PCR was used to compare the expression of TLR7, TLR9, and PD-L1. Gene expression was normalized with GAPDH and then compared to the expression levels in murine macrophage cell line RAW64.7 that was used as a positive control (1%). Data are presented as mean ± SEM for triplicate determinations. Data shown are representative of two independent experiments showing similar results.
6 Supplemental Figure 6 A C D Relative expression normalized to Rps Relative expression normalized to Rps CD11b + cells CD6/Mrc1 x V7 1V7 +1V7 M1-like macrophage related genes x NOS 1V7 CCL5/RANTES x V7 M-like macrophage related genes x Ym1/Chil3 1V7 qpcr x CD163 1V7 Relative expression normalized to Rps CCL3/MIP1α x B 1V7 Antigen-presentation machinary CD4 x x x V7 TNF 1V7 Fizz/Retnla 1V7 x x x CD8 IL-1β Arg1 1V7 1V7 1V7 x1-3 1 x CD86 1V7 IL-1a 1V7 PD-L1 x x V7 IL-1b 1V7 Supplemental Figure 6. Expression of genes related to antigen presenting function and M1 macrophages is upregulated following 1V7 exposure ex vivo. (A) Experimental protocol. CD11b+ TAMs were isolated from day 14 SCC7-tumors using CD11b MicroBeads (Miltenyi Biotec) and treated with 1 μm 1V7 or vehicle overnight. Quantitative RT-PCR was performed. Primers and probes, which were purchased from Thermo Fisher Scienticfic, are listed in Supplemental Table 4. (B) Antigen-presentation machinery. (C) M1-like macrophage related genes. (D) M-like macrophage related genes. Data are presented as mean ± SD of triplicates. P<.5, P<.1, and P<.1 (one-tailed unpaired t test).
7 Supplemental Figure 7 A Implant SCC7 cells α-asialo-gm1 Ab i.p. α-pd-1 mab i.p. 1V7 i.t. treatment Days B Injected site n.s. n.s. + α-asialo+ +1V7+α-PD-1 α-asialo+1v7+α-pd Supplemental Figure 7. NK cells are partially involved in tumor suppression effects by the combination therapy. (A) Experimental protocol of NK cell depletion. SCC7-bearing mice were treated with the combination therapy of 1V7 and anti-pd-1 agent. Anti-asialo GM1 rabbit polyclonal antibody (5 μl/injection) or rabbit IgG polyclonal antibody was injected on days -1, 1, 5, 9, 13, and 17. (B) Tumor growth at the injected site was monitored. P<.1; n.s., non-significant by two-way repeated measures ANOVA with Bonferroni post hoc test (n=4-5/group).
8 Supplemental Figure 8 1V7 + α-pd-1 CD45 CD CD8 CD IFNγ IFNγ CD8 CD CD8+.8 IFNγ +.17 CD8 Supplemental Figure 8. Representative flow cytometric plots of tumor infiltrating CD8 + T cells. Tumors (injected and uninjected sites) were harvested on day 1 from SCC7-bearing mice treated with 1V7 and/or anti-pd-1 as described in Figure 1A. The single cell suspensions of tumor cells and splenocytes were prepared and stained for CD45, CD8, and intracellular IFNγ to identify tumor infiltrating IFNγ + CD8 + T cells. Fixation/Permeabilization Solution kit (BD Biosciences) were used for intracellular IFNγ staining. Representative plots of cells after staining with isotype antibodies are shown on the lower panel.
9 Supplemental Figure 9 A CD45 Lymph node CD CD45+.1 CD3 CD CD3+.1 CD8 CD CD8+.1 HPV tetramer CD8 CD8 1V7+αPD-1 B Number of HPV + CD8 + cells/mouse Lymph node 1V7+αPD-1 C Number of HPV + CD8 + cells/mm 3 Tumor V7+αPD-1 Supplemental Figure 9. The combination treatment increased HPV-tetramer positive CD8 + T cells in the dlns and the tumors HPV-positive MEER cells were implanted s.c. in both flanks of C57BL/6 mice (n=5/group). The combination treatment with anti-pd-1 and 1V7 was given as described in Figure A. One day after the final 1V7 injection, tumors and draining lymph nodes (dlns) at injected sites were harvested. The single cell suspensions of LN cells and tumor cells were incubated with antibody cocktail (CD45, CD3, CD8 and HPV tetramer). itag Tetramer/PE - H- Db HPV 16 E7 (RAHYNIVTF) (MBL international Corporation, MA) was used to detect HPV specific CD8 + T cells. (A) Representative flow cytometric plots. (B) Number of CD8 + HPV + T cells in the dln. (C) Number of CD8 + HPV + T cells in the tumor at injected sites. Data are presented as mean ± SEM P<.5, one-tailed Welch s t test.
10 Supplemental Figure 1 A i.p. injection nmol /injection 1V7 (/week) B i.p. injection 8 nmol /injection 1V7 (/week) 8 nmol /injection 1V7 (3/week) Supplemental Figure 1. Systemic administration of 1V7. (A and B) 1 5 B16 melanoma cells were implanted in the right flank of C57BL/6 mice. Mice were intraperitoneally (i.p.) treated with 1V7 ( nmol/injection or 8 nmol/injection) twice a week or three times a week. (A) nmol/injection of 1V7 twice a week. (B) 8 nmol/injection of 1V7 twice a week or three times a week. P<.5, and P<.1 by two-way ANOVA, Bonferroni post hoc test.
11 Supplemental Figure 11 Tumor volume mm 3 CTRL Anti-PD-1 plus CTRL-ODN SD-11 Anti-PD-1 plus SD-11 /6 3/6 Anti-PD-1 SD-11 Days Supplemental Figure 11. Graphical representation of mean tumor volumes over time CT6 tumor cells were injected s.c. in the flank of Balb/c mice on day (n=5-7 mice/group). Anti-PD-1 and SD-11 were both started five days after tumor implantation. Anti-PD-1 and SD-11 were administered every 4 days from day 5 to. SD-11 (5 µg/injection) or CTRL-ODN (non-cpg ODN control) was given i.t. and anti- PD-1 (clone RMP1-14, 5 µg/injection) was i.p. administered. CT6 bearing mice received concomitant treatment with anti-pd-1 and SD-11 which led to incomplete tumor rejections (5% rejection). As we previously reported (Wang et al. PNAS 16, Fig A, (3)), anti-pd-1 was given at day 7 and SD-11 at day 19 after tumor implantation which resulted in 1% rejection. Data shown are representative of three independent experiments showing similar results.
12 Supplemental Figure 1 A B C D E 1 WT %Survival WT MyD88 -/- TLR7 -/- TLR9 -/ MyD88-/- TLR7 -/- TLR9 -/- Supplemental Figure 1. MyD88/TLR signal pathways play roles in anti-tumor immunity. 1 6 HPV + MEER cells were implanted s.c. in C57BL/6 WT, Myd88-/-, Tlr7-/- and Tlr9-/- mice (n=14-/group) and tumor growth was monitored. (A) WT, (B) Myd88-/-, (C) Tlr7-/- and (D) Tlr9-/- mice. (E) Kaplan-Meier survival curve.
13 Supplemental Table 1. M1 and M related gene expression in tumor tissues treated with i.t. 1V7 or vehicle (extracted from ncounter PanCancer Immune Profiling Panel) a) Gene M1 or M related Control mean (log) 1V7 mean (log) log Fold Change Raw p Adjusted p (BH) b) Ccl E-5.1 Ccr Cd Tnf Ccl M1 Il1b Nos Il1a Cxcl Cd Ido Ptgs Il Il1b Ccl Cxcl Pparg Irf Il1 M Chil Arg Ccl Ccl Cd Mrc1/Cd a) The genes were analyzed from ncounter PanCancer Immune Profiling Panel (NanoString). b) Adjusted p-values by Benjamini-Hochberg procedure.
14 Supplemental Table. Antibodies used in flow cytometry analysis Antibody Clone Color Cat# Source CD3ε 145-c11 APC ebioscience CD4 RM4-5 efluor ebioscience CD8α FITC 5533 BD Biosciences CD8α efluor ebioscience CD11b M1/7 efluor ebioscience CD45 3-F11 PE/Cy BioLegend F4/8 BM8 APC ebioscience CD6 C68C PE BioLegend IFNγ XMG1. APC ebioscience CD4 1C1 PE 1-41 ebioscience CD8 16-1A1 FITC 1476 BioLegend PD-L1 (CD74, B7-H1) 1F.9G PE 1438 BioLegend MHC Class II (I-Ek) S FITC ebioscience CD16/CD3 (FcR).4G Purified BD Biosciences
15 Supplemental Table 3. Antibodies used for immunofluorescent staining Primary antibody Dilution Cat# Source Rat anti-mouse F4/ ebiosciences Rabbit anti-mouse CD6 ab64693 abcam Rat anti-mouse CD BD bioscience Secondary antibody DyLight 549 Goat anti-rat IgG Jackson immunoresearch Alexa 488 Goat anti-rabbit IgG Jackson immunoresearch
16 Supplemental Table 4. Primers and probes used in quantitative RT-PCR analysis Gene Primer and probe number a) Nos Mm445_m1 Ccl5/Rantes Mm1347_m1 Ccl3/Mip1α Mm44159_m1 Tnf Mm44358_m1 Il-1β Mm4348_m1 Il-1a Mm434165_m1 Il-1b Mm188989_m1 Cd6 Mm13936_m1 Ym1/Chil3 Mm657889_mH Cd163 Mm47491_m1 Fizz/Retnla Mm44519_m1 Arg1 Mm475988_m1 Cd4 Mm441891_m1 Cd8 Mm71166_m1 Cd86 Mm444543_m1 Tlr7 Mm44659_m1 Tlr9 Mm446193_m1 Pd-l1 Mm4554_m1 Rps Mm3488_g1 Gapdh Mm _g1 a) The primers and probes were purchased from Thermo Fisher Scientific.
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