Treg Cells in Live CD45+ Cells
14.1
Cyclo + ACT 0.25
B
Naive Cyclo Cyclo + ACT
PMN-MDSCs & M-MDSCs in Live CD45+CD11bHi MHCII- Cells
Cyclo + ACT
CTLs, B Cells, NK Cells in Naive Tumors before ACT
0.61 0.61
Foxp3 FITC
CD4 PE Cy7
Ly6G PE Cyclo + ACT
+ anti-CSF1R
CD64 APC Macrophage Numbers
0
Activation Phenotypes of Injected OT1 T cells
10.2
% of Max
1.45
Figure S1. Immune Landscape of SCC Tumors at Different Stages Post-ACT Treatment, Related toFigure 1
(A) Flow cytometry quantifications of tumor infiltrating CD8+ CTLs, CD19+ B cells and NK1.1+ NK cells in a naive HRASG12Vskin tumor before ACT treatment.
(B) Flow cytometry and numerical analysis of tumor infiltrating CD4+Foxp3+Regulatory T (Treg) cells in a naive HRASG12Vskin tumor, HRASG12Vskin tumor receiving one dose of cyclophosphamide (Cyclo), or cyclophosphamide plus ACT treatment.
(C) Flow cytometry and numerical analysis of tumor infiltrating CD11b+Ly6G+Ly6Clopolymorphonuclear (PMN) or CD11b+Ly6G-Ly6Chimonocytic (M)-myeloid-derived suppressor cells (MDSCs) in a naive HRASG12Vskin tumor, HRASG12Vskin tumor receiving one dose of cyclophosphamide (Cyclo), or cyclophosphamide plus ACT treatment± Gr1 blocking Ab to deplete MDSCs.
(D) Flow cytometry and numerical analysis of infiltrating CD11b+MHCII+CD64+tumor-associated macrophages (TAMs) in a naive HRASG12Vskin tumor, HRASG12Vskin tumor receiving one dose of cyclophosphamide (Cyclo), or cyclophosphamide plus ACT treatment± CSF1R blocking Ab to deplete TAMs.
(E) Flow cytometry analyses of T cell activation markers (CD25, CD69, CD44 and CD62L) in the OT-I T cells at the first and second weeks after ACT. The naive CD8+ T cells from donor spleen were used as negative control.
Figure S2. Single-Cell RNA-Seq Analysis of SC Signatures of ACT-Surviving Tumor Cells and Tumor Cells from Naive HRASG12VSkin Tumors, Related toFigure 2
(A) Sorting strategy for purifying live, non-epithelial lineage negative, mCherry+epithelial cells from tumors remaining after ACT treatment.
(B) Heatmap of RNA-seq analyses of individual ACT-surviving tumor cells from (A), revealing transcriptome similarities between single cells as measured by Pearson’s correlation coefficient matrix.
(C) t-SNE plots showing expression of various epithelial-specific keratin markers in ACT survivors. Krt18 is expressed specifically by SCC basal cells and is seen in the C1 cluster. Krt1, Krt10, Krt6, and Krt16 are markers of suprabasal cells of well-differentiated SCCs and are seen in C2, C3, C4 clusters.
(D) Sorting strategy for purifying integrin a6hiTGFb-reporter+tSCs (green), as well as non-TGFb-responding basal progenitors (red) and suprabasal cells (black) from naive HRasG12Vskin tumors.
(E) Single cell RNA-seq of individual tumor cells. Shown are unbiased clustering of transcriptomes of FACs purified individual integrin a6hiTGFb reporter+tSCs (C1), non-TGFb-responding basal progenitors (C2), and suprabasal cells (C3). Each cell is represented as a dot, colored by clustering algorithm and plotted on the t-SNE graph.
(F) t-SNE plots showing enriched expression of SC signature genes in the C1 representing the integrin a6hiTGFb-reporter+tSCs.
(G) t-SNE plot showing the absence of immune cell specific markers, including Cd45 for pan immune cells, Cd11b for myeloid cells, Cd11c for dendritic cells and Cd207 for Langerhan cells.
Figure S3. TGF-b-Responding Cells in ACT-Treated Tumors Are tSCs, Related toFigure 3
(A) Representative IMF and quantification of Ki67 (green), TGFb-reporter (red) and Integrin a6 (blue) in tumor at 1 week post-ACT. Three tumors and > 150 cells from each of two sagittal sections per tumor were analyzed. Scale bars, 50 mm.
(B) Representative IMF and quantification of EdU (green), TGFb-reporter (red) and Integrin a6 (blue) in tumor at 1 week post-ACT after two pulses of EdU at 24 hr and 12 hr before collection. Three tumors and > 150 cells from each of two sagittal sections per tumor were analyzed. Scale bars, 50 mm.
(C) (Top left) Experimental scheme for tSC lineage tracing. (Top right), quantifications of tSC markers expressed by Tomato+ cells following short-term tamoxifen treatment. (Bottom) Representative IMF images confirming that the TGFb-reporter drives CreER activity specifically in SCs, which are positive for CD44 and CD34. Note that most tSCs (CD44Hiand CD34Hi) are marked by Tomato. > 150 cells from each of two sagittal sections per tumor were analyzed. Scale bars, 50 mm.
(D) Lineage-tracing of TGFb-responding tSCs following ACT therapy. Construct design (Top), experimental scheme (middle), and flow cytometry based quantification (bottom) from lineage-traced tumor at 1 week post-ACT.
(E) Flow cytometry quantifications of integrin a6hiTGFb-reporter+tSCs in tumors that are treated with control or blocking Abs targeting Gr1 or CSF1R for one week, then followed by ACT plus the same Ab treatment for another week.
(F) t-SNE plots showing the low expression of PDL1 (Cd274), and broad distribution of Cd47 across different tumor populations.
Figure S4. CD80 Expression by tSCs in Relapsed SCCs after ACT, DMBA/TPA-induced Mouse Skin SCCs, and Human Skin SCCs, Related to Figure 4
(A) Immunofluorescence and quantifications of CD80 (green) and pSMAD2+(blue) cells in Tomato+tumor cells that relapsed following ACT treatment (red). Data are from 3 tumors and two sagittal sections (> 150 cells each) for each tumor analyzed at each time point. All scale bars = 50 mm.
(B) Immunofluorescence co-labeling of CD80 (green), pSMAD2/3 (red) and Keratin 14 (blue) in the skin epithelium of a DMBA/TPA induced tumor generated on wild-type C57/BL6 mice (left) or Rag2 / mice (right), which lack a functional adaptive immune system.
(C) Immunofluorescence labeling of CD80 (red) and Keratin 14 (green) epithelial cancer basal cells in (left) xenografts of the human skin SCC line A431 on SCID mice or (right) cells that have undergone metastasis to the lung following injection of A431 skin SCC cells into the tail vein of SCID mice.
Figure S5. Cd80 Silencing by shRNA Has a Similar Impact on Tumorigenesis as CRISPR-Mediated Cd80 Ablation, Related toFigure 5 (A) Flow cytometry quantification of proliferation marker (Ki67) in cytotoxic T cells that infiltrated CD80(+) or CD80(-) PDVC57 C57/Bl6 SCCs generated from engraftment into C57BL/6 immune competent mice.
(B) Isogenically matched, PDVC57 SCC cells were transduced with LV expressing Scrambled shRNA or Cd80 shRNA and engrafted onto WT C57/Bl6 mice. Note that Cd80 silencing diminished tumorigenesis, analogous to what we observed with CRISPR/Cas-mediated ablation of Cd80. Data are mean± SEM. ***p < 0.001, unpaired Student’s t test, n = 6.
(C) Cells as in (A) were engrafted onto Rag2/ mice, which lack an adaptive immune system. Note that loss of adaptive immunity eliminates the tumorigenesis defects from CD80 silencing. n = 15; data are mean± SEM.
(D) Cells as in (A) were engrafted onto wild-type mice that had been immunodepleted for CD8+T cells. Note that loss of cytotoxic T cells eliminated the tumorigenic defects resulting from Cd80 silencing in the SCC cells. n = 15; data are mean± SEM.
(E) Flow cytometry quantifications of the CD8+T cells infiltrating Scrambled shRNA or Cd80 shRNA transduced tumors (percentage of total CD45+cells).
***p < 0.001.
OVA 257-264 SIINFEKL
OT1 Purify CD8+ T Cells
Label with CFSE
T Cell Proliferation (% of Total CD8+ T Cells)
0 20 40 60 80
T Cell Proliferation (% of Total CD8+ T Cells)
0 20 40 60 80
T Cell Proliferation (% of Total CD8+ T Cells)
0
Relative Contribution of CTLA4, CD28 or PDL1 to Tumor CD80-Mediated T Cell Supression Experimental Scheme for SCC/T Cell Co-culture to
Dissect the Involvement of CTLA4, CD28 or PDL1
**
Figure S6. Immunosuppressive Effects of tSC-CD80 Depends on T Cell Surface Receptor CTLA4, but Not CD28 or PDL1, Related toFigure 6 (A) Scheme showing the experimental setup for SCC cell co-culture with activated OT-I cytotoxic T cells to dissect the relative contributions of putative CTL receptors for the surface CD80 on SCC tSCs.
(B) Measurement of OT-I T cell proliferation as monitored by flow cytometry quantification of CFSE dye dilution in activated primary OT-I T cells when co-cultured with CD80(+) or CD80(-) PDVC57 SCC cells, with or without CTLA4 blocking Abs. Data are mean± SEM. **p < 0.005, unpaired Student’s t test, n = 6.
(C) Measurement of OT-I T cell proliferation as monitored by flow cytometry quantifications of CFSE dye dilution in activated primary OT-I T cells when co-cultured with CD80(+) or CD80(-) PDVC57 SCC cells, with or without CTLA4 blocking Abs, CD28 agonist Abs or CD28 antagonist Abs. Data are mean± SEM. **p < 0.005, unpaired Student’s t test, n = 6.
(D) Measurement of OT-I T cell proliferation as monitored by flow cytometry quantifications of CFSE dye dilution in activated primary OT-I CD8+ T cells when co-cultured with CD80(+) or CD80(-) PDVC57 SCC cells, with or without PDL1 blocking Abs, which eliminate possible interactions between PDL1 on the surface of PDVC57 tumor cells and either PD1 on the surface of CD8+ T cells or tumor CD80 (CIS interactions). See text for further details. Data are mean± SEM. **p < 0.005, unpaired Student’s t test, n = 6.
*p < 0.1, **p < 0.01.
Figure S7. CD80 on TGF-b-Responding tSCs Acts through CTLA4 on CTLs to Enable tSCs to Survive ACT and Evade Immune Attack, Related toFigure 7
(A) CD80(+) or CD80(-) PDVC57 SCC cells were transplanted into C57/BL6 mice treated with either isotype control or CTLA4 blocking Abs, and tumor weight was analyzed at 4 weeks post-grafting. Data are mean± SEM.
(B) Scheme showing the experimental setup for generating OT-I T cell-specific CTLA4 silencing for use in ACT.
(C) Flow cytometry analysis of T cell exhaustion markers (CTLA4, PD1, Tim3 and LAG3) and proliferation marker (Ki67) in the injected OT-I T cells that had been transduced with control or Ctla4 shRNA and then collected at 2 week post-ACT. The naive CD8+ T cells from donor spleen were used as negative control. These data are from the same experiment shown inFigure 7B. CTLA4(+) OT1 and Naive OT1 data as control groups in this experiment are intentionally duplicated here.
(D) Flow cytometry quantifications of integrin a6hiTGFb-reporter+tSCs in mice treated with control or Ctla4 shRNA transduced OT-I T cells at 1 week post-ACT.
(E) Flow cytometry based quantification of lineage-traced (GFP+), TGFb-responding (mCherry+) Cd80-null tSCs in Cd80-null tumors following 1 week post-ACT.
Note that CreER was selectively activated in naive tSCs at a time when all differentiated tumor cells were GFP(-). Thus, had the tSCs present in ACT-treated tumors been derived from de-differentiation of differentiated tumor cells, they would be mCherry+ but many would be GFP(-).
(F) Changes in tumor size following depletion of injected OT-I T cells after ACT treating the TGFb-responding tSC specific CD80 KO tumors. Tumor volumes are normalized to their original size. Data are mean± SEM.
(G) Volume changes of the relapsed Scr sgRNA or Cd80 sgRNA transduced tumors following ACT. n = 12 for control; n = 7 for tSC-specific targeting of Cd80. Data are mean± SEM.
(H) Immunofluorescence for CD80 (green), integrin a6hi(blue) and TGFb-reporter (red) to examine tumor cells that relapsed following ACT treatment. Prior to treatment, Cd80 was depleted specifically in TGFb-responding tSCs within the tumors of these mice. Note that, whereas majority of a6+TGFb-reporter+tSCs in these relapsed tumor are CD80 negative (arrowhead), a few TGFb-reporter+tSCs are CD80 positive (arrow). All scale bars represent 50 mm.
**p < 0.01, ***p < 0.001.