Supplement Figures S1-S4 from T-cell Homing Therapy for Reducing Regulatory T Cells and Preserving Effector T-cell Function in Large Solid Tumors

Fig. S1. CD4+ T cell infiltration in mouse and human tumor sections. Fig. S2. Correlations between the numbers of infiltrated murine T cells and the ratios of costimulatory and coinhibitory receptors. Fig. S3. Quantitative PCR analysis of CXCL9 and CXCL10 mRNA expression in tumor cells that were treated with doxorubicin. Fig. S4. Induction of CXCL9 and CXCL10 at mRNA levels in LLC and Mel2549 tumors after the treatment with IL-12 plus doxorubicin plus T cell infusion.</p

Overexpression of PD-L1 is associated with higher expression of EGFR and immune-related genes, and sensitivity to EGFR inhibitors.

<p>(A) Lung cancer cell lines (N186) from Cancer Cell Line Encyclopedia (CCLE) database were divided into two groups according to median expression of PD-L1 at Log₂ Robust Multi-array Average (RMA), 5.087. Mean Log₂ RMAs of PD-L1 expression of PD-L1^high and PD-L1^low groups were 7.171 ±1.508 and 4.578 ± 0.2630, respectively. (B) EGFR expression of PD-L1^high and PD-L1^low lung cancer cell lines, and correlation between EGFR and PD-L1 expression (C) The half maximum inhibitory concentration (IC₅₀) of Erlotinib for PD-L1^high lung cancer cell lines (N51) and PD-L1^low lung cancer cell lines (N40). (D) Expression of MHC-I and genes associated with MHC-I antigen presentation from PD-L1^high and PD-L1^low lung cancer cell lines. (E) Expression of MHC-II and genes associated with MHC-II antigen presentation from PD-L1^high and PD-L1^low lung cancer cell lines. (F) Expression of genes associated with inflammation from PD-L1^high and PD-L1^low lung cancer cell lines. PD-L1^high lung cancer cell lines are associated with higher expression of EGFR and sensitivity to erlotinib (B and C). In addition, PD-L1^high lung cancer cell lines express genes associated with MHC-I and II antigen presenting pathways higher than PD-L1^low lung cancer cell lines (D and E). Significantly higher expression of IL-6 and positive regulators for IFNγ signaling pathway such as STAT1, STAT3, and IRF9 were associated with PD-L1^high lung cancer cell lines, while the expression of negative regulators for IFNγ signaling pathway such as IRF2BP2 and SIN3A were significantly lower in PD-L1^high lung cancer cell lines (F). P values were calculated by Wilcoxon-Man-Whitney test and summarized as follows, ****: <i>p</i> < 0.0001, ***: <i>p</i> = 0.0001 to 0.001, **: <i>p</i> = 0.001 to 0.01, *: <i>p</i> = 0.01 to 0.05, ns: <i>p</i> > 0.05.</p

Supplementary Figures 1 through 9 and Supplementary Materials and Methods from Induction of NKG2D Ligands on Solid Tumors Requires Tumor-Specific CD8⁺ T Cells and Histone Acetyltransferases

Fig. S1. Gradual reduction of Rae-1 during tumor progression. Fig. S2. Induction of Rae-1 by chemotherapy in vitro but not in vivo. Fig. S3 Effector molecule perforin expression in tumors after indicated treatments. Fig. S4 Rae-1 expression is not induced by IL-12 plus doxorubicin in normal tissues. Fig. S5 Accumulation of NKG2D+CD8+ T cells in tumors after IL-12 plus doxorubicin treatment. Fig. S6 Immunochemistry staining of Rae-1 and TUNEL assay in tumor sections. Fig. S7 Rae-1 induction by IL-12 plus doxorubicin only occurs on tumor cells in vivo. Fig. S8 Antitumor cytotoxicity of IL-12 plus doxorubicin abolished by depletion of CD8+ T cells, but not CD4+ T cells or NK cells. Fig. S9 Enrichment of NK cells, CD4+ T cells, or CD8+ T cells in splenocytes collected from BALB/c mice treated with IL-12 plus doxorubicin.</p

Differential effects on MHC-II in lung cancer cell lines by IFNγ and EGFR inhibitors.

<p>Various lung cancer cell lines were treated with IFNγ at a concentration of 1000 pg/ml in the presence or absence of erlotinib at concentration of 10 μM for 24 hours, and assessed for surface expression of MHC-II. (A) The average MFI ± SD of MHC-II staining were represented as bar graphs. (B and C) Lung cancer cell lines were divided into two groups according to the half maximum effective concentration, EC₅₀ (EC₅₀ <8 vs EC₅₀ ≥ 8) of erlotinib, and average MFI of MHC-II for individual cell line was represented as a single dot for further statistical analysis to assess the effect of IFNγ and/or erlotinib on the expression of MHC-II. There was only minimal baseline expression of MHC-II in untreated lung cancer cell lines regardless of sensitivity to erlotinib (B), but the treatment with IFNγ significantly up-regulated the expression of MHC-II more often on lung cancer cell lines sensitive to erlotinib compared to lung cancer cell line resistant to erlotinib (C). The addition of erlotinib to untreated or IFNγ treated lung cancer cell lines did not significantly altered the expression of MHC-II in most cell lines (B and C). Abbreviations used in this experiment are as follow, E: erlotinib, I: IFNγ. The experiment shown here was performed in triplicates, and a representative of three independent experiments. P-value less than 0.05 (*) was determined as being significant, “ns” represents “not statistically significant”.</p

Lung Cancer Cell lines used in the evaluation of PD-L1 protein expression.

<p>Lung Cancer Cell lines used in the evaluation of PD-L1 protein expression.</p

Differential effects on MHC-I in lung cancer cell lines by IFNγ and EGFR inhibitors.

<p>Various lung cancer cell lines were treated with IFNγ at a concentration of 1000 pg/ml in the presence or absence of erlotinib at concentration of 10 μM for 24 hours, and assessed for surface expression of MHC-I. (A) The average mean fluorescence intensities (MFIs) ± standard deviation (SD) of MHC-I staining were represented as bar graphs.(B and C) For further statistical analysis, lung cancer cell lines were divided into two groups according to the half maximum effective concentration, EC₅₀ (EC₅₀ <8 vs EC₅₀ ≥ 8) of erlotinib, and average MFI of MHC I for individual cell line was represented as a single dot for further statistical analysis to assess the impact of treatment with IFNγ and/or erlotinib on MHC I expression within group of lung cancer cell lines sensitive to erlotinib (EC₅₀ <8) or resistant to erlotinib (EC₅₀ ≥8) There was a trend toward higher expression of MHC-I in untreated lung cancer cell lines sensitive to erlotinib (BB), and the treatment with IFNγ further up-regulated the expression of MHC-I more often on lung cancer cell lines sensitive to erlotinib compared to lung cancer cell line resistant to erlotinib (C). The addition of erlotinib to untreated or IFNγ treated lung cancer cell lines did not significantly altered the expression of MHC-I in most cell lines regardless of sensitivity to erlotinib (B and C). Abbreviations used in this experiment are as follow, E: erlotinib, I: IFNγ. The experiment shown here was performed in triplicates, and a representative of three independent experiments. P-value less than 0.05 was determined as statistically significance. “NS” represent “statistically non-significant”.</p

EGFR inhibitor down-regulates the PD-L1 expression on lung cancer cell lines.

<p>Various lung cancer cell lines were treated with IFNγ at a concentration of 1000 pg/ml in the presence or absence of erlotinib at concentration of 10 μM for 24 hours, and assessed for surface expression of PD-L1. (A) The average MFIs± SD of PD-L1 staining were represented as bar graphs in (A) (B and C) Lung cancer cell lines were divided into two groups according to EC₅₀ (EC₅₀ <8 vs EC₅₀ ≥ 8) of erlotinib, and average MFI of PD-L1staining for individual cell line was represented as a single dot in dot plots for further statistical analysis to assess the influence of IFNγ and/or erlotinib on PD-L1 expression. There was a trend toward higher expression of PD-L1 in untreated lung cancer cell lines sensitive to erlotinib, and erlotinib treatment down-regulated PD-L1 expression on lung cancer cell lines sensitive to erlotinib but not statistically significant (B). The treatment with IFNγ significantly up-regulated the expression of PD-L1 proteins on lung cancer cell lines sensitive to erlotinib, and the addition of erlotinib significantly inhibited IFNγ-induced PD-L1 overexpression on lung cancer cell lines sensitive to erlotinib (C). Abbreviation used in this experiment is as follows. E: Erlotinib, I: IFNγ. The experiment shown here was performed in triplicates, and a representative of three independent experiments. (D) Various lung cancer cells were cultured in the presence or absence of erlotinib at a concentration of 10 μM for 24 hours, and the total RNA was isolated for Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR) performed in quadruplicates to evaluate the expression of PD-L1 transcripts. The relative PD-L1 expression of individual lung cancer cell line treated with erlotinib to untreated control was represented as a single dot. Lung cancer cell lines were again divided into two groups according to EC₅₀ (EC₅₀ <8 vs EC₅₀ ≥ 8) of erlotinib. Erlotinib down-regulated PD-L1 expression at the transcript level, and inhibitory effect was more pronounced in lung cancer cells sensitive to EGFR inhibitors. P-value less than 0.05 was determined as statistically significance. “NS” represent “statistically non-significant”.</p

The correlation between expression of immune-related genes and PD-L1 of lung cancer cell lines from CCLE.

<p>The correlation between expression of immune-related genes and PD-L1 of lung cancer cell lines from CCLE.</p

EGFR inhibitor increases antigen specific T cell mediated tumor killing.

<p>(A) The expression of HLA-A2 on HLA-A2⁺ lung cancer cell line, H441, was confirmed using flowcytometry. (B) Mart-1 (M27) specific cytotoxic T cell line (CTL), 1007G, was stained with anti-CD3 and Mart-1 (M27)/HLA-A2 tetramer to confirm the antigenic specificity. More than 90% of T cells expressed T cell receptors specific for Mart-1(M27)/HLA-A2 tetramers. (C) H441 cells pretreated with or without erlotinib in the presence or absence of Mart-1(M27) or pp65 control peptide were used as target cells for standard cytotoxic T cell (CTL) assay using Mart-1(M27) specific CTLs as effector T cells. Percent Specific cytotoxicity was calculated as follows: [(test release-spontaneous release)/(maximum release-spontaneous release)] × 100. Mart-1 (M27) specific CTL efficiently lysed H441 only in the presence of Mart-1 peptide but not control peptide, and the treatment of erlotinib significantly increased T cell mediated killing. The experiment shown here was performed in quadruplicates, and a representative of two independent experiments.</p

The expression of immune-related genes associated with PD-L1^High vs PD-L1^Low expressing lung cancer cell lines from CCLE.

<p>The expression of immune-related genes associated with PD-L1^High vs PD-L1^Low expressing lung cancer cell lines from CCLE.</p