Supplementary Figure 1. Flow cytometry panels used for BD Canto (A) and BD Fortessa (B).

Transcription

1 Intra Immune nalysis Surface Supplementary Figure 1. Flow cytometry panels used for D Canto () and D Fortessa (). Name Fluorochrome ID F488 PE PerCp-Cy5.5 PC Paclue PE-Cy7 PC-H7 Lympho* 1 CD56 CD8 CD16 CD3 CD45 CD19 CD4 T1* 2 CD4 CD38 HL-DR CD45R CCR7 CD3 CD8 T2 3 CD45 Tim-3 CD8 LG3 CD3 PD-1 CD4 T2 iso 4 CD45 iso CD8 iso CD3 iso CD4 T3 5 CD4 Tim3 CD8 CD45RO CD3 PD-1 CD45R T4 6 CD11a CD69 CD8 CXCR3 CD3 PD-1 CD4 Myelo* 7 CD45 CD66b HL-DR CD33 CD14 CD123 CD16 Tumor* 8 CD45 PD-L2 CD66b PD-L1 CD33 EpCM CD14 Tumor iso* 9 CD45 iso CD66b iso (PD-L1) CD33 EpCM CD14 T1 1 CD45 CTL-4 CD8 FOXP3 CD3 PD-1 CD4 T2 11 CD45 iso CD8 FOXP3 CD3 iso CD4 T3 12 CD45 Tim-3 CD8 FOXP3 CD3 Ki67 CD4 Sorting 1 CD45 CD56 CD8 CD33 EpCM CD4 CD16

2 % T im -3 + /P D -1 + % T im -3 + /P D -1 + Supplementary Figure 2. PD-1/TIM-3 co-expression by CD8 + T cells () and CD4 + T cells () based on clinical features. C D 8 T IM 3 + /P D *** ** *** *** * s m o k e r n e v e r s m o k e r s q u a m o u s a d e n o c a r c in o m a n o r m a l lu n g K R S n e ith e r E G F R T C + T C -/IC + n e g a tiv e C D 4 T IM 3 + /P D *** ** *** ** * s m o k e r n e v e r s m o k e r s q u a m o u s a d e n o c a r c in o m a n o r m a l lu n g K R S n e ith e r E G F R T C + T C -/IC + n e g a tiv e

3 % P D -L 1 (E p C M ) % P D -L 1 in E p C M (flo w ) Supplementary Figure 3. Percent of PD-L1 + epithelial cells (EpCM + ) detected by flow correlates with the percent of PD-L1 in tumor cells by IHC (). oth correlate with hot and cold cluster breakdown () % P D -L 1 T C ( IH C ) r =.5225 p =.5 PD-L1+ (EpCM) 8 ** h o t c o ld

4 % P D -1 + % P D -1 + Supplementary Figure 4. Strong correlation between %PD-1 and %TIM-3 expression by both CD8 + T cells () and CD4 + T cells (). 1 CD8 8 6 r =.7969 p < % T IM -3 CD4 r =.5695 p < % T IM -3

5 n u m b e r o f m u ta tio n s Supplementary Figure 5. Number of mutations detected by OncoPanel sequencing highly correlates with patient reported smoking status in pack-years r =.5297 p = p a c k -y e a rs

6 Supplementary Figure 6. Significantly differentially regulated genes by Nanostring in hot versus cold immnuophenotypic clusters. Log2 fold change Lower confidenc e limit Upper confidenc e limit P-value FDR Gene.sets D E Cell Functions, T-Cell Functions C4P E Complement GZM E Cell Functions, Cytotoxicity FOS E TNFSF E Chemokines, TNF Superfamily TOLLIP E-5.78 C E-5.78 Complement CXCL E-5.78 Chemokines, Cytokines, Pathogen Defense, Regulation, T-Cell Functions CDK E-5.78 CXCL Chemokines, Regulation, T-Cell Functions VEGFC CREP CXCL Chemokines ITG dhesion CTSH IRC Cell Cycle FOXJ STT Chemokines, Regulation CCL Chemokines EGR Senescence, T-Cell Functions TNFSF Chemokines, TNF Superfamily PL2G Regulation PRKCE Macrophage Functions IL6R Cytokines VCM dhesion, Regulation TTK SIGIRR CD1C T-Cell Functions CCL Chemokines, Regulation PK ICM dhesion, Regulation STT Chemokines, Regulation, T-Cell Functions CCND Cell Cycle C Complement NUP Cell Cycle RORC Cell Functions FCER IL Interleukins, T-Cell Functions PRG Pathogen Defense FCGR Regulation CCL Chemokines, Regulation COL Regulation RRD Cell Functions REPS Cell Functions

7 T L TLS S s score c o re TLS score C D 1 9 e x p r e s s io n ( n a n o s t r in g ) S P s c o r e ( IH C ) SP CD3 medium large medium large large small small large Supplementary Figure 7. TLS size was accessed for quantification (). Percent of CD19 + cells defined by flow correlates with the Nanostring counts for CD19 gene expression () and cell count by IHC (C). TLS score highly correlates with abundance of cells quantified by flow (D) but not with hot or cold immunophenotype (E). Scale bars measuring TLS size to the tenth of a μm are overlayed onto image. 1 8 C 6 r =.6612 p = r =.6598 P< C D c e lls in t o t a l liv e (f lo w ) C D c e lls in t o t a l liv e (f lo w ) D 6 r =.5689 p =.57 E % C %CD19+ D ccells e lls (of(liv live e cells) c e lls ) hot cold

8 Supplementary Figure 8. No significant differences between Ig light chains between tumor cells and normal lung cells or between IL-1 + cells and IL-1 - cells. bsolute counts Relative contribution

9 Supplementary Figure 9. Immune parameters used for clustering in Figure 4. immune parameter 1 %EpCM+ cells (live cells) 2 %CD45+ cells (live cells) 3 %CD3+ T cells (CD45+) 4 %CD19+ T cells (CD45+) 5 %CD56+ NK cells (CD45+) 6 %CD33+ monocytes (CD45+) 7 %CD66b+ granulocytes (CD45+) 8 %CD16+ NK cells 9 %CD8+ T cells (CD3+) 1 CD8/CD4 T cell ratio 11 %TIM-3+ (CD4+ T cells) 12 %PD-1+ (CD4+ T cells) 13 %TIM-3+ (CD8+ T cells) 14 %PD-1+ (CD8+ T cells) 15 %CD4+ T cells (CD3+)