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. 2022 Nov 1;10(11):e005548. doi: 10.1136/jitc-2022-005548

Figure 2.

Figure 2

Multidimensional immune-profiling of tumor suspensions that show ex vivo responsiveness to bsAbs or anti-PD-1 treatment. (A) tSNE plots of pooled treatment conditions from the four responsive patients showing cell-type annotation (treatment of 96 hours; n patients=4, n treatments=4, total n analyzed samples=16). (B) tSNE plots showing identified cell types for pooled patient samples, split by treatment (left). Bar plots showing the proportion of each identified cell type out of all the cells in each treatment (patients are pooled, right). (C) Proportion of CD8+ T and CD45– cells from the four responsive tumor suspension in different treatment conditions. Clustering was conducted using FlowSOM (*p<0.05, **p<0.01, one-way ANOVA with multiple comparisons). (D) Median marker intensity of Ki67 and HLA-DR on the activated CD8+ population in the four responsive tumor suspensions in different treatment conditions. Clustering was conducted using FlowSOM (*p<0.05, **p<0.01, ***p<0.001, one-way ANOVA with multiple comparisons). (E) Heatmap of top significantly differentially regulated proteins, obtained by running a linear model using limma, from the multiplex supernatant analysis, in the four responsive tumor suspensions across different treatment versus control comparisons. (F) Normalized protein expression (NPX) of indicated soluble markers in the four responsive tumor suspensions which show significant differences (FDR <0.05) in different treatment versus control comparisons (BGN: comparing PD1-LAG3 with all other treatments; FGF21: comparing PD1-TIM3 or PD1-LAG3 with isotype or anti-PD1; GALNT7: comparing PD1-TIM3 with all other treatments; SLAMF8: comparing PD1-TIM3 with PD1-LAG3) measured by Olink analysis. ANOVA, analysis of variance; bsAbs, bispecific antibodies; BGN, Biglycan; CD40L, CD40-ligand; CXCL9, chemokine (C-X-C motif) ligand 9; FDR, false discovery rate; FGF21, fibroblast growth factor 21; GALNT7, GalNAc transferase 7; IFN, interferon; IL-17A, interleukin-17A; LAG-3, lymphocyte-activation gene 3; LOD, limit of detection; LTA, lymphotoxin; PD-1, programmed cell death protein 1; SLAMF8, SLAM Family Member 8; TIM-3, T cell immunoglobulin and mucin-domain containing-3; TNFRSF4, TNF Receptor Superfamily Member 4; tSNE, t-distributed stochastic neighbor embedding.