Journal: medRxiv
Article Title: TNF blockade with certolizumab improves the efficacy of anti-PD-1 and anti-CTLA-4 combination therapy for melanoma
doi: 10.64898/2026.02.11.26346073
Figure Lengend Snippet: a , Diagram summarizing the analysis strategy. The phenotype of circulating immune cells of TICIMEL patients was compared to the one of patients treated with nivolumab and ipilimumab alone (MELANFα clinical trial). T cell responses in the blood of patients of all three cohorts (ipi/nivo; ipi/nivo/certo and ipi/nivo/inflix) were analysed at W0 and W6 by CITE-Sequencing (CITE-Seq) and flow cytometry. b-i , Peripheral blood monuclear cells isolated from the blood of patients (8 patients per cohort) before treatment initiation (week 0, W0) and 6 weeks after the beginning of treatments (week 6, W6) was anlayzed by CITE-Seq. b , Unified Manifold and Approximation and Projection (UMAP) of CD8 T cells of all patients and time points, identifying 8 clusters of CD8 T cells [7 clusters [C0-C7] of CD8 T cells and 1 cluster of Mucosal Associated Invariant T cells (MAIT)]. c , Dotplot showing the expression of key gene markers in the identified CD8 subclusters (proportion and average expression). d , Trajectory analysis using slingshot and showing 3 possible endpoints. e , Proportion of cells from cluster 2 between W0 and W6 per treatment cohort. *p<0.05 Wilcoxon. f , A differential gene expression analysis was performed to compare W6 and W0 cells from cluster 2, separating each treatment cohort. Over-representation analysis (ORA) was then performed on the 3 lists of differentially expressed genes (DEG) to identify the 10 most significantly deregulated pathways after ipi/nivo, ipi/nivo/certo or ipi/nivo/inflix (see Extended data Fig. 2). The bar plot shows the fold enrichment of the 21 identified pathways in cells from cluster 2 after ipi/nivo; ipi/nivo/certo or ipi/nivo/inflix. g , Genes contributing to the identification of pathways depicted in “f” were identified. Those, which were differentially modulated (log2 fold change (W6/W0) of the gene expression) between at least two treatment cohorts were selected and plotted on a heatmap. Gene induction or downregulation is shown per patient and treatment cohort. On the left side, yellow squares identified a significant difference in gene expression comparing two treatment cohort at a time (p<0.05, Wilcoxon). h , Boxplot showing the log2 fold change (W6/W0) in the expression of genes from the heatmap in “g” and involved in T cell activation and metabolism ( JMJD6, MYH9, GCH1 ), and differentiation ( FOXO1, ICOS, TCF7 ) per treatment cohort. i , Heatmap showing the modulation in the activity of IRF1, IRF2, IRF3 and STAT1 transcription factors (TF) in cluster 2 cells between W0 and W6 per patient and treatment cohort (DecouplR package). On the left side, yellow squares identified a significant difference between the evolution of TF activities comparing two treatment cohorts at a time (p<0.05, Wilcoxon; ns: non-significant). j , CD8 T cells were isolated from W0 and W6 PBMC of patients before polyclonal restimulation in the presence of vesicular transport inhibitors. IFN-γ production was assessed by flow cytometry. CR: complete response; PR: partial response; SD: stable disease; PD: progressive disease reflecting the best response over the two years of follow-up for TICIMEL patients and response at 12 weeks for MELANFα patients. Statistics: the Wilcoxon matched-pairs signed-rank test was applied to data sets with n>10. For CITE-Seq analyses, one patient from the ipi/nivo cohort was excluded from the analyses in Fig. 2g-i because fewer than 10 cells were detected in cluster 2 at one time point.
Article Snippet: The capacity of CD8 T cells to produce IFN-γ was assessed on CD8 T cells isolated from PBMC (CD8 MicroBeads, human, Miltenyi Biotech).
Techniques: Sequencing, Flow Cytometry, Isolation, Expressing, Gene Expression, Activation Assay, Activity Assay
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![a , Diagram summarizing the analysis strategy. The phenotype of circulating immune cells of TICIMEL patients was compared to the one of patients treated with nivolumab and ipilimumab alone (MELANFα clinical trial). T cell responses in the blood of patients of all three cohorts (ipi/nivo; ipi/nivo/certo and ipi/nivo/inflix) were analysed at W0 and W6 by CITE-Sequencing (CITE-Seq) and flow cytometry. b-i , Peripheral blood monuclear cells isolated from the blood of patients (8 patients per cohort) before treatment initiation (week 0, W0) and 6 weeks after the beginning of treatments (week 6, W6) was anlayzed by CITE-Seq. b , Unified Manifold and Approximation and Projection (UMAP) of CD8 T cells of all patients and time points, identifying 8 clusters of CD8 T cells [7 clusters [C0-C7] of CD8 T cells and 1 cluster of Mucosal Associated Invariant T cells (MAIT)]. c , Dotplot showing the expression of key gene markers in the identified CD8 subclusters (proportion and average expression). d , Trajectory analysis using slingshot and showing 3 possible endpoints. e , Proportion of cells from cluster 2 between W0 and W6 per treatment cohort. *p<0.05 Wilcoxon. f , A differential gene expression analysis was performed to compare W6 and W0 cells from cluster 2, separating each treatment cohort. Over-representation analysis (ORA) was then performed on the 3 lists of differentially expressed genes (DEG) to identify the 10 most significantly deregulated pathways after ipi/nivo, ipi/nivo/certo or ipi/nivo/inflix (see Extended data Fig. 2). The bar plot shows the fold enrichment of the 21 identified pathways in cells from cluster 2 after ipi/nivo; ipi/nivo/certo or ipi/nivo/inflix. g , Genes contributing to the identification of pathways depicted in “f” were identified. Those, which were differentially modulated (log2 fold change (W6/W0) of the gene expression) between at least two treatment cohorts were selected and plotted on a heatmap. Gene induction or downregulation is shown per patient and treatment cohort. On the left side, yellow squares identified a significant difference in gene expression comparing two treatment cohort at a time (p<0.05, Wilcoxon). h , Boxplot showing the log2 fold change (W6/W0) in the expression of genes from the heatmap in “g” and involved in T cell activation and metabolism ( JMJD6, MYH9, GCH1 ), and differentiation ( FOXO1, ICOS, TCF7 ) per treatment cohort. i , Heatmap showing the modulation in the activity of IRF1, IRF2, IRF3 and STAT1 transcription factors (TF) in cluster 2 cells between W0 and W6 per patient and treatment cohort (DecouplR package). On the left side, yellow squares identified a significant difference between the evolution of TF activities comparing two treatment cohorts at a time (p<0.05, Wilcoxon; ns: non-significant). j , CD8 T cells were isolated from W0 and W6 PBMC of patients before polyclonal restimulation in the presence of vesicular transport inhibitors. IFN-γ production was assessed by flow cytometry. CR: complete response; PR: partial response; SD: stable disease; PD: progressive disease reflecting the best response over the two years of follow-up for TICIMEL patients and response at 12 weeks for MELANFα patients. Statistics: the Wilcoxon matched-pairs signed-rank test was applied to data sets with n>10. For CITE-Seq analyses, one patient from the ipi/nivo cohort was excluded from the analyses in Fig. 2g-i because fewer than 10 cells were detected in cluster 2 at one time point.](https://med-rxiv-images-cdn.bioz.com/dois_ending_with_73/10__64898_slash_2026__02__11__26346073/10__64898_slash_2026__02__11__26346073___F2.large.jpg)