murine cd39  (Miltenyi Biotec)

Journal: Journal for immunotherapy of cancer

Article Title: TMED inhibition suppresses cell surface PD-1 expression and overcomes T cell dysfunction.

doi: 10.1136/jitc-2024-010145

Figure Lengend Snippet: Figure 4 TMED expression in CD8 T cells positively correlates with T cell dysfunction and lack of immunotherapy response in patients. (a) Representative flow cytometry histograms of CD39-PE-Vio770 MFI of resting or anti-CD3-activated OT-I/Cas9 CD8 T cells carrying either an sgRNA targeting Tmed10 or a non-targeting control sgRNA. (b) Quantification of samples in (a) for CD39-PE-Vio770 abundance. Each data point indicates data obtained with CD8 T cells from an independent spleen. Error bars denote SD. Statistical analysis was performed with a one-way analysis of variance, followed by a Tukey post hoc test. (c) Correlation plot of TMED complex expression and T cell dysfunction signature expression in CD8 T cells in melanomas from patients.12 Each data point indicates mean expression across all CD8 T cells in an individual patient. The best fit line is shown with the shaded region denoting the 95% CI. (d) TMED complex expression in exhausted CD8 T cells and other CD8 T cells in 31 single-cell RNA sequencing datasets. Each data point indicates the mean expression of the TMED complex across all exhausted CD8 T cells or all CD8 T cells in each dataset. In the boxplots, the center line, box edges and whiskers denote the median, IQR and the rest of the distribution respectively, with outliers being shown separately. Statistical testing was performed by the Wilcoxon rank-sum test. (e) TMED complex expression in CD8 T cells of responders (R) and non-responders (NR) to ICB, respectively.12 Each data point indicates expression of the TMED complex in single CD8 T cells. In the boxplots, the center line, box edges and whiskers denote the median, IQR and the rest of the distribution, respectively, with outliers being shown separately. Statistical testing was performed by the Wilcoxon rank-sum test. (f) ROC curve analysis of patients from the Sade-Feldman cohort who had their tumor biopsied before the start of ICB treatment, using TMED complex expression to distinguish R from NR patients. (g) As in (f) but for patients whose biopsy was taken after the onset of therapy. (h) Kaplan-Meier survival curve of patients who received TIL therapy with a TIL product with high (50% highest expressors) or low (50% lowest expressors) expression of the TMED complex. Statistical testing was performed by log-rank test. (i) ROC curve analysis of patients from the TIL cohort, using TMED complex expression in the TIL product to distinguish patients who failed to survive for more than 1 year from those who did. (j) TMED complex expression in patients who survived for more than 1 year after TIL infusion (R) and those who did not (NR). In the box plots, the center line, box edges and whiskers denote the median, IQR and the rest of the distribution, respectively, with outliers being shown separately. Statistical testing was performed by Student’s t- test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. ICB, immune checkpoint blockade; MFI, mean fluorescence intensity; ROC, receiver-operating characteristic; TIL, tumor-infiltrating lymphocytes; AUC, area under the ROC curve.

Article Snippet: Antibodies against murine and human PD- 1 (both PE- conjugated), murine and human CD137 (both APCconjugated), murine CD8a (FITC- conjugated), murine CD39 (PE- Vio770- conjugated), murine CD45 (APCVio770- conjugated; all Miltenyi Biotec), murine CTLA- 4 (PE- Cy7- conjugated, BioLegend), murine PD- L1 (BV711conjugated, BD) human CD8A (BB515- conjugated, BD) and human IgG Fc (PE- conjugated, BioLegend).

Techniques: Expressing, Flow Cytometry, Control, RNA Sequencing, Fluorescence