Extended Data Fig. 2: Tumor PD1 + TCF1+lin- CD4 T cells retain proliferation and differentiation capacity.
From: Differentiation fate of a stem-like CD4 T cell controls immunity to cancer
Representative CTV staining and expression of selected differentiation markers after five days of culturing TCF1+ lin- and CD39 + CD4 T cells in unstimulated (U) or a) Th0 (green, TCF1+ n = 11, CD39+ n = 8) d) Eomes (red, TCF1+ n = 9, CD39+ n = 6) and e) TFH (pink, TCF1+ n = 6, CD39+ n = 6) stimulating conditions. Summary plots show the frequency of cells within each population positive for the indicated marker on day 5. Each point represents an individual patient. Multiple conditions were performed with the same patient depending on cell sorting numbers. Median ± 95% CI are represented and analyzed by two-sided unpaired Mann–Whitney U test. U vs. S within the same population were compared across conditions. b) Frequency of original plated cells undergoing division for TCF1+ or CD39 + CD4 T cells in Th0 conditions. Median is represented and analyzed by two-sided unpaired Mann-Whitney U test. c) Frequency of TCF1+ or CD39 + CD4 T cells expressing the indicated marker on day 5 under Th1 (TCF1+ n = 13, CD39+ n = 2) or Treg stimulation (TCF1+ n = 10, CD39+ n = 5). Median ± 95% CI are represented and analyzed by two-sided unpaired Mann–Whitney U test. f) Summary plots represent the frequency of cells within each sorted population (TCF1+ lin- or CD39+) expressing the indicated transcription factor after 5 days of culture in each of the respective conditions tested. Cytokine stimulation conditions were the following: Th0 (IL-2), Th1 (IL-2 and IL-12), Treg (IL-2, TGF-beta, and anti-IFN gamma), Tfh (Activin A, IL-12), or Eomes (IL2, IL-12, IL-4, aIFNg). g-h) Representative plots of various markers on sorted TCF1+ lin- or CD39 + CD4 T cells after 5-days of 1:1 co-culture with patient matched dendritic cells (n = 7 patients for TCF1+ lin- and n = 4 patients for CD39+). Medians are shown and analyzed by two-sided unpaired Mann–Whitney U test. i) Frequency of original plated cells undergoing division for TCF1+ or CD39 + CD4 T cells after DC co-cultures. Medians are shown and analyzed by two-sided unpaired Mann–Whitney U test. j) Representative plot of TBET expression on TCF1+ lin- CD4 T cells in DC co-cultures with exogenous IL-12 (no IL12 n = 7, IL12 n = 4). Mean ± s.d. are shown and analyzed by two-sided unpaired Mann–Whitney U test between each division. k) Experimental design to test the capacity of PD1 + CD39 + CD4 T cells to proliferate with 4000U/ml of exogenous IL-2. l) Proliferation and FOXP3 expression on sorted CD39 + CD4 T cells after 5-days of CD3/CD2/CD28 bead stimulation with 10U/ml (low, n = 3) or 4000U/ml (high, n = 4) of exogenous IL-2. Medians are shown and analyzed by two-sided unpaired Mann–Whitney U test. m) UMAP projections of the distribution of TCR clonotypes corresponding to cells in the Treg (blue) or Eomes (red) clusters. Summary bar graph shows the cluster distribution of the 3 most dominant TCR clonotypes across all clusters for one patient. The number of cells sharing the respective TCR clonotype is indicated below. n) Correlation matrix showing the number of clonotypes shared by each population in the tumor for both patients.
