Figure 1.
Activation of human CD8+ T cells via the Wnt pathway generates cells with a naive phenotype.
CD8+ T cells from peripheral blood of healthy donors were either untreated or polyclonally activated in the presence of TWS119 (Wnt pathway activator) prior to their analysis. (A) Evaluation of β-catenin protein expression by Western blot analysis from fresh CD8+ T cells or CD8+ T cells activated for 6 h in the presence of anti-CD3 and IL-2, with or without TWS119. β-actin protein level was used as a loading control. (B) Representative dot plot analysis of CD45RA and CD62L surface expression on CD8+ T cells either fresh (left) or cultured 5 days with anti-CD3 and IL-2, with (middle) or without (right) TWS119. Sub-populations were defined according to isotype controls and population density. (C) Percentage of CD8+ T cells in each CD45RA/CD62L populations defined in panel B, for 11 healthy donors. * P = 0.001; ** P = 0.002.
Figure 2.
Activation of human CD8+ T cells in the presence of TWS119 favors a young/memory phenotype.
CD127 and CD133 expression in CD8+ T cell sub-populations defined by expression of CD45RA and CD62L (see Figure 1A) following treatment with TWS119. (A) Representative histograms of total CD8+ T cells expressing CD127 or CD133 after a 5-day anti-CD3/IL-2 activation with or without TWS119. (B–C) Percentage of CD8+ T cells expressing, respectively, CD127 (B) and CD133 (C) in total CD8+ T cell population (Total), and in each CD45RA/CD62L-defined population from Figure 1A, for 9 healthy donors in B and 5 in C. (D) Percentage of CD8+ T cells expressing both CD127 and CD133 markers in total CD8+ T cell population (total) and in each CD45RA/CD62L-defined population from Figure 1A for 5 healthy donors. * P<0.004.
Figure 3.
A minimal concentration of 2.5 µM of TWS119 is required to generate T cells with a young/memory phenotype.
Expression of CD45RA and CD62L on CD8+ T cells from peripheral blood of healthy donors after activation of the Wnt pathway with TWS119 dose-response (0.5 to 5 µM). (A) Expansion of viable CD8+ T cells activated with anti-CD3/IL-2 after five days of culture with or without 5 µM TWS119, illustrated by final cell concentration. Initial cell concentration was 1×106 cell/mL. (B) Representative dot plot analysis for surface expression of CD45RA and CD62L on CD8+ T cells activated with anti-CD3/IL-2 with or without increasing concentrations of TWS119. (C) Percentage of total CD8+ T cells in each CD45RA/CD62L population for 6 healthy donors. (D) Percentage of CD8+ T cells expressing CD127 in total CD8+ T cell population in a TWS119 dose-response, in each population defined by expression of CD45RA and CD62L in Figure 1A (for 6 donors).
Figure 4.
Activation of the Wnt pathway in human CD8+ T cells modulates secretion of effector cytokines/chemokines and secreted factors.
Cytokine secretion profile of total CD8+ T cells from peripheral blood of healthy donors following the Wnt pathway activation with TWS119. Cytokine secretion was measured by CBA with a panel of 27 cytokines in supernatant of total CD8+ T cells cultured with anti-CD3/IL-2 and TWS119 and reactivated with anti-CD3/anti-CD28 and TWS119 (data from 3 healthy donors). Only cytokines considered positive for secretion (>50 pg/mL and double or higher the “No Stimulation” value) are presented (negative for IL-1ß, -4, -6, -7, -9, -10, -12p70, -17a, -21, CD40L, TNF-RI, CXCL9 (MIG) and VEGF).
Figure 5.
Human CD8+ TIL activated via the Wnt pathway acquire a young/memory phenotype.
CD8+ T cells isolated from lung TIL were cultured with TWS119 and expression of CD45RA, CD62L, CD127 and CD133 was determined by flow cytometry. (A) Representative flow cytometry analysis for CD45RA and CD62L expression on CD8+ TIL freshly-isolated from lung cancer tumors (representative of 4 tumors). (B) Representative analysis of CD45RA/CD62L expression on CD8+ TIL following 5 days of anti-CD3/IL-2 activation, with or without TWS119. Compilation graph from 4 patients for the CD45RAint/CD62L+ defined population (right panel). (C) Representative histogram analysis of CD127 (left) or CD133 (right) expression in total CD8+ TIL following 5 days of anti-CD3/IL-2, with or without TWS119. (D) Compilation graphs for CD127 and CD133 expression (percentage and MFI as indicated) on total activated CD8+ TIL. (E) Characterization of CD127 and CD133 expression by different CD45RA/CD62L-defined CD8+ TIL, following treatment with TWS119. Specifically, expression of CD127 and CD133 on CD45RAint/CD62L+ and CD45RA−/CD62L− CD8+ TIL populations is shown, after culture with TWS119.
Figure 6.
Activation of the Wnt pathway in human CD8+ TIL from lung cancer tumors with TWS119 diminishes secretion of effector cytokines.
Cytokine secretion profile of CD8+ TIL from lung cancer tumors after activation of the Wnt pathway with TWS119. Cytokine secretion was measured by CBA with a panel of 27 cytokines in supernatant of total CD8+ TIL cultured with anti-CD3/IL-2 and TWS119 and reactivated overnight with anti-CD3/anti-CD28 and TWS119 (for 3 lung cancer tumors). Only cytokines considered positive for secretion (>50 pg/mL and double or higher the “No Stimulation” value) are presented (negative for IL-1ß, -4, -7, -9, -10, -12p70, -17a, -21, CD40L, LT-α, TNF-RI, CXCL9 (MIG), CXCL10 (IP-10) and VEGF).
Figure 7.
Treatment of human CD8+ T cells with TWS119 protects against apoptosis.
Molecular analysis of anti- and pro-apoptosis actors in CD8+ T cells following activation of the canonical Wnt pathway. (A) Densitometric profile of Western blot analysis of the Bcl-2 protein. Data are presented as ratios of Bcl-2 protein expression (normalized with β-actin) and fold changes between CD8+ T cells cultured with and without TWS119 for 2 healthy donors (representative of 5). (B) Western blot analysis of the cleaved form of caspase-3 (2 fragments) in total CD8+ T cells following 5 day anti-CD3/IL-2 activation with or without TWS119 for 2 healthy donors (representative of 5).