Activation of human CD8⁺ T cells in the presence of TWS119 favors a young/memory phenotype.

<p>CD127 and CD133 expression in CD8⁺ T cell sub-populations defined by expression of CD45RA and CD62L (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041074#pone-0041074-g001" target="_blank">Figure 1A</a>) following treatment with TWS119. (<b>A</b>) 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>B–C</b>) Percentage of CD8⁺ T cells expressing, respectively, CD127 (<b>B</b>) and CD133 (<b>C</b>) in total CD8⁺ T cell population (Total), and in each CD45RA/CD62L-defined population from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041074#pone-0041074-g001" target="_blank">Figure 1A</a>, for 9 healthy donors in B and 5 in C. (<b>D</b>) 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041074#pone-0041074-g001" target="_blank">Figure 1A</a> for 5 healthy donors. * <i>P</i><0.004.</p

Stimulation of Wnt/ß-Catenin Pathway in Human CD8⁺ T Lymphocytes from Blood and Lung Tumors Leads to a Shared Young/Memory Phenotype

<div><p>Cancer can be treated by adoptive cell transfer (ACT) of T lymphocytes. However, how to optimally raise human T cells to a differentiation state allowing the best persistence in ACT is a challenge. It is possible to differentiate mouse CD8⁺ T cells towards stem cell-like memory (T_SCM) phenotype upon TCR stimulation with Wnt/ß-catenin pathway activation. Here, we evaluated if T_SCM can be obtained from human mature CD8⁺ T cells following TCR and Wnt/ß-catenin activation through treatment with the chemical agent 4,6-disubstituted pyrrolopyrimidine (TWS119), which inhibits the glycogen synthase kinase-3β (GSK-3β), key inhibitor of the Wnt pathway. Human CD8⁺ T cells isolated from peripheral blood or tumor-infiltrating lymphocytes (TIL), and treated with TWS119 gave rise to CD62L⁺CD45RA⁺ cells, indicative of early differentiated stage, also expressing CD127 which is normally found on memory cells, and CD133, an hematopoietic stem cell marker. T_SCM cells raised from either TIL or blood secreted numerous inflammatory mediators, but in lower amounts than those measured without TWS119. Finally, generated T_SCM CD8⁺ T cells expressed elevated Bcl-2 and no detectable caspase-3 activity, suggesting increased persistence. Our data support a role for Wnt/ß-catenin pathway in promoting the T_SCM subset in human CD8⁺ T cells from TIL and the periphery, which are relevant for ACT.</p> </div

Activation of the Wnt pathway in human CD8⁺ T cells modulates secretion of effector cytokines/chemokines and secreted factors.

<p>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).</p

Human CD8⁺ TIL activated via the Wnt pathway acquire a young/memory phenotype.

<p>CD8⁺ T cells isolated from lung TIL were cultured with TWS119 and expression of CD45RA, CD62L, CD127 and CD133 was determined by flow cytometry. (<b>A</b>) Representative flow cytometry analysis for CD45RA and CD62L expression on CD8⁺ TIL freshly-isolated from lung cancer tumors (representative of 4 tumors). (<b>B</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 CD45RA^int/CD62L⁺ defined population (right panel). (<b>C</b>) 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. (<b>D</b>) Compilation graphs for CD127 and CD133 expression (percentage and MFI as indicated) on total activated CD8⁺ TIL. (<b>E</b>) Characterization of CD127 and CD133 expression by different CD45RA/CD62L-defined CD8⁺ TIL, following treatment with TWS119. Specifically, expression of CD127 and CD133 on CD45RA^int/CD62L⁺ and CD45RA⁻/CD62L⁻ CD8⁺ TIL populations is shown, after culture with TWS119.</p

Activation of human CD8⁺ T cells via the Wnt pathway generates cells with a naive phenotype.

<p>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. (<b>A</b>) 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>B</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. (<b>C</b>) Percentage of CD8⁺ T cells in each CD45RA/CD62L populations defined in panel B, for 11 healthy donors. * <i>P</i> = 0.001; ** <i>P</i> = 0.002.</p

Treatment of human CD8⁺ T cells with TWS119 protects against apoptosis.

<p>Molecular analysis of anti- and pro-apoptosis actors in CD8⁺ T cells following activation of the canonical Wnt pathway. (<b>A</b>) 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>B</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).</p

A minimal concentration of 2.5 µM of TWS119 is required to generate T cells with a young/memory phenotype.

<p>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). (<b>A</b>) 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>B</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. (<b>C</b>) Percentage of total CD8⁺ T cells in each CD45RA/CD62L population for 6 healthy donors. (<b>D</b>) 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041074#pone-0041074-g001" target="_blank">Figure 1A</a> (for 6 donors).</p