7 research outputs found
Stimulation of Wnt/Ă-Catenin Pathway in Human CD8<sup>+</sup> 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<sup>+</sup> T cells towards stem cell-like memory (T<sub>SCM</sub>) phenotype upon TCR stimulation with Wnt/Ă-catenin pathway activation. Here, we evaluated if T<sub>SCM</sub> can be obtained from human mature CD8<sup>+</sup> T cells following TCR and Wnt/Ă-catenin activation through treatment with the chemical agent 4,6-disubstituted pyrrolopyrimidine (TWS119), which inhibits the glycogen synthase kinase-3ÎČ (GSK-3ÎČ), key inhibitor of the Wnt pathway. Human CD8<sup>+</sup> T cells isolated from peripheral blood or tumor-infiltrating lymphocytes (TIL), and treated with TWS119 gave rise to CD62L<sup>+</sup>CD45RA<sup>+</sup> cells, indicative of early differentiated stage, also expressing CD127 which is normally found on memory cells, and CD133, an hematopoietic stem cell marker. T<sub>SCM</sub> cells raised from either TIL or blood secreted numerous inflammatory mediators, but in lower amounts than those measured without TWS119. Finally, generated T<sub>SCM</sub> CD8<sup>+</sup> T cells expressed elevated Bcl-2 and no detectable caspase-3 activity, suggesting increased persistence. Our data support a role for Wnt/Ă-catenin pathway in promoting the T<sub>SCM</sub> subset in human CD8<sup>+</sup> T cells from TIL and the periphery, which are relevant for ACT.</p> </div
Activation of human CD8<sup>+</sup> T cells in the presence of TWS119 favors a young/memory phenotype.
<p>CD127 and CD133 expression in CD8<sup>+</sup> 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<sup>+</sup> T cells expressing CD127 or CD133 after a 5-day anti-CD3/IL-2 activation with or without TWS119. (<b>BâC</b>) Percentage of CD8<sup>+</sup> T cells expressing, respectively, CD127 (<b>B</b>) and CD133 (<b>C</b>) in total CD8<sup>+</sup> 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<sup>+</sup> T cells expressing both CD127 and CD133 markers in total CD8<sup>+</sup> 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
Activation of the Wnt pathway in human CD8<sup>+</sup> T cells modulates secretion of effector cytokines/chemokines and secreted factors.
<p>Cytokine secretion profile of total CD8<sup>+</sup> 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<sup>+</sup> 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 âNo Stimulationâ value) are presented (negative for IL-1Ă, -4, -6, -7, -9, -10, -12p70, -17a, -21, CD40L, TNF-RI, CXCL9 (MIG) and VEGF).</p
Activation of human CD8<sup>+</sup> T cells via the Wnt pathway generates cells with a naive phenotype.
<p>CD8<sup>+</sup> 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 ÎČ-catenin protein expression by Western blot analysis from fresh CD8<sup>+</sup> T cells or CD8<sup>+</sup> T cells activated for 6 h in the presence of anti-CD3 and IL-2, with or without TWS119. ÎČ-actin protein level was used as a loading control. (<b>B</b>) Representative dot plot analysis of CD45RA and CD62L surface expression on CD8<sup>+</sup> 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<sup>+</sup> T cells in each CD45RA/CD62L populations defined in panel B, for 11 healthy donors. * <i>P</i>â=â0.001; ** <i>P</i>â=â0.002.</p
Treatment of human CD8<sup>+</sup> T cells with TWS119 protects against apoptosis.
<p>Molecular analysis of anti- and pro-apoptosis actors in CD8<sup>+</sup> 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 ÎČ-actin) and fold changes between CD8<sup>+</sup> 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<sup>+</sup> T cells following 5 day anti-CD3/IL-2 activation with or without TWS119 for 2 healthy donors (representative of 5).</p
Human CD8<sup>+</sup> TIL activated via the Wnt pathway acquire a young/memory phenotype.
<p>CD8<sup>+</sup> 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<sup>+</sup> TIL freshly-isolated from lung cancer tumors (representative of 4 tumors). (<b>B</b>) Representative analysis of CD45RA/CD62L expression on CD8<sup>+</sup> TIL following 5 days of anti-CD3/IL-2 activation, with or without TWS119. Compilation graph from 4 patients for the CD45RA<sup>int</sup>/CD62L<sup>+</sup> defined population (right panel). (<b>C</b>) Representative histogram analysis of CD127 (left) or CD133 (right) expression in total CD8<sup>+</sup> 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<sup>+</sup> TIL. (<b>E</b>) Characterization of CD127 and CD133 expression by different CD45RA/CD62L-defined CD8<sup>+</sup> TIL, following treatment with TWS119. Specifically, expression of CD127 and CD133 on CD45RA<sup>int</sup>/CD62L<sup>+</sup> and CD45RA<sup>â</sup>/CD62L<sup>â</sup> CD8<sup>+</sup> TIL populations is shown, after culture with TWS119.</p
A minimal concentration of 2.5 ”M of TWS119 is required to generate T cells with a young/memory phenotype.
<p>Expression of CD45RA and CD62L on CD8<sup>+</sup> T cells from peripheral blood of healthy donors after activation of the Wnt pathway with TWS119 dose-response (0.5 to 5 ”M). (<b>A</b>) Expansion of viable CD8<sup>+</sup> T cells activated with anti-CD3/IL-2 after five days of culture with or without 5 ”M TWS119, illustrated by final cell concentration. Initial cell concentration was 1Ă10<sup>6</sup> cell/mL. (<b>B</b>) Representative dot plot analysis for surface expression of CD45RA and CD62L on CD8<sup>+</sup> T cells activated with anti-CD3/IL-2 with or without increasing concentrations of TWS119. (<b>C</b>) Percentage of total CD8<sup>+</sup> T cells in each CD45RA/CD62L population for 6 healthy donors. (<b>D</b>) Percentage of CD8<sup>+</sup> T cells expressing CD127 in total CD8<sup>+</sup> 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