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

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

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

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

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Additional file 2: of Effect of neoadjuvant chemotherapy on the immune microenvironment in non–small cell lung carcinomas as determined by multiplex immunofluorescence and image analysis approaches

Figure S2. Characterization of immune cell phenotyping using marker panel 1. Cell phenotypes were defined as malignant cells expressing PD-L1 (AE1/AE3 + PD-L1+), T lymphocytes (CD3; pan T-cell marker including helper T cells CD3 + CD4+, cytotoxic T cells CD3 + CD8+, and other CD3+ T cells), helper T cells (CD3 + CD4+), cytotoxic T cells (CD3 + CD8+), tumor-associated macrophages (TAMs; CD68+), and TAMs expressing PD-L1 (CD68 + PD-L1+). Each type of cell is shown as individual images by marker and by a composite image. (PPTX 102487 kb

Additional file 7: of Effect of neoadjuvant chemotherapy on the immune microenvironment in non–small cell lung carcinomas as determined by multiplex immunofluorescence and image analysis approaches

Table S2. Median densities of tumor-associated immune cells in NSCLCs from patients who received neoadjuvant chemotherapy (NCT) or did not receive NCT (non-NCT), by tumor compartment and histology (N = 112). (DOCX 23 kb