Activation of Wnt signaling arrests effector differentiation in human peripheral and cord blood-derived T lymphocytes

Abstract

The canonical Wnt/β-catenin signaling pathway plays an important role in thymocyte development and T cell migration, but little is known about its role in naive-to-effector differentiation in human peripheral T cells. We show that activation of Wnt/β-catenin signaling arrests human peripheral blood and cord blood T lymphocytes in the naive stage and blocks their transition into functional T effector cells. Wnt signaling was induced in polyclonally activated human T cells by treatment either with the glycogen synthase kinase 3β inhibitor TWS119 or the physiological Wnt agonist Wnt-3a, and these T cells preserved a naive CD45RA(+)CD62L(+) phenotype compared with control-activated T cells that progressed to a CD45RO(+)CD62L(-) effector phenotype, and this occurred in a TWS119 dose-dependent manner. TWS119-induced Wnt signaling reduced T cell expansion, as a result of a block in cell division, and impaired acquisition of T cell effector function, measured by degranulation and IFN-γ production in response to T cell activation. The block in T cell division may be attributed to the reduced IL-2Rα expression in TWS119-treated T cells that lowers their capacity to use autocrine IL-2 for expansion. Collectively, our data suggest that Wnt/β-catenin signaling is a negative regulator of naive-to-effector T cell differentiation in human T lymphocytes. The arrest in T cell differentiation induced by Wnt signaling might have relevant clinical applications such as to preserve the naive T cell compartment in Ag-specific T cells generated ex vivo for adoptive T cell immunotherapy.

Figure 1. Treatment with TWS119 causes activation of canonical β-catenin-Wnt signaling

(A) Western blot analysis of β catenin expression in CD3+ T cells cultured with DMSO or 7 μM TWS119 with or without activation for 6 hours. 293T cells were used as a positive control for β catenin expression and GAPDH served as loading control. Immunoblot data from one of 2 independent experiments. (B) qRT-PCR analysis of Wnt target genes in activated CD3+ T cells cultured with DMSO or 7 μM TWS119 for 8 hours. Fold change in expression of genes was calculated with respect to 0 hours and the data are shown in a log scale. Data summarize mean ± SD of 5 independent experiments. (C) Time course of Wnt target gene expression in activated T cells treated with DMSO or 7 μM TWS119 for 0, 2, 5 and 8 hours analyzed by qRT-PCR. Data from one of 2 independent experiments.

Figure 2. TWS119 treatment blocks transition of CD45RA+ cells into CD45RO+ cells and preserves a subset of phenotypically naïve T cells

(A) Flow cytometry analysis of naïve and effector T cell markers - CD62L, CD45RA and CD45RO - on freshly isolated CD3+ T cells at baseline (left panels) and after activation and 7 days culture with DMSO or different doses of TWS119 (right panels). Plot from one of 3 independent experiments. (B) Analysis of CD45RO and CD45RA expression on CD3+ T cells, further selected for CD45RA or CD45RO, activated and cultured with DMSO or 3 μM TWS119 for 7 days. Plot from one of at least 3 independent experiments (C) Comparison of CD45RA, CD45RO or CD62L expression on CD3+ CD45RA+ selected cells after activation and culture with DMSO or 3 μM TWS119 for 7 days. Data summarize mean ± SD of 4 independent experiments. (D) Comparison of CD45RA, CD45RO or CD62L expression on CD45RA+ selected cells, further selected for CD4 or CD8, after activation and culture with DMSO or 3 μM TWS119 for 7 days. Data summarize mean ± SD of 3 independent experiments.

Figure 3. TWS119 treatment impairs acquisition of T cell effector function

(A and B) Evaluation of IFNγ production and degranulation by CD3+ cells cultured for a week in DMSO or 3 μM TWS119 by flow cytometry. (A) Representative plot and (B) percentages of DMSO or TWS119 treated CD3+ cells that express CD107 and intracellular IFNγ in response to PMA/ionomycin stimulation. Data summarize mean ± SD of at least 4 independent experiments. (C) ELISA assessment of IFNγ release in supernatant of PMA/ionomycin stimulated T cells that had been treated for a week with DMSO or 3 μM TWS119. Data summarize mean ± SD of 3 independent experiments. (D and E) Evaluation of cytotoxic function and cytokine production of antigen-specific T cells cultured with DMSO or 3 μM TWS119 cells with CD19+ Raji cells at 5:1 ratio for 5 days. Non-transduced (NT) DMSO or TWS119 treated T cells served as negative controls. (D) Representative plot of CD20+ residual tumor cells in coculture of CAR CD19 redirected T cells with Raji cells. Data from one of 2 independent experiments. (E) Cytokines in the 24 hours supernatant of coculture of CAR T cells with Raji cells assessed by CBA. Data summarize mean ± SD of 3 independent experiments.

Figure 4. Treatment of T cells with TWS119 reduces cell expansion by blocking proliferation

(A) Cell counts of CD3+ CD45RA+ and CD3+ CD45RO+ cells activated and cultured with DMSO or 3 μM TWS119 for 1 week. Data summarize mean ± SD of at least 3 independent experiments. (B) Assessment of live cell population, denoted by percentage of Annexin^negative 7AAD^negative cells, at the end of 1 week of culture with DMSO or 3 μM TWS119. Data summarize mean ± SD of 3 independent experiments. (C and D) Evaluation of T cell proliferation by CFSE dilution of CD3+ CD45RO+ and CD3+ CD45RA+ cells activated or left non-activated and cultured with DMSO or 3 μM TWS119 for 7 days. Percentages of dividing cells after activation represent the CFSE^low cells. Data summarize mean ± SD of at least 3 independent experiments.

Figure 5. PHA activation of T cells or induction of Wnt signaling by Wnt-3a produces similar effects as OKT3/anti-CD28 activated, TWS119-treated T cells

(A) Expression of CD62L, CD45RA and CD45RO in T cells activated with 5 μg/ml PHA and cultured with DMSO or 3 μM TWS119 for 7 days. (B) Cells were stained with CFSE and cultured with DMSO or 3 μM TWS119 and the extent of proliferation was measured in terms of CFSE dilution by day 7 of culture. (C) Expression of CD62L, CD45RA and CD45RO in T cells activated and cultured with or without Wnt-3a (5 μg/ml) for 7 days. (D) Cell counts at the 7 days of culture with or without Wnt-3a (5 μg/ml) were determined. Data summarize mean ± SD of 3 independent experiments

Figure 6. TWS119 treatment inhibits expression of activation markers CD25 and CD69 but not production of IL-2

(A) Evaluation of CD25 and CD69 expression by CD3+ cells treated for a week with DMSO (solid line, open circles and triangles) or 3 μM TWS119 (dotted line, closed circles and triangles) by flow cytometry at the indicated time points. Data summarize mean ± SD of 3 independent experiments. (B and C) Evaluation of IL-2 production by CD3+ cells cultured for a week in DMSO or 3 μM TWS119 by intracellular staining. (B) Representative plot and (C) percentages of DMSO or TWS119 treated CD3+ cells that express intracellular IL-2 in response to PMA/ionomycin stimulation. Data summarize mean ± SD of 5 independent experiments. (D) ELISA assessment of IL-2 release in supernatant of PMA/ionomycin stimulated T cells that had been cultured for a week with DMSO or 3 μM TWS119. Data summarize mean ± SD of 3 independent experiments. (E) Evaluation of CD25 expression on CD3+ CD45RA+ cells treated with DMSO or 3 μM TWS119 for a week. Plot from one of 3 independent experiments.

Figure 7. Cytokines can overcome the block in proliferation induced by TWS119 treatment but it leads to a concomitant loss of the naïve T cell phenotype

(A) Evaluation of proliferation of CD3+ CD45RA+ cells cultured for a week with DMSO or 3 μM TWS119 in the absence or presence of IL-15 (5 ng/ml), IL-7 (10 ng/ml) or IL-2 (50 U/ml) using CFSE dilution assay. Plots show the extent of proliferation in terms of CFSE dilution by day 7 of culture. (B) Analysis by flow cytometry of CD45RA, CD45RO or CD62L expression by these cells at the end of 7 days culture in the absence or presence of the cytokines. Dot plots from one of 3 independent experiments.

Figure 8. TWS119 treatment blocks transition of CD45RA+ cells into CD45RO+ cells in cord blood derived T cells

(A) Representative dot plots and (B) percentage of cells expressing CD45RA, CD62L and CD45RO for CD3+ T cells isolated from CBMCs and cultured with DMSO or 3 μM TWS119 for 7 days. Data summarize mean ± SD of 5 independent experiments. (C and D) Assessment of CBMC-derived CD3+ T cell proliferation using CFSE dilution assay after a week of culture with DMSO or 3 μM TWS119. Data summarize mean ± SD of 3 independent experiments.