Inhibition of TGF-β1 signaling promotes central memory T cell differentiation

Abstract

This study affirmed that isolated CD8(+) T cells express mRNA and produce TGF-β following cognate peptide recognition. Blockage of endogenous TGF-β with either a TGF-β-blocking Ab or a small molecule inhibitor of TGF-βRI enhances the generation of CD62L(high)/CD44(high) central memory CD8(+) T cells accompanied with a robust recall response. Interestingly, the augmentation within the central memory T cell pool occurs in lieu of cellular proliferation or activation, but with the expected increase in the ratio of the Eomesoderm/T-bet transcriptional factors. Yet, the signal transduction pathway(s) seems to be noncanonical, independent of SMAD or mammalian target of rapamycin signaling. Enhancement of central memory generation by TGF-β blockade is also confirmed in human PBMCs. The findings underscore the role(s) that autocrine TGF-β plays in T cell homeostasis and, in particular, the balance of effector/memory and central/memory T cells. These results may provide a rationale to targeting TGF-β signaling to enhance Ag-specific CD8(+) T cell memory against a lethal infection or cancer.

Figure 1

Temporal-dependent in vitro production of TGF-β, IFN-γ and IL-2 by isolated CD8⁺ T cells following cognate peptide stimulation. Splenic CD8⁺ T cells from TCR transgenic mice for the nucleoprotein of influenza virus NP68 (F5 Tg-mice) were isolated by magnetic beads, and stimulated with 10⁻⁴ µg/ml of cognate peptide (NP68 peptide), 1.0 µg/ml of H2Db-dimer X and 2.0 µg/ml of anti-CD28. (A) TGF-β mRNA as measured by quantitative PCR at the indicated time points. (B) TGF-β1, (C) IFN-γ and (D) IL-2 production in the T cell culture supernatant as measured using appropriate cytokine ELISA assays. Data represent the mean ± SE of triplicate samples from three independent experiments.

Figure 2

Blockade of TGF-β signaling increased central memory T cell phenotype. Splenic CD8⁺ T cells isolated from F5 mice were pretreated with either the anti-TGF-β mAb (0.1–10 µg/ml) or TGF-β receptor I kinase inhibitor, SD208 (0.3–3.0 µM), for 1 hr prior to stimulation with 10⁻⁴ µg/ml of the cognate peptide (NP68 peptide), 1.0 µg/ml of H2Db-dimer X and 2.0 µg/ml of anti-CD28 and all analyses were performed 72 h later. (A) Flow cytometric analyses of F5-CD8⁺ T cells, as determined by anti-CD62L and either anti-CD44 (upper panel) or anti-CD127 (lower panel) are shown. (B) Total number of CD62L^high/CD44^high central memory (solid bars) versus CD62L^low/CD44^high effector memory (open bars) CD8⁺ T cells recovered and assessed by trypan-blue exclusion. *, p<0.05 (0.1 µg/ml vs 1.0 and 10 µg/ml anti-TGF-β mAb); ***, p<0.001 (untreated vs. 0.1 µg/ml vs 1.0 and 10 µg/ml anti-TGF-β mAb) (C) IFN-γ production as measured by ELISA. **p<0.01 untreated vs. 0.1 µg/ml anti-TGF-β mAb); ***, p<0.001 (untreated vs. 1.0 and 10 µg/ml anti-TGF-β mAb) (D) SD208 effects on the phenotypic changes of F5-CD8⁺ T cells, as determined by anti-CD62L and anti-CD44. (E) Total number of CD62L^high/CD44^high central memory (solid bars) versus CD62L^low/CD44^high effector memory (open bars) CD8⁺ T cells recovered and assessed by trypan-blue exclusion. *, p<0.05 (untreated vs 0.3 µM SD208); **, p<0.05 (untreated vs 1 µM SD208); ***, p<0.001 (untreated vs. 3 µM SD208). (F–H) Splenocytes from C57BL/6 mice were pre-incubated with various doses of SD208 (0.3–3.0 µM) for 1 hr, and stimulated with 2.5 µg/ml of anti-CD3 and 1.25 µg/ml of anti-CD28 and all analyses were done 72 hr later. (F) Flow cytometric analyses of CD8⁺ and CD4⁺ T cells stained with anti-CD62L and anti-CD44 antibodies. Numbers in each upper right quadrant denote percentage of cells. (G and H) Total number of (G) CD8⁺ or (H) CD4⁺ CD62L^high/CD44^high central memory (solid bars) versus CD62L^low/CD44^high effector memory (open bars) T cells recovered following stimulation with cognate peptide. Cell numbers were determined by trypan-blue exclusion and the total number of CD8⁺, CD4⁺, CD62L^low/CD44^high, CD62L^high/CD44^high cells were determined based on flow cytometry data. (G) CD8⁺ - *, p<0.05 (0.3 vs 1.0 µM & 1.0 vs. 3.0 µM SD208); ***, p<0.001 (untreated vs. 0.3, 1.0 and 3.0 µM; 0.3 vs. 3.0 µM SD208). (H) CD4⁺ - ***, p<0.001 (untreated vs. 0.3, 1.0 and 3.0 µM SD208). Data are the mean ± SE of triplicate samples of at least three independent experiments. Statistical significance was measured by the one-way ANOVA followed by Tukey's multiple comparison test.

Figure 3

Exogenous TGF-β inhibits central memory CD8⁺ T cell differentiation. Splenocytes from F5 mice were pre-incubated with 0.1–5.0 ng/ml recombinant human TGF-β1 for 1 hr and then stimulated with 10⁻⁴ µg/ml cognate peptide for 72 hr. (A) Flow cytometric analysis of the phenotypic changes 72 hr after peptide stimulation. Cells were stained with anti-CD8a, CD62L and CD44 (upper panel in A) or CD127 (lower panel in A). Numbers in each quadrant denote percentage of cells. (B) IFN-γ production in the culture supernatant measured by ELISA. ***, p<0.001 (untreated vs. 0.1, 0.5, 1.0 and 5.0 ng/ml rhTGF-β). (C) Number of CD62L^high/CD44^high central memory (solid bars) versus CD62L^low/CD44^high effector memory (open bars) CD8⁺ T cells recovered following stimulation with cognate peptide. Total viable cells were determined by trypan-blue exclusion and the total number of CD8⁺, CD62L^low/CD44^high/CD8⁺, CD62L^high/CD44^high CD8⁺ cells were determined based on flow cytometry data. Data are the mean ± SEM of triplicate samples from three independent experiments. (D) Apoptotic cells, as determined by staining with anti-CD8a and Annexin-V antibodies, after exogenous TGF-β pre-incubation and peptide stimulation. **, p<0.01 (untreated vs. 1.0 ng/ml rhTGF-β). ***, p<0.001 (untreated vs. 0.1, 0.5 and 5.0 ng/ml rhTGF-β). Data are the mean ± SE of triplicate samples from three independent experiments. Statistical significance was measured by the one-way ANOVA followed by Tukey's multiple comparison test.

Figure 4

TGF-β blockade of central memory T cell differentiation is not mediated through SMAD or MAPK super family signaling pathways. (A) CD8⁺ T cells were pre-incubated with SD208 (0.3–3.0 µM) for 1 hr, then treated with 5 ng/ml of recombinant human TGF-β1. Cells were harvested 1 hr later and the phosphorylation of the Erk, P38 MAPK and JNK signal transduction pathways was examined using Western immunoblots. (B) Splenocytes from SMAD2 conditional knockout (CKO) mice were pre-incubated with 1–10 µg/ml TGF-β mAb for 1 hr, stimulated with anti-CD3 and anti-CD28 for 72 hr and then stained with anti-CD8a, CD4, CD44 and CD62L antibodies and analyzed by flow cytometry. Numbers in the upper right quadrant denote percentage of cells. (C) Splenocytes from F5 mice were pre-incubated with 3 µM of SD208 for 1 hr, stimulated with cognate peptide, and p70 and S6 phosphorylation in the TORC1 pathway was examined by Western immunoblot at the indicated time points.

Figure 5

Change of transcriptional factors T-bet and Eomes following TGF-β blockade. Isolated splenic CD8⁺ T cells from F5 mice were pre-incubated with 1 µg/ml of TGF-β mAb for 1 hr and then stimulated with 10⁻⁴ µg/ml of cognate peptide (NP68 peptide), 1.0 µg/ml of H2Db-dimer X and 2.0 µg/ml of anti-CD28. (A) T-bet mRNA levels, (B) Eomes *, p<0.05 (untreated; open circle vs. 1.0 µg/ml anti-TGF-β MAb; filled circle @ 72 hrs) (C) Eomes/ T-bet mRNA calculated ratios. ***, p<0.001 (untreated vs. 1.0 µg/ml anti-TGF-β mAb @ 48 and 72 hrs). (D) T-bet and Eomes protein expressions measured by intracellular FACS and calculated ratio at 96 hr post-peptide. (E) Eomes/ T-bet protein ratios calculated ratios. **, p<0.01, ***, p<0.001 (untreated vs. 0.1, 1 and 10 µg/ml anti-TGF-β mAb) Statistical significance was determined using the two-tailed unpaired Student’s t-test (A, B and C) or the one-way ANOVA followed by Tukey's multiple comparison test (E).

Figure 6

Increased central memory phenotype alters in vivo proliferation upon recall response. Splenocytes from F5 mice were pre-incubated with 3 µM of SD208 or vehicle for 1 hr, stimulated with 10⁻⁴ µg/ml of cognate peptide for 96 hr and then stained with anti-CD8, CD62L and CD44. (A) CD62L and CD44 expression on CD8 T cells 96 hr after in vitro stimulation. (B) In vivo distribution of adoptively transferred F5 memory CD8⁺ T cells after 3 days and prior to rF-NP68-TRICOM vaccination. Mice (3/group) were euthanized and the peripheral blood, spleen and inguinal lymph nodes were analyzed for NP68 dextramer staining. Each dot represents a single mouse and the horizontal line indicates the mean. NS: no statistical significance. (C) A representative FACS plot for each group. The numbers in the top right quadrant denote the percentage of CD62L^high/CD44^high cells among CD8⁺/dextramer⁺ T cells from splenocyte. (D) A representative CFSE dilution for each group of CD8⁺/dextramer⁺ T cells from splenocyte. (E–G) Recall response 3 days after rF-NP68-TRICOM challenge. Peripheral blood from rF-NP68-TRICOM vaccinated mice (n=3/group) (no adoptive transfer, adoptive transfer with vehicle treated-cells and adoptive transfer with SD208-treated-cells) was collected and analyzed for NP68 dextramer staining. (E) A representative FACS plot for each group. The numbers in the top right quadrant denote the percentage of dextramer⁺ cells among CD8⁺ T cells. (F) Each dot represents a single mouse and the horizontal line indicates the mean. (G) A representative CFSE dye dilution for each group of CD8⁺/dextramer⁺ T cells from a single experiment. Three separate experiments were carried out and the increased proliferation in the SD208-treated T cells was statistically significant (p<0.01) as measured using the one-way ANOVA followed by Tukey's multiple comparison test.

Figure 7

Increased central memory T cells in human PBMCs following TGF-β blockade. (A) CD62L expression on gated CD8⁺/CD45RO⁺ cells 6 days after anti-CD3 stimulation. Numbers in the upper and lower quadrants denote percentage of CD62L^high and MFI of CD62L expression on CD8⁺ T cells, respectively. (B) Total number of CD62L^high/CD45RO⁺ central memory (solid bars) versus CD62L^low/CD45RO⁺ effector memory (open bars) CD8⁺ T cells recovered following stimulation with anti-human CD3. The number of viable cells was determined by trypan-blue exclusion and the total number of CD8⁺, CD62L^low/CD45RO⁺, CD62L^high/ CD45RO⁺ cells was determined based on flow cytometry data. *, p<0.05 (untreated vs. 0.3 µM SD208); **, p<0.01 (untreated vs. 1.0 µM SD208); ***, p<0.001 (untreated vs. 3.0 µM SD208). (C) IFN-γ production in the culture supernatant as measured by ELISA. Data are the mean ± SE of triplicate samples of at least three independent experiments. Statistical significance was measured using the one-way ANOVA followed by Tukey's multiple comparison test.