Differential responses of human regulatory T cells (Treg) and effector T cells to rapamycin

Abstract

Background: The immunosuppressive drug rapamycin (RAPA) promotes the expansion of CD4(+) CD25(high)Foxp3(+) regulatory T cells via mechanisms that remain unknown. Here, we studied expansion, IL-2R-gamma chain signaling, survival pathways and resistance to apoptosis in human Treg responding to RAPA.

Methodology/principal findings: CD4(+)CD25(+) and CD4(+)CD25(neg) T cells were isolated from PBMC of normal controls (n = 21) using AutoMACS. These T cell subsets were cultured in the presence of anti-CD3/CD28 antibodies and 1000 IU/mL IL-2 for 3 to 6 weeks. RAPA (1-100 nM) was added to half of the cultures. After harvest, the cell phenotype, signaling via the PI3K/mTOR and STAT pathways, expression of survival proteins and Annexin V binding were determined and compared to values obtained with freshly-separated CD4(+)CD25(high) and CD4(+)CD25(neg) T cells. Suppressor function was tested in co-cultures with autologous CFSE-labeled CD4(+)CD25(neg) or CD8(+)CD25(neg) T-cell responders. The frequency and suppressor activity of Treg were increased after culture of CD4(+)CD25(+) T cells in the presence of 1-100 nM RAPA (p<0.001). RAPA-expanded Treg were largely CD4(+)CD25(high)Foxp3(+) cells and were resistant to apoptosis, while CD4(+)CD25(neg) T cells were sensitive. Only Treg upregulated anti-apoptotic and down-regulated pro-apoptotic proteins. Treg expressed higher levels of the PTEN protein than CD4(+)CD25(neg) cells. Activated Treg+/-RAPA preferentially phosphorylated STAT5 and STAT3 and did not utilize the PI3K/mTOR pathway.

Conclusions/significance: RAPA favors Treg expansion and survival by differentially regulating signaling, proliferation and sensitivity to apoptosis of human effector T cells and Treg after TCR/IL-2 activation.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. The phenotype and sensitivity to apoptosis of CD4+CD25high T cells expanding±RAPA.

A. Gating strategy used to designate CD25high and CD25inter/low T cells within the CD3+CD4+ T cell population. Note that after 6 weeks of culture+RAPA, nearly all cells are CD25high. B. Left panel, percentages of expanded CD4+CD25neg or CD4+CD25+ T cells±RAPA after 3 weeks in culture. Data are means±SD cells in 10 cultures initiated with MACS-purified CD4+ T-cell subsets. Right panels, ANX V binding to fresh, uncultured MACS-isolated CD4+CD25− and CD4+CD25high T cells. A representative dot blot is shown. C. ANX V binding to CD4+CD25− T cells in 3 week cultures±RAPA. The data are mean percentages±SD of ANX V+ cells in 10 cultures. Note that only few CD4+CD25neg cells expanding in cultures with no RAPA bind ANX V, while all CD4+CD25neg cells present in +RAPA cultures bind ANX V. D. ANX V binding to CD4+CD25neg and CD4+CD25high T cells after 3 weeks of culture+1 nM RAPA. A representative dot blot is shown. E. Western blots of CD4+CD25high and CD4+CD25neg T cells±RAPA showing caspase-3 activation only in the CD4+CD25neg T-cell subset +RAPA.

Figure 2. Expansion and suppressor activity of CD25+ T cells cultured in presence of rapamycin.

A. Expansion rates of in vitro-expanded MACS purified human CD4+CD25neg, CD4+CD25+ and sorted CD4+CD25high T cells cultured with irradiated feeder cells, anti-CD3/CD28 Ab-coated beads and 1,000 IU/mL of IL-2 in the absence of RAPA. Cell counts were performed at weekly intervals throughout culture period. The expansion rate is the ratio of absolute cell counts after culture vs. counts of the purified (by AutoMACS or sorting) cells before culture. The data are means±SD of experiments performed with cells of 10 NC. B. Results of the same experiments as in A but performed+1 nM RAPA. C. Expansion after culture for 3 weeks of CD4+CD25+ and CD4+CD25neg T cells+RAPA used at various concentrations. The data are mean fold expansion±SD from 6 experiments with cells of different NC. Note the highest fold expansion of CD4+CD25high T cells in the presence of 1 nM RAPA (p<0.001). Differences in expansion between CD4+CD25high and CD4+CD25neg T cells were also significant at p<0.0001. D. Enrichment of the cultures in CD4+CD25high T cells at different concentrations of RAPA. The gating strategy was to include all CD4+ T cells in the gate, then to re-gate on CD4+CD25high Treg. A representative dot blot of 5 experiments with cells of different NC is shown.

Figure 3. Phenotypic analysis of CD4+CD25+ T cells cultured±1 nM RAPA.

A. MACS-purified CD4+CD25+ cells were cultured for 3 weeks, and then their phenotype was assessed by flow cytometry. Culture conditions±1 nM RAPA were as described in Materials and Methods. Freshly-isolated CD4+CD25high T cells were similarly phenotyped. The flow cytometry data, obtained by gating on CD4+CD25high T cells in fresh PBMC or+RAPA cultures, are mean percentages±SD from experiments with cells of 6 NC. The asterisks indicate significant differences at p<0.001 in the percentage of positive cells relative to freshly isolated Treg.

Figure 4. Suppression of proliferation of CD4+CD25neg or CD8+CD25neg responder cells with T cells cultured±1 nM RAPA.

A. Fresh MACS-purified CD4+CD25neg or CD8+CD25neg T cells were used as CFSE-labeled responders. Autologous MACS-isolated CD4+CD25+ T cells cultured in presence of anti-CD3/CD28 beads and 1,000 IU/mL IL-2 in the presence or absence of 1 nM RAPA for 3 weeks were added at the 1∶1 ratio to responder cells. Gates were set on CD4+ and CFSE+ cells and analyzed using the ModFit program as described in Materials and Methods. Suppression of responder cell proliferation is indicated as %. A representative experiment of 10 performed is shown. B. The mean percentages±SD of proliferation suppression in CD4+CD25neg or CD8+CD25neg responder cells by suppressor cells generated in 3 week cultures±1 nM RAPA (n = 10). The p value is for differences between suppression mediated by S from R0 vs. S from R1 cultures. C. Suppression levels mediated by Treg expanded with different concentrations of RAPA. A representative experiment of 6 performed with cells of different NC is shown.

Figure 5. Expression levels the anti-apoptotic (Bcl-2 and Bcl-xL) and the pro-apoptotic (Bax) proteins in CD4+CD25+ or CD4+CD25neg T cells measured before and after culture in the presence or absence of RAPA.

The data are mean fluorescence intensity (MFI) mean values (±SD) for Bcl-2, Bcl-xL and Bax expression levels obtained from measurements with freshly isolated T-cell subsets or T-cell subsets cultured with RAPA or without RAPA. The ratios of Bcl-2/Bax or Bcl-xL/Bax are above the histograms. T cell subsets were separated by MACS from PBMC of 10 NC and cultured as described in Materials and Methods.

Figure 6. Activation of STAT5/3 proteins in CD4+CD25neg and CD4+CD25high T cells cultured at different RAPA concentrations.

Mean fluorescence intensity (MFI) of phosphorylated STAT5 in CD4+CD25neg and CD4+CD25high T cells in A, and of phosphorylated STAT3 in B. T cells were tested by flow cytometry immediately after isolation and after short-term (4 h) culture with different RAPA concentrations (1 nM or 50 nM). Before analysis, the cells were briefly stimulated with 150 IU/mL IL-2 (A and B) or with 150 IU/mL IL-6 (C and D) and stained for phosphorylated STAT5 or phosphorylated STAT3, respectively. The gate was set on CD3+CD4+CD25neg or CD3+CD4+CD25high cells, respectively. Results obtained with T cells of 6 different NC are shown.

Figure 7. Activation of STAT5/3 proteins in fresh (untreated) and RAPA-expanded CD4+CD25neg and CD4+CD25high T cells.

Mean fluorescence intensity (MFI) of phosphorylated STAT5 in CD4+CD25neg in A, and CD4+CD25high T cells in B and of phosphorylated STAT3 in CD4+CD25neg in C and CD4+CD25high T cells in D. PBMC-derived T cells were sorted into CD4+CD25neg and CD4+CD25+ populations and tested by flow cytometry immediately after isolation and after culture in the presence of 1 nM RAPA (at baseline unstimulated, in medium) or were briefly stimulated with 150 IU/mL IL-2 (A and B) or with 150 IU/mL IL-6 (C and D) just before analysis. In addition, T cells were cultured for the indicated periods of time and stimulated with IL-2 and IL-6 just before flow cytometry analysis. The gate was set on CD3+CD4+CD25neg or CD3+CD4+CD25high T cells, respectively. RAPA was added to T-cell cultures at the indicated concentrations. Data obtained with T cells of 6 NC are shown. Fold expansion and percent suppression mediated by these cells are shown in Table 4 .

Figure 8. PTEN expression and signaling in fresh and RAPA-expanded CD4+CD25high and CD4+CD25neg T cells.

A. Percentages of PTEN+ cells tested fresh (no RAPA) or after culture +RAPA for the indicated time periods. B. The MFI for PTEN expression in fresh and RAPA-expanded T cell cultures. C. Fold expansion and % suppression mediated by the same CD4+CD25high and CD4+CD25high T cell subsets cultured±RAPA on week 3. The data in A–C are means±SD from 6 experiments with cells of normal donors. Asterisks indicate significant differences between CD4+CD25high and CD4+CD25neg cells at p<0.001. C. Results of Western blots comparing expression levels of phospho-Akt, phospho-4EBP-1 and phospho-p70S6 in CD4+CD25high and CD4+CD25neg T cell subsets±RAPA. T cells were either unstimulated or stimulated with bead-coated anti-CD3/CD28 Abs and 100 IU/mL of IL-2 for 2 h and cultured in the presence of RAPA for the indicated time periods. The cells were harvested and used for Western blots. D. Flow cytometry histograms showing expression of phospho-4EBP-1 and phospho-p70S6 in freshly-isolated CD4+CD25high and CD4+CD25neg T cells subsets. Cells were stimulated as described above for 24 h and cultured in the presence of RAPA. They were harvested, permeabilized, stained and examined by flow ctyometry. Results are from one out of 3 experiments performed.