The effect of mammalian target of rapamycin inhibition on T helper type 17 and regulatory T cell differentiation in vitro and in vivo in kidney transplant recipients

Abstract

Sirolimus (SRL) is a promising alternative to calcineurin inhibitors, such as tacrolimus (TAC), in kidney transplant recipients (KTRs), but the immunological benefits of conversion from calcineurin inhibitors to SRL are not fully investigated. In the present study, we evaluated the effect of conversion from TAC to SRL on the T helper type 17/regulatory T (Th17/Treg) axis in three separate studies. First, the effect of SRL on the Th17/Treg axis was evaluated in vitro using peripheral blood mononuclear cells (PBMCs). Second, the effect of conversion from TAC to SRL on the Th17/Treg axis was studied in KTRs. Finally, the effect of SRL on CD8(+) Treg cells was evaluated. In vitro analysis of PBMCs isolated from KTRs showed that SRL suppressed Th17 cell differentiation but TAC did not. Conversion from TAC to SRL markedly decreased the number of effector memory CD8(+) T cells and significantly increased the proportion of CD4(+) and CD8(+) Treg cells compared with TAC in KTRs. SRL treatment induced the CD8(+) Treg cells, and these cells inhibited the proliferation of allogeneic CD4(+) T cells and Th17 cells. In conclusion, conversion from TAC to SRL favourably regulates Th17 and Treg cell differentiation in KTRs. These findings provide a rationale for conversion from TAC to SRL in KTRs.

Keywords: T cells; cytokines; mTOR; regulatory T cells; transplantation.

Figure 1

Effects of tacrolimus (TAC) or sirolimus (SRL) on CD4⁺ T cells isolated from the peripheral blood mononuclear cells (PBMCs) of healthy donors and cultured under T helper type 0 (Th0) -polarizing conditions. Human PBMCs were isolated from healthy subjects (n = 4) and pre-incubated with TAC (0·1, 1, or 10 ng/ml) or SRL (0·1, 1, or 10 ng/ml) as indicated. They were then cultured under Th0-polarizing conditions (anti-CD3, 1 μg/ml and anti-CD28, 1 μg/ml) for 48 hr. The percentage of CD4⁺ T cells secreting interleukin-17 (IL-17), interferon-γ (IFN-γ), or IL-4 was measured by flow cytometry. (a) The percentages of IFN-γ⁺/CD4⁺ T cells, IL-4⁺/CD4⁺ T cells, IL-17⁺/CD4⁺ T cells, and CD25⁺FOXP3⁺ T cells within the PBMC populations isolated from healthy controls. ^#P < 0·05 and ^##P < 0·01 versus culture under Th0-polarizing conditions alone. (b) SRL- and TAC-mediated suppression (%) of IFN-γ⁺/CD4⁺ T cells, IL-4⁺/CD4⁺ T cells, IL-17⁺/CD4⁺ T cells, and CD25⁺FOXP3⁺ T cells in the PBMC populations isolated from healthy controls. ^#P < 0·05 and ^##P < 0·01 versus culture under Th0-polarizing conditions alone. Bars represent the mean ± SD.

Figure 2

Effects of tacrolimus (TAC) or sirolimus (SRL) on CD4⁺ T helper type 17 (Th17) cells and CD4⁺ regulatory T (Treg) cells isolated from the peripheral blood mononuclear cells (PBMCs) of kidney transplant recipients (KTRs) and cultured under Th17-polarizing conditions. PBMCs isolated from KTRs (n = 5) were pre-incubated with TAC (10 ng/ml) or SRL (10 ng/ml) as indicated, and then cultured under Th17-polarizing conditions for 48 hr. The percentage of CD4⁺ T cells producing interleukin-17 (IL-17) or Foxp3 was measured by flow cytometry. (a) SRL- and TAC-mediated suppression (%) of IL-17⁺ CD4⁺ T cells and CD25⁺ FOXP3⁺ CD4⁺ T cells in the PBMC populations isolated from KTRs. ^#P < 0·05 and ^##P < 0·01. (b) The expression of IL-17 and Foxp3 mRNA measured using real-time PCR. ^##P < 0·01. (c) Production of IL-17 and IL-22 by Th17 cells and secreted into the culture supernatant. ^##P < 0·01. Bars represent the mean ± SD.

Figure 3

Effects of tacrolimus (TAC) or sirolimus (SRL) on the expression of mammalian target of rapamycin (mTOR) and signal transducer and activator of transcription (STAT3) proteins in CD4⁺ T cells isolated from the peripheral blood mononuclear cells (PBMCs) of healthy donors (n = 3). (a) Immunoblotting of p-mTOR, mTOR, p-STAT3(705), STAT3, p-Akt and Akt in CD4⁺ T cells pre-treated with SRL (10 ng/ml) and then cultured under T helper type 17 (Th17) -polarizing conditions for 1 hr. (b) Stimulation of CD4⁺ T cells under Th17-polarizing conditions activated the phosphorylation of p-mTOR, mTOR, p-STAT3(705), STAT3, p-Akt and Akt as detected by Western blotting and shown by the ratio of phosphorylated to total proteins. ^##P < 0·01. Bars show the mean ± SD results in three patients, in one of three independent experiments.

Figure 4

Effect of converting from tacrolimus (TAC) to sirolimus (SRL) on CD4⁺ T-lymphocyte subpopulations within the peripheral blood mononuclear cell (PBMC) population isolated from kidney transplant recipients (KTRs). Distribution of the T helper type 1 (Th1) [interferon (IFN-γ)], Th17 [interleukin-17 (IL-17)], Th2 (IL-4) and regulatory T (Treg) CD4⁺ subpopulations within the total PBMC population in KTRs (n = 5) before and after conversion from TAC to SRL. PBMCs from KTRs before and after conversion from TAC to SRL were stimulated for 4 hr in vitro with PMA and ionomycin in the presence of GolgiStop. The percentage of CD4⁺ T cells producing IFN-γ, IL-4, or IL-17 was measured by flow cytometry. (a) The percentage of IFN-γ⁺ CD4⁺ T cells within the PBMC populations isolated KTRs before and after conversion from TAC to SRL. (b) The percentage of IL-4⁺ CD4⁺ T cells within the PBMC populations isolated KTRs before and after conversion from TAC to SRL. **P < 0·01 versus before conversion. (c) The percentage of IL-17⁺ CD4⁺ T cells within the PBMC populations isolated KTRs before and after conversion from TAC to SRL. **P < 0·01 versus before conversion. (d) The percentage of CD25⁺ FOXP3⁺ T cells within the PBMC populations isolated KTRs before and after conversion from TAC to SRL. Bars represent the mean ± SD.

Figure 5

Conversion from tacrolimus (TAC) to sirolimus (SRL) increases the immunomodulatory potential of CD8⁺ regulatory T (Treg) cells in kidney transplant recipients (KTRs). Distribution of naive, central memory (Tcm), effector memory (Tem) and regulatory CD8⁺ T-cell subpopulations within the total peripheral blood mononuclear cell (PBMC) population in KTRs (n = 5) before and after conversion from TAC to SRL. PBMCs isolated from KTRs before and after conversion from TAC to SRL were stimulated for 4 hr in vitro with PMA and ionomycin in the presence of GolgiStop. CD8⁺ lymphocytes were stained with monoclonal antibodies against CD45RA and CCR7, which resulted in the identification of four subsets. (a) The percentage of CD45RA⁺ CCR7⁺ CD8⁺ T cells (Tnaive/CD8⁺ T) within the PBMC populations isolated KTRs before and after conversion from TAC to SRL. (b) The percentage of CD45RA⁻ CCR7⁺ CD8⁺ T cells (Tcm/CD8⁺ T) within the PBMC populations isolated KTRs before and after conversion from TAC to SRL. (c) The percentage of CD45RA⁻ CCR7⁻ CD8⁺ Tcells (Tem/CD8⁺ T) within the PBMC populations isolated KTRs before and after conversion from TAC to SRL. **P < 0·01 versus before conversion. (d) The percentage of CD8⁺ Treg/CD8⁺ cells (CD8⁺ CCR7⁺ CD8⁺ T) within the PBMC populations isolated from KTRs before and after conversion from TAC to SRL. *P < 0·05 versus before conversion. Bars represent the mean ± SD.

Figure 6

Suppression of effector T-cell proliferation by CD8⁺ regulatory T (Treg) cells induced with sirolimus (SRL). (a) Peripheral blood mononuclear cells (PBMCs) were collected from healthy donors (n = 4), plated at a density of 2 × 10⁵ per well, and then stimulated with an anti-CD3 antibody, interleukin-15 (IL-15; 50 ng/ml), and SRL. On Day 3, the cells were harvested, stained with antibodies specific for CD8, CCR7 and Foxp3 or isotype, and then subjected to flow cytometry. (b) CD8⁺ Treg-mediated suppression of the proliferation of effector T cells within the PBMC population isolated from healthy donors was measured using a [³H]thymidine incorporation assay. Briefly, CD4⁺ T cells were stimulated with anti-CD3 (1 μg/ml) and T-cell-depleted irradiated antigen-presenting cells (APC) in the absence or presence of CD8⁺ Treg cells (CD8⁺ CCR7⁺) that were differentiated by exposure to 1 μg/ml plate-bound anti-CD3, IL-15 and SRL. The cells were cultured for 3 days. [³H]Thymidine was added for the final 8 hr before harvesting. The incorporation of [³H]thymidine into the CD4⁺ T cells was measured using a liquid β-scintillation counter. CD8⁺ Treg cells were isolated as CD8⁺ CCR7⁺ cells. The purity of all the T-cell subsets was > 95% as determined by FACS analysis. *P < 0·05 versus Nil; ^#P < 0·05 versus anti-CD3. (c) IL-17 levels in the supernatants of the cultures described in (b). **P < 0·01 versus Nil; ^##P < 0·01 versus anti-CD3. Bars represent the mean ± SD.