Contrasting effects of cyclosporine and rapamycin in de novo generation of alloantigen-specific regulatory T cells

Abstract

The outcome of T-cell-mediated responses, immunity or tolerance, critically depends on the balance of cytopathic versus regulatory T (T(reg)) cells. In the creation of stable tolerance to MHC incompatible allografts, reducing the unusually large mass of donor-reactive cytopathic T effector (T(eff)) cells via apoptosis is often required. Cyclosporine (CsA) blocks activation-induced cell death (AICD) of T(eff) cells, and is detrimental to tolerance induction by costimulation blockade, whereas Rapamycin (RPM) preserves AICD, and augments the potential of costimulation blockade to create tolerance. While differences between CsA and RPM in influencing apoptosis of activated graft-destructive T(eff) cells are apparent, their effects on graft-protective T(reg) cells remain enigmatic. Moreover, it is unclear whether tolerizing regimens foster conversion of naïve peripheral T cells into alloantigen-specific T(reg) cells for graft protection. Here we show, using reporter mice for T(reg) marker Foxp3, that RPM promotes de novo conversion of alloantigen-specific T(reg) cells, whereas CsA completely inhibits this process. Upon transfer, in vivo converted T(reg) cells potently suppress the rejection of donor but not third party skin grafts. Thus, the differential effects of RPM and CsA on T(eff) and T(reg) cells favor the use of RPM in shifting the balance of aggressive to protective type alloimmunity.

Figure 1. RPM but not CsA induces Foxp3 expression in CD4⁺Foxp3⁻ T cellsin vitro

(A) FACS-sorted CD4⁺Foxp3⁻(GFP⁻) cells from naïve Foxp3GFP knock-in mice were stimulated with plate-bound anti-CD3 and anti-CD28 alone or in the presence of TGF-β1 (1 ng/mL) or RPM (20 nM) for 3 days. Foxp3 induction was monitored by the GFP signal by FACS. The percentages of the Foxp3⁺(GFP⁺) cells within the total CD4⁺ T-cell population are indicated. (B) Kinetics of Foxp3 induction by different doses of RPM. Error bars represent two measurements of Foxp3 message (relative to that of GAPDH) by real-time PCR in cells stimulated in vitro as above. (C) Anti-TGF-β1 blocked the induction of Foxp3 message by RPM. The neutralizing antibody (20 μg/mL) was added at the beginning of the 3-day culture. (D) Differential effects of RPM and CsA on TGF-β induction of Foxp3⁺(GFP⁺) cells. FACS-sorted CD4⁺Foxp3⁻(GFP⁻) cells were stimulated as in (A) in the presence of TGF-β1 (1 ng/mL) with increasing doses of RPM or CsA. After 3 days, total cells were gated for Annexin V staining. The percentages of GFP⁺ cells in Annexin V negative population were plotted against the drug doses in (E). Data represent three independent experiments.

Figure 1. RPM but not CsA induces Foxp3 expression in CD4⁺Foxp3⁻ T cellsin vitro

(A) FACS-sorted CD4⁺Foxp3⁻(GFP⁻) cells from naïve Foxp3GFP knock-in mice were stimulated with plate-bound anti-CD3 and anti-CD28 alone or in the presence of TGF-β1 (1 ng/mL) or RPM (20 nM) for 3 days. Foxp3 induction was monitored by the GFP signal by FACS. The percentages of the Foxp3⁺(GFP⁺) cells within the total CD4⁺ T-cell population are indicated. (B) Kinetics of Foxp3 induction by different doses of RPM. Error bars represent two measurements of Foxp3 message (relative to that of GAPDH) by real-time PCR in cells stimulated in vitro as above. (C) Anti-TGF-β1 blocked the induction of Foxp3 message by RPM. The neutralizing antibody (20 μg/mL) was added at the beginning of the 3-day culture. (D) Differential effects of RPM and CsA on TGF-β induction of Foxp3⁺(GFP⁺) cells. FACS-sorted CD4⁺Foxp3⁻(GFP⁻) cells were stimulated as in (A) in the presence of TGF-β1 (1 ng/mL) with increasing doses of RPM or CsA. After 3 days, total cells were gated for Annexin V staining. The percentages of GFP⁺ cells in Annexin V negative population were plotted against the drug doses in (E). Data represent three independent experiments.

Figure 2. The influence of RPM and CsA upon alloantigen-driven extrathymic de novo generation of Foxp3⁺ cells

(A) Naïve CD4⁺GFP⁻ cells from Foxp3GFP mice (H-2^b, CD45.1⁺CD45.2⁺) were enriched by FACS sorting. The pre- (upper) and post-sort (lower) FACS plots, and the scheme of adoptive cell transfer are shown. BDF1 hosts (H-2^b,d, CD45.2⁺) were injected (i.v.) with 10 million of sorted C57BL/6 CD4⁺GFP⁻ cells, and treated on days 0, 1, 2 with HBSS, RPM (3 mg/kg, i.p.), CsA (20 mg/kg, s.c.), alone or together with anti-CD154 (MR1, 0.25 mg, i.p.). (B) C57BL/6 CD4⁺ T cells residing in the spleens of BDF1 hosts were analyzed on day 4 via FACS gating on the CD45.1⁺ population. The percentages of induced Foxp3⁺(GFP⁺) cells within the gated C57BL/6 CD4⁺ T cells are indicated. Note that induced T_reg cells (RPM and/or anti-CD154 treatment in B) express lower levels of GFP than natural T_reg cells (upper panel in A). The absolute numbers of GFP⁺ cells recovered from the spleens of animals under different treatments were presented in parenthesis. See Supplementary Tables 1a and b for detailed calculations. Data are representative of the results obtained in 10 different experiments.

Figure 3. Differences between T_eff cells and de novo generated T_reg cells in cell cycle progression and susceptibility to apoptosis

(A) BDF1 hosts injected with CFSE-labeled CD4⁺CD25⁻ cells from naïve C57BL/6 mice were treated with RPM+anti-CD154 to induce de novo conversion of T_reg cells as described in Methods. Lymph node cells from BDF1 hosts were stained with anti-Foxp3 and the gated H-2D(d)⁻CD4⁺ (C57BL/6) fraction was analyzed by FACS on day 4. The oval gate indicates the small contaminating natural T_reg population within the starting CD4⁺CD25⁻ pool, which has higher Foxp3 expression than de novo generated T_reg cells. (B) Foxp3⁺ cells were induced in the early cell divisions. The percentages of C57BL/6 Foxp3⁺ cells induced 4 days after adoptive transfer into BDF1 hosts and subsequent drug treatment were plotted as a function of the number of cell divisions. (C) Anti-CD154, in conjunction with RPM, preferentially inhibits the proliferation of Foxp3⁻ T_eff cells. Day 4 CFSE dilution profiles of H-2D(d)⁻CD4⁺ T_eff cells (Foxp3⁻ gating in A) and T_reg cells (Foxp3⁺ gating in A) with frequencies of divided and nondivided cells are shown. (D) The converted GFP⁺ T_reg cells, but not GFP⁻ T_eff cells, are more resistant to apoptosis as demonstrated by Annexin V negative staining. T_reg cells were de novo generated from GFP⁻ naïve T cells of the knock-in mice upon adoptive transfer into BDF1 hosts and subsequent RPM+anti-CD154 treatment. Total H-2D(d)⁻CD4⁺ T cells were gated for Annexin V staining. Data are representative of three different experiments.

Figure 4. De novo generated T_reg cells exert alloantigen-specific suppression and donor-selective graft protection

(A) In vitro MLR. Naïve CD4⁺GFP⁻ T_eff cells from the knock-in mice (H-2^b) were stimulated with Mitomycin C-treated DBA/1 (H-2^q) or DBA/2 (H-2^d) splenocytes for 4 days. CD4⁺GFP⁺ induced T_reg (iT_reg) cells were FACS-sorted and added at varying ratios to T_eff cells in the MLR. T-cell proliferation in these cultures, as measured by the mean values of incorporated thymidine of triplicate wells, is compared to that of MLR cultures with T_eff alone (normalized as 100%). (B) Protection of donor but not third party skin grafts by iT_reg cells. C57BL/6 RAG-1-deficient mice were simultaneously transplanted on the opposite sides of the flank with allogeneic tail skin grafts from DBA/1 and DBA/2 mice. CD4⁺GFP⁻ T_eff cells (2 × 10⁵) alone (dotted line) or together with CD4⁺GFP⁺ iT_reg cells (3 × 10⁴) (solid line) were then transferred by tail vein injection. P<0.01 in Kaplan-Meier survival analysis is considered statistically significant.

Figure 5. RPM induces long-term skin allograft survival with concomitant de novo generation of Foxp3⁺ cells

(A) RPM, but not CsA, induces long-term skin graft survival in an adoptive transfer model. FACS-sorted CD4⁺GFP⁻ T cells (2 × 10⁵, from naïve knock-in mice) were transferred into C57BL/6 RAG-1-deficient mice receiving allogeneic tail skin grafts from DBA/2 donor. Mice were either not treated, or treated with RPM (3 mg/kg, i.p., daily for the first 3 days and then every other day for 11 days) or CsA (20 mg/kg, s.c, daily for 14 days). (B) Cells from spleens and graft-draining lymph nodes were examined for GFP expression by FACS on day 18 and 30. The percentages of GFP⁺ cells among transferred CD4⁺ T cells were indicated. Data represent the mean values of two individual mice from each group.