Induction of apoptosis and modulation of activation and effector function in T cells by immunosuppressive drugs

Abstract

Immunosuppressive drugs (ISD) are used for the prevention and treatment of graft rejection, graft-versus-host-disease (GVHD) and autoimmune disorders. The precise mechanisms by which ISD interfere with T cell activation and effector function or delete antigen-specific T cells are defined only partially. We analysed commonly used ISD such as dexamethasone (DEX), mycophenolic acid (MPA), FK506, cyclosporin A (CsA), rapamycin (RAP), methotrexate (MTX) and cyclophosphamide (CP) for apoptosis-induction and modulation of activation and effector function in human peripheral T cells, cytotoxic T cell lines (CTL) and Jurkat T cells. Of all drugs tested only CP and MTX prevented antigen-specific proliferation of T cells and decreased cytotoxicity of alloantigen specific CTL lines by direct induction of apoptosis. MTX and CP also slightly increased activation-induced cell death (AICD) and CD95-sensitivity. In contrast, all other drugs tested did not induce T cell apoptosis, increase CD95-sensitivity or AICD. CsA and FK506 even prevented AICD by down-modulation of CD95L. DEX, MPA, CsA, FK506 and RAP inhibited activation of naive T cells, but were not able to block proliferation of activated T cells nor decrease cytotoxic capacity of CTL lines. These results show that ISD can be classified according to their action on apoptosis-induction and inhibition of proliferation and would favour a rational combination therapy to delete existing reactive T cells and prevent further T cell activation.

Fig. 1

Flow cytometric analysis of apoptosis induction in activated T cells by three different methods. Activated HLA-A1-restricted CTL were incubated with Apo-1 (1 μg/ml), DEX (10⁻⁷m), MPA (100 ng/ml), CsA (100 ng/ml), FK506 (100 ng/ml), RAP (10 ng/ml), MTX (1000 ng/ml) and CP (1000 ng/ml) for 72 h. Percentage specific apoptosis was determined by measuring FSC/SSC, propidium iodide-stained DNA content (Nicoletti) and binding of annexin V-FITC to externalized phosphatidylserine. Data represent the mean of three different experiments. □, FSC/SSC; , Nicoletti; ▪, annexin V.

Fig. 2

MTX and CP induce cell death in Jurkat cells. Jurkat cells were incubated with increasing concentrations of MTX, CP, MPA, CsA, FK506, RAP and DEX and specific apoptosis was calculated after 24, 48 and 72 h by FACS analysis measuring FSC/SSC ▪, 24 h; □, 48 h; , 72 h. (a). Jurkat cells were incubated with increasing amounts of Apo-1 MoAb in the absence or presence of DEX, MPA, CsA, FK506, RAP, MTX and CP. (b) Viability was assessed by FACScan analysis after 12 h measuring FSC/SSC. ♦, Medium; •, dexamethasone 10⁻⁷ M1; ▴, MPA 100 ng/ml; ×, CsA 100 ng/ml; *, FK506 100 ng/ml; ♦, RAP 10 ng/ml; ▵, MTX 1000 ng/ml; □, CP 1000 ng/ml. Results are representative of four distinct experiments showing similar effects.

Fig. 3

Modulation of AICD of Jurkat cells by ISD. Jurkat cells were incubated with immobilized OKT3 (α-CD3, 100 μg/ml) or medium and ISD DEX (10⁻⁷m), MPA (100 ng/ml), CsA (100 ng/ml), FK506 (100 ng/ml), RAP (10 ng/ml) MTX (1000 ng/ml) and CP (1000 ng/ml). (a) After 24 h specific apoptosis was determined by FACS analysis measuring FSC/SSC. □, Medium; ▪, OKT3 (100 μg/ml). (b) After 4 h immunoblot analysis for CD95L and β-actin levels was performed; % specific apoptosis is the mean of one experiment of four performed and the gels shown are representative of four different experiments for both CD95L and β-actin.

Fig. 3

Modulation of AICD of Jurkat cells by ISD. Jurkat cells were incubated with immobilized OKT3 (α-CD3, 100 μg/ml) or medium and ISD DEX (10⁻⁷m), MPA (100 ng/ml), CsA (100 ng/ml), FK506 (100 ng/ml), RAP (10 ng/ml) MTX (1000 ng/ml) and CP (1000 ng/ml). (a) After 24 h specific apoptosis was determined by FACS analysis measuring FSC/SSC. □, Medium; ▪, OKT3 (100 μg/ml). (b) After 4 h immunoblot analysis for CD95L and β-actin levels was performed; % specific apoptosis is the mean of one experiment of four performed and the gels shown are representative of four different experiments for both CD95L and β-actin.

Fig. 4

Influence of ISD on T cell proliferation and apoptosis. Purified CD4⁺ and CD8⁺ human T cells were either stimulated with α-CD3 (1 μg/ml) and α-CD28 (1 μg/ml) MoAbs for 3 days or left untreated and were cultured together with DEX, MPA, CsA, FK506, RAP, MTX and CP. (a) At day 3 cells were labelled with ³H]thymidine and harvested after 18 h. (b) At day 4 specific apoptosis was determined by FACScan analysis measuring FSC/SSC. Experiments were performed three times and standard deviations were always <5% of the mean value. ▪, Medium; , dexamethasone 10⁻⁷m; , MPA 100 ng/ml; , CsA 100 ng/ml; , FK506 100 ng/ml; , RAP 10 ng/ml; □, MTX 1000 ng/ml; , CP 1000 ng/ml.

Fig. 5

MTX and CP accelerate CD95-mediated apoptosis of stimulated T cells. CD4⁺ and CD8⁺ human T cells were stimulated with α-CD3 (1 μg/l) and α-CD28 (1 μg/ml) MoAbs for 6 days and afterwards incubated with increasing concentrations of APO-1 in the absence or presence of DEX (10⁻⁷m), MPA (100 ng/ml), CsA (100 ng/ml), FK506 (100 ng/ml), RAP (10 ng/ml), MTX (1000 ng/ml) and CP (1000 ng/ml). After 24 h cells were double-stained with MoAb CD45RA-FITC and CD45RO-PE and specific apoptosis was determined by FACScan analysis measuring FSC/SSC. Values are the mean of triplicates and represent one of four different experiments. ♦, Medium; ▪, dexamethasone; ▴, MPA; ○, CsA; ×, FK506; •, RAP; □, MTX; ▵, CP.

Fig. 6

ISD do not accelerate AICD in human peripheral T cells. Purified human CD4⁺ and CD8⁺ T cells were activated for 6 days with OKT3 (100 ng/ml) and then restimulated with PMA (50 ng/ml) and ionomycin (1 μg/ml) for 16 h (a) or 7 h (b) in the presence or absence of DEX (10⁻⁷m), MPA (100 ng/ml), CsA (100 ng/ml), FK506 (100 ng/ml), MTX (1000 ng/ml) or CP (1000 ng/ml). (a) Viability was assessed by FACScan analysis measuring FSC/SSC. ▪, Medium; , dexamethasone; , MPA; , CsA; , FK506; , MTX; □, CP. (b) Immunoblot analysis for CD95L and β-actin (protein loading control) levels; % specific apoptosis is the mean of one experiment of three performed and the gels shown are representative of three different experiments for both CD95L and β-actin.

Fig. 6

ISD do not accelerate AICD in human peripheral T cells. Purified human CD4⁺ and CD8⁺ T cells were activated for 6 days with OKT3 (100 ng/ml) and then restimulated with PMA (50 ng/ml) and ionomycin (1 μg/ml) for 16 h (a) or 7 h (b) in the presence or absence of DEX (10⁻⁷m), MPA (100 ng/ml), CsA (100 ng/ml), FK506 (100 ng/ml), MTX (1000 ng/ml) or CP (1000 ng/ml). (a) Viability was assessed by FACScan analysis measuring FSC/SSC. ▪, Medium; , dexamethasone; , MPA; , CsA; , FK506; , MTX; □, CP. (b) Immunoblot analysis for CD95L and β-actin (protein loading control) levels; % specific apoptosis is the mean of one experiment of three performed and the gels shown are representative of three different experiments for both CD95L and β-actin.

Fig. 7

MPA, MTX and CP inhibit proliferation of preactivated antigen-specific T cells. PBMC from healthy HLA-A1⁻ donors (naive T cells) or HLA-A1 specific CTL (activated T cells) were stimulated with HLA-A1⁺.721 cells in the absence or presence of DEX (10⁻⁷m), MPA (100 ng/ml), CsA (100 ng/ml), FK506 (100 ng/ml), RAP (10 ng/ml), MTX (1000 ng/ml) or CP (1000 ng/ml). At day 5 cells were labelled with ³H]thymidine and harvested after 18 h (a). Cytokine production of.721 stimulated HLA-A1⁻ PBMC was determined at day 2 after antigen stimulation (b). Data are representative of three experiments.

Fig. 8

MTX and CP decrease cytotoxicity and induce apoptosis of allo-antigen specific CTL. HLA-A1 specific CTL were incubated at day 5 after the last restimulation with in DEX (10⁻⁷m), MPA (100 ng/ml), CsA (100 ng/ml), FK506 (100 ng/ml), RAP (10 ng/ml), MTX (1000 ng/ml), CP (1000 ng/ml) or medium. After 24 h a standard chromium release assay was performed. The HLA-A1-positive cell lines C1R.A1 and P1.A1/hβ₂m and the untransfected cell lines C1R and P1.HTR were used as ⁵¹Cr-labelled targets (a). ♦, Medium; ▪, dexamethasone; ▴, MPA; ×, CsA; *, FK506; •, RAP; ▵, MTX; □, CP. At 24 and 48 h after ISD incubation specific apoptosis was determined by FACScan analysis measuring FSC/SSC (b). □, 24 h; ▪, 48 h. One representative experiment out of six performed is shown and data represent the mean value of triplicates.