Immunosuppressive agents differently suppress T cell subpopulations

Purpose: The recent discovery of a CD4+ T cell subtype characterized by the secretion of interleukin IL-17 (Th-17 cells) had a major impact on our understanding of immune processes not readily explained by the Th-1/Th-2 paradigm. We investigated the efficacy of each member of the main immunosuppressive substance classes to specifically suppress activated lymphocyte sub-populations. Methods: Heterotopic BN-Lew heart transplantations were performed. Six days after transplantation, spleens were recovered and splenocytes were isolated. Equal amounts of donor and recipient splenocytes were incubated for 24h at 37°C. Unidirectional ELISPOT assays for Th-1 (IFNg), Th-2 (IL-4), Th-17 (IL17) and Treg subtypes (TGFβ) were performed to identify the frequency of activated T cell subsets. Drugs were added at concentrations which are considered therapeutic trough levels in patients: calcineurin-inhibitor (tacrolimus; 15ng/ml), mTOR-inhibitor (rapamycin: 15ng/ml), pyrimidine synthesis inhibitor (FK778: 120ug/ml), purin synthesis inhibitor (MMF: 5ug/ml), JAK3-inhibitor (R333: 3000ng/ml), and Syk-inhibitor (R406: 3000ng/ml). The in vivo situation was simulated by adding 10% albumin to the culture medium. The specific suppressive potency on the above T cell subsets was assessed for each drug. Results: Total spot frequencies were 2731±963 for untreated cells and were significantly decreased by tacrolimus (148±66), rapamycin (1860±701), FK778 (66±100), MMF (751±449), R333 (14±14), and R406 (11±9) (p=0.001 for rapamycin vs untreated and p<0.001 for all other treatment groups vs untreated). Tacrolimus decreased spot frequency of Th-1, Th-2 and Th-17 cells significantly to 3±3%, 15±12%, and 30±18%, respectively compared to untreated cells (p<0.001). However, no decreasing effect on TGFβ secreting cells was observed (102±43%). FK778 showed a significant decrease of all investigated subtypes (2±4% Th-1, 2±2% Th-2, 2±2% Th-17, and 5±4% TGFβ), similar to R333 and R406 (Th-1: 0% and 0%, Th-2: 5±5% and 4±4%, Th-17: 2±2% and 4±4%, and TGFβ producing cells 3±3% and 4±4%, respectively). MMF was less potent than the above drugs, but still effective in decreasing Th-1 (35±17%), Th-2 (17±17%), and Th-17 cells (17±17%) and also decreased Treg subtypes (11±6%). Rapamycin showed no significant suppressive potency on Th-1 (95±33%) and Th-2 cells (77±31%); however, Th-17 cells were suppressed significantly (53±20%; p<0.001), whereas TGFβ producing cells were only mildly effected (60±26%). Conclusion: FK778, R333, and R406 demonstrate the most potent but unselective effect on all investigated T cell subpopulations. MMF was also unselective, but far less potent. The suppressive effect of rapamycin was most potent on Th-17 cells, whereas the Th-1, Th-2, and TGFβ producing cell population seemed to be unaffected. Tacrolimus showed a selective, very potent effect on Th-1, Th-2, and Th17 without decreasing TGFβ.