Rapamycin, unlike cyclosporine A, enhances suppressive functions of in vitro-induced CD4+CD25+ Tregs.
BACKGROUND
A growing body of data shows that CD4(+)CD25(+) regulatory T cells (Tregs) can induce transplantation tolerance by suppressing immune responses to allograft antigens. However, both the generation and the suppressive capacity of CD4(+)CD25(+) Tregs can be substantially affected by different immunosuppressive drugs used in clinical transplantation. The goal of this study was to compare the effects of cyclosporine A and rapamycin on the induction and suppressive functions of human CD4(+)CD25(+) Tregs in vitro.


METHODS
CD4(+)CD25(+) Tregs were induced in two-way mixed lymphocyte reaction (MLR) in the presence of rapamycin (Treg-Rapa) or cyclosporine A (Treg-CsA). Tregs were identified in MLR cultures by flow cytometry using anti-CD4, anti-CD25, anti-CTLA-4, anti-CD122, anti-GITR mAbs and ant-PE-FOXP3 staining sets. Suppressive capacity of induced Tregs was evaluated by their capability to inhibit anti-CD3 Ab-triggered proliferation of peripheral blood mononuclear cells (PBMCs), as measured by flow cytometry. The concentration of TGF-beta1 in culture supernatants was measured by enzyme-linked immunosorbent assay.


RESULTS
Although both rapamycin and cyclosporine A suppressed the induction of CD4(+)CD25(+) Tregs during MLRs, this effect was significantly more pronounced in cells cultured with cyclosporine. On the other hand, only rapamycin significantly decreased the percentage of CD4(+)CD25(+) Tregs which expressed GITR, a negative regulator of Treg's suppressive capacity. Importantly, Treg-Rapa, unlike Treg-CsA, displayed significant suppressive activity and were capable of inhibiting the proliferation of anti-CD3 Ab-activated PBMCs. This activity was likely mediated by TGF-beta1.


CONCLUSIONS
Rapamycin, unlike cyclosporine A, does not inhibit the function of CD4(+)CD25(+) Tregs. This implies that rapamycin could contribute to the development of transplantation tolerance by promoting the induction of functional CD4(+)CD25(+) Tregs. Moreover, our results suggest that rapamycin could be combined with functional Tregs.
Bocian
Katarzyna
K
Department of Immunology, Faculty of Biology, University of Warsaw, Poland. kbocian@biol.uw.edu.pl
Borysowski
Jan
J
Wierzbicki
Piotr
P
Wyzgal
Janusz
J
Klosowska
Danuta
D
Bialoszewska
Agata
A
Paczek
Leszek
L
Górski
Andrzej
A
Korczak-Kowalska
Grazyna
G
D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D016572:Cyclosporine / Q000494:pharmacology*; D053450:Glucocorticoid-Induced TNFR-Related Protein; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D066298:In Vitro Techniques; D053645:Interleukin-2 Receptor alpha Subunit / Q000378:metabolism*; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000187:drug effects; D007959:Lymphocyte Culture Test, Mixed; D017475:Receptors, Nerve Growth Factor / Q000378:metabolism; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism; D020123:Sirolimus / Q000494:pharmacology*; D050378:T-Lymphocytes, Regulatory / Q000187:drug effects* / Q000276:immunology* / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000378:metabolism
D053450:Glucocorticoid-Induced TNFR-Related Protein; D007166:Immunosuppressive Agents; D053645:Interleukin-2 Receptor alpha Subunit; D017475:Receptors, Nerve Growth Factor; D018124:Receptors, Tumor Necrosis Factor; C491405:TNFRSF18 protein, human; D053773:Transforming Growth Factor beta1; D016572:Cyclosporine; D020123:Sirolimus