ABSTRACT
BACKGROUND: CTLA-4 immunoglobulin fusion proteins (CTLA4-Ig) suppress immune reactions by blocking the T-cell costimulatory CD28-CD80-86 pathway and are used in clinical trials for diseases featuring exaggerated T-cell reactivity including autoimmune diseases and allograft rejection. However, because CTLA4-Ig has been suspected to interfere with T regulatory (Treg) cell homeostasis and function, recently, substantial concerns on CTLA4-Ig's potentially antitolerogenic effects have been raised. METHODS: We tested immunoregulatory CTLA4-Ig explicitly for its effect on Treg cell numbers, frequencies and function in an in vitro murine major histocompatibility complex mismatched setting using C57BL/6 bone marrow-derived dendritic cells as stimulators of allogeneic Balb/c Foxp3 T cells, which allowed for tracing Treg cells in a straightforward fashion. RESULTS: The presence of CTLA4-Ig in mixed leukocyte reactions-while dampening the global proliferative response of allostimulated Balb/c T cells-resulted in a relative increase of the frequency of thymus-derived CD4CD25Foxp3 Treg cells with intact suppressive activity. This relative increase was caused by a selective inhibitory effect of CTLA4-Ig on proliferating conventional T cells, whereas the proliferative capacity of Treg cells in cell cultures remained unaffected. Additionally, in the presence of CTLA4-Ig, the frequency of apoptosis was decreased in these cells. CONCLUSION: Our findings unequivocally demonstrate that CTLA4-Ig does not negatively affect Treg cell frequencies and function in vitro.
Subject(s)
Immunoconjugates/pharmacology , T-Lymphocytes, Regulatory/cytology , Thymus Gland/cytology , Abatacept , Animals , Apoptosis , B7-1 Antigen/metabolism , B7-2 Antigen/metabolism , CD28 Antigens/metabolism , CD4-Positive T-Lymphocytes/cytology , Cell Proliferation , Cell Survival , Female , Interleukin-2 Receptor alpha Subunit/metabolism , Lymphocyte Activation , Lymphocyte Culture Test, Mixed , Major Histocompatibility Complex/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BLABSTRACT
Immunosuppressive cytotoxic T lymphocyte associated antigen-4 immunoglobulin fusion proteins (CTLA4-Ig) block the CD28:CD80/86 costimulatory pathway. On a cellular level, CTLA4-Ig is understood to dampen T cell responses. As a mechanism, CTLA4-Ig has been reported to affect dendritic cell (DC) function via inducing the immunosuppressive indoleamine 2,3 dioxygenase (IDO) pathway and promoting a DC regulatory phenotype. We here probed cellular mechanisms of CTLA4-Ig immunoregulation in an allogeneic setting using C57BL/6 splenic or bone marrow derived DCs (BMDCs) as stimulators of allogeneic Balb/c derived T cells. To address whether CTLA4-Ig immunosuppression affected DCs, we pre-exposed C57BL/6 splenic or BMDCs to CTLA4-Ig and removed unbound CTLA4-Ig before co-culture with allogeneic T cells. CTLA4-Ig disappeared rapidly (within 4 h) from the cell membrane by combined internalization and dissociation. These CTLA4-Ig pre-exposed DCs were fully capable of stimulating allogeneic T cell proliferation, suggesting that CTLA4-Ig does not impair the DC stimulatory capacity. Only the presence of CTLA4-Ig during DC/T cell co-culture resulted in the expected inhibition of proliferation. C57BL/6 splenic or BMDCs exposed to CTLA4-Ig did not display IDO activity. We conclude that CTLA4-Ig immunosuppressive activity does not depend on a DC regulatory phenotype but on its presence during DC/T cell interaction.
