Induction of T regulatory cells by cytotoxic T-lymphocyte antigen-2α on corneal endothelial cells.
OBJECTIVE
To determine whether murine corneal endothelial (CE) cells can promote the generation of T regulatory (Treg) cells in vitro.


METHODS
To induce Treg cells in vitro by CE cell lines, T cells exposed to CE cells were used as Treg cells. T cells exposed to CE cells in the presence of anti-mouse CD3 antibody were harvested and added to target bystander T cells in vitro. T-cell activation was assessed for proliferation by [(3)H]-thymidine incorporation. Expression of CD25 or Foxp3 on Treg cells was evaluated by flow cytometry. Expression of cytotoxic T-lymphocyte antigen-2 alpha (CTLA-2α) on CE cells was evaluated by flow cytometry, RT-PCR, immunohistochemistry, or in situ hybridization. Anti-CTLA-2α neutralizing antibodies, CTLA-2α siRNA, or pro-cathepsin L blocking proteins were used to abolish the CE-inhibitory function.


RESULTS
Cultured CE cells produced CTLA-2α on their surfaces, thereby enabling bystander CD4(+) T cells to be converted to Treg cells by TGFβ promotion. CE-induced Treg cells had immunosuppressive capacities by highly expressing CD25(high) and Foxp3. When mRNA downregulation (siRNA transfection), neutralizing antibodies, or blocking proteins were used to block CTLA-2α expression on CE cells, CE-induced Treg cells failed to acquire Treg function.


CONCLUSIONS
These findings indicate that cell surface CTLA-2α contributes to the CE-dependent suppression of bystander T cells. Thus, ocular resident tissue-exposed T cells can be induced to become regulators within the peripheral microenvironment.
Department of Ophthalmology and Visual Science, Tokyo Medical and Dental University Graduate School of Medicine and Dental Sciences, 1-5-45 Yushima, Bunkyo-ku, Tokyo, Japan. sunaoph@tmd.ac.jp
Sunao
S
Sugita
Yukiko
Y
Yamada
Shintaro
S
Horie
Orie
O
Nakamura
Kazumi
K
Ishidoh
Yoshimi
Y
Yamamoto
Satoru
S
Yamagami
Manabu
M
Mochizuki
D000818:Animals; D019138:Antibodies, Blocking / Q000494:pharmacology; D057134:Antibodies, Neutralizing / Q000494:pharmacology; D000943:Antigens, Differentiation / Q000502:physiology*; D015153:Blotting, Western; D056668:Cathepsin L / Q000037:antagonists & inhibitors; D002461:Cell Line, Transformed; D004728:Endothelium, Corneal / Q000187:drug effects / Q000276:immunology*; D004792:Enzyme Precursors / Q000037:antagonists & inhibitors; D005434:Flow Cytometry; D051858:Forkhead Transcription Factors / Q000378:metabolism; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D053645:Interleukin-2 Receptor alpha Subunit / Q000378:metabolism; D008213:Lymphocyte Activation / Q000502:physiology; D051379:Mice; D008810:Mice, Inbred C57BL; D034741:RNA, Small Interfering / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013601:T-Lymphocytes / Q000276:immunology; D050378:T-Lymphocytes, Regulatory / Q000276:immunology*; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism
D019138:Antibodies, Blocking; D057134:Antibodies, Neutralizing; D000943:Antigens, Differentiation; D004792:Enzyme Precursors; D051858:Forkhead Transcription Factors; C498833:Foxp3 protein, mouse; C508018:Il2ra protein, mouse; D053645:Interleukin-2 Receptor alpha Subunit; D034741:RNA, Small Interfering; D016212:Transforming Growth Factor beta; C102076:cytotoxic T-lymphocyte antigen-2, mouse; C063905:procathepsin L; D056668:Cathepsin L