Participation of pigment epithelium of iris and ciliary body in ocular immune privilege. 1. Inhibition of T-cell activation in vitro by direct cell-to-cell contact.
OBJECTIVE
To determine by what mechanism(s) iris and ciliary body (I/CB) pigment epithelial (PE) cells inhibit T-cell activation in vitro.


METHODS
Pure cultured I/CB PE cells were obtained from eyes of normal and CD95 ligand (CD95L)deficient mice and tested for their capacity to suppress T-cell activation in three different T-cell receptor (Tcr) ligand systems: mixed lymphocyte reactions, stimulation of Tcr transgenic T cells (D011.10) by specific antigen (ovalbumin), and ligation of the Tcr-associated CD3 molecule by anti-CD3 antibodies. Proliferation and secretion of cytokines (interferon [IFN]-gamma, interleukin [IL]-2, IL-4, and IL-10) were assessed as measures of T-cell activation. Suppressive influences of I/CB PE cells were determined on the basis of RT-PCR- detected cytokine genes expressed by I/CB PE cells, immunosuppression mediated by supernatants of cultured I/CB PE cells, direct contact between I/CB PE cells and T lymphocytes, and promotion of apoptosis among responding T cells. Attempts to reverse I/CB PE-dependent suppression of T-cell activation included the use of neutralizing antibodies to IL-10, tumor necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta, and the addition of exogenous IL-2 and IL-12.


RESULTS
Cultured mouse I/CB PE cells (including CD95L-deficient cells), which were more than 95% keratin positive, suppressed T-cell proliferation and secretion of IFN-gamma, IL-2, IL-4, and IL-10 in a dose-dependent fashion in all three Tcr ligand systems. Supernatants of cultured I/CB PE cells displayed little suppression activity, whereas cultures in which I/CB PE cells contacted responding T cells directly were profoundly immunosuppressive. Cultured I/CB PE cells expressed mRNA for TGF-beta1, TGF-beta2, IL-6, IL-10, and TNF-alpha, but not IL-4, IFN-gamma, proopiomelanocortin (POMC), and CD95L (Fas L). Antibodies to TGF-beta, IL-10, and TNF-alpha failed to reverse suppression mediated by I/CB PE cells. Moreover, neither exogenous IL-2 or IL-12 relieved the suppression.


CONCLUSIONS
Cultured I/CB PE cells, through direct cell-to-cell contact, prevent T cells from proliferating and secreting cytokines when stimulated through the Tcr for antigen by a mechanism that does not involve CD95 or apoptosis.
Yoshida
M
M
Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02114, USA.
Takeuchi
M
M
Streilein
J W
JW
D000818:Animals; D002450:Cell Communication / Q000502:physiology*; D002478:Cells, Cultured; D002924:Ciliary Body / Q000166:cytology*; D016207:Cytokines / Q000096:biosynthesis / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry; D007165:Immunosuppression Therapy; D020287:In Situ Nick-End Labeling; D007498:Iris / Q000166:cytology*; D008213:Lymphocyte Activation / Q000502:physiology; D007959:Lymphocyte Culture Test, Mixed; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D010857:Pigment Epithelium of Eye / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013601:T-Lymphocytes / Q000502:physiology*
D016207:Cytokines; D017931:DNA Primers; D012333:RNA, Messenger