Immunosuppressive Properties of Regulatory T Cells Generated by Incubation of Peripheral Blood Mononuclear Cells with Supernatants of Human RPE Cells

Abstract

PURPOSE.To determine whether supernatants of human retinal pigment epithelium (RPE) cells can convert CD4 þ T cells into regulatory T cells (Tregs) under Treg-induction conditions in vitro and in vivo.METHODS. Peripheral blood mononuclear cells were cocultured with supernatants from TGFb2-pretreated human RPE lines on anti-CD3-coated plates. Cells were then separated with a CD4 þ CD25 þ Treg isolation kit and cultured with supernatants from RPE, anti-CD3/CD28 antibodies, high-dose IL-2, and TGFb2. By flow cytometry sorting, CD25 þ CD45RA À Tregs were separated. Expressions of CD25 high , Foxp3, CD152, and TNFRSF 18 on Tregs were analyzed by flow cytometry. Cytokine production by Tregs was measured by ELISA. Proliferation of target T cells was assessed by [ 3 H]thymidine incorporation or CFSE incorporation. In addition, mouse RPE-induced Tregs were used for the in vitro assay and in vivo experimental autoimmune uveitis (EAU) models.RESULTS. Human RPE-induced Tregs expressed higher levels of the Treg markers CD25 high , Foxp3, CD152, and TNFRSF 18. In addition, RPE-induced Tregs included significant numbers of CD4 þ CD25 high Foxp3 high CD45RA À active effector Tregs that significantly suppressed the activation of Th1/Th17 cell lines, indicating that they have immunosuppressive properties. Furthermore, CD4 þ CD25 low Foxp3 low CD45RA À nonsuppressing cytokine-secreting T cells were removed from the in vitro-manipulated Treg population. Administration of mouse RPE-induced Tregs significantly suppressed ocular inflammation in mice with EAU. In addition, the Tregs suppressed retinal antigen-specific T cells in vitro.CONCLUSIONS. It is hoped that through the data provided in this study that Tregs might become useful as individualized therapeutic agents for ocular autoimmune diseases. (Invest Ophthalmol Vis Sci. 2012;53:7299-7309)

Peanut oral immunotherapy results in increased antigen-induced regulatory T-cell function and hypomethylation of forkhead box protein 3 (FOXP3)

“…either defined through CFSE lo or CD40L/CD69+ in response to specific antigen stimulation). Furthermore, our data show that the iTreg population was CD45RO+ (and CD45RA neg ), Helios lo/neg , CD49b+/LAG3+, Foxp3+, and IL-10+; therefore, the iTreg we identified with these markers are consistent with other studies 32–36 and are possibly Foxp3+ Tr1 cells 28,34,38,39 . Moreover, we demonstrated that the iTreg population induced through peanut OIT was specific in suppression of autologous Teff proliferation in response to peanut, not other antigens.…”

“…The method of identification of iTreg was consistent with other publications 32–36,49,68 in which iTreg were defined as proliferating CD4+CD25 hi Foxp3+ cells (i.e. either defined through CFSE lo or CD40L/CD69+ in response to specific antigen stimulation).…”

“…iTreg can be characterized as CD4+CD25 hi cells that proliferate in response to specific antigens (i.e., CFSE lo or CD40L/CD69+) 32–35 . iTreg can be Foxp3+ and/or TGF-β+ and/or IL-10+ 32,34,36,37,49,68 . Type 1 regulatory T cells (Tr1) release IL-10 28,38,39 and express CD4, CD49, and LAG3 on their surface 28,38,39 which differs from nTreg expressing CD4, CD25 hi , CD127 lo , and perhaps by Helios on their surface 25–27,40–43 .…”

Retinal Pigment Epithelial Cell Line Suppression of Phagolysosome Activation

“…Since there are many reports showing almost the same activity of ARPE-19 cell monolayers to cultured primary RPE [8][9][10][11], the conditioned media of cultured ARPE-19 cells were assayed to see whether they suppress phagolysosome activation in phagocytizing macrophages. The ARPE-19 cells were cultured to confluence, or kept subconfluent before changing the media to the serum-free media for an additional 24 hours of incubation.…”

“…These cells grow to a confluent monolayer, and have been used to study the effects of oxidants and cytokines involved in RPE changes related to age-related macular degeneration [8,9]. These cell lines when treated with TGF-β2 have been found to mediate the activation of Treg cells [10]. The activity in these studies have shown parallel effects as seen in the eye with AMD, or by primary RPE cell cultures.…”