Abstract
Purpose :
Limbal melanocytes (LMel) were previously reported to be involved in immune regulation of the corneal epithelial stem cell niche, but the underlying molecular mechanisms of their potent immunosuppressive effects have not been clarified. This study aimed at elucidating local immunomodulatory properties and molecular mechanisms by which LMels regulate T-cell activities within the limbal stem cell niche.
Methods :
Primary cultures of LMel and limbal mesenchymal stromal cells (LMSC) were established following their isolation by enzymatic digestion from human donor corneoscleral tissues. CD4+ T-cell blasts were isolated from peripheral blood mononuclear cells (PBMC) from variable donors and activated by CD3/CD28 Dynabeads with supplement of IL-2. The effects of LMel and LMSC on CD4+ T-cell proliferation, activation, differentiation and cytokine production were investigated by flow cytometry and cytometric bead arrays. Gene expression profiles of LMel and LMSC stimulated by activated CD4+ T-cells were analyzed by RT2 Profiler PCR arrays and qRT-PCR to identify immunosuppressive candidate factors.
Results :
LMels suppressed the proliferation, activation and cytokine production of CD4+ T-cell blasts, particularly upon direct contact, at ratios as low as 1:200 (LMel:T-cells), comparable to LMSCs. Moreover, LMels inhibited differentiation of naïve CD4+ T-cell blasts into Th1/ Th2 cells and promoted their differentiation into Foxp3+ induced regulatory T-cells (iTreg). These immunosuppressive effects were induced by pro-inflammatory cytokines (IL-1α, IFN-γ) secreted by activated CD4+ T-cells and accompanied by upregulation of multiple immunomodulatory candidate factors as revealed by PCR arrays. Blocking experiments using neutralizing antibodies or chemical inhibitors could effectively neutralize the immunosuppressive effects of IDO1, TGF-ß and TRAIL by LMel. siRNA-mediated silencing of IDO1 and TGF-ß in LMel abrogated suppression of CD4+ T-cell proliferation, activation and induction of iTreg, confirming their critical role in immune regulation.
Conclusions :
The findings suggest that LMel-mediated immune regulation of local T-cell activity in the limbal niche occurs through the combined effects of IDO1, TGF-ß and TRAIL. The potent immunosuppressive properties of LMel may hold great potential for tissue engineering and therapeutic applications.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.