Abstract
Purpose :
We have previously reported that inhibition of mast cell activation prolongs corneal graft survival following transplantation. The purpose of the current study is to investigate whether mast cells directly activate T cells to secrete IFNγ, a critical inflammatory molecule for their effector function.
Methods :
Single-cell suspensions of corneal tissue and peritoneal lavage were prepared to evaluate levels of major histocompatibility complex II (MHC II) expression (Mean Fluorescent Intensity; MFI) on FceR1+cKit+ mast cells using flow cytometry. Bone marrow cells were harvested from femurs and tibias of balb/c mice and cultured for 3-4 weeks in the presence of IL-3 (10 ng/ml) and SCF (50 ng/ml) to generate bone marrow mast cells. Alloprimed CD4+ T cells were sorted from the draining lymphoid tissues of C57BL/6 transplant recipients (grafted with Balb/c corneas) using flow cytometry. To evaluate the effect of mast cells on T cell stimulation, purified C57BL/6 CD4+ T cells were cultured with balb/c mast cells. Co-cultures of alloprimed CD4+T cells and antigen-presenting cells (APCs; CD90.2- from balb/c splenocytes) served as a positive control. Intracellular expression of IFNγ by CD4+ T cells was assessed after 48 hours using flow cytometry.
Results :
A high expression of MHC II was observed in mast cells harvested from ocular tissues (P<0.001) peritoneal fluid (P<0.001) and bone marrow (P<0.05), compared to isotype controls. Furthermore, mast cells upregulated MHC II expression by 2-fold following IFNγ stimulation (100 ng/ml), compared to unstimulated controls (P<0.05). Alloprimed CD4+T cells co-cultured with mast cells showed an approximate 3.5 fold increase in the expression of IFNγ, compared to T cells cultured alone (P<0.05). Similarly, allogenic APCs induced a 2-fold increase in the expression of IFNγ by CD4+ T cells (P<0.03), which was comparable to allogenic mast cells mediated T cell activation.
Conclusions :
Our data demonstrate that mast cells express antigen-presenting molecule, MHC II, and directly activate alloprimed T cells to secrete their signature molecule, IFNγ.
This is a 2021 ARVO Annual Meeting abstract.