Small Leucine-rich repeat proteoglycans regulate CD4⁺T lymphocytes - implications in the eye

Abstract

Purpose : Lumican, biglycan and decorin are small leucine-rich repeat proteoglycans (SLRPs) that form the bulk of the corneal stromal ECM, and increase in circulation during infections and inflammation. We hypothesize that these SLRPs, in their ECM-free form, interact with immune cells to regulate inflammation. Here we investigated the role of SLRPs in CD4⁺T cell mediated adaptive immune response using in vitro assays and in vivo inflammation models

Methods : Splenic CD4⁺T cells were activated in vitro with anti-CD3 and CD28 in the presence of recombinant (r) SLRPs, labeled with CFSE (CellTrace kit, Invitrogen), and assayed for dye dilution/proliferation by flow cytometry. Activation of CD4⁺T cell were assayed with anti-CD69 staining and flow cytometry. In a dermal inflammation model Lum^-/-, Bgn^-/- and WT mice (n=8/genotype) were sensitized with 0.5% dinitrofluoro benzene (DNFB), followed by 0.25% DNFB on the ear. All mouse studies were approved by IACUC. On day 5, CD4⁺T cells were analyzed in the ear tissues and draining lymph node (dLN) by flow cytometry, immunohistochemistry (IHC) and confocal microscopy.

Results : The rSLRPs suppressed CD4⁺T cell proliferation in vitro in a dose-dependent manner with >70% inhibition at 2.5 µg/ml (p<0.001). All three rSLRPs suppressed CD4⁺T activation (surface CD69) by 6h. Blocking β2 integrin (LFA-1 subunit) caused a loss of lumican and biglycan-mediated inhibition, suggesting these to function in an integrin-dependent manner. We are also localizing SLRPs in immunologoical synapses betwen DCs and T cells (figure). In an ear inflammation model, Lum^-/- and Bgn^-/- mice showed increased ear thickness, and higher CD4⁺T cells in tissue sections compared to wild types. The knockout mice also showed increased proliferating CD4⁺T cells in dLNs.

Conclusions : Our results suggest that stromal SLRPs may function as a natural brake for CD4⁺T cells by suppressing their proliferation and activation. These crosstalks involve SLRP interactions with LFA-1 integrins on T lymphocytes. In the future SLRP interactions may be manipulated to modulate CD4⁺T cells to control infection, inflammation and autoimmune diseases.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

 

Confocal image showing interaction of GFP⁺ OTII CD4⁺T cell (green) with mCherry⁺CD11c⁺ DC (orange) stimulated with OVA_323-339 peptide and polymerized actin (phalloidin, grey) at the immunological synapse between CD4⁺T cell and DC. DAPI (blue) stains the nucleus.