Impact of morphological features and chemical composition of tendon biomimetic scaffolds on immune recognition via Toll-like receptors.

Tendinopathies are a major worldwide clinical problem. The development of tendon biomimetic scaffolds is considered a promising, therapeutic approach. However, to be clinically effective, scaffolds should avoid immunological recognition. It has been well described that scaffolds composed of aligned fibers lead to a better tenocyte differentiation, vitality, proliferation and motility. However, little has been studied regarding the impact of fiber spatial distribution on the recognition by immune cells. Additionally, it has been suggested that higher hydrophilicity would reduce their immune recognition. Herein, polycaprolactone (PCL)-hyaluronic acid (HA)-based electrospun scaffolds were generated with different fiber diameters (in the nano- and micro-scales) and orientations as well as different grades of wettability and the impact of these properties on immunological recognition has been assessed, by means of Toll-like receptor (TLR) reporter cells. Our results showed that TLR 2/1 and TLR 2/6 were not triggered by the scaffolds. In addition, the TLR 4 signalling pathway seems to be triggered to a greater extent by higher PCL and HA concentrations, but the alignment of the fibers prevents the triggering of this receptor. Taken together, TLR reporter cells were shown to be a useful and effective tool to study the potential of scaffolds to induce immune responses and the results obtained can be used to inform the design of fibrous scaffolds for tendon repair.

The ligand-dependent suppression of TCR signaling by the immune checkpoint receptor LAG3 depends on the cytoplasmic RRFSALE motif.

Lymphocyte activation gene 3 (LAG3) is an inhibitory immune checkpoint receptor that restrains autoimmune and antitumor responses, but its evolutionarily conserved cytoplasmic tail lacks classical inhibitory motifs. Major histocompatibility complex class II (MHC class II) is an established LAG3 ligand, and fibrinogen-like protein 1 (FGL1), lymph node sinusoidal endothelial cell C-type lectin (LSECtin), and Galectin-3 have been proposed as alternative binding partners that play important roles in LAG3 function. Here, we used a fluorescent human T cell reporter system to study the function of LAG3. We found that LAG3 reduced the response to T cell receptor stimulation in the presence of MHC class II molecules to a lesser extent compared with the receptor programmed cell death protein 1. Analysis of deletion mutants demonstrated that the RRFSALE motif in the cytoplasmic tail of LAG3 was necessary and sufficient for LAG3-mediated inhibition. In this system, FGL1, but not LSECtin or Galectin-3, acted as a LAG3 ligand that weakly induced inhibition. LAG3-blocking antibodies attenuated LAG3-mediated inhibition in our reporter cells and enhanced reporter cell activation even in the absence of LAG3 ligands, indicating that they could potentially enhance T cell responses independently of their blocking effect.