T lymphocytes need less than 3 min to discriminate between peptide MHCs with similar TCR‐binding parameters

Abstract

T lymphocytes need to detect rare cognate foreign peptides among numerous foreign and self-peptides. This discrimination seems to be based on the kinetics of TCRs binding to their peptide–MHC (pMHC) ligands, but there is little direct information on the minimum time required for processing elementary signaling events and deciding to initiate activation. Here, we used interference reflection microscopy to study the early interaction between transfected human Jurkat T cells expressing the 1G4 TCR and surfaces coated with five different pMHC ligands of 1G4. The pMHC concentration required for inducing 50% maximal IFN-γ production by T cells, and 1G4-pMHC dissociation rates measured in soluble phase or on surface-bound molecules, displayed six- to sevenfold variation among pMHCs. When T cells were dropped onto pMHC-coated surfaces, rapid spreading occurred after a 2-min lag. The initial spreading rate measured during the first 45 s, and the contact area, were strongly dependent on the encountered TCR ligand. However, the lag duration did not significantly depend on encountered ligand. In addition, spreading appeared to be an all-or-none process, and the fraction of spreading cells was tightly correlated to the spreading rate and spreading area. Thus, T cells can discriminate between fairly similar TCR ligands within 2 min.

CD8 co-receptor enhances T cell activation without any effect on initial attachment

“…As previously, we used a human Jurkat T cell line expressing the 1G4 TCR (Brodovitch 2015). As this line does not express CD8 (CD8-), we used lentiviral transduction (Denham 2019) to produce a version that expresses CD8 (CD8+).…”

“…As a cell response, we measured the rate and extent of spreading during the first 15 minutes following contact with antigens. Indeed, it was previously found that the maximum spreading rate determined during the first 3 minutes after contact was robustly correlated to pMHC activation potency (Brodovitch 2015).…”

“…The wild type or H74A mutant of HLA-A2 heavy chain (residues 1-278) with C-terminal BirA tag and β2-microglobulin were expressed as inclusion bodies in E.coli, refolded in vitro in the presence of synthesized variants of the NY-ESO-1157−165 peptide SLLMWITQV (9V), and purified using sizeexclusion chromatography (Chen 2005, Aleksic 2010). The pMHCs used in this study, as in previously reported experiments (Brodovitch 2015, were 9V and variants 3A, 3Y, or 9L in complex with wild-type HLA-A2, or 9V in complex with the HLA-A2 mutant H74A.…”

“…Glass surfaces were prepared as previously described (Brodovitch 2015 by sequential cleaning with a mix of 70% sulfuric acid and 30% H202, coating with poly-L lysine (Sigma-Aldrich, 150,000-300,000 MW), activation with glutaraldehyde (2.5%), coupling with biotinylatedbovine serum albumin (100 µg/ml, Sigma) then neutravidin (10µg/ml), and finally coating with biotinylated pMHCs. Absolute calibration of pMHC surface density was done as previously described by labeling with an excess of Alexa Fluor 488 labeled anti-HLA antibody (Biolegend, San Diego) and fluorescence determination.…”

Biphasic mechanosensitivity of TCR mediated adhesion of T lymphocytes

“…Fig. 1D shows an example of single cell time-lapse RICM demonstrating that the cells on hard substrates lag behind in spreading already in the time window 0 to 5 minutes, a period shown earlier to be critical for antigen recognition (18). Fig.…”