In contrast, for the W2C8 TCR with an Ackon of 2.1 × 10−6 μm4s−1 and a koff of 3.6/s, to achieve a similar amount of cumulative

lifetime, it would require a pMHC surface density of more than 50 000/μm2 despite a slower off-rate (and a longer lifetime). Therefore, the apparently faster 2D off-rates of more potent interactions can be effectively compensated by greatly boosted 2D on-rates in terms of total confinement RXDX-106 molecular weight time as a result of fast serial TCR–pMHC engagement. It is well known that CD8/CD4 co-receptors greatly enhance T-cell responses to antigen stimulation [11, 34, 47]. However, the underlying mechanism is unclear. It has been proposed that CD8 binds to the same pMHC engaged with TCR to stabilize the TCR–pMHC interaction [47] and that co-receptors

(especially CD4) contribute to T-cell function by catalyzing the recruitment of Lck [47, 48]. SPR work using purified molecules reported discrepant results; some showed that CD8 enhances the TCR–pMHC interaction by reducing the off-rate [49] whereas others showed that TCR binds to pMHC independent of CD8 [50]. However, the work presented here and previous work by others [8, 51] demonstrate that CD8 significantly enhances pMHC tetramer staining of T cells. Tetramer technology is limited by low temporal resolution, low sensitivity, and difficulty to relate to intrinsic kinetic parameters [25]. Using the micropipette adhesion frequency method Fluorouracil cell line with much higher sensitivity and temporal resolution, we have recently shown that in the OT1 and F5 TCR transgenic mouse systems, surrogate APCs adhere to naïve T cells in a two-stage fashion [34]. The first stage (<1 s contact time) is dominated by the TCR–pMHC interaction and the second stage (>1 s contact time) includes a significant CD8-dependent adhesion increase. The second-stage adhesion increment results from cooperative TCR–pMHC–CD8 trimeric interaction that requires cell signaling via Src kinases. In this study, we have shown that this is a shared feature ALOX15 of the CD8+ hybridoma cells transfected with human TCRs.

However, in the gp209 system, the synergy indices Δ(/mpMHC) are much higher than what we previously observed, e.g. 0.2 μm2 (Fig. 6) and 0.023 μm2 [34] for the strongest interactions in this (19LF6) and the previous (OVA) studies, respectively. Interestingly, the much higher synergy indices correlate with the ∼ tenfold higher levels of CD8 than the gp209-specific TCRs expressed on the hybridoma cells (Fig. 1B). By comparison, the naïve T cells used in the previous study express ∼ twofold higher CD8 than OT1 TCR [34]. This suggests that the higher the CD8:TCR ratio, the greater the synergy. This study represents the first 2D kinetic analysis of recognition of a self-antigen by a panel of TCRs, which also differs from previous 2D kinetics studies using a single TCR to interact with a panel of variant pMHC ligands.