Kutuzov, M. | bioRxiv Science

bioRxiv2020

Determination of the molecular reach of the protein tyrosine phosphatase SHP-1

Immune receptor signalling proceeds by the binding (or tethering) of enzymes to their cytoplasmic tails before they catalyse reactions on substrates within reach. This is the case for the enzyme SHP-1 that, upon tethering to the inhibitory receptor PD-1, dephosphorylates membrane substrates to suppress T cell activation. Precisely how tethering regulates SHP-1 activity is incompletely understood. Here, we use surface plasmon resonance to measure binding, catalysis, and molecular reach for PD-1 tethered SHP-1 reactions. We find that the reach of PD-1--SHP-1 complexes is dominated by the 13.0 nm reach of SHP-1 itself. This is longer than an estimate from the structure of the allosterically active conformation (5.3 nm), suggesting that SHP-1 explores multiple active conformations. Using modelling, we show that when uniformly distributed, PD-1--SHP-1 complexes can only reach 15% of substrates but this increases to 90% when they are co-clustered. When within reach, we show that membrane recruitment increases the activity of SHP-1 by a 1000-fold increase in local concentration. The work highlights how molecular reach regulates the activity of membrane-recruited SHP-1 with insights applicable to other membrane-tethered reactions. Significance statementImmune receptors transduce signals by recruiting (or tethering) cytoplasmic enzymes to their tails at the membrane. When tethered, these enzymes catalyse reactions on other substrates to propagate signalling. Precisely how membrane tethering regulates enzyme activity is incompletely understood. Unlike other tethered reactions, where the enzyme tethers to the substrate, the substrate in this case is a different receptor tail. Therefore, the ability of the receptor-tethered enzyme to reach a substrate can be critical in controlling reaction rates. In this work, we determine the molecular reach for the enzyme SHP-1 and the receptor PD-1 to which it can tether, and show how molecular reach controls receptor signalling.

biophysics↗

bioRxiv2020

Human CD8+ T cells exhibit a shared antigen threshold for different effector responses

T cells recognising cognate pMHC antigens become activated to elicit a myriad of cellular responses, such as target cell killing and the secretion of different cytokines, that collectively contribute to adaptive immunity. These effector responses have been hypothesised to exhibit different antigen dose and affinity thresholds, suggesting that pathogen-specific information may be encoded within the nature of the antigen. Here, using systematic experiments in a reductionist system, where primary human CD8+ T cell blasts are stimulated by recombinant pMHC antigen alone, we show that different inflammatory cytokines have comparable antigen dose thresholds across a 25,000-fold variation in affinity. Although co-stimulation by CD28, CD2, and CD27 increased cytokine production in this system, the antigen threshold remained comparable across different cytokines. When using primary human memory CD8+ T cells responding to autologous antigen presenting cells equivalent thresholds were also observed for cytokine production and killing. These findings imply a simple phenotypic model of TCR signalling where multiple T cell responses share a common rate-limiting threshold and a conceptually simple model of antigen recognition, where the chance factor of antigen dose and affinity do not provide any additional response-specific information.

immunology↗