This week journal Cell published a very important paper from KC Garcia's lab at Stanford. The focus of the study was to understand and determine, if possible, all the parameters that makes a given peptide/MHC and its similar looking variants stimulatory versus non-stimulatory to a given TCR molecule.
First, they compared two TCR molecules specific for class II HLA-DR4-HIV(Gag164–183). While both TCR expressing clones, TCR6 and TCR11, could bind pHLA tetramer, only TCR6 "proliferated, secreted cytokines, and downregulated TCR in response to stimulation with" HLA-DR4-HIV(Gag164–183). Both TCR clones could respond directly or after transducing them into TCR αβ-deficient cell line to non-specific stimulation such as anti-CD3 (OKT3).
Similar observation were made using TCRs specific for class I HLA-B35-HIV(Pol448–456). Here too, both TCR clones designated as TCR589 and TCR55 could bind specific pMHC tetramer but only TCR589 showed dose-dependent secretion of IL-2 in response to peptide-pulsed APCs.
Next, the authors determined TCR/pMHC affinity by surface plasmon resonance. For TCR55 (non-stimulatory) and TCR589 (stimulatory) clones it was 17μM and 4μM KD, respectively. Surprisingly, for TCR6 (stimulatory) and TCR11 (non-stimulatory) clones affinity was 10μM and 1μM KD, respectively.
Analysis of TCR55 (non-stimulatory) and TCR589 (stimulatory) TCR/pMHC interaction on lipid bilayers by total internal reflection fluorescence (TIRF) microscopy showed similar mean dwell times of 10.1 ± 2.0 s and 8.7 ± 2.1 s, respectively. Structural analysis of the crystal structures of TCR589 and TCR55 in complex with HLA-B35-HIV(Pol448–456) also showed "no substantial differences in the conformations of the constant regions that would indicate differential allostery correlating with signaling."
Functional analysis of TCR proximal signaling molecules showed that stimulatory TCR589 but not TCR55 could up-regulate CD69, induce calcium release and recruit ZAP70 in response to HLA-B35-HIV(Pol448–456).
Next, they generated peptide library similar to HIV(Pol448–456) with potencies "ranging from full agonism to partial agonism to no detectable signaling". Importantly, "the peptides that elicited these disparate signaling outcomes were remarkably similar in sequence."
One common feature of agonist peptides such as pep20 and SQL that could signal TCR55 was that such interaction excluded CD45 with varying degrees of efficiency compared to non-stimulatory peptide ligands in "a cell-free system using giant unilamellar vesicles (GUVs) to eliminate effects of signaling in live cells".
In addition, another common feature of stimulatory peptide ligands were so called catch bond formation between TCR and stimulatory pMHC "under conditions of force using a Biomembrane Force Probe (BFP) bond lifetime assay". In contrast, non-stimulatory ligands formed slip bond formations.
In summary, this study indicates that "catch bond formation and CD45 exclusion as principal emergent properties differentiating stimulatory from non-stimulatory TCR-pMHC interactions" but "the same pMHC can be an agonist or non-agonist for different TCRs, although it is difficult to assign causality to either the TCR or the peptide-MHC. The formation of a catch or slip bond is a collective property of the entire TCR-pMHC interface."
My opinion: one drawback of this study is that not many labs can verify its conclusions. I have not even heard of some of the techniques they used here. I could only trust that the authors tried to interpret their data as unbiased as it is rationally possible. Another weakness of the study is a failure to identify parameters that control CD45 exclusion or catch bond formation when TCR interacts with pMHC. Basically, with the exception of few very high affinity ligands (< 2μM), we cannot predict in silico whether a given peptide will be stimulatory or non-stimulatory without actually doing an experiment on it.
posted by David Usharauli
Similar observation were made using TCRs specific for class I HLA-B35-HIV(Pol448–456). Here too, both TCR clones designated as TCR589 and TCR55 could bind specific pMHC tetramer but only TCR589 showed dose-dependent secretion of IL-2 in response to peptide-pulsed APCs.
Next, the authors determined TCR/pMHC affinity by surface plasmon resonance. For TCR55 (non-stimulatory) and TCR589 (stimulatory) clones it was 17μM and 4μM KD, respectively. Surprisingly, for TCR6 (stimulatory) and TCR11 (non-stimulatory) clones affinity was 10μM and 1μM KD, respectively.
Analysis of TCR55 (non-stimulatory) and TCR589 (stimulatory) TCR/pMHC interaction on lipid bilayers by total internal reflection fluorescence (TIRF) microscopy showed similar mean dwell times of 10.1 ± 2.0 s and 8.7 ± 2.1 s, respectively. Structural analysis of the crystal structures of TCR589 and TCR55 in complex with HLA-B35-HIV(Pol448–456) also showed "no substantial differences in the conformations of the constant regions that would indicate differential allostery correlating with signaling."
Functional analysis of TCR proximal signaling molecules showed that stimulatory TCR589 but not TCR55 could up-regulate CD69, induce calcium release and recruit ZAP70 in response to HLA-B35-HIV(Pol448–456).
Next, they generated peptide library similar to HIV(Pol448–456) with potencies "ranging from full agonism to partial agonism to no detectable signaling". Importantly, "the peptides that elicited these disparate signaling outcomes were remarkably similar in sequence."
One common feature of agonist peptides such as pep20 and SQL that could signal TCR55 was that such interaction excluded CD45 with varying degrees of efficiency compared to non-stimulatory peptide ligands in "a cell-free system using giant unilamellar vesicles (GUVs) to eliminate effects of signaling in live cells".
In addition, another common feature of stimulatory peptide ligands were so called catch bond formation between TCR and stimulatory pMHC "under conditions of force using a Biomembrane Force Probe (BFP) bond lifetime assay". In contrast, non-stimulatory ligands formed slip bond formations.
In summary, this study indicates that "catch bond formation and CD45 exclusion as principal emergent properties differentiating stimulatory from non-stimulatory TCR-pMHC interactions" but "the same pMHC can be an agonist or non-agonist for different TCRs, although it is difficult to assign causality to either the TCR or the peptide-MHC. The formation of a catch or slip bond is a collective property of the entire TCR-pMHC interface."
My opinion: one drawback of this study is that not many labs can verify its conclusions. I have not even heard of some of the techniques they used here. I could only trust that the authors tried to interpret their data as unbiased as it is rationally possible. Another weakness of the study is a failure to identify parameters that control CD45 exclusion or catch bond formation when TCR interacts with pMHC. Basically, with the exception of few very high affinity ligands (< 2μM), we cannot predict in silico whether a given peptide will be stimulatory or non-stimulatory without actually doing an experiment on it.
posted by David Usharauli
