Decision letter: Stimulation strength controls the rate of initiation but not the molecular organisation of TCR-induced signalling

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Millions of naïve T cells with different TCRs may interact with a peptide-MHC ligand, but very few will activate. Remarkably, this fine control is orchestrated using a limited set of intracellular machinery. It remains unclear whether changes in stimulation strength alter the programme of signalling events leading to T cell activation. Using mass cytometry to simultaneously measure multiple signalling pathways during activation of murine CD8+ T cells, we found a programme of distal signalling events that is shared, regardless of the strength of TCR stimulation. Moreover, the relationship between transcription of early response genes Nr4a1 and Irf8 and activation of the ribosomal protein S6 is also conserved across stimuli. Instead, we found that stimulation strength dictates the rate with which cells initiate signalling through this network. These data suggest that TCR-induced signalling results in a coordinated activation program, modulated in rate but not organization by stimulation strength. eLife digest Amongst the different types of cells the body uses to protect itself, killer T cells have an unique role: they can detect and neutralize cells that have been become dangerous for the organism – for example, cells which are cancerous or hijacked by viruses. In a healthy organism, T cells circulate through the body in an inactivated state. When a disease emerges, receptors at the surface of the cells can detect elements coming from harmful agents; this stimulation then triggers a molecular cascade inside the T cell that leads to activation. This system is relatively simple, pairing a finite number of receptors with a limited set of internal components. At the same time, the activity of T cells is finely regulated, and their activation tightly controlled: they must kill enough cells to stop the illness without causing excess damage. How this is accomplished is still unclear. A T cell can recognize harmful agents that bind its receptors with differing strengths, but how this variability in stimulation strength affects the signaling processes within the cell is still poorly understood. To investigate this question, Ma et al. used an approach called mass cytometry and analyzed the internal processes of mouse killer T cells receiving different strengths of stimulation. This investigation revealed little change in the patterns of signaling in response to signals of different strength. Instead, what differed was the proportion of T cells that became activated, and how fast this process took place: stronger stimulations led to a larger population of killer T cells being activated more rapidly. Overall, this work sheds light on how killer T cells fine-tune their response to illness using only a simple system to control their activation. Introduction Effector differentiation of a naïve CD8+ T cell begins when its T cell receptor (TCR) recognizes a peptide-MHCI ligand complex. If the interaction is strong enough, a cascade of signalling events follows that allows the naïve T cell to differentiate and expand into a pool of effector cells. Signal transduction downstream of the TCR involves a highly diverse network of post-translational protein modifications that ultimately drive transcriptional, translational, metabolic and cytoskeletal changes in the cell. It is estimated that fewer than 0.01% of naïve CD8+ T cells can recognize a particular foreign peptide-MHCI complex (Jenkins and Moon, 2012). Despite the diversity of rearranged TCRs on these naïve cells and the extensive range of antigenic peptides that may be presented, the intracellular machinery within each naïve T cell is able to sense the strength of the receptor-ligand interaction and mount an appropriate response. Previous work has demonstrated that the strength of stimulation a T cell receives upon binding a peptide-MHC ligand complex determines its fate in the thymus and its probability of activating in the periphery. During thymic selection, T cells that weakly recognize self-peptides are retained, while those that strongly recognize self-peptides undergo negative selection and are removed (Daniels et al., 2006; Hogquist et al., 1994; Juang et al., 2010; Prasad et al., 2009). In the periphery, the population response to stimuli of different strengths can vary in speed, magnitude and phenotype (Denton et al., 2011; King et al., 2012; Moreau et al., 2012; Ozga et al., 2016; Palmer et al., 2016; Zehn et al., 2009). Work from our group and others indicates that these observations may be explained by the fact that stimulation strength controls the rate with which individual cells activate transcriptional and proliferative processes (Hommel and Hodgkin, 2007; Richard et al., 2018). This then raises the question, how does stimulation strength impact signalling downstream of the TCR, and how does this relate to transcriptional activation (Balyan et al., 2018)? Many previous studies have examined signalling mediators and their coordinated network during naïve T cell activation (Kannan et al., 2012; Krishnaswamy et al., 2014; Voisinne et al., 2019). Signalling through the TCR (Courtney et al., 2018; Navarro and Cantrell, 2014) begins with LCK and Fyn-mediated tyrosine phosphorylation of ITAM motifs on the invariant CD3 subunits. This creates a high affinity binding site for ZAP70, which, upon phosphorylation and activation, leads to the generation of the LAT-SLP76 signalosome. From here, signalling activates multiple cascades including MAPK (MEK1/2-ERK1/2), PDK1-PI3K, calcium, and NFκB (including IκBα-p65) pathways, each amplified and propagated via a series of phosphorylation events or other post-translational modifications. Signal transduction pathways can be broadly categorized as digital, with distinct ‘on’ or ‘off’ outcomes, or analogue, giving rise to a graded response (Conley et al., 2016; Zikherman and Au-Yeung, 2015). In digital signalling, once the threshold for activation is surpassed, an output signal of constant intensity is produced. In analogue signalling, higher intensity of the originating stimulus results in a proportionally higher intensity of the output signal. Previous work has demonstrated that ligand potency determines the extent of signalling at various proximal and distal nodes (Palmer et al., 2016; Rosette et al., 2001). Studies focused on digital signalling nodes showed that stimulation strength affects the percentage of cells that phosphorylate ERK (Altan-Bonnet and Germain, 2005; Das et al., 2009; Tian et al., 2007), PKD2 (Navarro et al., 2014), IκBα and the p65 component of NFκB (Kingeter et al., 2010). Most of these studies have looked at each signalling molecule separately. It therefore remains unclear how ligand potency affects the coordination of signalling downstream of the TCR in naïve T cells. TCR-induced responses are rapid and often transient, and responding cell populations can be heterogeneous. Single-cell approaches are therefore well-suited to examining this system. Mass cytometry provides single-cell resolution, uses antibody-mediated measurements that can detect post-translational signalling protein modifications, and can achieve high-dimensionality through simultaneous measurement of up to 40 epitopes (Bandura et al., 2009; Bendall et al., 2011; Lou et al., 2007; Ornatsky et al., 2010). Previous mass cytometry studies of T cell signalling have demonstrated that small differences in proximal signalling molecules are propagated and amplified in downstream targets (Mingueneau et al., 2014) and that the interplay of ‘activatory’ versus ‘inhibitory’ signalling components determines the response of effector T cells to different antigen doses (Wolchinsky et al., 2014). In this study, we designed a mass cytometry panel probing surface receptors and elements of key signalling pathways (Figure 1) to examine the effect of stimulation strength on naïve CD8+ T cell responses. We used a minimal antigen presentation system to ask how modulating only the strength of the TCR-pMHC interaction affects signalling pathways without the influence of variable costimulatory factors or feedback from other cell types. Our multi-dimensional approach allowed us to determine how ligand potency impacts the synchronization of multiple parallel pathways. Through simultaneous measurement of S6 phosphorylation and early mRNA transcripts, we also examined the concurrent activation of these markers of translational and transcriptional processes. Our data suggest that the coordination of the TCR-induced signalling pathways that we tested does not vary with stimulation strength. Instead, strength of stimulation determines the rate with which T cells initiate this programme. Figure 1 Download asset Open asset Diagrammatic representation of TCR signalling pathways measured by mass cytometry panel. Cartoon depicts the TCR-related signalling pathways examined in this study in our minimal stimulation system wherein T cells present antigen to each other. Solid lines indicate evidence of direct and dotted lines suggested or indirect interaction. Signalling proteins and post-translational modifications directly measured by mass cytometry antibodies are coloured blue (proteins) or green/red (sites of phosphorylation events) and outlined in purple. The mass cytometry panel also profiled surface proteins TCRβ, CD8α, CD45, CD25 (IL2Rα), and CD44. Results Mass cytometry detects active T cell conjugates We used the OT-I TCR transgenic mouse on a recombination-activating gene (RAG)-deficient background as a model for evaluating the impact of stimulation strength on TCR signalling pathways. In this model, all peripheral CD8+ T cells recognize the ovalbumin peptide SIINFEKL. Variants of SIINFEKL with altered potency for the OT-I TCR allow manipulation of the strength of TCR stimulation (Daniels et al., 2006; Hogquist et al., 1994; Hong et al., 2018; Rosette et al., 2001). In this study, we used the high potency SIINFEKL (N4), intermediate potency SIITFEKL (T4), and low potency SIIGFEKL (G4) peptides, as well as an unrelated control peptide, ASNENMDAM (NP68). We designed a custom mass cytometry antibody panel to detect five surface markers of T cell identity and activation, eight phosphorylated signalling proteins with corresponding total proteins, and IκBα, which is degraded in response to stimulation (Figure 1; Materials and methods). The antibodies labelled key components of major signalling pathways, including proximal signalling (pZAP70[Y319]/pSyk[Y352], pSLP76[Y128], pLCK[Y505] and pPLCγ1[Y783]), the MAPK pathway (pERK1/2[T202/Y204]), the PDK1-PI3K and mTOR pathways (pAKT[S473], pS6[S235/S236]), the NFκB pathway (IκBα) and the IL2 pathway (pSTAT5[Y694]). All of these phosphorylation sites indicate active signalling with the exception of the inhibitory Y505 phosphorylation of LCK (D'Oro and Ashwell, 1999; Marth et al., 1988). Measurement of total proteins allowed us to determine whether changes in phospho-protein staining were due to differences in signalling or protein expression levels. We isolated naïve CD8+ T cells from OT-I Rag1-/- splenocytes before stimulating with ligands of various strengths. To monitor signalling while naïve CD8+ T cells transitioned to activated T cells, and to relate signalling to changes in mRNA and protein expression during this process, cells were stimulated for 1, 2, 4 and 6 hr. We used a minimal, controlled system of peptide addition, allowing T cells to present antigens to each other. We also added exogenous IL2 to mitigate effects of potency-dependent differences in paracrine IL2 (Au-Yeung et al., 2017; Denton et al., 2011; Marchingo et al., 2014; Tan et al., 2017; Verdeil et al., 2006; Voisinne et al., 2015) and provide all cells with an effector-promoting environment (Pipkin et al., 2010; Verdeil et al., 2006). This system was chosen in order to examine the cell-intrinsic effects of TCR stimulation strength on signalling pathways. Peptides were added at 1 µM since peptide titration revealed minimal differences in the percentage of cells phosphorylating S6 and ERK between 100 nM and 1 µM stimulation conditions (Figure 2—figure supplement 1). Using this reductionist stimulation system, we previously found that stimulation strength determined the rate with which naïve T cells initiated transcriptional activation but that cells activated by all ovalbumin-derived ligands were proliferating and expressing the effector molecule CD44 by two days (Richard et al., 2018). Stimulated cells were stained with metal-conjugated antibodies and markers for dead cells and DNA before profiling by mass cytometry. We gated events detected by the mass cytometer in a hierarchical manner to select single, living cells that were TCRβ+ and CD8α+ before examining individual signalling molecules (Figure 2a). While gating for single cells based on DNA content (Figure 2—figure supplement 2a), we noted that a substantial percentage of events contained more than one cell-equivalent of DNA, particularly among cells stimulated with the strongest peptide, N4 (Figure 2—figure supplement 2b). Separating events by DNA content revealed two distinct populations, such that events containing two cell-equivalents of DNA had higher phosphorylated and total protein staining (Figure 2b, Figure 2—figure supplement 2c–d). It is therefore likely that this population contained multiple cells (i.e. doublets), potentially even cells engaged in TCR-peptide-MHC interactions with their neighbours. After normalization of each phosphorylated protein to the DNA signal detected in the same mass cytometry event, signal intensities for events with two cell-equivalents of DNA had similar ranges to those of events with one for most markers (Figure 2—figure supplement 2e). This provided further evidence that events with two cell-equivalents of DNA were doublets. Supporting the hypothesis that these doublets represented actively conjugated cells, a greater proportion of doublets than singlets showed signalling behaviour. In addition, from 1 to 4 hr after stimulation, pSLP76 signal was higher in doublet events even after normalization, suggesting that SLP76 was preferentially phosphorylated in cells actively engaged in TCR-peptide-MHC interactions. Because it was not possible to discern which proteins were signalling in which cell in a multiplet event, for subsequent analyses we included only singlet events. Figure 2 with 2 supplements see all Download asset Open asset Mass cytometry measurements of signalling in singlet and doublet events. (a) Naïve CD8+ T cells were stimulated with 1 μM peptides of various potencies for 0, 1, 2, 4 and 6 hr before profiling by mass cytometry. Histograms depict pS6 signal. (b) Bubble plots of all signalling molecules after stimulation as in (a) in mass cytometry events with 1 or 2 cell-equivalents of DNA (singlets or doublets, respectively). The size of the bubbles denotes the percentage of positive cells, and the colour denotes the centred and scaled median intensity of each positive fraction. Results are representative of cells from six biological replicates measured in two independent experiments as detailed in Supplementary file 1. Ligand potency affects the kinetics of signalling protein activation We next examined the kinetics of individual signalling molecules within our mass cytometry data. Total levels of signalling proteins did not substantially change over a 6 hr stimulation, while expression of effector proteins CD44 and CD25 increased in a time- and potency-dependent manner (Figure 3—figure supplement 1). In the presence of exogenous IL2, ligand potency did not strongly influence the rate with which individual cells phosphorylated STAT5 (Figure 3—figure supplement 2a). In the absence of exogenous IL2, STAT5 phosphorylation was associated with ligand potency, such that weak G4-stimulated cells showed no STAT5 phosphorylation (Figure 3—figure supplement 2b), likely due to autocrine/paracrine IL2 rapidly secreted by strongly stimulated cells (Tan et al., 2017). The percentages of cells degrading IκBα or phosphorylating S6 or ERK1/2 were not impacted by the presence of exogenous IL2. The percentages of cells phosphorylating pAKT[S473] were subtly increased by IL2 particularly under stimulation with low potency ligands (Figure 3—figure supplement 2b). This may reflect the mechanism proposed by Ross et al. whereby JAK signalling induced by IL2 ultimately stimulates mTORC2 phosphorylation of AKT[S473] (Ross et al., 2016). Together, these data indicate the selectivity of IL2 effects on T cell signalling pathways. Phosphorylation of proximal, membrane-recruited mediators ZAP70 and PLCγ1 was only detectable in a small percentage of cells at any point during this time course, preventing further interpretation (Figure 2b). For LCK, the percentage of cells with inhibitory phosphorylation of pY505 decreased at 6 hr in a potency-dependent manner, suggesting that stronger stimuli resulted in greater LCK activity only at this late time point (Figure 3—figure supplement 2a). For SLP76, the high potency ligand N4 induced a greater percentage of signalling cells and greater signalling intensity within these cells between 1 and 4 hr, whereas signalling was minimal in cells stimulated with intermediate (T4) or low potency (G4) ligands (Figure 2b, Figure 3—figure supplement 2a). Examination of the kinetics of individual distal signalling molecules revealed two distinct patterns. We defined these as transient if the percentage of signalling cells increased and subsequently decreased during the time course of high potency stimulation, and sustained if maximal signalling was ongoing at 6 hr (Figure 3a). ERK1/2, AKT and IκBα displayed transient signalling. While ERK1/2 and AKT are phosphorylated in response to TCR stimulation, IκBα is degraded, releasing NFκB subunits and permitting their translocation to the nucleus (Paul and Schaefer, 2013). Therefore, a reduction in IκBα+ cells indicates active signalling by this node. For these three signalling mediators, the percentage of cells actively signalling was maximal at 2 hr when stimulated with the highest potency N4 peptide but was delayed until 4 or 6 hr when stimulated with the lower potency T4 or G4 peptides (Figure 3a). After 2 hr, signalling via these proteins declined in strongly stimulated cells, resulting in a convergence with more weakly stimulated cells. In addition, the maximum percentage of cells signalling through these nodes was substantially higher in strongly stimulated cells. This may indicate repeated node activation after high potency stimulation, such that a greater proportion of cells were signalling at any given time of measurement. Thus, for these transiently signalling proteins, ligand potency was associated with the maximal proportion of signalling cells and the speed with which this proportion was reached on a populational level. Figure 3 with 3 supplements see all Download asset Open asset Kinetics of selected signalling proteins and impact of MEK and mTOR pathway inhibitors on T cell activation parameters. (a) The percentage of cells positive for each marker is plotted against time. Results depict combined data from six biological replicates measured in two independent experiments as detailed in Supplementary file 1. Points represent the mean and error bars depict the SD. Data underlying plots are provided in Supplementary file 5. (b–c) Flow cytometry measurements of pS6[S235/236] and pERK1/2 after 2 hr of pre-treatment with DMSO vehicle control, rapamycin (Rapa) 200 nM, MEK162 (MEK) 0.5 μM, 1 μM and 5 μM, or combined rapamycin with MEK162 (R + M), followed by 4 hr stimulation with 1 μM N4 or NP68 peptides. Results are representative of 3 independent experiments. (d) The fraction of pS6+ cells in N4-stimulated conditions with the indicated inhibitor treatments versus DMSO (top). The median fluorescent intensity of pS6 among pS6+ cells in N4-stimulated conditions with the indicated inhibitor treatments (bottom). Lines represent the median. Results depict combined data from three independent experiments. In contrast to these transient signalling events, S6 phosphorylation induced by TCR stimulation was sustained within our time course (Figure 3a). Under N4 stimulation, there was a rapid initial increase in the percentage of pS6+ cells before a plateau. This pattern may be indicative of the signalling protein approaching saturation. The appearance of pS6+ cells was slower after stimulation with lower potency ligands, but the proportions of pS6+ cells approached convergence between N4, T4 and G4 stimulations at 6 hr. Thus, for S6, the rate with which cells active signalling, but not the maximal proportion of signalling cells was associated with ligand We a of pERK1/2 measurements in our mass cytometry with previous (Altan-Bonnet and Germain, 2005; Das et al., 2009; Tian et al., The extent of ERK1/2 phosphorylation in cells, as determined by the median marker was by ligand potency (Figure 3—figure supplement 3a). This that ERK1/2 digital signalling that is on a cell there is an ‘on’ or ‘off’ state. signal was also (Figure Figure 3—figure supplement The intensity of pS6 signal in pS6+ cells increased over time. to total S6 protein intensity this effect under strong and suggested that ligand potency may subtly the rate of S6 phosphorylation within individual cells (Figure 3—figure supplement in to the percentage of pS6+ cells at early time S6 phosphorylation at is by downstream of and downstream of et al., et al., 2007; et al., we were in how each of these pathways to its digital in strongly stimulated cells. To this we cells with inhibitors of et al., and mTOR and 2015) before stimulation with N4 peptides (Figure 3—figure supplement The phosphorylation of S6 decreased in response to rapamycin (Figure with little between doses of nM and 2 μM (Figure 3—figure supplement but the of pS6 and the percentage of pS6+ cells were not In while S6 phosphorylation also decreased within each cell in response to this response was between 0.5 and 5 μM, and at the highest the was In addition, even low doses of MEK162 the percentage of cells with phosphorylated S6 (Figure MEK162 and rapamycin resulted in of S6 phosphorylation (Figure the of these two substantially impacted CD44 expression at the of inhibitors used (Figure 3—figure supplement but a of was after 2 days (Figure 3—figure supplement Thus, simultaneous activity of the MEK and mTOR pathways is for phosphorylating and proliferative and MEK signalling is for S6 digital These data the coordinated of signalling downstream of the Ligand potency determines the of signalling cells but not the coordination of signalling events To of the simultaneous measurements in mass cytometry we next tested for of into all of the signalling markers measured et al., 2017; Figure Figure supplement 1). We defined in the high We then the of cells within these between and all stimulated conditions and methods). of revealed two signalling defined as pS6+ was most in the high potency cells. defined as pS6+ under all stimulation conditions with similar (Figure A and were by the and A and were transient, whereas and were that the high potency ligand N4 was of a greater of and A up to 4 hr after stimulation (Figure In of and increased at a similar rate between 1 and 6 hr after stimulation with all ovalbumin-derived ligands and were not associated with ligand Figure 4 with 2 supplements see all Download asset Open asset Examination of multi-dimensional in mass cytometry signalling data. (a) Mass cytometry stimulation time were further for that in with stimulation. was on two biological replicates as in Materials and were defined within the mass cytometry and of cells from each within each in the an individual At the of the to mass cytometry marker measurements with colour the intensity of each marker in each by was on these marker intensity At the of the to stimulation conditions with colour the change in in stimulated versus conditions within each that did not change in of are indicated in and populations by coloured of the underlying the are provided in Supplementary file 5. (b) of cells and in mass cytometry Results are combined data from six biological replicates measured in two independent experiments as detailed in Supplementary file 1. Points represent the mean and error bars depict the SD. Data underlying plots are provided in Supplementary file 5. as in (b) further by CD44 To these signalling we further their relationship with surface expression of the effector protein which was an to (Figure We examined the possible defined by of the markers and The strongest peptide, N4, was of a proportion of pS6+ cells after 1 hr of stimulation (Figure Figure supplement This was by an population of pS6+ cells, which reached its maximum 2 hr after stimulation. stimulated by the peptides T4 and G4 showed of these and their were For all a high of pS6+ cells was between 4 and 6 hr. CD44 by 4 hr after strong and intermediate stimulation and and by 6 hr after weak stimulation From these we the coordination of distal TCR-induced signalling. We that within our stimulation system, activating cells phosphorylate S6 and ERK1/2, followed by after which ERK1/2 followed by STAT5 in cells. In the early after stimulation, signalling cells CD44 at the same as cells but CD44 expression by hr. time and cells to sustained those activated with potency ligands to those stimulated with high potency ligand (Figure To this order of activation events, we activation of cells under each stimulation across all time based on their expression of pS6 (Figure supplement We then in what order these and CD44 activation events were initiated (Figure supplement We found that pS6 followed by and CD44. the of ERK1/2 phosphorylation to the most increase in S6 evidence that ERK activation phosphorylation (Figure The order of activation events was across stimulation conditions to of events, Materials and methods). The signalling molecules and IκBα were the of their order of activation particularly in weakly stimulated cells, but their changes the further suggested patterns between stimuli (Figure supplement