TCR signalling network organization at the immunological synapses of murine regulatory T cells.

Eur. J. Immunol. 2017. 0: 1–16 Marco van Ham et al. DOI: 10.1002/eji.201747041 1 Basic Molecular immunology and signaling Research Article TCR signalling network organization at the immunological synapses of murine regulatory T cells Marco van Ham∗1,Ren ´ eTeich ∗2, Lars Philipsen3, Jana Niemz2, Nicole Amsberg1, Josef Wissing1, Manfred Nimtz1,LotharGr ¨ obe2, Stefanie Kliche3,NadineThiel 2, Frank Klawonn1,5,MarioHubo 4, Helmut Jonuleit4, Peter Reichardt3,AndreasJ.M ¨ uller3,6, Jochen Huehn∗2and Lothar J¨ ansch∗1 1Cellular Proteomics, Helmholtz Centre for Infection Research, Braunschweig, Germany 2Experimental Immunology, Helmholtz Centre for Infection Research, Braunschweig, Germany 3Institute of Molecular and Clinical Immunology, Otto-von-Guericke University, Magdeburg, Germany 4Department of Dermatology, Johannes Gutenberg-University Mainz, Mainz, Germany 5Department of Computer Science, Ostfalia University of Applied Sciences, Wolfenbuettel, Germany 6Intravital Microscopy of Infection and Immunity, Helmholtz Centre for Infection Research, Braunschweig, Germany Regulatory T (Treg) cells require T-cell receptor (TCR) signalling to exert their immunosuppressive activity, but the precise organization of the TCR signalling network compared to conventional T (Tconv) cells remains elusive. By using accurate mass spectrometry and multi-epitope ligand cartography (MELC) we characterized TCR signalling and recruitment of TCR signalling components to the immunological synapse (IS) in Treg cells and Tconv cells. With the exception of Themis which we detected in lower amounts in Treg cells, other major TCR signalling components were found equally abundant, however, their phosphorylation-status notably discriminates Treg cells from Tconv cells. Overall, this study identified 121 Treg cell-specific phosphorylations. Short-term triggering of T cell subsets via CD3 and CD28 widely harmonized these variations with the exception of eleven TCR signalling components that mainly regulate cytoskeleton dynamics and molecular transport. Accordingly, conjugation with B cells indeed caused variant cellular morphology and revealed a Treg cell-specific recruitment of TCR signalling components such as PKCθ, PLCγ1 and ZAP70 as well as B cell-derived CD86 into the IS. Together, results from this study support the existence of a Treg cell-specific IS and suggest Treg cell-specific cytoskeleton dynamics as a novel determinant for the unique functional properties of Treg cells. Keywords: Immunological synapse rMELC rPhosphoproteome rTCR signalling rTreg Additional supporting information may be found in the online version of this article at the publisher’s web-site Introduction CD4+regulatory T (Treg) cells play an essential role in maintaining immune homeostasis and in preventing autoimmune reactivity of potentially self-reactive lymphocytes that have escaped Correspondence: Dr. Lothar J¨ ansch e-mail: [email protected] central tolerance mechanisms [1]. It is widely accepted that both Foxp3+Treg cells as well as their effector counterparts, Foxp3− conventional CD4+T (Tconv) cells, require activation via the T-cell receptor (TCR) for full acquisition of their immunosuppressive and effector functions, respectively [2–5]. TCR signalling ∗Shared first and senior authorship C 2017 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. www.eji-journal.eu This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. 2MarcovanHametal. Eur. J. Immunol. 2017. 0: 1–16 in Treg cells, thereby, seems to be differentially organized as in Tconv cells: Treg cells show reduced Ca2+flux and ERK1/2 phosphorylation upon TCR stimulation [6–8], and downstream signalling molecules such as Lck, LAT and PLCγ1 are essential for Treg cell suppressive capacity [9, 10]. In this line, a novel TCRmediated ADAP/integrin-independent PLCγ1 activation pathway was described to be required for suppression of Tconv cells by Treg cells [11]. Furthermore, Treg cells exhibit reduced S473 phosphorylation of Akt which seems a prerequisite for suppression [12]. Although the enzymatic activity of the tyrosine kinase ZAP70 seems to be dispensable for the suppressive phenotype of Foxp3+Treg cells [13], a single mutation within the SH2-domain of ZAP70 leads to impaired suppressive activities, which indicated its importance at the immunological synapse (IS) [14]. Finally, it has only recently been recognized that the recruitment of TCR signalling components into the IS is differentially organized in Treg cells and Tconv cells. The protein kinase PKCθis recruited to the IS in Tconv cells, in Treg cells, however, this kinase was found to be sequestered away from the IS, but still of importance to control suppression [15]. The spatial recruitment of signalling components during IS formation critically depends on cytoskeleton dynamics, which in turn is controlled by TCR activation and subsequent phosphorylation of proteins regulating cytoskeleton reorganization [5]. As part of these processes the microtubule-organizing centre (MTOC) is rapidly translocated in proximity to the IS. MTOC repositioning depends on LAT, ZAP70 and SLP76 [16] and is regulated by a cascade of distinct isoforms of the family of novel protein kinase C (nPKC) [17]. The MTOC serves as a platform to coordinate molecular movements from and to the IS through the support of microtubule (MT) motors [18]. For instance, TCR microclusters move along MTs towards the centre of the IS in a dynein-dependent manner [19], and hindrance of MTOC polarization, molecular transport and cytoskeleton dynamics prevent proper propagation of TCR signals [20]. It is now tempting to speculate that the localization of signalling modules within the IS may be instrumental for the formation of a Treg cell-specific IS and the suppressive phenotype of Treg cells. At this moment, however, the knowledge on how TCR signalling, protein recruitment and IS formation are differentially organized in Treg cells is far from complete. In the present study, we have now systematically studied the activity and spatial organization of TCR signalling components in ex vivo isolated Treg cells and Tconv cells by employing accurate mass spectrometry and microscopy supported by multi-epitope ligand cartography (MELC) [21, 22]. Together, our data reveal that TCR engagement harmonizes the activity status within the TCR signalling network, but in parallel induces a distinct diverging signalling pattern at regulators of cytoskeletal dynamics. This was accompanied by a variant recruitment of TCR signalling components into the IS upon T cell/B cell conjugation as well as variant CD86 uptake. Thus, results of this study substantiate the concept of a Treg cell-specific IS and suggest subset-specific cytoskeleton dynamics as a novel determinant for their suppressive phenotype. Results Major TCR signalling components are equally abundant in Treg cells and Tconv cells Although various TCR signalling components have already been identified that control differentiation and function of Treg cells [23], a comprehensive picture of the TCR signalling network organization is still missing. Our first goal was to analyse the overall protein abundances of TCR signalling components comparatively in ex vivo isolated, highly pure murine CD4+CD25+Treg cells and CD4+CD25−Tconv cells (Supporting Information Fig. 1A). These T cell subsets were spot-checked for major characteristics, i.e. expression of the transcription factor Foxp3 in Treg cells and IL-2 production in Tconv cells (Supporting Information Fig. 2). Next, total proteomes were extracted from sorted Treg cells and Tconv cells and quantitative peptide sequencing was performed using ex vivo iTRAQ labelling and accurate Fourier transform mass spectrometry (FTMS). Database searches were then restricted to high quality MS data and identified >4000 proteins in murine Treg cells and Tconv cells (Supporting Information Table 1). Regulatory data of CD3ε,PLCγ1, Foxp3, CD4 and CD25 (IL2Rα) were found in full accordance with literature, i.e. equal levels of CD3ε and PLCγ1 in both subsets and slightly reduced levels of CD4 and high abundances of Foxp3 and CD25 (IL2Rα) in Treg cells (Fig. 1A, Supporting Information Fig. 3 and Supporting Information Table 1). Cross-inspection of our data with former published proteomic studies [24–26] could now substantiate a series of proteins with Treg cell-specific abundances, although these proteins are not described to be directly involved in TCR signalling. This includes Helios, Aiolos, Sortin nexin-18, Ergic1, Annexin A4, CAPG, Stim2, NDRG1, Ladinin-1, Niban, S100A4 and Reticulocalbin-1 (Supporting Information Table 1). With respect to TCR signalling we mapped our proteome data with the support of interactome databases (GeneGo, MetaCore and IPA) and confirmed that the canonical TCR signalling pathway was completely covered. Notably, all TCR signalling components were found with similar abundances in both T cell subsets with the exception of Themis. In Treg cells, we detected Themis with reduced protein levels (Fig. 1A, Supporting Information Fig. 3 and Supporting Information Table 1), a finding which was confirmed by Western blotting and at the mRNA level by quantitative RT-PCR (Supporting Information Fig. 4A). Themis acts downstream of LAT and is required for IL-2 responses in Tconv cells [27]. Hence, we speculated that reduced Themis expression in Treg cells might be causal for known anergic signalling and impaired IL-2 responses. However, retroviral overexpression of Themis in ex vivo isolated Treg cells did neither restore IL-2 production nor abrogate suppressive capacity of Treg cells (Supporting Information Fig. 4B and 4C), suggesting that reduced Themis expression is not critical for the functional properties of Treg cells. In summary, our MS analysis revealed that major TCR signalling components are equally abundant in both T-cell subsets. C 2017 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. www.eji-journal.eu Eur. J. Immunol. 2017. 0: 1–16 Molecular immunology and signaling 3 Figure 1. Quantitative mass spectrometry determines the abundances and responsiveness of TCR signalling components. iTRAQ-based quantitative information of selected TCR signalling components and phosphorylation events in Treg cells and Tconv cells. Representative MS/MS-spectra and peptide sequences are given in Supporting Information Fig. 3. (A) Abundance: Treg cells and Tconv cells were freshly isolated from spleen and lymph nodes of female BALB/c mice and pooled. Proteomes were extracted and derived peptides from both subsets were differentially labelled by iTRAQ and quantitatively characterized by MS in two independent studies (32 mice/study). Only information of high-confident peptides were accepted (Supporting Information Table 1). Mean iTRAQ intensities of Treg cells and Tconv cells are presented; standard deviations of relative differences are CD3ε±0.080, PLCγ1±0.011, Foxp3 ±0.607 and Themis ±0.027 (colour codes are given in the left panels). (B,C) Responsiveness: Treg cells and Tconv cells were freshly isolated from spleen and lymph nodes of female BALB/c mice (580 mice). Cells were CD3/CD28-stimulated for 5 min in individual pools of cells derived from 25 to 30 mice each. Phosphopeptides from both subsets and stimulated/non-stimulated conditions were differentially iTRAQ labelled followed by 15 quantitative LC-MS experiments in one independent study. Only information of high-confident phosphopeptides were accepted (Supporting Information Table 2). (B) Phosphorylation sites of pre-activated TCR components (CD3ε,CD3ζ) and CD3/CD28-responsive down-stream components (ZAP70, SLP76) in Treg cells. (C) Phosphorylation sites at PKCθ, DBNL, MAPK14 and TIAM1, which discriminate Treg cells and Tconv cells (colour codes are given in the left panels). Identification of diverging TCR signalling components We next asked, whether equally abundant TCR signalling components might have a differential activity in Treg cells and Tconv cells. Protein phosphorylations constitute the key mechanism in signalling and we therefore aimed to profile the TCR signalling network by using quantitative phosphoproteomics. Our pivotal aim was to study the responsiveness of the TCR signalling components under rapid and robust activation conditions. For that, Treg cells and Tconv cells were ex vivo isolated and batch-wise either left untreated or stimulated for 5 min using anti-CD3 and anti-CD28 antibodies (Supporting Information Fig. 1B). Phosphorylated peptides of these four biological samples were then isolated and comparatively analysed by quantitative peptide sequencing using iTRAQ labelling and accurate FTMS. Data evaluation only considered high-confident phosphopeptides that iTRAQ-FTMS unambiguously detected in all four biological samples. This approach identified 3756 phosphorylations in freshly isolated Tconv cells and Treg cells (Supporting Information Table 2). About one-third of these phosphorylation sites could be assigned to major signalling pathways covering the TCR signalling network. To the best of our knowledge, we found all phosphorylations of the canonical TCR signalling pathway perfectly in line with the literature. For instance, phosphorylations at immunoreceptor tyrosine-based activation motifs (ITAMs) of Tconv cells (CD3ε, CD3ζ), as well as on proximal signal components (e.g. ZAP70 and SLP76) were found up-regulated after TCR engagement as expected (Fig. 1B and Supporting Information Fig. 3B). Interestingly, the profiles of the corresponding tyrosine phosphorylations at CD3εand CD3ζin Treg cells revealed first differences between both T cell subsets. They were found not inducible and with similar high intensities in non-stimulated and stimulated Treg cells C 2017 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. www.eji-journal.eu 4MarcovanHametal. Eur. J. Immunol. 2017. 0: 1–16 Figure 2. Phosphoproteomic profiling identifies converging and diverging TCR responses. For quantitative phosphoproteomics, ex vivo isolated Treg cells and Tconv cells were CD3/CD28-stimulated for 5 min or left non-stimulated and analysed by using quantitative MS. All regulation factors are given in log2 scale. (A) Box plots show regulations of all phosphorylation sites of proteins from stimulated Treg cells or Tconv cells in relation to non-stimulated cells. Numbers indicate the significantly upor down-regulated phosphorylation sites when assuming a normal distribution. (B) Comparison of upand down-regulated phosphorylation sites in Treg cells and Tconv cells (from A). (C) Scatter plot depicting regulations of all phosphorylated peptides in Treg cells (x-axis) and Tconv cells (y-axis). One hundred and twenty one phosphopeptides that are significantly differentially regulated between Treg cells and Tconv cells (see Supporting Information Fig. 5) are indicated by red dots. (D) Heat C 2017 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. www.eji-journal.eu Eur. J. Immunol. 2017. 0: 1–16 Molecular immunology and signaling 5 (Fig. 1B and Supporting Information Fig. 3B). This indicates a pre-activated phenotype of the TCR signalling network in freshly isolated Treg cells, which likely is a consequence of tonic interactions with antigen-presenting cells in vivo [28]. Using Western blotting analysis we could indeed confirm the enhanced level of phosphorylated CD3ζin non-stimulated Treg cells in comparison to non-stimulated Tconv cells (Supporting Information Fig. 5). Nevertheless, this pre-activation did not impair the responsiveness of the TCR signalling network in Treg cells as illustrated by the induced Y492 phosphorylation of ZAP70 (Fig. 1B and Supporting Information Fig. 3B). In a global view, TCR triggering in Treg cells causes similar numbers of induced and repressed phosphorylation events, whereas Tconv cells showed a higher number of significantly up-regulated phosphorylations (Fig. 2A). Several TCR components exhibited similar phosphorylation responses in both T cell subsets, although the number of jointly upand down-regulated proteins in Treg cells and Tconv cells is limited (Fig. 2B). This includes the aforementioned ZAP70 as well as known TCR scaffold proteins SASH3/Sly, raftlin, Lad/TSAd and LIME1, indicating an overall similar architecture of the signal networks. The vast majority of CD3/CD28-responding phosphorylations exhibited subset-specific regulations and a comparative inspection revealed a total of 121 phosphorylations (Supporting Information Fig. 6) that discriminate Treg cells and Tconv cells in the first five min of signalling (Fig. 2C and Supporting Information Table 2, sheet3). From a mechanistical angle these Treg cell-specific regulations can be grouped in six classes (Fig. 2C and D). For instance, the deubiquitinase CYLD, which plays a role in the IS recruitment of Lck, was induced at its inhibitory S414 site only in Tconv cells (class I). Likewise, class II phosphorylations are predominantly induced only in Treg cells; comprising the activation site T538 of PKCθ(Fig. 1C) as well as components of gene regulation (ISW1 and ROA1) and vesicle trafficking (AP3δ1and α-PIX). In a side-by-side inspection of both subsets, the majority of 121 phosphorylations (>60 %), showed an opposite regulatory behaviour under the same stimulation condition (Fig. 2D, classes V and VI). However, when non-stimulated Tconv cells served as the reference for normalization, protein-specific phosphorylations became apparently similar after five min stimulation (Fig. 2E, compare lanes five min). This convergence of phosphorylations is most apparent but not restricted to class V and VI proteins. For instance, the abundance of phosphorylated T538 of PKCθ(class II) was at its lowest in freshly isolated Treg cells, but converged to similar activation level after TCR engagement in both stimulated subsets. Thus, equal ex vivo TCR stimulation of Treg cells and Tconv cells notably harmonize the post-translational status of the TCR signalling network. Nonetheless, a distinct set of eleven TCR signalling components were not harmonized by TCR engagement and indicated an even diverging signalling pattern in Treg cells and Tconv cells (Fig. 2F and MS data for DBNL, MAPK14 and TIAM in Fig. 1C). This group is constituted by known TCR signalling components and, from a systematical view, is enriched for one functional class (Table 1): Beside the hematopoietic lineage-specific phosphatase PTPN18 and the Transducin-like enhancer of split 3 (TLE3), both involved in T cell differentiation [29–32], all other nine components are associated with cytoskeleton dynamics [19, 33–39]. Many of those proteins were found with induced phosphorylations only in Tconv cells, whereas these responses are even repressed in Treg cells. This includes DC1L1/LIC1, a subunit of the dynein complex, that is supposed to determine cargo load [40]. In contrast, Treg cell-specific induced phosphorylations were identified at SMCR8 and TIAM, of which the latter is required for LFA-1 integrin activation [41]. In conclusion, ex vivo Treg cells exhibited a pre-activation TCR phenotype. Their phosphorylation status discriminates them from Tconv cells, but can be largely harmonized under precise TCR activation conditions ex vivo. For that, phosphorylations showed opposite regulations in Treg cells and Tconv cells and the global phosphorylation signatures widely converge in only 5 min. Still, a set of eleven components that are mostly reported as regulators of the cytoskeleton identified diverging nodes in the two subsetspecific TCR signalling networks. In this context, protein network analyses based on induced phosphorylated proteins actually confirmed the close interconnectivity between TCR signalling and the cytoskeleton (Supporting Information Fig. 7A). Thereby, the responding Tconv cell network revealed the superior enrichment of “TCR signalling components”, whereas the responding Treg cell network revealed the superior enrichment of “cytoskeleton dynamics”. Indeed, network analyses restricted to the 121 subsetspecific phosphorylations strengthened the hypothesis that mainly TCR signalling and cytoskeleton dynamics discriminate Treg cells from Tconv cells (Supporting Information Fig. 7B). Treg cells reveal unique cell polarization and MTOC positioning but do not recruit PKCθto the IS Diverging signalling components indicated a variant regulation of cytoskeletal organization in the two T cell subsets that may orchestrate T cell motility, conjugation, IS formation as well as  maps showing regulations of all 121 significantly regulated phosphorylation sites in Tconv cells and Treg cells. Heat maps are ordered according to boxes I–VI in (C). (E) Heat map illustrating relative abundances of the 121 significantly regulated phosphorylation sites in non-stimulated and CD3/CD28-activated Tconv cells and Treg cells with non-stimulated Tconv cells as baseline. Note the generally converging signalling in both subsets resulting in almost comparable abundances after 5 min of stimulation. (F) Heat map illustrating 16 phosphorylation site intensities that discriminate Treg cells from Tconv cells after five min of stimulation (left panel; Tconv cells minus Treg cells; >1 on the log2 base). Of those, eleven TCR signalling components showed diverging phosphorylation site regulations (right panel and Table 1). Data is pooled from ten independent groups of each 25–30 mice for nonstimulated and 12 independent pools of each 25–30 mice for CD3/CD28-stimulated Treg cells and Tconv cells. Phosphopeptides were isolated and differentially labelled followed by quantitative MS of 15 fractions in one independent study. In total, 580 mice, 4.4 ×108cells and 1.1 ×108cells/condition were analysed (see also Fig. 1 and Supporting Information Table 2). C 2017 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. www.eji-journal.eu 6MarcovanHametal. Eur. J. Immunol. 2017. 0: 1–16 Table 1. Intrinsic Treg cell-specific phosphoresponses Uniprot Trivial names Site Treg cells Tconv cells Function Node STMN1 Stathmin S16 − ++++ Cytoskeletal reorganization tubulin / MTOC [33] CaMKII [53] TLE3 TLE3 S217 − ++++ Transcriptional co-repression TCF / Lef [31] FoxA1 / HDAC1/2 [32] DC1L1 LIC1 S412 − ++++ Vesicle transport Dynein [19] TCR microcluster dynamics tubulin/MTOC [34] RHG15 ArhGAP15 S51 − ++++ cytoskeletal reorganization small GTPases (Rac) [78] Pak1/2 [54] PTN18 PTPN18 Y381 −− +++ tyrosine phosphatase Lck / ZAP70 [29] FLP-1/PTP-HSCF differentiation immune cells Lyn [30] M3K3 MAP3K3 S337 − +++ T cell differentiation / activation IFN-γ[79] / NFκB [80] MEKK3 PLCγ[55] DBNL Drebrin-like S277 −− +++ T cell activation / IS formation ZAP70 / p38 / PLCγ[35, 36] HIP-55 cytoskeletal reorganization HPK1 / NFAT [37] MK14 MAPK14 Y182 −−−− ++ induced Treg cell generation NFAT / Akt / ERK [81] p38αcytoskeletal reorganization TGFβ/ ILs [82] ML12B MLC20 S20 −−− ++ cytoskeletal reorganization PKC / ILK [83] Myl12b actin / p38 [38] SMCR8 SMCR8 S416 ++ −− vesicle transport RABs [39] TIAM1 TIAM1 S1437 ++ −−− integrin signalling 5-1 / 14-3-3 / Rac [41] cytoskeletal reorganization PKC / Rac [70] List of 11 TCR signalling components that were not harmonized upon CD3/CD28-stimulation and that revealed an even diverging signalling pattern in Treg cells and Tconv cells. Regulations of phosphorylation sites in Treg cells and Tconv cells are given by “+” and “−” signs (−/+: 0.00–0.50; −−/++: 0.50–1.00; −−−/+++: 1.00–1.50; −−−−/++++:>1.50 (log2 scale)), known functional roles in T cells and major nodes are listed. effector functions. Here, we focused on conjugation and IS formation. For that, Treg cells and Tconv cells were isolated from ovalbumin (OVA)-TCR transgenic mice (DO11.10), IS formation was induced by co-incubation with OVA peptide-loaded ex vivo isolated B cells, and polarization dynamics of BT-pairs were studied by microscopy. Global inspection of BT-pairs already demonstrated a subset-specific morphology phenotype. Along cell conjugation the shape of Tconv cells was found to be more elongated as those from Treg cells, which exhibited a more rounded morphology upon conjugation (Fig. 3A). We next considered only those BT-pairs, which had established a mature actin-enriched IS as indicated by phalloidin. Cells were additionally stained for the microtubule network and we found the distance between MTOC and IS significantly diminished in conjugated Treg cells as compared to Tconv cells. To score differences within the repositioning of the MT network and especially the MTOC in Treg cells and Tconv cells, we determined the polarization index [42] upon BTpairing and confirmed a significant Treg cell-specific polarization phenotype and MTOC positioning (Fig. 3B). MTOC positioning is regulated by PKC family members [16], and PKCθwas already reported to delocalise from the Treg cellIS [15]. Interestingly, PKCθwas identified as a Treg cell-specific component following TCR engagement in our phosphoproteomic approach (class II). Thus, we next investigated PKCθlocalization dynamics in relation to MTOC positioning (Supporting Information Fig. 1C). IS formation was studied by using anti-CD3/antiCD28-coated coverslips, and confocal spinning disk microscopy confirmed the MTOC positioning phenotype. In Treg cells, this MTOC positioning phenotype was not accompanied with a notable recruitment of PKCθto the IS in the first 30 min (Fig. 3C and D). Thus, PKCθis not actively delocalized away from the Treg cellIS but instead is not recruited at all. In contrast, for Tconv cells we could confirm IS recruitment of PKCθthat became more obvious after 3D reconstruction (Fig. 3C and D). Interestingly, PKCθ recruitment into the Tconv cells-IS was only found transiently after ten min, and already 30 min after TCR activation the majority of cells lost the polarized PKCθdistribution (Fig. 3C). Thus, MTOC positioning in Tconv cells is accompanied by a transient IS recruitment of PKCθ, whereas an even more polarized positioning of the MTOC towards the IS in Treg cells could not attract PKCθinto the synaptic compartment. Treg cells establish a unique transport phenotype of TCR signalling network components at the IS Since we (i) revealed Treg cell-specific and diverging signalling of cytoskeleton regulators and (ii) characterized a variant MTOC positioning and impaired PKCθrecruitment in Treg cells, we next aimed to study the spatial organization of selected TCR signalling components in stimulated Treg cells and Tconv cells. For this, we employed the multi-epitope ligand cartography (MELC) technology [21, 22], and evaluated a total of about 300 OVA-specific BT-pairs using a panel of 25 antibodies (see Material and Methods, Supporting Information Fig. 1C and Supporting Information Video 1). This approach allowed subcellular localization analyses C 2017 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. www.eji-journal.eu Eur. J. Immunol. 2017. 0: 1–16 Molecular immunology and signaling 7 Figure 3. Subset-specific cytoskeletal rearrangement and PKCθdistribution in Treg cells and Tconv cells. (A) B cells from BALB/c mice were loaded with OVA323-339 peptide and co-incubated for 10 min with Treg cells or Tconv cells from DO11.10 mice and analysed by immunofluorescence microscopy. Representative images from one out of two independent experiments using six mice each; staining: tubulin (green) and F-actin (red). Dashed lines show distances from MTOC to IS. Scale bars define 5 μm. (B) Quantification of the polarization index revealed altered polarization of Treg cells after B cell engagement. Polarization index was calculated as follows: distance of MTOC to IS divided by the distance of the uropod to the IS [42] (n=16 for Treg cells and n=20 for Tconv cells; median is indicated by the horizontal line; *p<0.05, Mann– Whitney U test). Each dot represents one analysed cell and representative data of one of two independent experiments using six mice each is shown. (C, D) PKCθis transiently recruited to the IS only in Tconv cells. Purified Treg cells and Tconv cells were seeded on anti-CD3/anti-CD28 antibodies coated coverslips, fixed after indicated time points and analysed using spinning disk microscopy (n=3; four mice per experiment). (C) PKCθdistribution was calculated by dividing proximal localization by distal localization (pooled data: n=101 for Treg cells and n=136 for Tconv cells; median is indicated by the horizontal line; *p<0.05, Mann– Whitney U test) Each dot represents one analysed cell and data shown is pooled from three independent experiments using four mice each. (D) Representative 3D images of one Treg cell (upper panel) and one Tconv cell (lower panel) 10 min after seeding on coverslips. PKCθis shown in green, and nuclei (blue) and F-actin (red) were used for counter staining; substrate area is indicated by a gray dashed line. Scale bars define 5μm. of key TCR signalling components as well as their specific phosphovariants. Following 30 and 120 min after BT-pair formation, robust fluorescence intensities could be detected and quantified to calculate the abundances of components in seven distinct spatial regions. Our segmentation strategy, which was based on the position of pSLP76 in the actin-rich IS (green dot, Fig. 4A and B), could define the SMAC and cSMAC as well as the entire IS that spanned the width of the SMAC and protruded five pixels into both the B and T cell (Fig. 4A and B, and Supporting Information Fig. 8). Stable BT-pairs were defined based on signals from CD3ε, CD45R, F-actin, pSLP76 and pLAT and allowed recording the position and size of a mature IS (Fig. 4A). At first glance, the basic IS architecture of BTregand BTconvpairs appeared similar as indicated by the global intensity pattern of the inspected TCR signalling components (Fig. 4C, compare Treg cells versus Tconv cells at 30 and 120 min). The MELC analyses also confirmed the accumulation of activated proximal TCR signalling components, i.e. pCD3ζ, pZAP70, pLck, pLAT, pSLP76 and pPLCγ1 at the IS, SMAC and cSMAC. This accumulation was found to further increase after 120 min and coincided with the enrichment of the corresponding total protein levels within these regions as well (Fig. 4C). Statistical analyses of the BT-pairs revealed a number of striking differences in IS organization: (i) Treg cells were notably impaired in recruiting additional CD3ε,Lck,SLP76 and PLCγ1 after the formation of a mature IS at 30 min. In contrast, Tconv cells were able to continue this recruitment over a period of 30 to 120 mins more efficiently (Fig. 4D, relative differences over time). Interestingly, both subsets likely continue signalling along this period as observed by the consecutive accumulation of the corresponding phosphovariants. (ii) Significantly lower amounts of PLCγ1, CD3εand pZAP70 were detected at the Treg cell-IS (Fig. 4E, relative differences in subsets). Thereby, pZAP70 was mostly affected after 30 min but remained reduced in the cSMAC, and PLCγ1andCD3εwere found significantly reduced only after 120 min (Fig. 4E, and representative images in Fig. 4F and G). (iii) Instead, significantly higher amounts of CD45, Lck, SLP76 and Fyn as well as pERK, pPLCγ1 and pLAT were localized at the Treg cell-IS 30 min after conjugation (Fig. 4E). This argues for a more rapid maturation of the Treg cell-IS compared to the Tconv cell-IS. (iv) Only Treg cells were able to recruit CD86 and CD80 to their IS compartment (Fig. 4E and Supporting Information Fig. 6), as described previously [43]. Thus, CD86 transendocytosis, as a part of the Treg cell suppressive phenotype, was now demonstrated 30 min after IS formation and did not indicate any exhaustion after 120 min. In conclusion, MELC analyses showed different recruitment of TCR signalling components into the IS of Treg cells versus Tconv cells at early time points, which, however, ultimately resulted in a more alike distribution at 120 min. Still, the key signalling components CD3ε, ZAP70, PLCγ1 and CD86 remained differentially distributed at the IS and could explain Treg celland Tconv cellspecific phenotypic responses. Discussion TCR signalling controls differentiation and function of Treg cells [23]. Although previous studies already had described some differences in TCR signalling between Treg cells and Tconv cells [44], a comprehensive picture of the TCR signalling network C 2017 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. www.eji-journal.eu 8MarcovanHametal. Eur. J. Immunol. 2017. 0: 1–16 Figure 4. Activation-dependent recruitment of TCR signalling components to the IS. BTpairs were generated as in Fig. 3, and pairs were fixed after 30 or 120 min and analysed by sequential staining (multi-epitope ligand cartography, MELC). (A) Representative image showing a BT-pair with pSLP76 (green), CD3ε (red) and F-actin (gray) staining that was used to define the different compartments of the IS (IS circled by dotted line, SMAC framed by dashed line and cSMAC framed by solid line). (B) Segmentation strategy of the IS compartments as determined by the position of pSLP76 staining (green dot), which was used to define a three pixel (0.86 μm) wide SMAC region stretched over the synapse as defined by F-actin. The cSMAC was defined as the middle 33 % of the width of the SMAC. The entire IS area was defined as an oval spanning the width of the SMAC and protruding five pixel (1.43 μm) beyond the SMAC into either cell. (C) Relative intensities of all analysed (phospho)proteins in BTregand BTconv-pairs 30 and 120 min after pair formation determined in the B cell (B) and T cell (T) without the IS, the B cell (Bs) and T cell (Ts) part of the IS without SMAC, the entire IS, the complete SMAC (S) and the cSMAC (cS). Note the gradual enrichment of both total TCR-signalling proteins and their phosphorylated variants in the IS (IS, S and cS). (D,E) Relative differences of all analysed (phospho)proteins in BT-pairs in the IS (i.e. IS, S and cS). Only statistically significant differences are shown (p<0.05, two-tailed t-test). The relative differences and p-values for all BT-pair compartments are given in Supporting Information Fig. 8. (F,G) Representative images showing the localization of proteins used to define the IS compartments of BTreg- (F) and BTconv- (G) pairs (upper images; CD3ε(red) and pSLP76 (green), Foxp3 (blue) marks only Treg cells) and representative images showing differences between BTregand BTconv-pairs regarding reduced PLCγ1 (green) and pZAP70 (red) localization within the IS and Ts, and the cSMAC, respectively (lower images). F-actin is shown in gray. A photomontage of BT-pairs (BTreg vs. BTconv) that includes information of all investigated proteins and phosphoproteins obtained by MELC is given in Supporting Information Video 1. Representative images given in A,F,G are from one of two independent experiments with eight mice each and of 51 BTregversus 130 BTconv-pairs at 30 min and 28 BTregversus 62 BTconv-pairs at 120 min analysed, heat maps shown in C-E represent pooled data from all analysed BT-pairs. organization was missing. Results of this study now suggest a Treg cell-specific activation of cytoskeleton regulators and a subsequent Treg cell-specific recruitment of TCR signalling components to the mature IS. This constitutes a mechanistic model of how functionally distinct TCR signalling is realized in both T cell subsets. First, the proteome analyses determined largely identical abundances of TCR signalling components within Treg cells and Tconv cells. This observation is widely in line with recent data from proteome studies of human and murine Treg cells, although those studies did not specifically focus on TCR signalling [24–26]. The present proteomic approach identified low abundance of Themis in Treg cells, a molecule known to play a role in the early LAT signalosome in Tconv cells [27]. Overexpression of Themis, however, could neither restore IL-2 expression nor reduce the suppressive C 2017 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. www.eji-journal.eu Eur. J. Immunol. 2017. 0: 1–16 Molecular immunology and signaling 9 capacity of Treg cells in vitro. Nevertheless, Themis levels play a role in T cell development and survival [45–48], and Themis has been reported to modulate IL-10 expression [49], aspects that were not addressed in this study. Thus, protein abundances seemed not to be the key determinant of functionally distinct TCR signalling networks. In contrast, this first phosphoproteome study on primary murine T cells detected numerous Treg cell-specific phosphorylations in the TCR signalling network. Ex vivo Treg cells displayed a pre-activated phosphorylation status of key TCR signalling molecules (e.g. CD3ε,CD3ζand SLP76). Initially, this appeared counterintuitive since a diminished tyrosine phosphorylation of CD3ζin peripheral human Treg cells has been reported [7, 8]. However, T cells residing in secondary lymphoid organs - as used in our study - continuously receive “tonic” receptor stimuli [50] that can result in priming the down-stream signal network as described for Tconv cells [28], and this now could be confirmed here for Treg cells as well. The functional role of this priming needs further investigation, but it did not abrogate Treg cells’ responsiveness since we found Y492 in ZAP70 equally well phosphorylated in ex vivo isolated T cell subsets and equally well inducible upon TCR engagement. This site is a known substrate site of Lck and serves as an autophosphorylation site of activated ZAP70 as well [51]. The CD3εimmunoreceptor tyrosine-based activation motif (ITAM), however, was already found phosphorylated in ex vivo isolated, non-stimulated Treg cells. Hence it is fair to speculate that the signal amplification mechanism involving the dissociation of ZAP70 from the TCR complex, as very recently described for Tconv cells [52], is already induced in non-stimulated Treg cells. This would support ZAP70 function in inside-out signalling and adhesion of Treg cells, which did not require its kinase activity [13]. In total, 121 Treg cell-specific phosphorylations define known and novel down-stream components of the TCR signalling network. Identical stimulation conditions thereby converge the phosphopatterns significantly within five min. More noteworthy, harmonizing of both TCR signalling networks is predominantly realized by counter-regulated signalling. This involved well-known proximal TCR signalling components like PKCθ,SLP76 and GRP2/CalDAG-GEFI, and substantiates the model of Treg cells having a variant activation status in vivo. Furthermore, this demonstrates that Treg cells in vivo integrate signals from multiple receptors and have the capacity to reverse the direction of TCR-mediated regulations. It was exciting to detect even a diverging TCR signalling under the applied stimulation conditions in a set of eleven proteins. These components have been related previously to distal parts of the TCR signalling network. Nine of these are regulators of cytoskeleton dynamics and molecular transport [19, 33, 37, 53–55] and indeed cellular polarity, MTOC positioning and signal component recruitments then confirmed a cell-polarization as well as an IS assembly phenotype of Treg cells. Such an IS phenotype is still mostly defined by PKCθthat plays a pivotal role in MTOC dynamics and IS formation in T cells [56]. Zanin-Zhorov and colleagues recently observed PKCθto be delocalized six min after Treg cell-IS formation and gave the first evidence for a Treg cell-specific IS organization [15]. Here, we could now confirm this phenotype and further demonstrate that in contrast to Tconv cells, PKCθis not recruited at all to the Treg cell-IS, whereas other TCR components were found efficiently recruited. Actually, in Tconv cells PKCθis recruited only transiently to the IS by so far unknown antagonistic translocation mechanisms. This finding must be further elucidated e.g. in bi-cellular conjugations, but likely is under control of post-translational modifications. During TCR-induced signalling, PKCθintegrates co-stimulatory signals from CD3 and CD28, and GLK (germinal centre kinase-related protein kinase) can phosphorylate PKCθat T538, which coordinates its activity and localization [57, 58]. Interestingly, we found this site solely inducible in Treg cells. Furthermore, PKCθlocalization might depend on sumoylations [59], which was not addressed in the present study. But what is the role of delocalized PKCθin the context of Treg cell-IS functions? In Tconv cells, upon TCR stimulation PKCθsupports NF-κB activation [60] and can maintain phosphorylated RelA, the p65 subunit of NF-κB, in the nucleus [61]. Interestingly, Treg cells were found to accumulate phosphorylated p65 at the late IS, although its functional role there remains elusive. In the same pathway, PKCθis described as an interacting antagonist of the deubiquitinase CYLD, which acts on Lck to support its IS localization [62, 63]. We found CYLD only responding in Tconv cells, indicating that the PKC/CYLD axis is differentially organized in Treg cells. A more systematic inspection of the Treg cell-IS was corroborated by MELC analyses. When compared to Tconv cells, about one-third of the here-inspected components displayed significantly different recruitment dynamics to the mature IS. Phosphovariants of TCR signalling components were equally recruited into the IS of Treg cells and Tconv cells as expected, however, Tconv cells could recruit TCR signalling components more efficiently. It is tempting to speculate that a limited recruitment of these components can dampen TCR signalling in Treg cells. Indeed, ZAP70 phosphorylated at Y318, a site unambiguously needed in T cell activation [64], was reduced in the IS in BTreg-pairs at 30 min, and this holds true for the cSMAC even after 120 min. Furthermore, we observed reduced levels of PLCγ1 in the Treg cell-IS 120 min after BT-pair initiation. Mechanistically this could be explained by the differentially regulated S210 in the P1 domain of SLP76 that facilitates the interaction with the SH3 domain of PLCγ1 and is required for TCR-mediated activation [65]. LAT competes for PLCγ1 interaction via this SH3 domain, and this PLCγ1/LAT complex is restricted to the cell membrane in resting Jurkat T cells [66]. Thus interfering with SLP76/PLCγ1 interaction through S210 phosphorylation could result in aberrant complex recruitment via LAT to the Treg cell-IS. Here, one should note that, due to limitations in the antibody panel used in the MELC analyses, we most probably underestimate the number of proteins that show differences in IS recruitment. For instance also Lymphocyte Cytosolic Protein 1 (LCP1 or L-plastin/PLSL) might be differentially recruited into the Treg cell-IS. Phosphorylation at S5 supports its translocation to the IS and therewith its contribution to cell motility in Tconv cells [67]. This site was indeed induced in Tconv cells as expected, however, in Treg cells the S5 C 2017 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. www.eji-journal.eu 16 MarcovanHametal. Eur. J. Immunol. 2017. 0: 1–16 77 Grubbs, F. E., Sample criteria for testing outlying observations. Ann. Math. 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Abbreviations: BT-pair: B cell and T cell pair ·BTreg-pair: B cell and Treg cell pair ·BTconv-pair: B cell and Tconv cell pair ·FTMS: Fourier transform mass spectrometry ·IS: immunological synapse ·ITAM: immunoreceptor tyrosine-based activation motif ·iTRAQ: isobaric tags for relative and absolute quantification ·MELC: multi-epitope ligand cartography ·MS: mass spectrometry ·MT: microtubule ·MTOC: microtubule-organizing centre ·OVA: ovalbumin ·P1 domain: prolinerich domain ·ROI: region of interest ·SH domain: Src homology domain · SMAC: supramolecular activation cluster ·cSMAC: central-SMAC ·TCR: T cell receptor ·Tconv cell: conventional T cell ·Treg cell: regulatory T cell Full correspondence: Dr. Lothar J¨ ansch, Cellular Proteomics, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig, Germany Fax: +49-831-6181-2655 e-mail: [email protected] Received: 15/3/2017 Revised: 28/6/2017 Accepted: 14/8/2017 Accepted article online: 17/8/2017 C 2017 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. www.eji-journal.eu