Separate signaling events control TCR downregulation and T cell activation in primary human T cells

Univ São Paulo, Inst Biomed Sci, Dept Immunol, BR-05508000 São Paulo, Brazil

Expression of the CD45 tyrosine protein phosphatase is required for the response of functional lymphocytes to stimulation through the antigen receptor. One or more of its substrates may therefore be essential for signal transduction during lymphocyte activation. We have studied the phosphorylation of the closely related lck, fyn, and c-src tyrosine protein kinases in leukemic murine T-cell lines that have lost the expression of CD45. The phosphorylation of the lck kinase at an inhibitory site of tyrosine phosphorylation, Tyr-505, was increased by two-, six-, and eightfold in three different cell lines. Phosphorylation of the fyn kinase at the homologous site, Tyr-531, was unaltered in one of these cell lines, but increased by 2.5-fold in the two others. The phosphorylation of p60c-src at the homologous tyrosine was essentially unchanged in the one CD45-negative cell line in which it was examined. The expression of CD45 therefore regulates the phosphorylation and potentially the activity of the lck and fyn tyrosine protein kinases, but the effect on the lck kinase is much greater than on the fyn kinase. This finding and the observation that CD45 had no effect on the phosphorylation of p60c-src suggest that CD45 exhibits polypeptide substrate specificity in vivo. Additionally, these findings are consistent with the hypothesis that the unresponsiveness of CD45-negative lymphoid cells to antigenic stimulation is due largely to hyperphosphorylation of the lck kinase.

We recently demonstrated antagonism for hapten-reactive T cells by altered hapten ligands. Here we investigated partial peptide- or hapten-agonism and effects of antigen stimulation on the expression of TCR and the CD8 coreceptor using a set of DNP- or TNP-peptide-induced, H-2Kb-restricted mouse CTL clones. Various Kb-binding TNP- and DNP-peptides acted as partial agonists, cross-reactively stimulating individual clones for cytotoxicity and IFN-gamma secretion, but failing to induce proliferation or TNF-alpha production. Full agonism, i.e. activation of all possible functions, was usually restricted to those hapten-peptide combinations used for the induction of the respective clones. Our data imply distinctive kinetic optima for TCR antigen contacts in the induction of the various T cell effector functions. Down-regulation of TCR was efficiently induced by full, but with one exception not by partial, agonists, indicating the independence of cytotoxicity or IFN-gamma secretion from TCR modulation. On the other hand, a reduction of TCR expression induced by full agonists was usually not accompanied by synchronous down-modulation of CD8 as reported by others for human T cells. In fact, three of four full agonists and all partial agonists markedly enhanced rather than reduced the expression of CD8. Increased CD8 surface levels enhanced cytolytic potential and increased cross-reactivity patterns of individual clones. Brefeldin A blocked this CD8 induction by partial agonists, and in the case of full agonists resulted in a parallel reduction of both, TCR and CD8. Thus, antigenic stimulation of mouse T cells initially down-modulates CD8 together with TCR, but the loss of coreceptor is over-compensated by a signal for increased CD8 export.

The T cell antigen receptor (TCR).CD3 complex contains several distinct but related signal transduction modules termed "Reth motifs": one each in the cytoplasmic domains of CD3-gamma, -delta, and -epsilon chains and three in the CD3-zeta polypeptide (zeta A, zeta B, and zeta C). Cross-linking of individual motifs expressed in chimeric molecules leads to early and late T cell activation events. Although the activated T cell receptor associates with nonreceptor tyrosine kinases, the sites of interaction with kinases and other potential effector molecules have not been fully mapped. Here we show that phosphatidylinositol 3-kinase (PI 3-kinase) preferentially associated with the zeta chain membrane proximal motif zeta A. Maximal PI 3-kinase/zeta A association occurred following TCR.CD3 activation and was dependent upon phosphorylation of both tyrosine residues in zeta A. The association of PI 3-kinase was specific for zeta A and could be ranked zeta A >> zeta C > zeta B. Phosphorylation of the zeta A motif on tyrosine residues occurred in response to TCR.CD3 cross-linking in vivo. These results indicate that T cell activation leads to assembly of an intracellular signaling complex: recruitment of a tyrosine kinase, phosphorylation of zeta A, and association of PI 3-kinase. These data also support a model in which different Reth motifs of the TCR.CD3 complex recruit distinct signal transduction molecules. Thus, the subdomains of the T cell antigen receptor zeta chain may serve different roles during T cell maturation and antigen-driven activation.

The purpose of our study is to determine whether and how lymphocyte-specific protein tyrosine kinase (Lck) may exhibit novel functions in cancer cell mitochondria. Mitochondria are responsible for producing cellular energy via oxidative phosphorylation (OXPHOS) of glucose and fatty acids. Compared to normal differentiated cells, cancer cells rely more on aerobic glycosis than mitochondrial OXPHOS. This shift in energy metabolism allows more resources to be converted into biomass for cancer cell's uncontrolled growth and proliferation. Numerous mechanisms contribute to altered mitochondrial activity, including phosphorylation of mitochondrial proteins. Recent studies showed that cytoplasmic protein tyrosine kinases (PTK) also translocate to the mitochondria and regulate mitochondrial functions. It represents an important non-canonical pathway of how PTK modulates mitochondrial activity. As a Src-family PTK, Lck is essential for T cell activation by transmitting signals downstream of cell surface receptors. Overexpression and aberrant activity of Lck have been reported in both blood malignancies and solid tumors. We showed previously that Lck exhibited additional oncogenic property when translocated to the nucleus (BBRC 417:1058). To explore the possibility that Lck may also translocate to mitochondria, we examined human Jurkat T cell leukemia and its Lck-deficient derivative Jcam cell line. Subcellular fractionation and microscopy both confirmed the presence of Lck in Jurkat mitochondria. Compared to Jcam, Jurkat cells exhibit high levels of mitochondrial protein tyrosine phosphorylation, disrupted mitochondrial morphology, and reduced OXPHOS. Subsequent mass spectrometry and in situ proximity ligation assay identified CR6-interacting factor 1 (CRIF1) as a novel Lck-interacting partner in mitochondria. CRIF1 interacts with mitoribosome and Tid1 to facilitate translation and membrane insertion of mitochondrion-encoded proteins, respectively. Compared to Jcam, Jurkat cells express less mitochondrial proteins and show reduced CRIF1-Tid1 interaction. Knocking down CRIF1 in Jcam also results in decreased mitochondrial protein level and mitochondrial dysfunction. Interestingly, CRIF1 is not phosphorylated on tyrosine by Lck, suggesting the presence of other Lck substrates in mitochondria. All together, these results support the role of mitochondrial Lck as a negative regulator of CRIF1 through competitive binding and independent of its kinase activity. Disrupted mitochondrial protein translation and membrane insertion then lead to reduced OXPHOS and abnormal morphology. These findings raise the possibility of targeting mitochondrial Lck to redirect the metabolic shift in cancer cells. Citation Format: Shahrooz Vahedi, Fu-Yu Chueh, Chao-Lan Yu. Lymphocyte-specific protein tyrosine kinase (Lck) interacts with CR6-interacting factor 1 (CRIF1) in mitochondria to repress oxidative phosphorylation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3041. doi:10.1158/1538-7445.AM2015-3041

A Mouse Model for Immunotherapeutic Reversal of Leukemia-Induced T Cell Dysfunction

Understanding the parameters involved in T cell activation has been complicated by the discovery of partial T cell agonists. Altered peptide ligands (APL) have recently shown that different subsets of T cell responses can be selectively activated by certain peptides, which define a hierarchy of T cell activation. For cytotoxic T cells, this hierarchy ranges from sensitizing target cells for lysis through proliferation to effector cell induction. The degree of TCR down-regulation mediated by APL-MHC interactions correlates well with the induction of specific T cell effector functions. This suggests that the potential agonist response induced by a given peptide occurs at different triggering thresholds. To examine the relative agonist and antagonist functions of different peptides, we have investigated the ability of lymphocytic choriomeningitis virus glycoprotein-derived APL to induce or inhibit a range of effector functions in naive CD8+ T cells. By this, we have defined a hierarchy of peptides that display a range of properties from strong agonist to no agonist function. At each level, peptides that were ranked lower in this hierarchy were able to interfere or antagonize the induction of effector functions by higher ranking peptides. We have therefore shown that this spectrum of peptides ranging from strong to no agonist function has an inverse gradient from strong antagonist to no antagonist function. Moreover, the ability of the different peptides to inhibit TCR internalization correlated with their ranking within the hierarchy. These findings support the model that antagonists are effectively preventing TCR oligomerization and functional TCR triggering.

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 Supramolecular signaling assemblies are of interest for their unique signaling properties. A µm scale signaling assembly, the central supramolecular signaling cluster (cSMAC), forms at the center of the interface of T cells activated by antigen-presenting cells. We have determined that it is composed of multiple complexes of a supramolecular volume of up to 0.5 µm3 and associated with extensive membrane undulations. To determine cSMAC function, we have systematically manipulated the localization of three adaptor proteins, LAT, SLP-76, and Grb2. cSMAC localization varied between the adaptors and was diminished upon blockade of the costimulatory receptor CD28 and deficiency of the signal amplifying kinase Itk. Reconstitution of cSMAC localization restored IL-2 secretion which is a key T cell effector function as dependent on reconstitution dynamics. Our data suggest that the cSMAC enhances early signaling by facilitating signaling interactions and attenuates signaling thereafter through sequestration of a more limited set of signaling intermediates. eLife digest Cells receive dozens of signals at different times and in different places. Integrating incoming information and deciding how to respond is no easy task. Signaling molecules on the cell surface pass messages inwards using chemical messengers that interact in complicated networks within the cell. One way to unravel the complexity of these networks is to look at specific groups of signaling molecules in test tubes to see how they interact. But the interior of a living cell is a very different environment. Molecules inside cells are tightly packed and, under certain conditions, they interact with each other by the thousands. They form structures known as ‘supramolecular complexes’, which changes their behavior. One such supramolecular complex is the ‘central supramolecular activation cluster’, or cSMAC for short. It forms under the surface of immune cells called T cells when they are getting ready to fight an infection. Under the microscope, the cSMAC looks like the bullseye of a dartboard, forming a crowd of signaling molecules at the center of the interface between the T cell and another cell. Its exact role is not clear, but evidence suggests it helps to start and stop the signals that switch T cells on. The cSMAC contains two key protein adaptors called LAT and SLP-76 that help to hold the structure together. So, to find out what the cSMAC does, Clark et al. genetically modified these adaptors to gain control over when the cSMAC forms. Clark et al. examined mouse T cells using super-resolution microscopy and electron microscopy, watching as other immune cells delivered the signal to switch on. As the T cells started to activate, the composition of the cSMAC changed. In the first two minutes after the cells started activating, the cSMAC included a large number of different components. This made T cell activation more efficient, possibly because the supramolecular complex was helping the network of signals to interact. Later, the cSMAC started to lose many of these components. Separating components may have helped to stop the activation signals. Understanding how T cells activate could lead to the possibility of turning them on or off in immune-related diseases. But these findings are not just relevant to immune cells. Other cells also use supramolecular complexes to control their signaling. Investigating how these complexes change over time could help us to understand how other cell types make decisions. Introduction T cell activation is governed by spatiotemporal organization of signal transduction across scales. At the nanoscale, receptors form clusters of dozens of molecules that can coalesce into microclusters and are commonly associated with active forms of signaling intermediates (Boyle et al., 2011; Hu et al., 2016; Lillemeier et al., 2006; Schamel et al., 2005; Sherman et al., 2011; Varma et al., 2006; Yokosuka et al., 2005). This association suggests that such receptor clusters mediate efficient T cell signaling. Larger, µm scale assemblies were first described at the center and periphery of T cells activated by antigen-presenting cells (APC) for the TCR, PKCθ and LFA-1, talin, respectively, as central and peripheral supramolecular activation clusters (cSMAC and pSMAC) (Grakoui et al., 1999; Monks et al., 1998; Monks et al., 1997). µm scale of assemblies, in particular in the form of supramolecular protein complexes, provides unique biophysical and signaling properties (Banani et al., 2016; Li et al., 2012; Shin and Brangwynne, 2017). Supramolecular protein complexes play critical roles in viral sensing (Cai et al., 2014), inflammation (Franklin et al., 2014), embryonic development (Brangwynne et al., 2009), protein folding in cancer (Rodina et al., 2016), nuclear ubiquitinoylation (Marzahn et al., 2016), and chromatin compaction (Larson et al., 2017). Such complexes are readily observed by fluorescence microscopy, held together by a network of multivalent protein interactions and often have distinct phase properties (Banani et al., 2016; Li et al., 2012; Shin and Brangwynne, 2017). The cSMAC has many properties of such supramolecular protein complexes: It contains various multivalent signaling intermediates (Balagopalan et al., 2015), prominently LAT (linker of activation of T cells), components of this complex including LAT and PKCθ exchange with the remainder of the cell to a moderate extent and slowly (Roybal et al., 2015), and components of this complex can be assembled into supramolecular structures in vitro (Su et al., 2016). Therefore, understanding biophysical properties of the cSMAC and how it regulates T cell activation is of substantial importance. cSMAC function is controversial despite decades of work. Limitations in investigating the cSMAC are that its properties are largely unresolved and that its composition and/or assembly have not been systematically manipulated inside live T cells. Based on association of cSMAC formation with T cell activation conditions, the cSMAC has been proposed to enhance T cell signaling, terminate it, not be related to signaling or only upon weak stimulation or at late time points (Čemerski et al., 2008; Freiberg et al., 2002; Grakoui et al., 1999; Lee et al., 2002; Monks et al., 1997). cSMAC formation is often associated with efficient T cell activation conditions, fitting with a role in enhancing T cell signaling. Accumulation of signaling intermediates at the T cell:APC interface center is substantially reduced by blockade of the costimulatory receptor CD28 (Singleton et al., 2009; Wülfing et al., 2002), in regulatory T cells (Zanin-Zhorov et al., 2010), during thymic selection (Ebert et al., 2008), or in the absence of the signal amplifying kinase Itk (IL-2 inducible T cell kinase) (Singleton et al., 2011). To determine cSMAC properties, we have used stimulated emission depletion (STED) super-resolution microscopy and correlative light electron microscopy (CLEM). The cSMAC was composed of multiple complexes of supramolecular dimensions and associated with extensive membrane undulations. To investigate cSMAC function, we have systematically manipulated the localization of three adaptor proteins in live primary T cells: LAT (Balagopalan et al., 2015) is an integral component of the cSMAC. SLP-76 (SH2 domain-containing leucocyte protein of 76 kD) (Koretzky et al., 2006) is associated with it only during the first minute of T cell activation (Roybal et al., 2015). Grb2 (growth factor receptor-bound 2) (Jang et al., 2009) association with the cSMAC is less prevalent (Roybal et al., 2015). Interface recruitment of all three adaptors was diminished upon attenuation of T cell activation by costimulation blockade and Itk-deficiency as was IL-2 secretion, a critical T cell effector function. By fusing these adaptors with various protein domains with a strong interface localization preference we brought them back to the interface under the attenuated T cell activation conditions and restored cSMAC formation. Such restoration enhanced IL-2 secretion but only when executed to the extent and with dynamics seen under full stimulus conditions. Results μm scale LAT accumulation at the center of the T cell APC interface is associated with efficient T cell activation To investigate the function of µm scale protein accumulation at the center of the T cell:APC interface, we first cataloged protein localization events that were consistently associated with efficient T cell activation. We attenuated T cell activation through costimulation blockade (Singleton et al., 2009; Wülfing et al., 2002) and Itk deficiency. The 5C.C7 T cell receptor (TCR) recognizes the moth cytochrome C (MCC) 89–103 peptide presented by I-Ek. In the restimulation of in vitro primed 5C.C7 T cells with CH27 B cell lymphoma APCs and MCC peptide IL-2 amounts in the supernatant were reduced upon blockade of the CD28 ligands CD80 and CD86 (‘costimulation blockade’) and in T cells from Itk knock out 5C.C7 TCR transgenic mice, in particular at lower peptide concentrations (Figure 1A). As IL-2 amounts in T cell culture supernatants are determined by the difference between IL-2 generation and consumption we also determined IL-2 mRNA levels. Even at an MCC peptide concentration of 10 µM the level of IL-2 mRNA in T cells was significantly (p<0.001) reduced to less than 50% upon costimulation blockade and Itk-deficiency (Figure 1B). 10 µM MCC was used for the remainder of the study. To more precisely relate the determination of IL-2 amounts in T cell culture supernatants to IL-2 mRNA generation, we determined the time course of both (Figure 1—figure supplement 1). IL-2 mRNA generation occurred during the first six hours of T cell activation, consistent with transient nuclear localization of NFkB and previous data establishing that APC contact times of less than one hour are sufficient to commit a primed T cells to proliferation (Iezzi et al., 1998). We used IL-2 mRNA generation for the remainder of the study because of its greater sensitivity to stimulus attenuation. Figure 1 with 4 supplements see all Download asset Open asset CD28 and Itk regulate IL-2 secretion and signaling organization. (A) In vitro primed 5C.C7 T cells, wild type or Itk-deficient (‘Itk ko’), were activated by CH27 APCs and the indicated concentration of MCC peptide in the absence or presence of 10 µg/ml anti-CD80 plus anti-CD86 (‘full stimulus’ or ‘costimulation blockade’). IL-2 levels in the supernatant are given relative to stimulation of wild type 5C.C7 T cells under full stimulus conditions with 10 µM MCC with SEM. 4–8 experiments were averaged per condition. Statistical significance as determined separately for each MCC peptide concentration by 1-way ANOVA is indicated. (B) Relative levels of IL-2 mRNA are given upon 5C.C7 T cell activation similar to A with only 10 µM MCC. 3–18 experiments were averaged per condition. Statistical significance as determined by 1-way ANOVA is indicated. (C) Wild type and Itk-deficient ('Itk ko') 5C.C7 T cells expressing the indicated sensors were activated by CH27 B cell APCs (10 µM MCC) in the absence or presence of 10 µg/ml anti-CD80 plus anti-CD86 ((‘full stimulus’ or ‘costimulation blockade’) and percentage occurrence of patterns of interface enrichment (Figure 1—figure supplement 2A) is given in shades of red from −40 to 420 s relative to tight cell coupling. Cluster trees are given in pink.Sensors used and source data for panel Care given in Figure 1—figure supplements 2B–4, Figure 1—source data 1. Figure 1—source data 1 Sensors used in Figure 1C,D. Publications describing the sensors used in Figure 1C,D and representative 5C.C7 T cell imaging data are given. https://cdn.elifesciences.org/articles/45789/elife-45789-fig1-data1-v2.pdf Download elife-45789-fig1-data1-v2.pdf We characterized T cell signaling organization as extensively described before (Ambler et al., 2017; Roybal et al., 2015; Singleton et al., 2009). Briefly, in vitro primed 5C.C7 T cells are retrovirally transduced to express fluorescent signaling intermediates or sensors, FACS sorted to low expression as close as possible to endogenous signaling intermediate concentrations and imaged in three dimensions over time during restimulation with APC and 10 µM MCC peptide (‘full stimulus’). In image analysis the frequency of occurrence of geometrically quantified µm scale subcellular distributions that represent underlying cell biological structures is determined (Figure 1—figure supplement 2A) (Roybal et al., 2013). Of particular interest here are accumulation at the center of the T cell APC interface (‘central’), the cSMAC, and accumulation in a µm deep ‘invagination’ at the center of the interface that likely mediates termination of early central signaling (Singleton et al., 2006). Upon costimulation blockade most sensors that displayed frequent central accumulation upon full T cell stimulation, in particular during the first two minutes of cell coupling, did less so. In Itk-deficient 5C.C7 T cells sensors with only transient early central accumulation in wild type T cells lost much of that accumulation (Figure 1C,D; Figure 1—figure supplements 2B–4, Figure 1—source data 1). Both phenotypes are indicative of reduced cSMAC formation. Efficient IL-2 secretion thus was associated with µm scale central signaling localization. LAT as a key cSMAC component displayed µm scale central localization (Figure 2A) in a biphasic pattern. At the time of tight cell coupling under full stimulus conditions 49 ± 6% of cell couples showed central LAT accumulation. After 2 min of cell coupling central LAT accumulation was only found in about 25% of cell couples (Figure 2B) and remained stable at that level. In the absence of Itk, the initial peak of central LAT accumulation was significantly (p=0.005) diminished to 26 ± 6% of cell couples with central LAT accumulation. Such reduction was more pronounced (11 ± 4%, p<0.001) upon costimulation blockade (Figure 2B; Figure 2—source data 1). Combining costimulation blockade and Itk deficiency yielded the least interface LAT accumulation (Figure 2B). Impaired activation of cytoskeletal transport processes is a likely contributor to diminished central LAT accumulation upon costimulation blockade and Itk deficiency, as enhancement of actin dynamics with active Rac and Cofilin (Roybal et al., 2016) significantly (p<0.001, Figure 2C; Figure 2—source data 1)(Roybal et al., 2016) increased central and overall LAT accumulation. Figure 2 with 2 supplements see all Download asset Open asset LAT localization and activation is regulated by costimulation and Itk. (A) An interaction of a LAT-GFP-transduced 5C.C7 T cell with a CH27 APC (10 μM MCC) is shown at the indicated time points (in minutes) relative to the time of formation of a tight cell couple. Differential interference contrast (DIC) images are shown in the top row, with top-down, maximum projections of 3-dimensional LAT-GFP fluorescence data in the bottom row. LAT-GFP fluorescence intensities are displayed in a rainbow-like false-color scale (increasing from blue to red). The scale bar corresponds to 5 µm. A corresponding video is available as Figure 2—Video 1. (B) The graphs display the percentage of cell couples with LAT accumulation in the indicated patterns (Figure 1—figure supplement 2A, ‘periphery’ is the sum of asymmetric and peripheral) relative to tight cell couple formation for wild type or Itk-deficient 5C.C7 T cells activated with CH27 APCs (10 µM MCC) in the absence or presence of 10 µg/ml anti-CD80 plus anti-CD86 (‘costimulation blockade’) as indicated. 47–77 cell couples from 2 to 5 independent experiments were analyzed per condition, 226 total. A statistical analysis is given in Figure 2—source data 1. (C) Itk-deficient 5C.C7 T cells were activated with CH27 APCs (10 µM MCC) in the presence of 10 µg/ml anti-CD80 plus anti-CD86 and 250 nM constitutively active Cofilin plus 1 µM constitutively active Rac1 as protein transduction reagents. LAT interface accumulation is given as in B. 30 cell couples from a single experiment were analyzed. Statistical significance is given in Figure 2—source data 1. (D) 5C.C7 T cells were activated as in B for the indicated times. Band intensities of α-LAT pY191 blots (Figure 2—figure supplement 1) as normalized to the 1 min time point under full stimulus conditions are given. 5–7 experiments were averaged per condition. Statistical significance as determined separately for each time point by 1-way ANOVA is indicated. Figure 2—source data 1 Statistical significance of differences in LAT accumulation under different T cell activation conditions is given for the indicated patterns as determined by proportion’s z-test. No entry indicates p>0.05. 0.000 indicates p<0.0005. Gray scale is used to visualize the level of significance. https://cdn.elifesciences.org/articles/45789/elife-45789-fig2-data1-v2.pdf Download elife-45789-fig2-data1-v2.pdf Attenuation of T cell activation was associated with diminished LAT phosphorylation at Y191 (Figure 2D; Figure 2—source data 1) upon costimulation blockade, in particular in combination with Itk deficiency. At 2, 5, and 10 min after tight cell coupling LAT phosphorylation was significantly (p≤0.02) reduced in Itk-deficient 5C.C7 T cells upon costimulation blockade compared to wild type 5C.C7 T cells under full stimulus conditions by 39%, 61%, and 70%, respectively. The cSMAC consists of multiple complexes of supramolecular dimensions and is associated with extensive membrane undulations To determine cSMAC properties, we stained 5C.C7 T cell:CH27 B cell APC couples for LAT and LAT phosphorylated at Y191 (‘pLAT’) and imaged them using STED super-resolution microscopy (Figure 3A). Multiple LAT/pLAT complexes formed in the cSMAC and/or beyond, on average four per cell. LAT complexes were significantly larger (p=0.04) in the cSMAC region with a supramolecular volume of 0.23 ± 0.03 µm3 than in T cells without a cSMAC (0.12 ± 0.01 µm3)(Figure 3B). pLAT complexes were similarly larger in the cSMAC region with volumes of 0.25 ± 0.03 µm3 (cSMAC) versus 0.13 ± 0.01 µm3 (non-cSMAC, p=0.007)(Figure 3B). Figure 3 with 3 supplements see all Download asset Open asset The cSMAC consists of multiple smaller complexes and is associated with extensive membrane undulations. (A) Two representative STED midplane images are given of 5C.C7 T cells activated by CH27 APCs (10 µm MCC) for 4.5 min and stained with α-LAT pY191. Staining fluorescence intensity is given in rainbow-like false-color scale (increasing from blue to red). The T cell outline is given in yellow. The scale bars correspond to 1 µm. (B) For experiments as in A LAT and LAT pY191 cluster size is given separately for cell couples with central or diffuse LAT accumulation as indicated (number of cell couples analyzed in two independent experiments in parentheses). Statistical significance as determined separately for ‘LAT’ and ‘pLAT’ by Student’s test is indicated. (C) Midplane sections are given for two EM tomograms from a CLEM experiment. 5C.C7 T cells expressing LAT-GFP (top) or LAT V3-GFP (bottom)(see Figure 4B) were activated by CH27 APCs under full stimulus (top) or costimulation blocked (bottom) conditions, respectively. Upon formation of a cell couple with central LAT-GFP or LAT V3-GFP accumulation cell were fixed and processed for EM. The T cell plasma membrane at the cellular interface is traced in blue. Videos of the entire EM tomogram reconstructions are given as Figure 3—Videos 1 and 2. (D) In cell couples processed as in C membrane undulations were determined as the ratio of the length of the plasma membrane (‘length’) to a straight-line interface diameter of the same region (‘diameter) in single images of EM sections. In cell couples with central LAT-GFP (black symbols) or LAT V3-GFP (red symbols) accumulation the interface center (‘cSMAC’) and periphery (‘pSMAC’) were measured separately. Peripheral regions were measured twice per cell, once to the left and once to the right of the central region. In control cell couples without central LAT-GFP (black symbols) or LAT V3-GFP (red symbols) accumulation (‘no the entire interface was analyzed. couples are from two independent experiments per condition. Statistical significance as determined by 1-way ANOVA is indicated To understand how LAT as a protein could the formation of multiple supramolecular complexes, we related cSMAC formation to plasma membrane with CLEM (Figure Figure supplement 1). live cell imaging of 5C.C7 T cells activated by CH27 B cell APCs we fixed upon of central LAT and processed them for electron microscopy We used two conditions, a full stimulus and activation of 5C.C7 T cells expressing a protein of LAT with the PKCθ upon costimulation blockade as an to enhanced cSMAC formation upon attenuation of T cell activation, as in in the In EM sections we measured the extent of membrane undulations in cSMAC and interface regions as the ratio of the plasma membrane length to the straight-line diameter of the region. This ratio was significantly (p<0.001) larger in the cSMAC ± than in the of the same T cell ± In control cell couples without cSMAC formation the ratio as measured across the entire interface was as ± as that in regions of cell couples with central LAT (Figure The data are consistent with diminished membrane undulations in 5C.C7 T cells upon costimulation blockade with a ratio of ± (Roybal et al., 2016). The cSMAC thus is a cellular region membrane undulations and multiple supramolecular complexes are associated with enhanced of signaling intermediates. To the for cSMAC in the remainder of these we used microscopy to determine central protein accumulation as a of cSMAC formation. of LAT with protein domains with pronounced interface localization preference LAT localization To determine cSMAC function, we to its formation upon costimulation blockade and in Itk-deficient T cells. To we to how cSMAC components interact within the a supramolecular complex could function through formation of protein interactions or by enhancing signaling through complex formation such that the same proteins can be assembled using and protein interaction of the large number of cSMAC components of which are (Figure 1) we the as most Impaired cSMAC formation upon costimulation blockade and Itk-deficiency at least in be by a reduction in the number of protein interaction that is the of key components such as LAT as by diminished phosphorylation (Figure We be to cSMAC formation by enhancing through the of protein interaction this and protein interaction the live cell signaling through the formation of the central region of increased protein be We increased LAT by three protein PKCθ or The PKCθ is for central interface accumulation of PKCθ et al., it central localization by (Figure supplement 1A). The domains strong central accumulation only within the first minute of cell coupling (Figure supplement as consistent with the localization of full length (Figure 1—figure supplement The interface accumulation on the first two minutes of cell coupling without a central preference (Figure supplement 1A). the of protein interaction domains to LAT is to their initial experiments to this remained as in the expression of LAT and the three LAT proteins was by FACS for the same level of proteins were at ± the endogenous level of LAT in T cells (Figure supplement with change to endogenous LAT levels in the transduced T cells. of LAT to the PKCθ yielded efficient central accumulation that was over the entire imaging time under all conditions at levels 50% of cell couples with central accumulation (Figure s after tight cell coupling such central accumulation was significantly Figure data 1) more frequent than central accumulation of LAT under full stimulus at time of LAT to the PKCθ thus central LAT accumulation the levels seen for LAT under condition. in formed upon LAT expression membrane undulations were similarly enhanced as in T cells expressing LAT (Figure cSMAC properties. Figure 4 with 4 supplements see all Download asset Open asset LAT localization can be by with protein domains with a strong interface localization (A) A of LAT-GFP is given (top) with LAT accumulation data under full stimulus conditions from Figure 2B) as a for the of the (B) top are given for the three proteins of LAT with protein localization domains as indicated. imaging data are given in the wild type or Itk-deficient 5C.C7 T cells transduced to express the LAT indicated on the top of the were activated with CH27 APCs (10 µM MCC) in the absence or presence of 10 µg/ml anti-CD80 plus anti-CD86 (‘costimulation blockade’) with different T cell activation conditions given in as indicated. the percentage of cell couples that displayed accumulation of the LAT with the indicated patterns as in Figure relative to tight cell couple formation in and red accumulation of LAT-GFP in or the central interface respectively, under the same T cell activation conditions Figure 2B) for For costimulation blocked conditions representative imaging data are given similar to Figure are available as Figure cell couples from 2 to 5 independent experiments were analyzed per condition, total. Statistical analysis is given in Figure data 1. Figure data 1 Statistical significance of differences in accumulation of as compared to LAT under different T cell activation conditions is given for the indicated patterns as determined by proportion’s z-test. No entry indicates p>0.05. 0.000 indicates p<0.0005. Gray scale is used to visualize the level of significance. Download of LAT to the domains yielded different on the T cell activation conditions. Upon a full T cell stimulus LAT in diminished central and overall accumulation compared to LAT that was across many time points (Figure Figure data that LAT not enhance assembled signaling Upon the three attenuated LAT consistently enhanced central most at most time for costimulation blockade in wild type and Itk cells (Figure Figure data 1). LAT accumulation upon attenuated T cell stimulation in was largely from LAT accumulation under full stimulus conditions Figure data 1) and central accumulation was enhanced only between 1 and 3 min after tight cell coupling. of LAT to the thus for close reconstitution of full LAT localization upon attenuated T cell of LAT to the LAT but was less (Figure Figure data 1). Upon the three attenuated T cell LAT enhanced central and overall LAT accumulation but consistently the same extent as LAT under full stimulus conditions. For in Itk-deficient 5C.C7 T cells upon costimulation blockade interface accumulation in was consistently enhanced for time point and from upon expression of LAT accumulation at the interface center only increased from a of to To that overall interface accumulation of the LAT

The intracellular events that regulate lymphocyte proliferation upon antigen encounter and the ability of the cells to respond to subsequent stimulation and to differentiate into effector cells remain largely to be understood. Several studies have linked T cell proliferation with the maintenance of antigen responsiveness and with the ability of the cells to differentiate and to acquire proper effector functions. The aim of my Ph.D. research project was to investigate the role of the TCR, CD28, and IL-2 generated intracellular events dictating CD4+ T lymphocyte proliferation and differentiation. In a model of CD3-induced clonal anergy, we have shown that antigen responsiveness was uniquely regulated by an IL-2/IL-2R-induced signalling event, which was delivered independently of IL-2-driven cell proliferation, and which was Rapamycin-sensitive. This indicates that proliferation and antigen responsiveness are independently regulated and that the latter specifically requires intact signalling through mTor, the mammalian target of Rapamycin. Moreover, we have shown that proper activation of p70S6k, one the known target of mTor, might play a crucial role in the maintenance of T lymphocyte responsiveness. We have also investigated the role of mTor in a model of in vitro antigen driven naive T cell differentiation. In this model, blocking mTor activity by the addition of Rapamycin during T cell activation, allowed comparable T cell expansion, but completely prevented polarization of effector cells. Together our results indicate that the intracellular events that dictate T cell proliferation are distinct from the intracellular signals that modulate the functional phenotype of activated T lymphocytes and suggest that, while mTor-dependent signalling is dispensable for T cell proliferation, it is primarily involved in the acquisition of proper T cell effector functions.

The activation kinetics of MAPK Erk are critical for T cell development and activation. In particular, sustained Erk signaling is required for T cell activation and effector functions, such as IL-2 production. Although Raf-1 triggers transient Erk activation, B-Raf is implicated in sustained Erk signaling after TCR stimulation. In this study, we show that B-Raf is dephosphorylated on its inhibitory serine 365 upon TCR triggering. However, it is unknown how B-Raf activation is coupled to the TCR. Using mass spectrometry, we identified protein kinase D-interacting substrate of 220 kDa (Kidins220)/ankyrin repeat-rich membrane spanning protein, mammalian target of rapamycin, Rictor, Dock2, and GM130 as novel B-Raf interaction partners. We focused on Kidins220, a protein that has been studied in neuronal cells and found that it associated with the pre-TCR, αβTCR, and γδTCR. Upon prolonged TCR stimulation, the Kidins220-TCR interaction was reduced, as demonstrated by immunoprecipitation and proximity ligation assays. We show that Kidins220 is required for TCR-induced sustained, but not transient, Erk activation. Consequently, induction of the immediate early gene products and transcription factors c-Fos and Erg-1 was blocked, and upregulation of the activation markers CD69, IL-2, and IFN-γ was reduced. Further, Kidins220 was required for optimal calcium signaling. In conclusion, we describe Kidins220 as a novel TCR-interacting protein that couples B-Raf to the TCR. Kidins220 is mandatory for sustained Erk signaling; thus, it is crucial for TCR-mediated T cell activation.

Death receptor 3 (DR3) is a tumor necrosis factor receptor superfamily member (TNFRSF25), which is minimally expressed on resting conventional T cells (though readily inducible upon cell activation), yet highly expressed on resting FoxP3+ regulatory T cells (Treg). We recently demonstrated that activation of DR3 with an agonistic antibody (4C12) leads to selective expansion and activation of Treg in healthy mice and suppression of graft-versus-host disease (GVHD) in recipient mice when donor mice are treated. However, given the long antibody half-life and concomitant safety concerns, along with the lack of a humanized agonistic antibody to DR3, both human and murine fusion proteins incorporating the natural DR3 ligand TL1A (TL1A-Ig) have been developed. Herein, we show that DR3 activation with 4C12 or with TL1A-Ig, with or without the addition of low dose IL-2 to the treatment regimen, led to a significant expansion of murine Treg in spleen, lymph nodes, and peripheral blood. Bioluminescent imaging revealed peak Treg expansion around day 7–8, with return to near baseline after 2–3 weeks. In addition to expansion, all DR3 agonist treatment regimens led to increased activation of Tregs, with significant upregulation of the activation markers ICOS, KLRG-1, PD-1, and CD103, and the proliferation marker Ki-67. The near absence of activated Treg populations in control treated spleens was also detected on tSNE analysis of flow cytometry data. Subtly different patterns of splenic Treg activation by the different DR3 agonists were noted in both tSNE analysis of flow cytometry data and RNA-sequencing analysis. However, upregulation of gene transcripts which play important roles in cell proliferation, trafficking, activation, and effector function were observed regardless of the DR3 agonist treatment regimen used. In the major MHC-mismatch model of hematopoietic cell transplantation, DR3 agonist-mediated expansion and activation of Tregs in donor mice led to a significant improvement in GVHD in recipient mice. These data provide important preclinical information regarding the outcome of DR3 activation with an agonistic antibody or natural ligand and provide insight into the therapeutic use of this approach to reduce GVHD in recipients and improve outcomes of hematopoietic cell transplantation.

The earliest event in T cell receptor(TCR) signaling is the activation of the Src family kinase(SFK) Lck. Phosphorylation of a conserved tyrosine in the C-terminal tail of Lck inhibits its kinase activity and is reciprocally regulated by the protein tyrosine kinase, C-terminal Src kinase(Csk), and the protein tyrosine phosphatase, CD45. Using a chemical-genetic approach, our lab has generated a novel analog-sensitive dominant inhibitory allele of Csk(CskAS) that can be specifically inhibited by a small molecule. Surprisingly, inhibition of the CskAS allele leads to rapid T cell activation in the absence of any TCR stimulation, indicating that Lck activity is dynamically regulated by an equilibrium between Csk and CD45 activities and that there is an adaptive response to the reduced basal signaling imposed by the dominant inhibitory CskAS. Moreover, mutating the SH2 and SH3 domains of CskAS results in reduced T cell activation upon inhibition of CskAS, suggesting that protein-protein interactions are important for this ligand-independent activation. We have identified Dok-1(downstream of tyrosine kinases-1), an adaptor protein that inhibits tyrosine kinase signaling, as a candidate in controlling the adaptive response responsible for ligand-independent T cell activation. Dok-1 is hyperphosphorylated and interacts with Csk via its SH2 domain upon CskAS inhibition. We suggest that Dok-1 is part of a negative feedback loop that acts to dampen TCR signals.

The non-receptor tyrosine kinase LCK belongs to the SRC family of kinases. SRC family kinases are proto-oncogenes that have long been known to play key roles in cell proliferation, motility, morphology and survival. Here we show that LCK regulates the function of the type III receptor tyrosine kinase FLT3 in murine pro-B cells. We observed that expression of LCK significantly enhances the colony forming capacity of the constitutively active FLT3 mutant FLT3-ITD (internal tandem duplication). Furthermore, cells expressing LCK developed tumor earlier compared to cells transfected with empty control vector. Staining of the tissues from mouse xenografts showed higher Ki67 staining in cells expressing LCK suggesting that expression of LCK enhances the FLT3-ITD-mediated proliferative capacity. LCK expression did not affect either FLT3-WT or FLT3-ITD -induced AKT, ERK1/2 or p38 phosphorylation. However, LCK expression significantly enhanced FLT3-ITD-mediated STAT5 phosphorylation. Taken together, our data suggest that LCK cooperates with oncogenic FLT3-ITD in cellular transformation.

In Vitro Formation of a Multi-Protein Complex Involved in TCR Signaling

Ligation of the Fc gamma R on natural killer (NK) cells results in the tyrosine phosphorylation of multiple substrates critical for intracellular signaling and activation of NK cell effector functions. However, it remains unclear which nonreceptor protein-tyrosine kinases (PTK) participate in this process. In this report we demonstrate that Fc gamma R ligation induced the tyrosine phosphorylation and increased the catalytic activities of both syk family PTKs, ZAP-70, and syk. The phosphorylation of ZAP-70 and syk was enhanced markedly by overexpression of wild-type lck but not by a kinase-inactive mutant, suggesting that early Fc gamma R-initiated activation of lck results in the subsequent regulation of syk family PTKs. The regulatory interplay between src and syk family PTKs was emphasized further by the observation that lck overexpression enhanced the association of ZAP-70 with the zeta chain of the Fc gamma R complex. Additional analyses indicated that lck induced the subsequent tyrosine phosphorylation of phospholipase C (PLC)-gamma 2. Interestingly, the regulatory effects of lck on ZAP-70, syk, and PLC-gamma 2 could not be replaced by overexpression of either fyn or src, demonstrating a selective role for lck in effectively coupling Fc gamma R stimulation to critical downstream signaling events. Taken together, our results suggest not only that Fc gamma R stimulation on NK cells is coupled to the intracellular activation of both ZAP-70 and syk, but that the src family member, lck, can selectively regulate this tyrosine kinase cascade.