Abstract 002: Dynamic T Cell-Antigen Presenting Cell Interactions and Direct T Cell Activation Within the Vascular Wall During Hypertension

The present studies were carried out to characterize the cellular interactions involved in the activation and function of the antigen- specific and antigen-nonspecific T suppressor (Ts) cells that regulate the IgG responses of Lyb-5-B cells. The in vitro activation of both Lyt- 1+2- antigen-nonspecific Ts cells and Lyt-1-2+ antigen-specific Ts cells was shown to require the interaction of accessory cells and antigen-primed T cells. It was further demonstrated that this interaction was major histocompatibility complex (MHC)-restricted in that T cell recognition of I-A-encoded determinants on accessory cells was required for Ts cell activation. The activation of antigen-primed (A X B)F1 T cells with antigen in the presence of parentA or parentB accessory cells resulted, respectively, in the generation of parentA- restricted or parentB-restricted Ts cells. ParentA-restricted F1 Ts cells suppressed the responses generated by (A X B)F1 T helper (Th) cells cooperating with parentA (B + accessory) cells but did not suppress responses by the same (A X B)F1 Th cell population cooperating with parentB (B + accessory) cells. Neither parentA-restricted Ts cells alone nor parentB-restricted Ts cells alone suppressed the responses of (A X B)F1 (B + accessory) cells, whereas a mixture of these two Ts cell populations was able to significantly suppress the responses of F1 (B + accessory) cells. In contrast, responses of (A X B)F1 leads to parentA Th cells (restricted to recognizing parentA but not parentB MHC determinants on F1 cells) and (A X B)F1 (B + accessory) cells was suppressed by parentA-restricted Ts cells but not by parentB-restricted Ts cells. Collectively these findings suggest that the Ts cell populations characterized here do not function by directly inhibiting the activity of Th cells, B cells or accessory cells of a given MHC genotype, but rather that they appear to function through a unique mechanism involving highly specific inhibition of the interaction between MHC-restricted Th cells and the (B + accessory) cells required for these responses.

DOCA salt‐induced hypertension is associated with oxidation of tetrahydrobiopterin (H4B) and uncoupling of eNOS. We sought to examine whether administration of H4B precursor sepiapterin restores eNOS function. Bioavailable NO detected by electron spin resonance (ESR) was markedly reduced in aortas of DOCA‐salt hypertensive mice. Pre‐incubation with sepiapterin (10 æmol/L for 30 min) failed to increase NO in hypertensive aortas while it augmented NO production from control vessels, implicating a hypertension‐associated deficiency in sepiapterin reductase (SPR), the rate‐limiting enzyme for sepiapterin conversion to H4B. Indeed, western analysis revealed a decrease in SPR expression in whole aortic homogenates of hypertensive mice. The rabbit anti‐serum for SPR was assessed using antibody clearance, and proved specific and reproducible in detecting SPR monomer of 28 kD in aortic homogenates. Differential western analysis using isolated pure endothelial cells and aortas denuded of endothelium indicated that SPR was only downregulated in the endothelium. Chronic infusion of sepiapterin (10 mg/kg/day) upregulated SPR expression in control mice while had mixed responses in hypertensive mice. In summary, administration of sepiapterin is ineffective in recoupling eNOS in hypertension, likely due to a loss in SPR at baseline and a failure of substrate‐induced upregulation. This may explain the previously reported inconsistency of sepiapterin in modulating endothelium‐dependent vasorelaxation in different models of vascular diseases. This work is supported by American Heart Association and American Diabetes Association Grants 0435189N and 7‐04‐RA‐16, and NIH Grants HL077440 and HL081571 (all to H Cai)

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

Neutrophils are short-lived effector cells of the innate immune system that form the first line of defense against microbial pathogens.1 Recent studies show that, by migrating to bone marrow, these cells can also induce (VACV)-specific T cell responses.2 Neutrophils polarize to distinct phenotypes in response to environmental cytokine/chemokine signals. When granulocyte macrophage-colony stimulating factor (GM-CSF) is present, neutrophils can acquire an antigen-presenting cell (APC) phenotype (by upregulating MHC class II and CD80/CD86 costimulatory molecules) to trigger CD8 T cell activation3; when transforming growth factor (TGF)-β is found in a tumor context, tumor-associated neutrophils polarize from the anti-tumor N1 to the pro-tumor N2 subtype, whose ability to activate CD8 T cells is impaired.4 In mice infected with the New York vaccina virus strain (NYVAC), we can alter neutrophil recruitment. For example, manipulation of this vector can activate NFκB, which generates a specific cytokine/chemokine milieu at the infection site; this influences the migration of distinct neutrophil subtypes (Nα and Nβ).5 Nβ neutrophils are more lobulated and hypersegmented than the Nα subtype, which confirms neutrophil ability to acquire a distinct phenotype in specific cytokine/chemokine conditions. The APC markers CD80 and CD86 are upregulated in Nβ cells, which show a greater capacity to generate antigen-specific CD8 T cell responses than Nα cells.5 Although Nβ neutrophils present antigen to CD8 T effector cells to induce their activation, they cannot activate naive CD8 T cells ex vivo.5 Other APC-activating signals such as CD40, which aids dendritic cell (DC) stimulation of CD8 T cells, could help neutrophil activation of naive CD8 T cells. In vivo studies confirmed that neutrophils do not prime VACV-specific CD8 T cell responses in the absence of myeloid DC,2 and that neutrophils transport processed antigens and cross-prime naive CD8 T cells in vivo to generate vigorous antigen-specific CD8 T cell responses.6 These earlier findings suggest that antigen transport after peritoneal VACV infection is due not to direct neutrophil infection, but rather to neutrophil uptake of apoptotic infected cells in which antigen has been processed. These neutrophils then migrate to secondary lymphoid organs such as the spleen, where they might cross-present antigens; when typical APC such as DC are present, the neutrophils could prime naive CD8 T cells (Fig. 1). Whether Nβ neutrophils cross-present antigens from virus-infected cells more efficiently than Nα neutrophils remains to be determined. Figure 1. Model of neutrophil-dependent CD8 (T)cell activation. Apoptotic vaccinia virus (VACV)-infected macrophages (Mϕ) are engulfed by neutrophils (Nβ) that cross-prime CD8 T cells with the help of dendritic cells (DC). After infection with a human immunodeficiency virus (HIV) antigen-bearing NYVAC, murine neutrophils help to enhance CD8 T cell responses to non-secreted HIV antigens,5 demonstrating a potential role in vaccine-induced immune responses and confirming their capacity to cross-prime antigens from virus-infected cells. Although neutrophils increase antigen-specific CD8 T cell responses, this is not the case for CD4 T cell responses.5 It is possible that neutrophils prime CD4 T cells with difficulty, and probably downregulate CD4 T cell activation, which would explain the reduction in antigen-specific CD4 T cell responses. Another study of vaccine immunization confirmed that neutrophils generate poor antigen-specific CD4 responses, as their contacts with DC in the draining lymph nodes are very brief, which alters the quality of DC-T cell interactions.7 An in vivo study of the mechanism of neutrophil-dependent control of T cell subset responses to VACV-delivered antigens would be of major interest for the generation of VACV-based vaccines that preferentially induce CD4 and CD8 T cell responses. How Nα and Nβ subtypes interact with typical APC and T cell subsets must be defined. Inducing pathogen-specific T cell responses is one of the most important goals for achieving effective vaccines. The distinct ability of neutrophil subtypes to activate CD8 T cells suggests that virus-based vaccine efficacy might be improved by defining which cytokines/chemokines induce this functional switch, which would allow modulation of the Nβ/Nα neutrophil ratio after infection.

Limited efficiency of endogenous interleukin-7 levels in T cell reconstitution during HIV-1 infection: will exogenous interleukin-7 therapy work?

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

We analyzed the mechanism by which accessory cells support the induction of the proliferation of human peripheral blood T cells by a monoclonal anti-CD3 antibody, OKT3. Cross-linking of T cell receptor/CD3 complex by anti-CD3 coupled to latex beads and the addition of IL-1 are not enough to induce the IL-2 production and proliferation of T cells extensively depleted of accessory cells, while the addition of both the culture supernatant of macrophages or a monoblastic cell line, U937 cells, and the paraformaldehyde-fixed macrophages or U937 cells which had been precultured with interferon-gamma before fixation into the culture of the T cells with anti-CD3-latex did induce the T cell proliferation. Lack of the addition of either one of these did not induce the response. These results indicate that the signal(s) delivered by soluble factors released from the accessory cells and that delivered by the physical interaction between accessory cells and T cells are both required for the induction of IL 2 production and proliferation of T cells by anti-CD3-latex. Importantly, the macrophages or U937 cells had to be cultured with Con A-stimulated lymphocyte culture supernatant or IFN-gamma prior to fixation with paraformaldehyde, suggesting that a molecule(s) inducible on accessory cells surface by IFN-gamma or other lymphokine is necessary for the effective accessory cell-T cell interaction to induce the T cell response. It was further revealed that the activity of the culture supernatant of accessory cells may be mediated synergistically by IL 1 and a certain other factor(s) and was actually shown to be replaced by the combined addition of rIL-1 and rIL-6 but not by rIL-1 alone. The experimental system described here will be very useful for dissecting the accessory functions for T cell activation.

Cardiovascular diseases (CVDs) are one of the leading causes of mortality worldwide.1 It has been so for decades, notwithstanding a wide array of – mostly preventive – treatment modalities targeting known risk factors, such as hyperlipidemia, type 2 diabetes mellitus, hypertension, or obesity. Recent technical and conceptual advances have unveiled important contributions of the immune system in the pathophysiology of a variety of CVDs such as atherosclerosis, ischemic stroke, chronic heart failure, and other myocardial conditions like myocardial ischemia and reperfusion, viral myocarditis, and cardiac transplantation.2–4 In many of these disorders, so-called danger-associated molecular patterns (DAMPs), released from necrotic tissue and dying cells, can lead to the activation of certain immune cell populations such as monocytes/ macrophages, granulocytes, and T cells, thus aggravating ongoing inflammatory processes at the lesion site. Dendritic cells (DCs) are key modulators of immunity, pivotal in directing innate and adaptive immune responses against microbial, viral, but also modified self-antigens present at the sites of injury. Given the tissue trauma underlying various CVDs, it is not surprising that recent observations have allocated a regulatory role for DCs in CVD-associated immune responses. Interestingly, nondiseased arteries of young individuals were seen to host a network of resident vascular DCs (CD1a+ S100+ lag+ CD31− CD83− CD86−),5 representing a phenotype related to Langerhans cells in the skin. In agreement, monocyte-derived CD11c+ CD68+ dendritic cells could be detected in the atherosclerosis-prone lesser curvature and aortic sinus in inbred atherosclerosis-susceptible (C57Bl/6), but not resistant mouse strains (balb/c).6 Murine vascular resident DCs express an immature phenotype with low expression of costimulatory molecules, and are present in the subendothelial space with occasional probing into the vascular lumen. DCs have …

Central tolerance ensures autoreactive thymocytes are either eliminated via clonal deletion or are diverted to the regulatory T-cell lineage, thus minimizing T cell autoimmunity. To undergo central tolerance, thymocytes must enter the medulla to test their TCRs for autoreactivity against the diverse self-antigens displayed by medullary antigen presenting cells (APCs). While CCR7 is known to promote thymocyte medullary entry and negative selection against some self-antigens, our previous studies implicate CCR4 in these processes, raising the question of whether CCR4 and CCR7 play distinct or redundant roles in central tolerance. Here, we use synchronized positive selection assays and 2-photon timelapse microscopy to demonstrate that CCR4 and CCR7 are expressed sequentially after positive selection and promote medullary accumulation and central tolerance of distinct post-positive selection thymocyte subsets. CCR4 is upregulated within hours of positive selection signaling and promotes medullary entry and clonal deletion of immature post-positive selection thymocytes. In contrast, CCR7 is expressed several days later and promotes medullary entry and negative selection of mature thymocytes. Unlike CCR7 ligands, which are expressed by medullary thymic epithelial cells, CCR4 ligands are expressed by hematopoietic APCs, and we find that CCR4 enforces thymocyte tolerance to self-antigens presented by activated thymic APCs. Our findings demonstrate that CCR7 expression is neither necessary nor sufficient for thymocyte medullary entry and support a revised model of central tolerance in which CCR4 and CCR7 promote early and late stages of central tolerance, respectively, via interactions with distinct APC subsets. Supported by a grant from the NIH/NIAID (R01AI104870)

CD40 receptor is a member of TNF-receptor superfamily, broadly expressed on the surface of antigen-presenting cells (APC) including B cells, dendritic cells (DC), macrophages, monocytes, and non-APC like platelets, endothelial cells and even some tumor cells. CD40 is essential for APC activation and signalling via ligation of CD40 ligand located on helper T cells and CD40 on APC to mediate licensing (activating) of APC. Ligation of CD40 on DCs, for example, can induce increased surface expression of costimulatory and MHC molecules, production of pro-inflammatory cytokines, and enhanced T cell activation. CD40 ligation on resting B cells can increase its antigen-presenting function and proliferation. In contrast to its critical role in the induction of effective innate and adaptive immune responses, CD40 signalling on certain malignant cells, particularly B cell lymphomas, triggers tumor apoptosis. The dual function of CD40 signalling provide unique opportunities for the use of agonistic CD40 antibodies in cancer immunotherapy. Indeed, several CD40 monoclonal antibodies are now in different stages of clinical development, some shown promising preliminary results in combination with other cancer immunotherapies. However, we are lacking preclinical models to evaluate efficacy of therapeutic CD40 agonistic antibodies, which usually don’t cross-bind to mouse targets. To fill this gap, we developed a human CD40 knock-in model (CD40 HuGEMM), of which mouse exon 2-5 are replaced by human counterparts. These KI mice express a chimeric CD40 with human extracellular domain &amp; mouse trans-membrane and intracellular sequences. We have characterized the human CD40 expression of CD40 HuGEMM on a broad spectrum of mouse cells, such as lymphocytes, macrophages, DCs, and endothelial cells. In addition, we confirmed that human CD40 antibodies bind to the chimeric m/hCD40 recombinant protein, and lead to increased expression of MHCII on the B cells. Finally, the in vivo efficacy study indicates that human CD40 antibody treatment lead to robust anti-tumor response and contribute to better survival of the mice. Taken together, our CD40 HuGEMM provides a powerful preclinical model to assess the efficacy of the human-specific CD40 agonistic antibodies. It may also serve as a model for proof of concept studies of CD40 agonistic antibodies with other therapies. Citation Format: Daniel Xianfei He, Lei Zheng, Ruilin Sun, Annie Xiaoyu An, Jian Fei, Henry Qixiang Li, Davy Xuesong Ouyang. Ex vivo functional characterization and in vivo efficacy validation of human CD40 agonistic antibodies in the human CD40 knock-in model (CD40 HuGEMM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4976.

Pulmonary hypertension is a cardiovascular disorder manifested by elevated mean arterial blood pressure (>20 mmHg) together with vessel wall stiffening and thickening due to alterations in collagen, elastin, and smooth muscle cells. Hypoxia-induced (type 3) pulmonary hypertension can be studied in animals exposed to a low oxygen environment for prolonged time periods leading to biomechanical alterations in vessel wall structure. This study introduces a novel approach to formulating a reduced order nonlinear elastic structural wall model for a large pulmonary artery. The model relating blood pressure and area is calibrated using ex vivo measurements of vessel diameter and wall thickness changes, under controlled pressure conditions, in left pulmonary arteries isolated from control and hypertensive mice. A two-layer, hyperelastic, and anisotropic model incorporating residual stresses is formulated using the Holzapfel-Gasser-Ogden model. Complex relations predicting vessel area and wall thickness with increasing blood pressure are derived and calibrated using the data. Sensitivity analysis, parameter estimation, subset selection, and physical plausibility arguments are used to systematically reduce the 16-parameter model to one in which a much smaller subset of identifiable parameters is estimated via solution of an inverse problem. Our final reduced one layer model includes a single set of three elastic moduli. Estimated ranges of these parameters demonstrate that nonlinear stiffening is dominated by elastin in the control animals and by collagen in the hypertensive animals. The pressure-area relation developed in this novel manner has potential impact on one-dimensional fluids network models of vessel wall remodeling in the presence of cardiovascular disease.

In recent years, monoclonal antibodies have been successfully used in clinical trials to block or activate key mediators of immune checkpoint pathways, including CTLA4, PD-1, PD-L1, CD40 and others. Combination therapy can promote antigen release and T cell priming, T cell activation and homing, which helps to overcome tumor immune-evasive mechanisms and maximize efficacy, ultimately benefitting most patients. However, along the IO drug development process, in vivo efficacy models have always been a rate-limiting step, especially to test combination therapy using two IO antibodies. The transmembrane protein receptor CD40 is a member of the tumor necrosis factor (TNF) receptor super family and is involved in co-stimulation of immune cells. CD40 is expressed by antigen-presenting cells (APCs) including dendritic cells (DCs), B-cells, macrophages, and monocytes. It has the ability to "wake" DCs to prime effective cytotoxic T-cell responses. Thus, effective anti-CD40 antibodies provide an ideal therapy alternative in combination with other IO antibodies. To evaluate the in vivo efficacy and safety (immune-related adverse events) of CD40 and PD-1 combination antibodies, we developed a double humanized B-hPD-1/hCD40 mouse model. In this model, mouse PD-1 gene exon 2, and CD40 gene exons 2-7 were replaced with their human counterparts. Murine colon cancer MC38 cells were subcutaneously implanted into homozygous B-hPD-1/hCD40 mice. We compared the effect among single hPD-1 antibody Keytruda, single hCD40 antibody Selicrelumab and combination of the two. Combination treatment of Selicrelumab and Keytruda show higher inhibitory effects than single antibody treatments. The blood chemistry assays showed that the CD40 antibody had no toxicity effects compared with the control group. Based on these findings, we conclude that the B-hPD-1/hCD40 double-humanized mouse model is a powerful tool for in vivo efficacy and safety evaluation of hPD-1 and hCD40 antibodies for combination therapy. Citation Format: Yanan Guo. In vivo efficacy and safety evaluation of anti-human PD-1 and CD40 mAbs using double humanized PD-1/CD40 mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3219.

CD3/CD28 costimulation-induced NF-KB activation, is not mediated by protein metallothionein 2A and FAS associated death domain T he process of activating T cells is intricate and requires close collaboration. It is among the immunological response's most significant occurrences. In a physiologically normal state, lymphocytes are at rest. The potency of signals received by the T-cell receptor (TCR/CD3) and a series of co-stimulatory receptors, the most notable of which is CD28, determines the capacity of naïve T cells to multiply and acquire potential roles [1] . Concurrent ligation of these receptors sets off signals that The immune response depends on T cell activation, which is triggered by signals from receptors such as CD28 and TCR/CD3, resulting in T cell proliferation and programmed cell death (AICD). This control avoids disorders like immunity and cancer. These receptors are stimulated by Antigen-Presenting Cells (APCs), which set off a signaling cascade that activates the important transcription factor NF-κB. Degrading inhibitory proteins is necessary for NF-κB activation, which permits it to reach the nucleus and regulate gene expression. Methods: Semi-quantitative RT-PCR was used to assess the levels of metallothionein 2A mRNA in primary T cells to validate the results of the microarray analysis. Five-to six-week-old male C57BL/6 wild-type mice were used in the investigation. Mouse primary T cells from lymph nodes were suspended aseptically, and anti-CD3/CD28 was used to activate the cells. The cells were transfected with plasmid DNA using a Gen Pulser, and the T cells were separated by magnetic cell sorting. Following the synthesis of cDNA from total RNA, microarray analysis was used to assess variations in gene expression. The microarray results were validated by RT-PCR. Western blotting was used to confirm protein expression, and flow cytometry with CD69 was used to measure cell death. Immunoreactive bands were visible in co-immunoprecipitation assays using monoclonal anti-MT2A and FADD antibodies. Results: This work focused on FAS-associated Death Domain (FADD), MT2A, and NF-κB gene expression profiles in mouse primary T cells before and after anti-CD3/CD28 stimulation, utilizing microarray analysis. The results shed light on these genes' functions in AICD and T cell activation. The cascade triggers the activation of transcription factors necessary for T cell proliferation and cytokine production, such as NF-κB. Degradation of inhibitory IκB proteins is necessary for NF-κB activation, which permits NF-κB to reach the nucleus and control gene transcription. The involvement of PLC-γ1 in CD3/CD28-induced NF-κB activation has been highlighted by recent studies. The processes that determine whether T cells divide or undergo apoptosis are still unknown, despite advances. To compare the gene expression of FADD, MT2A, and NF-κB in mouse primary T cells before and after anti-CD3/ CD28 stimulation, this work used microarray analysis. The purpose of our research is to shed light on these genes' functions in T cell activation and activation-induced cell death (AICD).

GRAIL (gene related to anergy in lymphocytes), is an E3 ubiquitin ligase with increased expression in anergic CD4+ T cells. The expression of GRAIL has been shown to be both necessary and sufficient for the induction of T cell (T) anergy. To date, several subsets of anergic T cells have demonstrated altered interactions with antigen-presenting cells (APC) and perturbed TCR-mediated signaling. The role of GRAIL in mediating these aspects of T cell anergy remains unclear. We used flow cytometry and confocal microscopy to examine T/APC interactions in GRAIL-expressing T cells. Increased GRAIL expression resulted in reduced T/APC conjugation efficiency as assessed by flow cytometry. Examination of single T/APC conjugates by confocal microscopy revealed altered polarization of polymerized actin and LFA-1 to the T/APC interface. When GRAIL expression was knocked down, actin polarization to the T/APC interface was restored, demonstrating that GRAIL is necessary for alteration of actin cytoskeletal rearrangement under anergizing conditions. Interestingly, proximal TCR signaling including calcium flux and phosphorylation of Vav were not disrupted by expression of GRAIL in CD4+ T cells. In contrast, interrogation of distal signaling events demonstrated significantly decreased JNK phosphorylation in GRAIL-expressing T cells. In sum, GRAIL expression in CD4+ T cells mediates alterations in the actin cytoskeleton during T/APC interactions. Moreover, in this model, our data dissociates proximal T cell signaling events from functional unresponsiveness. These data demonstrate a novel role for GRAIL in modulating T/APC interactions and provide further insight into the cell biology of anergic T cells.

Several receptors and counter-receptor pairs on T cells and on antigen- presenting cells (APCs) deliver costimulatory signals to T cells during antigen presentation. The CD28 receptor on T cells with its ligand B7 represents one of the best characterized and most important examples of this costimulation. We show here that interleukin 12 (IL-12), a cytokine also produced by APCs (monocyte/macrophages and B cells) and active on T and natural killer cells, has a strong synergistic effect with the B7/CD28 interaction in inducing proliferation and cytokine production in both mitogen-activated and freshly isolated peripheral blood T cells. Together with anti-CD28 antibodies, IL-12 induces proliferation of T cells to levels higher than those obtained with IL-2 stimulation and it is effective at IL-12 concentrations 100- to 1,000- fold lower than effective concentrations of IL-2. The proliferative effect of anti-CD28 and IL-12 is resistant to moderate doses of cyclosporin A and is largely independent of endogenous IL-2, IL-12, in synergy with anti-CD28 or B7-transfected cells, is most effective in inducing interferon gamma (IFN-gamma) production, but production of tumor necrosis factor alpha and granulocyte/macrophage colony- stimulating factor is also observed. IL-12-induced IFN-gamma production in peripheral blood mononuclear cells is inhibited by the chimeric molecule CTLA-4 immunoglobulin, which prevents binding of CD28 to B7, suggesting that endogenous B7 on the mononuclear cells and IL-12 cooperate in inducing IFN-gamma production. IL-10 inhibits both IL-12 production and B7 expression on monocytes. These two effects are largely responsible for the ability of IL-10, acting on accessory cells, to inhibit IFN-gamma production by lymphocytes, because anti- CD28 antibodies and IL-12 can reverse the inhibitory effect of IL-10 on IFN-gamma production. Our results in vitro suggest that the synergy between B7 and IL-12, a surface antigen and a soluble product of APCs, respectively, plays a role in regulating T cell activation and immune response in the microenvironment of inflamed tissues.