In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. CD BB; TNFSR9 is an activation-induced surface receptor that through costimulation effects provide antigen-primed T cells with augmented survival, proliferation and effector functions as well as metabolic advantages.
These immunobiological mechanisms are being utilised for cancer immunotherapy with agonist CDbinding and crosslinking-inducing agents that elicit CD intracellular signaling. In this study, side-by-side comparisons show that provision of CD costimulation in-cis with regard to the TCR-CD3-ligating cell is superior to that provided in-trans in terms of T cell activation, proliferation, survival, cytokine secretion and mitochondrial fitness in mouse and human.
Here we report that the superiority of cis versus trans CDcostimulation is readily observed in vivo and is relevant for understanding the immunotherapeutic effects of CAR T cells and CD agonistic therapies currently undergoing clinical trials, which may provide costimulation either in cis or in trans. In turn, when CD is ligated with its natural ligand CDL, BBL or an agonist monoclonal antibody, it provides costimulatory signals for T cell proliferation, antiapoptosis, cytokine secretion, chromatin remodeling, and mitochondrial fitness 5.
The immunobiology of CDCDL is mostly involved in antiviral cytotoxic T lymphocyte CTL responses as concluded from experiments in knock-out mouse strains 6 , 7 and from observations in patients deficient in CD 8 , 9 , In human cancer patients, an antibody directed to CD termed urelumab, shows signs of clinical activity but induces serious liver inflammation in about one-fifth of treated patients at optimal doses 17 , To circumvent liver toxicity, targeting strategies to restrict CD exposure and crosslinking to the tumor microenvironment are already under clinical development as single agents or in combination with PD-L1 checkpoints inhibitors 5 , 19 , Such agents are mainly bispecific constructs targeting a molecule expressed by tumor cells, enabling CDcostimulation in cis 19 , 21 , 22 , or by stromal cells in the tumor microenvironment, such as fibroblast activated protein FAP enabling CDcostimulation in trans Previous evidence had shown that CD28 ligation in cis is far superior to that provided in trans with respect to TCR stimulation To assess the consequences of CDcostimulation in cis or trans with respect to antigen-presentation, we experimentally compare the provision of CDcostimulation in cis or in trans with respect to the source of CD3-TCR ligation.
CDcostimulation in cis resulted in a more prominent induction of surface CD25 Fig. Representative histograms of the geometric mean fluorescence intensity for each condition.
Summary data are given. Summary data are given as the difference of the value between each costimulation condition subtracted from the anti-CD3 value in each case. Source data are provided as Source Data file. Such superiority of cis-costimulation was preserved in a range of different densities of CD3 coating of the cis and trans beads and when using different bead to T cell ratios Supplementary Fig. Of note, surface PD-1 expression was also more pronounced following cis-costimulation as compared to trans costimulation Supplementary Fig.
Conceivably T cell priming in vivo occurs first under the influence of CD28 costimulation. Superiority of cis-costimulation was also clearly observed in these CD28 prestimulated lymphocytes in terms of both activation markers and cytokine secretion Supplementary Fig.
The control of these lymphocyte functions has been related to the transcription factors T-bet and Eomes Recently, studies have reported on the critical metabolic control of CDcostimulation on T cell mitochondrial mass and function 28 , 29 , As shown here, cis-costimulation rendered higher mitochondrial mass assessed by mitotracker staining Fig.
In addition to CD agonist antibodies as artificial ligands, we tested CDL-Fc coated microbeads covalently built to compare cis- versus trans costimulation Fig. Statistical significance was determined with the Wilcoxon test two-sided. Considered together, these results indicate that all functional activities attributed to CD ligation on T cells are more potently induced when CD and CD3 stimulation are provided in cis by the microbeads experimentally resembling antigen-presenting cells.
Superiority of cis-costimulation could be explained by different access to the beads. Representative frames in Supplementary Fig.
Quantitative data supporting this notion are provided in Supplementary Fig. An additional analysis of the duration of individual transient beads to T cell contacts showed that contact times lasted equally in the different conditions Supplementary Fig.
In the same line, using beads coated with anti-CD7 mAb that binds to T cells without providing detectable costimulation did not impair costimulation given in cis by the CD3 CD beads Supplementary Fig.
Bispecific monoclonal antibodies were used to study cis and trans CDcostimulation as provided by tumor cells on which CD3-TCR and CD ligation could take place redirected by such bispecific protein constructs. Numbers indicate geometric mean fluorescence intensity, gMFI. APV BsAbs. Statistical significance was determined with paired t tests two-sided. These experiments exclude less frequent intercellular interactions as a trivial reason for trans inferiority.
APV BsAb. Independently of the source of stimulation, coated anti-CD mAb Fig. Data are representative of at least three independent experiments. Shown data are representative of two independent donors with similar results. Data are representative of at least two independent donors.
The relative presence of the tumor cell clones in the xenografted tumors are shown in an inset in the experimental layout. Summary data are given as the bioluminescence photon flux subtracted from the tumors prior to the experiment background. Each pair of dots represents a single mouse bearing a cis tumor and a trans tumor in the left and right flank, respectively. RLU relative luciferase units. In d and f - g samples derive from the same experiment and loading control gels were processed in parallel.
Moreover, p65 nuclear translocation was more prominently observed in cis versus trans by confocal microscopy Fig. Of note, coinjection resulted in 9-day established tumors that combined comparable percentages of each variant, thereby representing the trans costimulation conditions. Interestingly, the most prominent features were the induction of genes involved in cell cycle control and replication as well as in DNA damage repair Fig.
In accordance to the transcriptomic analysis, a more intense proliferation was observed as a result of cis-costimulation compared to trans costimulation conditions. The horizontal bars denote the different pathways based on the z-score. Orange color indicates upregulation, while red color indicates downregulation.
Cumulative data are shown as the difference between the value of cis and trans conditions to which the anti-CD3 background was subtracted in each case. The division peaks are numbered from 1 to 3.
Statistical significance was determined with paired t test two-sided for d , e , g and h and one-way Anova one-sided with Holm-Sidak multiple comparison test for f. These results indicate that cis versus trans CDcostimulation provided replicative advantages, while preserving the integrity of genomic DNA undergoing replicative stress during clonal expansion.
These experiments conclude that cis-costimulation superiority is conserved between human and mouse. Furthermore, we compared the cytotoxicity of OT-1 cells in culture against B OVA cells. Supplementary Fig. Follow-up of adoptively transferred T cells over time indicated that cis-costimulated lymphocytes dominated over trans-costimulated lymphocytes in terms of numbers as late as 12 and 20 days following adoptive transfer Fig.
These findings were recapitulated in T cells present in liver, spleen, lymph nodes and bone marrow when mice were sacrificed twenty days after competitive adoptive transfer Fig. A mixture of ratio of in vitro cis-costimulated CD Data show gMFI. Each dot represents a single mouse. Data show geometric mean fluorescence intensity gMFI. A mixture of ratio of cis-costimulated OT-1 CD Statistical significance was determined with paired t test two-sided.
To address potential mechanisms, we differentially studied CD Clear differences suggesting less accumulation of DNA damage in cis CDcostimulated cells was found in lymphocyte suspensions from the different organs Fig. These intriguing results suggest that cis-costimulation during priming functionally reprograms T cells and protect them from replication stress.
Next, we sought to perform experiments of competitive infiltration of in vivo engrafted B Experiments as those graphically described in Fig. These differences were more striking following boost intravenous inoculations. Type regedit into Start. This will search for the Registry Editor command. Click regedit. It's a collection of blue blocks at the top of the Start window. Click Yes when prompted. Doing so will open the Registry Editor window.
Expand the "Services" folder. This will result in a long list of folders appearing. Scroll down and select the "sppsvc" folder. Click this folder to prompt its contents to display on the right side of the window. Select the "Start" file. It's near the bottom of the list of files that's on the right side of the window. Click the Edit tab. This option is near the top-left corner of the Registry window.
A drop-down menu will appear. Click Modify. It's at the top of the drop-down menu. Type 4 into the window. This value change will disable the activation notifications. Method 3. This circular arrow-shaped icon is at the bottom of the Settings window. Click Activation. It's a tab on the left side of the window. Activate your copy of Windows.
You can do this in one of two ways, depending on whether you have to buy a key or already upgraded for free on a different computer: [1] X Trustworthy Source Microsoft Support Technical support and product information from Microsoft. The PIs, in particular PI 4,5 P2, play multiple and versatile roles in the activities of numerous proteins, especially those localized in the PM, as exemplified by large number of ion channels whose activities are tightly regulated by its binding to PI 4,5 P2 18 , In addition, the interaction of PI 4,5 P2 with many membrane receptors modulates their function In this study, we were interested in the role of PI 4,5 P2 in TCR triggering, a process that has remained poorly understood despite several decades of extensive studies and accumulated information on its protein participants.
The exact same strategy has previously been used for the PM targeting of the functional protein units, which revealed various key aspects of the TCR triggering mechanism 50 , The precise reason for the apparent discrepancy with our present work is currently not clear. Recent studies revealing that the activation of TCR upon the dynamic 2D binding with pMHC in physiological conditions is likely to occur through mechanisms that are quite different from those observed when the receptor is stimulated by antibody crosslinking 54 , On the other hand, the differential effects of MCID expression on the pMHC- and soluble anti-CD3-elicited TCR activation suggests that these effects are not due to TCR-unrelated alterations such as a higher Lck activity 56 or positive feedback effects from down-stream signaling events such as a stronger calcium influx Additionally, in our opinion, the effects of the phosphatase on TCR activation were not due to diminished cell contacts, owing to results from a previous study that showed that the reduction of PI 4,5 P2 levels in T cells promotes rather than inhibits the contacts between T cells and APCs 57 , which could be a result of decreased T cell membrane rigidity These studies also showed that when presented in the form of lipid arrays, the phosphatidylinositol monophosphates, PI3P, PI4P and PI5P, displayed higher relative binding affinities to BRS than phosphatidylinositol bisphosphates and trisphosphates.
However, when incorporated into artificial lipid bilayer membranes, the binding of PI 4,5 P2 to BRS was increased drastically over the phosphatidylinositol monophosphates and emerged as the ligand with the highest relative affinity among the tested PIs 39 , The results of our present study conducted in vivo further substantiate this proposal.
Indeed, it has recently been proposed that the specific molecular interactions and surface electrostatic potential may conjointly promote the CD3CD association with the PM. In addition, in a previous study based on a focal decrease of the availability at the immunological synapse, phosphatidylserine PS has been put forward as an anionic lipid responsible for the CD3CD binding to the PM inner leaflet Although such a functional role for PS remains controversial 51 , 59 , we think that PI 4,5 P2 and PS could work cooperatively 60 , possibly along with cholesterol 61 to contribute to the assembly of the TCR-lipid nanodomains whereby CD3CD association with the PM inner leaflet is promoted.
In conclusion, our study strongly suggests that PI 4,5 P2 is a key regulator of the TCR membrane dynamics and controls of TCR activation and initiation of signaling pathway. In addition, our results provide new insights on the TCR triggering mechanism, especially on the dynamic dissociation of CD3CD from the PM inner leaflet upon the receptor engagement.
All other chemical reagents were from Sigma—Aldrich. All fluorescent-protein chimera were generated upon different conventional molecular biology strategies as summarized in Table S1.
In some cases, PCR fragments were inserted into linearized vector upon in-fusion methods Clontech , in presence of cloning enhancer, according to the manufacturer guidelines. All constructs were verified by sequencing MWG Biotech. Digestion was stopped with addition of 0.
Stable cell lines were obtained by electroporation using Amaxa nucleofactor Lonza according to manufacturer protocols 3A9m protocol B solution V and selected upon resistance to puromycin 1. Cells were sorted according to their positivity to surface labeling by C4H3 antibodies Facsvantage, Becton Dickinson. APC monolayers were generated by seeding 5. The proteins were then blotted with specific antibodies followed by chemiluminescence detection ECL, Amersham Pharmacia biotech.
For the quantification of signals in immunoblots, we measured the chemiluminescence signals using a Bioimage analyzer LAS and its dedicated software MultiGauge, Fujifilm. Data analysis was performed with FlowJo Tree star and the median intensity of fluorescence was plotted vs time after exclusion of dead cells and cell debris. Fixation and permeabilization 0. Fixed cells were transferred on ice, and all subsequent steps were performed on ice with pre-chilled solutions.
Cells were washed twice in ice-cold PBS. Cells were then blocked in a blocking buffer containing PBS and 0. Labelling was evaluated either using flow cytometer or by confocal microscopy.
The actin cytoskeleton architecture was imaged on a Zeiss LSM confocal microscope equipped with a sensitive and super-resolved Fast-AiryScan detector. FLIP imaging was adapted from Between the bleaching periods, the cells were imaged with low light intensity. We recorded one image every 1. Plasma membrane fluorescence decay was measured in a 5 pixel-width segmented line. Average fluorescence in the region was quantified with a homemade ImageJ macro available on request and plotted versus time.
Five wells were pooled 2. Indifferently, BD PBX loaded T cell calcium responses were evaluated by time lapse recording either by a flow cytometer or videomicroscopy. Data analysis was performed with FlowJo software and the median intensity of fluorescence was plotted vs. Specific calcium response amplitude was determined upon specific thresholds depending on experimental stimuli set according to our previous study.
Percentage of responding cells, the average response amplitude, the average response time fraction and the average burst duration were automatically calculated and tabulated in excel data sheets. Results were plotted as dot plots, limited to calcium responses above threshold. Fluorescence signal was recorded for additional seconds as described in T cells were seeded onto polylysine coated Lab-Tek slides Nunc.
Overnight metabolic treatments were kept during measurements. The detailed procedure for svFCS acquisition and analysis has been extensively described in 46 , 69 , 70 , For each series, the 20 recordings were selected in order to remove unexpected fluctuations visible on the intensity fluctuations upon a unique criteria of fluorescence fluctuation around a stationary regime of the count rate or unexpected shape of the ACF Auto-Correlation Function. This is repeated for 10 to 15 series, and the mean diffusion time, which is weighted in accordance to the number of kept ACF per series, is calculated.
Each FCS diffusion law was obtained from at least individual recordings. Finally, to estimate the statistical significance between two diffusion laws, testing hypotheses were performed independently for t 0 and D eff accounting for their variances, returning a value of the probability of false alarm PFA 37 , These percentages can be considered as the probability of the chance the compared values are originated from the same statistical process.
The overall statistical analysis is summarized in a 2D color-coded table t 0 vs D eff. All individual measurements were carried out on a minimum of 20 different cells. The data were fitted by equation 3 as previously described 71 , 73 :. All statistical analyses were performed using GraphPad Prism 5.
A non- parametric two-tailed unpaired Mann-Whitney test was used, unless otherwise stated. An amendment to this paper has been published and can be accessed via a link at the top of the paper. Smith-Garvin, J. T cell activation. Annu Rev Immunol 27 , — Malissen, B. Early T cell activation: integrating biochemical, structural, and biophysical cues. Annual review of immunology 33 , — Weiss, A. Signal transduction by lymphocyte antigen receptors.
Cell 76 , —74 Dorfman, J. Nat Immunol 1 , —35 Nika, K. Constitutively active Lck kinase in T cells drives antigen receptor signal transduction. Immunity 32 , —77 Krogsgaard, M. Nature , —43 Gil, D. Recruitment of Nck by CD3 epsilon reveals a ligand-induced conformational change essential for T cell receptor signaling and synapse formation. Cell , —12 Davis, S.
The kinetic-segregation model: TCR triggering and beyond. Nature immunology 7 , Xu, C. Regulation of T cell receptor activation by dynamic membrane binding of the CD3epsilon cytoplasmic tyrosine-based motif. Cell , —13 Shi, X. Nature , — Guo, X. BsAb comprise binding sites for two different target antigens for example, CD3 and a cell surface antigen and can directly cross-link T cells and antigen-expressing cells.
This results in a major histocompatibility complex MHC - and TCR-independent activation and triggering of effector mechanisms of the cross-linked immune cell. In the current study, peripheral human Tregs were isolated in a two-step procedure to high purity Figure 1a. Highly pure, freshly isolated or expanded human Tregs do not display a cytotoxic potential. One representative donor d , e as well as summary of four g or five f independent donors T cell to tumor cell ratio is shown.
Statistical significance was determined using one-way analysis of variance with Bonferroni multiple comparison test. To obtain sufficient Treg material for a clinical application, in vitro expansion of this rare cell population is inevitable. Redirecting expanded Tregs with a bsAb does not result in tumor-cell elimination.
Next, we wanted to exclude that an additional costimulatory signal is necessary to trigger the cytotoxic potential of Tregs. Analysis of three different donors Figure 2a revealed that although Tregs become activated However, we cannot rule out the possibility that apoptosis in Teffs is induced indirectly for example, via cytokine deprivation.
As shown in Figure 2b , injection of expanded Tregs in combination with tumor cells and bsAb did not reduce tumor outgrowth in mice, substantiating that Tregs are not capable of eliminating co-injected cancer cells. Nevertheless, despite the lack of cytotoxic activity bsAb-redirected Tregs hold a great suppressive potential.
The same observation holds true for Tregs antigen-specifically engineered with chimeric immune receptors, which instead of contributing to tumor-cell killing efficiently suppress the antitumor reaction of Teffs in vivo.
Highly pure, expanded Tregs are not cytotoxic in vivo and display a potent suppressive capacity. Tumor size was measured weekly.
The in vivo experiment was performed in parallel with previously presented work. One representative donor out of five independently performed experiments is depicted.
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