Functional Anatomy of T Cell Activation and Synapse Formation (Annual Review of Immunology Book 28)

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The fluorescent probes were excited with the use of an argon ion laser and a HeNe laser. To evaluate colocalization of proteins and their position within the IS, unprocessed images were analyzed with the linescan function of the MetaMorph program Molecular Devices, Downingtown, PA. Briefly, a reference line was drawn along the T-B cell contact site. The software calculates the mean red and green fluorescence intensities along the reference line for four pixels of width and plots the measurements with respect to their position within the selected portion of the membrane.

Eight random fields were analyzed for each donor and each treatment condition. Images were processed using the MetaMorph software. The external solution was in mM : NaCl, 4. The pipette solution was in mM : potassium aspartate, 8. All solutions were — mosM. Statistical analyses were performed with Student's t -test paired or unpaired.

YFP-tagged KCa3. Their pharmacological and electrophysiological properties were investigated and compared with native KCa3. Overall, the KCa3. Furthermore, KCa3. Chandy Fig. I , current; V , voltage. B : KCa3. Our results revealed an upregulation of KCa3. Similar membrane capacitance, a measure of cell surface area, was measured in transfected and nontransfected cells: 1.

This indicated that there was a significant increase in the number of functional KCa3. However, the degree of this increase may be accentuated or diminished if differences in channel open probability were great between the control and transfected groups. Currents were obtained by ramp depolarization as described in Fig. Inset : representative images of a control top and transfected bottom cell are shown. Both cells were from the same donor and in the same image.

A , bottom : there was a significant increase in KCa3. Data are the average of 5 cells from 2—4 donors. Currents, elicited by ramp depolarization as described in Fig. After we confirmed that functional KCa3. Furthermore, upon T cell activation, extensive cytoskeletal reorganization takes place, resulting in F-actin accumulation at the contact point; as such, it can serve as a marker of IS formation 3 , 5. This time frame was sufficient for channel expression and insertion in the plasma membrane, as demonstrated by the electrophysiological and microscopy experiments described in the previous section Fig.

Moreover, the cells were used at this early time point to avoid overexpression, which could possibly affect the function of the T cells.

Antigen Presentation, T Cell Activation and Deactivation

As shown in Fig. Interestingly it appears that F-actin is concentrated mostly on the periphery of the T cell-bead contact point after 30 min of activation as previously shown, whereas KCa3. These studies indicate that KCa3. Further studies are needed, however, to elucidate the location of KCa3. To induce the formation of the supramolecular activation complex, we used superantigen-loaded B cells that closely mimic the APCs found in vivo.

T cells not conjugated with beads display uniform distribution of KCa3. The three-dimensional T-bead interface reconstruction xz projection is shown under the corresponding two-dimensional 2D image. The reconstructed portion of the T-B cell complex is indicated by a box in the 2D merged image. DIC, differential interference contrast.


T cells were maintained in a heated chamber, and the nm-to nm ratio was monitored throughout the experiment. After a short equilibration period, SEB-loaded B cells were added in the chamber. A : human T cells were loaded with the ratiometric dye fura 2 and stimulated with SEB-pulsed B cells for 15 min.

Arrows indicate point of introduction of the B cells into the bath. These data are representative of a total of 35 activated T cells. Unstimulated cells cells not contacting a B cell showed little or no response. Scale bars correspond to s. Experiments in B and C were performed at It has been shown that KCa3. As a control, we performed identical experiments with resting T cells.

In these cells, Kv1. To ensure that the decrease in number of activated T cells responding to antigen presentation was not due to a decrease in T-B cell conjugate formation, we compared the number of T-B cell conjugates that form in activated T cells in the presence and absence of TRAM Overall, these data confirm in our experimental setting that KCa3. Moreover, they indicate that blockade of KCa3. It still remains to be determined whether KCa3. Confocal microscopy experiments were performed to study the KCa3. Our data indicate that KCa3.

Notably, KCa3. Overall, KCa3. Our data therefore suggest that functional KCa3. The three-dimensional T-B cell interface reconstructions are shown under the corresponding 2D images. The area used for the reconstruction is marked in the 2D merged images by a box. The line scan analyses of KCa3. These images are representative of the results obtained from 5 donors for control experiments and 2 donors for pretreatment experiments.

Overall, these results indicate that KCa3.

Mechanisms behind Functional Avidity Maturation in T Cells

To more precisely define the kinetics of KCa3. The results obtained in these studies confirmed the observations in the fixed microscopy studies, although a certain degree of variability in the kinetics of KCa3. In the majority of conjugates imaged, the channels were recruited at the IS within 40 s to 2 min 5 of 7 conjugates Fig.

Only in two conjugates was a longer time necessary for channel recruitment. It is not uncommon for T cells to undergo serial stimulation by APCs during the activation process However, because of the multiple encounters and transient nature of this polarization, these experiments were not included in the overall analysis.

In addition, we observed two distinct patterns of retention in the IS. These data further define the dynamics of KCa3. Top rows : brightfield images. Bottom rows : corresponding images of KCa3. The location of the B cells is indicated by a white line around the cell membrane. Snapshot sequence corresponds to supplemental movie S1 note that the online version of this manuscript contains supplemental data. We observed that at time , before the transfected T cell comes in contact with an antigen presenting cell APC , KCa3.

After min, an APC comes in contact with the T cell, and, on contact, the channels begin to accumulate at the immunological synapse that forms at the T cell-APC interface where they remain for over 25 min. At min, the channels began to redistribute along the cell membrane, and they acquire a uniform distribution by min. This is characterized by an extensive reorganization of the actin cytoskeleton and specific membrane TCR and adhesion molecules and signaling proteins to the T cell-APC contact interface 5 , 9 , In the present study, we demonstrate that KCa3.

To our knowledge, this is the first report showing KCa3. This was made necessary by the fact that, to our knowledge, no specific anti-KCa3. The availability of a specific antibody would have allowed us to study the behavior of native KCa3. Furthermore, from a technical point of view, this would have excluded the limitation inherent in the low efficiency of transfection of primary T cells i. Recombinant channels instead carry the risk of overexpression, which we avoided as much as possible by using the cells as soon as expression was seen.

In our experiments, the transfected T cells expressed on average a KCa3. This is comparable to the level of expression of native KCa3. It is also possible that the YFP tag could have hindered other molecular interactions, although it did not compromise the ability of the channel to enter and exit the IS.

Still, a YFP protein is extremely photostable, thus allowing detailed and prolonged live cell imaging experiments, minimizing the risk of photobleaching. Furthermore, these studies with recombinant channels set the stage for future structural-functional studies, that will allow the determination of the channel protein sequence or sequences necessary for its recruitment in the IS and thus the possible mechanisms driving this process. This allowed us to express these channels in primary human T lymphocytes and perform localization experiments to demonstrate that KCa3.

The former have been used as surrogate APCs, and they have been shown to induce reorganization of F-actin and accumulation of structural proteins at the bead-T cell contact area These experiments were performed in human T cells preactivated by exposure to PHA for 72 h. This intervention was shown to induce expression of KCa3.

This reflects the mixed T cell population that comprises T cells freshly isolated from the blood and includes T cells at different degrees of activation and development. Overall, the results presented reveal that KCa3. Furthermore, fixed microscopy experiments, representative snapshots of the process, revealed that KCa3. We observed that KCa3. To further substantiate our data, we also performed time-lapse microscopy experiments.

Intriguingly, we also noted two patterns of recruitment: a sustained recruitment and a shorter lived one. It is well known that, in activated T cells, KCa3. In agreement with the literature, we observed that blockade of KCa3. Interestingly, we observed that, when KCa3. Thus our results suggest that KCa3. Furthermore, it suggests that the functionality of the channel is not integral to its migration to the IS.

Similarly, we have observed that Kv1. The functional consequences of KCa3. It has already been shown that Kv1. Moreover, it is commonly believed that formation of the IS occurs to provide close proximity between various elements of the T cell activation machinery and thus more efficient signaling among them 9. As a result, it is quite possible that recruitment of KCa3. Consequently, modulation of KCa3. In view of this, we propose that the functional relevance of KCa3.

The costs of publication of this article were defrayed in part by the payment of page charges. Section solely to indicate this fact. We thank M.

Functional anatomy of T cell activation and synapse formation.

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Multifocal structure of the T cell – dendritic cell synapse

Find articles by Cox, D. Find articles by Hartwig, J. Find articles by Irvine, D. Find articles by Luscinskas, F. Find articles by Geha, R. First published September 6, - More info. The clinical phenotype of WAS includes susceptibility to infection, allergy, autoimmunity, and malignancy and overlaps with the symptoms of dedicator of cytokinesis 8 DOCK8 deficiency, suggesting that the 2 syndromes share common pathogenic mechanisms. We determined that the guanine nucleotide exchange factor activity of DOCK8 is essential for the integrity of the subcortical actin cytoskeleton as well as for TCR-driven WASp activation, F-actin assembly, immune synapse formation, actin foci formation, mechanotransduction, T cell transendothelial migration, and homing to lymph nodes, all of which also depend on WASp.

The integrity of the actin cytoskeleton is important for T cell migration into tissues, defense against pathogens, and immunosurveillance 1. The Wiskott-Aldrich syndrome WAS protein WASp plays an important role in the organization and function of the actin cytoskeleton in hematopoietic cells 2. T cells deficient in WASp have low baseline F-actin levels, impaired T cell receptor—driven TCR-driven actin assembly, immune synapse IS formation, actin foci formation and mechanotransduction, and defective migration into tissues 2 , 6.

WIP also binds to actin 2. Several observations suggest that DOCK8 regulates actin cytoskeleton—dependent functions. WASp deficiency, which results in the X-linked WAS, and DOCK8 deficiency, which causes autosomal recessive hyper IgE syndrome, share clinical and laboratory findings that include recurrent sinopulmonary infections, viral skin infections, eczema, elevated serum IgE levels, food allergies, and an increased risk for malignancy and autoimmune diseases 14 — IP with an IgG isotype Ab was used as a negative control.

Data in A — D represent 3 independent experiments each. Images in E are representative of 50 cells that were examined in 2 independent experiments. Similar results were obtained with mouse splenic T cells data not shown. Data in C , E , and F represent 4 independent experiments. Ctrl, control. Data are representative of 3 independent experiments in B and 2 independent experiments in C and D.

We performed quantitative analysis of activated WASp by calculating the ratio of the intensity of the WASp band in the CSAb immunoprecipitates to that in total lysates. Results are representative of 2 experiments involving 2 patients and 2 controls.

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Residue S is in red, and the region affected by the SP mutation is in magenta. Results are representative of 3 experiments. The experiment was performed and the results expressed as described in A. Results in E — I are representative of 3 experiments using 3 mice from each strain. CDC42 has been shown to play an important role in WASp activation and actin reorganization 24 — 27 , along with subsequent T cell activation, cytokine secretion, polarization, and chemotaxis 28 — Modeling the SP mutation suggests that the Pro residue probably fails to form similar interactions, which may reduce the affinity to CDC42 Figure 4C.

Furthermore, structural dynamics simulation suggests that the SP mutation diminishes the interaction with CDC42 indirectly by inducing local conformational changes data not shown. To evaluate the ultrastructure of the actin cytoskeleton, purified T cells were spread on anti-CD3—coated coverslips, their apical surface removed, and the attached cell membranes processed for electron microscopy EM. Shown are the cytoskeletal actin fibers associated with the cytoplasmic side of the adherent plasma membranes.

Results are representative of 2 experiments with T cells from 1 mouse of each strain. More than 50 cells were examined in each experiment. Scale bar: nm. Results are representative of 3 independent experiments using 3 mice from each strain. Results are expressed as the increase in the mean fluorescence intensity MFI of F-actin from the baseline time 0.

TCR ligation triggers foci formation at the IS, which leads to invadopodia-like T cell membrane protrusions that push into the target cell 6.

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In addition, TCR-driven actin remodeling induces mechanical strain at the IS, reflected by the phosphorylation of the mechanosensory molecule CasL the pCas isoform expressed in lymphocytes at the Y residue 6. To better mimic the physiologic interactions between T cells and vascular endothelium, T cells were plated on a superantigen-loaded endothelial cell monolayer, and cell-cell contacts were examined by spinning-disc confocal microscopy.

Orange indicates merging. The extracellular striated pattern observed in B phalloidin panel on the left is due to the F-actin fluorescence contributed by the endothelial cell cytoskeleton. Each data point represents a value obtained for a single cell. Similar results A — G were obtained in 2 independent experiments.

Dynamic rearrangements of the actin cytoskeleton, including actin foci formation, are important for T cell motility and transcellular diapedesis Migration of T cells from blood vessels into tissues occurs under physiological shear flow conditions and involves sequential interactions between adhesion molecules expressed on T cells and endothelial cells, including selectin-mediated rolling and tethering, followed by chemokine-induced integrin-mediated firm adhesion and arrest, spreading, and TEM Results are representative of 3 independent experiments using 2 mice each per strain.

Arrows indicate fully spread cells scored with 2 points, and solid triangles indicate partially spread cells scored with 1 point. Round, unspread cells were scored with no points to calculate the mean spreading score of cells adherent to ICAM-1 in 5 fields of view at 0.

A DOCK8-WIP-WASp complex links T cell receptors to the actin cytoskeleton

Duplicate coverslips were assessed in 3 independent studies. DOCK8 is essential for T cell homing to lymph nodes. Circulating T cells enter lymph nodes LNs by migrating across high endothelial venules The homing index was calculated as the ratio of Alexa Fluor — to Alexa Fluor —labeled cells and normalized to the ratio of WT controls.

Similar results were obtained when the cell-labeling dyes were reversed data not shown. Migration of DOCK8-deficient T cells through 3D spaces that constrain cell movement results in catastrophic cell death that becomes evident after 12 hours Results in A — D are representative of 2 independent experiments each using 5 recipients per donor strain.

T cell extravasation across the endothelial cell lining is essential for the recruitment of effector T cells to sites of infection, immunosurveillance, and tolerance It has been established that circulating T cells constitutively express VLA-4 integrins in conformations that recognize VCAM-1 under flow conditions In this respect, DOCK8 is known to interact with talin and protein tyrosine kinase 2 PYK2 , both of which play a role in integrin-mediated cell adhesion 19 , 50 — Decreased contact with the substrate due to defective spreading, defective formation of actin foci, podosome equivalents important for pushing through endothelial cells, and reduced mechanotransduction may all contribute to the impaired TEM and defective homing to LNs of DOCK8-deficient T cells.

Moreover, we show that DOCK8-deficient T cells share with WASp-deficient T cells a number of actin cytoskeletal defects that include an attenuated subcortical actin cytoskeleton, reduced F-actin content, defective TCR-driven actin assembly, actin foci formation and mechanotransduction, and impaired TEM and homing to LNs. These defects probably contribute to the susceptibility of DOCK8-deficient and WASp-deficient patients to infections, allergy, autoimmunity, and malignancy. The generated F-actin is stabilized by binding to WIP and is essential for actin cytoskeleton—dependent T cell functions.

These include actin foci formation at the immune synapse, mechanotransduction, and TEM into LNs and tissues, both of which are critical for T cell—mediated immune surveillance. CT mutation that introduces a stop codon in exon 9 of the endogenous Dock8 gene is described in Supplemental Figure 1.