Cells lines included Jurkat T cell clones E6.1 (Weiss et al., 1984) (obtained from ATCC) and JA3 (Moretta et al., 1985), and Raji B cells. A E6-1 line expressing high levels of LFA-1 was obtained by transduction with lentiviruses encoding LFA-1 subunits CD11a and CD18. Expression vectors for CD11a and CD18 were generated by insertion of their respective full-length genes into the lentiviral expression vector pHR. Genes were synthesized including terminal STOP codons and flanked by a 5′ MluI restriction exonuclease site and GCCACC Kozak sequence, and a 3′ NotI restriction site (GeneArt Gene Synthesis, ThermoFisher Scientific). Fragments were inserted into pHR vector via MluI + NotI restriction digestion and ligation by T4 DNA ligase. Lentiviruses were produced in HEK 293T cells at 3 × 10⁵/ml, 2 ml/well in 6-well plates in DMEM (#11960044) + 10% FBS (#15595309), 1% Glu (#25030-024), 1% Pen/Strep (#15140-122), all from Thermo Scientific. GeneJuice transfection was performed with vectors pHR-CD11a and pHR-CD18, pP8.91, and pMD.G. After 3 days of culture, supernatant was removed, spun at 500g for 5 min and passed through a 0.45 μm syringe filter. This virus solution was mixed with polybrene and spun down with Jurkat cells at 800 g for 90 min (RT, dec 6, acc 4). Cells were grown for >48 h at 37°C, 5% CO₂ and passaged twice before stocks were frozen. Transduction efficiency was near 100% such that no selection was necessary. Experiments were performed with E6.1 LFA1^high cells from two transductions and the transduced cells were not used more than 10 passages. We used the validated ATCC E6.1 as a control for experiments in this paper. We note that E6.1 cells transduced with LifeAct-GFP (Fritzsche et al., 2017) and CXCR4-HaloTag (Felce et al., 2020) using the same lentiviral system showed no change in LFA-1 expression or IS organization compared to the untransduced ATCC E6.1 cells (unpublished observations).

Cells were cultured in RPMI 1640 medium (Life Technologies, #31870074) supplemented with 10% (vol/vol) FBS, 2 mM L-glutamine and 50 U/ml of Penicillin-Streptomycin at a max density of 1.5 × 10⁶/ml.

Raw counts for publicly deposited RNA-seq datasets (as used in Felce et al., 2021; Jurkat E6.1: GEO: GSE145453, Expression Atlas: E-MTAB-2706, GEO: GSE93435; Primary T cells: GEO: GSE122735, NCBI SRA: SRP026389, Expression Atlas: E-MTAB-3827) were normalized for gene length and ACTB mRNA counts. Fold difference was calculated as the mean normalized count in Jurkat samples relative to each primary T cell sample.

To assess surface expression of CD11a and CD3ε on Jurkat cells, 0.25 × 10⁶ cells/sample were washed and blocked with 2% FBS/PBS for 30 min at 4°C and then stained with anti-CD3ε-Alexa Fluor 488 (BioLegend, #317310) and anti-CD11a (ThermoFisher, #MA11A10) conjugated in house with Alexa Fluor 647 (ThermoFisher, #A20006), at 10 μg/mL for 30 min at 4°C. Finally, cells were washed three times in 2% FBS/PBS, fixed in 2% PFA/PBS, analyzed by LSRFortesa (BD Biosciences) with BD FACSDiva software and plotted using FlowJo version 10.

Flow cytometric analysis of surface CD69 was carried out using FITC anti-CD69, clone FN50 (BioLegend, #310904) at 1 μg/ml for 30 min at 4°C. Samples were analyzed with Guava Easy Cyte Cytometer (Millipore) and plotted using FlowJo version 6.1.1.

IL-2 intracellular staining flow cytometry was carried out using APC-labeled anti-human IL-2, clone MQ1-17H12 (BioLegend, #500310), at 0.125 μg/5 × 10⁵ cells. Samples were analyzed using a Becton Dickinson FACS CANTO II with BD FACSDiva 6.0 software.

For all experiments unstained cells and isotype controls were performed for background correction and gating.

Planar Supported Lipid Bilayers (SLBs) were formed as previously described (Saliba et al., 2019). In brief, glass coverslips were cleaned with piranha solution (30% H₂O₂ and 70% H₂SO₄), rinsed extensively, dried, negatively charged through plasma cleaning, and assembled with a six-channel sticky-Slide VI 0.4 (Ibidi, #80608). SLBs were formed by filling each channel with a suspension of small unilamellar vesicles composed of 1,2-dioleoyl-sn-glycero-3-[N-(5-amino-1-carboxypentyp) succinyl] (12.5% mol) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-cap biotinyl (0.05% mol) in 1,2-dioleoyl-sn-glycero-3-phosphocholine at a total lipid concentration of 0.4 mM (Avanti Polar Lipids). SLBs were blocked with 5% casein (Sigma-Aldrich) in PBS containing 100 μM NiSO₄. His-tagged proteins were incubated on bilayers for additional 20 min to obtain histidine-tagged–conjugated mouse ICAM1 at 200 molecules/μm², histidine-tagged–conjugated UCHT1-Fab′ fragments at 30 molecules/μm², and histidine-tagged–conjugated human CD80 at 100 molecules/μm². Unbound proteins were flushed out with HEPES Buffered Salin Solution supplemented with 0.1% Human Serum Albumin (Calbiochem). Protein concentrations required to achieve desired densities on bilayers were calculated from calibration curves constructed from flow-cytometric measurements of bead-SLB, compared with reference beads containing known numbers of the appropriate fluorescent dyes (Bangs Laboratories). Bilayers were continuous liquid disordered phase as determined by fluorescence recovery after photobleaching with a 10 μm-bleach spot on an FV1200 confocal microscope (Olympus).

Jurkat cells were incubated at 37°C on SLB containing fluorescently labeled ICAM1-Alexa Fluor-405 and UCHT1-Fab’-CF568 and unlabeled CD80. After 15 min of incubation cells were fixed with 4% electron microscopy grade formaldehyde in PBS for 30 min at RT and washed three times with PBS. For intracellular staining, fixed cells were permeabilized and blocked with 0.01% Triton X-100 (Sigma-Aldrich, #X100-5ML) in blocking buffer (5% BSA/PBS) for 1 h at RT, washed three times with PBS, and stained with anti-CD3ζ Alexa Fluor-647 (SantaCruz, #SC-1239 AF647), anti-pTyr Alexa Fluor-488 (Biolegend, #309306) at 10 μg/mL in 5% BSA/PBS for 1 h at RT followed by three times washing with PBS. Cells were also stained with Alexa Fluor-405 Phalloidin (Thermo Fisher Scientific, # A30104) to highlight actin cytoskeleton.

Conjugates between Jurkat cells and SEE-pulsed Raji B cells were carried out as previously described (Finetti et al., 2009). Raji cells were pulsed for 2 h with 10 μg/ml SEE (Toxin Technology, Sarasota, FL, United States) and labeled with 10 μM Cell Tracker Blue (Molecular Probes) for the last 20 min. Conjugates between T cells and unpulsed B cells were used as negative controls. SEE-pulsed or unpulsed Raji B cells were mixed with Jurkat T cells (1:1) and conjugates analyzed 15 min after their formation. Samples were allowed to adhere for 15 min on poly-L-lysine (Sigma-Aldrich)-coated wells of diagnostic microscope slides (ThermoFisher Scientific), then fixed by immersion in methanol for 10 min at -20°C. Following fixation, samples were washed in PBS and incubated with anti-pTyr (Cell Signaling, #8954) at 10 μg/mL and anti-CD3ζ at 15 μg/mL (SantaCruz, #sc-1239) in PBS 1X overnight at 4°C. After washing in PBS, samples were incubated for 45 min at room temperature with anti-rabbit Alexa-Fluor-488- and anti-mouse Alexa-Fluor-555-labeled secondary antibodies (ThermoFisher Scientific, #A11008 and #A211422, respectively).

TIRFM was performed on an Olympus IX83 inverted microscope equipped with a 4-line (405, 488, 561, and 640 nm laser) illumination system. The system was fitted with an Olympus UApON 150 × 1.45 NA objective, and a Photomertrics Evolve delta EMCCD camera to provide Nyquist sampling. Analysis of TIRFM images was performed with ImageJ (National Institute of Health). Mean fluorescence intensities of ICAM-1, CD3ε, CD3ζ, pTyr and phalloidin were calculated as the sum of intensities in each pixel in the cell spreading area divided by the spreading area of the corresponding cell. The spreading area was determined by thresholding the IRM (Interference Reflection Microscopy) images of each cell.

Intensity compactness of CD3ε clusters represents how compact the intensity signal from each CD3ε cluster is within the defined area. If the CD3ε clusters are concentrated around the center of the defined area, the intensity compactness is closer to 1. If the CD3ε clusters are more sparsely and heterogeneously distributed within the defined area, the value goes toward 0. The area to calculate the intensity of compactness was defined by the cSMAC of Jurkat cells. The ImageJ plugin to calculate the intensity compactness was kindly provided by Prof. Jérémie Rossy (Biotechnology Institute Thurgau, University of Konstanz, Switzerland).

Confocal microscopy imaging of Jurkat cells on SLB was carried out on a Zeiss LSM980 (Zeiss, Germany) using a 63 × 1.40 NA oil immersion objective. Cells were stained as for TIRF microscopy and 3D confocal imaging was carried out at 200 nm z-steps. The orthogonal views and 3D reconstruction of images was performed using ImageJ (National Institute of Health).

Confocal microscopy of cell–cell conjugates was carried out on a Zeiss LSM700 (Zeiss, Germany) using a 63x/1.40 oil immersion objective. Images were acquired with pinholes opened to obtain 0.8 μm-thick sections. Detectors were set to detect an optimal signal below the saturation limits. Images were processed with Zen 2009 image software (Carl Zeiss, Jena, Germany). The quantitative co-localization analysis of pTyr with CD3ζ in E6.1 LFA-1^high cells was performed on median optical sections using ImageJ and JACoP plug-in to determine Manders’ coefficient M1 (Manders et al., 1992). Scoring of conjugates for pTyr clustering at the IS was based on the concentration of the respective staining solely at the T-cell:APC contact. The recruitment index of pTyr was calculated as either the relative fluorescence at the T-cell:APC contact site compared to the mean fluorescence of three membrane regions with the same area outside of the contact (Figure 3B) or the relative fluorescence at the T-cell:APC contact site compared to the entire cell, either excluding (Figure 3D) or including (Figure 3C) the fluorescence of the constitutive intracellular pTyr pool. 3D confocal imaging of conjugates was carried out on a Leica TCS SP8 (Leica, Germany) at 220 nm z-steps. The orthogonal views and 3D reconstruction of images were performed using ImageJ (NIH).

For immunoblot experiments 12-well plates were coated with 5 μg/ml of anti-CD3ε mAb, clone OKT3 (BioLegend, #317302) and 1 μg/ml of ICAM1 Fc (R&D System, #720-IC) in PBS 1X overnight at 4°C or for 2 h at 37°C, followed by extensive rinse with PBS 1X. Cells (2.5 × 10⁶ cells/sample) were pelleted, resuspended in serum-free RPMI-1640 at the density of 1 × 10⁶ cells/ml, then seeded on non-activating (ICAM-1) or activating (ICAM-1 + anti-CD3ε) plates and incubated for 5 or 20 min at 37°C, 5% CO₂. Unstimulated samples were included as negative controls.

For the quantification of T-cell activation Jurkat cells (0.15 × 10⁶ cells/sample) seeded on either plate-bound anti-CD3ε or anti-CD3ε + ICAM-1 and incubated for 16 h at 37°C, 5% CO₂. Surface CD69 was quantified by flow cytometry. T cells activated with 100 ng/ml PMA and 500 ng/ml A23187 were used as positive control. For IL-2 measurements, 10⁶ cells were activated on plate-bound anti-CD3ε mAb, clone OKT3 (BioLegend, #317302) and anti-CD28 mAb, clone CD28.2, all at 5 μg/ml in PBS 1X (BioLegend, #302902) for 6 h. After 1 h incubation, the protein transport inhibitor cocktail containing brefeldin A and monensin (eBioscience, #00-4980-93) was added (2 μl/ml). Membrane permeabilization was done by BD cytofix/cytoperm fixation/permeabilization kit (BD Bioscience, #554714) according to the manufacturer’s protocol prior to processing samples for flow cytometry.

Cells (2.5 × 10⁶ cells/sample) were pelleted, washed twice in ice-cold PBS and lysed in 25 μl lysis buffer (1% Triton X-100 in 20 mM Tris-HCl, pH 8.0, 150 mM NaCl) in the presence of Protease Inhibitor Cocktail Set III (Calbiochem) and 0.2 mg of the phosphatase inhibitor sodium orthovanadate (Sigma-Aldrich). Quantification of total protein content was carried out using the Quantum Protein Assay Kit according to manufacturer’s protocol (EuroClone). Post-nuclear supernatants were denatured in 4X Bolt^TM LDS Sample Buffer (Invitrogen) supplemented with 10X Bolt^TM LDS Sample Buffer (Invitrogen) for 5 min at 100°C. Proteins were subjected to SDS-PAGE on Bolt^TM Bis-Tris Mini Protein Gels (Invitrogen) and transferred to nitrocellulose (GE HealthCare) under wet conditions. Blocking was performed in 5% non-fat dry milk in PBS 0.02% Tween-20. Membranes were incubated with anti-p-ZAP70 Y319 (Cell Signaling, #2701) and anti-p-Erk1/2 T202/Y204 (Cell Signaling, #9101) primary antibodies at RT followed by secondary horseradish-peroxidase (HRP)-conjugated antibodies for 45 min at RT. Secondary antibodies were detected by using SuperSignal^TM West Pico Plus Chemiluminescent Substrates (ThermoFisher Scientific). Membranes were stripped with ReBlot Plus Mild Antibody Stripping Solution 10X (Chemicon) and reprobed with anti-ZAP70 (Merck Millipore, #05-253) and anti-Erk2 (SantaCruz, #sc-1647) control antibodies. Blots were scanned using a laser densitometer (ET-M2170; EPSON, Suwa, Yapan) and quantified using ImageJ (NIH).

Jurkat cells were loaded with the fluorescent calcium indicator Fura- 2 acetoxymetyl ester (Fura-2/AM) to evaluate changes of cytosolic free calcium concentration as described (Gamberucci et al., 1994). Briefly, after Fura-2-AM (3 μM) loading for 30 min at RT, the cells were maintained in medium (140 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 1 mM CaCl₂, 10 mM glucose, 15 mM Hepes buffer, pH 7.4). Aliquots were rapidly centrifuged and resuspended in fresh nominally Ca²⁺ free medium at the density of 2 × 10⁶ cells/1 ml: cytosolic Ca²⁺ basal levels were obtained by adding Na-ethylene glycol bis(B-amino ethyl ether)-N,N,N0,N0 tetraacetic acid (EGTA) to nominally Ca²⁺ free medium to eliminate possible calcium traces; reticular Ca²⁺ was evaluated after depletion of the stores with anti-CD3ε mAb, clone OKT3 at 1.3 μg/ml (BioLegend, #317302) followed by cross-linking with anti-mouse IgG at 10 μg/ml. The subsequent Ca²⁺ influx through the plasma membrane channels was evaluated by re-adding 1.7 mM CaCl₂.

Fura-2 fluorescence was measured using a Varian Cary Eclipse fluorescence spectrophotometer (Palo Alto, CA, United States) (excitation wavelengths, 340 and 380 nm; emission, 510 nm) equipped with magnetic stirring and temperature control set at 35°C. At the end of each experiment, digitonin (20 mg/ml) and EGTA (10 mM) (Sigma-Aldrich) were added in order to measure maximal (Rmax) and minimal (Rmin) ratio (340/380) fluorescence values, respectively. The equation [Ca²⁺]_i = (R-R_min)/(R_max-R)Sf^∗Kd was used to convert the Fura-2 ratio values to intracellular calcium concentrations.

Total [Ca²⁺]_i release over time was quantified by measuring the Area Under the Curve (AUC) that represents the area enclosed under the curve of the [Ca²⁺]_i versus the time for 4 min after OKT3 addition; AUC was calculated using GraphPad Prism version 7.00 (La Jolla, CA, United States).

The number of repeats and the number of cells analyzed is specified in each figure legend. Statistical analyses were performed using GraphPad Software (La Jolla, CA, United States). Pairwise or multiple comparisons among values with normal distribution were carried out by using Student’s t-test (paired or unpaired) and one-way ANOVA with Tukey’s post hoc test, whereas values without Gaussian distribution were analyzed with Mann–Whitney test or Kruskal–Wallis test. Statistical significance was defined as: ns p > 0.05, ^∗p ≤ 0.05, ^∗∗p ≤ 0.01, ^∗∗∗p ≤ 0.001, ^****p ≤ 0.0001.

Transcriptomic comparison of E6.1 cells against primary CD4⁺ T cells using RNA-seq (public datasets as used in Felce et al., 2021) revealed that primary T cells have 8.7 ± 5.5 times more ITGAL mRNA (encoding CD11a) and 7.1 ± 2.6 times more ITG2B mRNA (encoding CD18) than E6.1 cells. To investigate whether the amount of surface LFA-1 influences the core architecture of the IS formed by two most commonly used Jurkat cell lines, E6.1 (Weiss et al., 1984) and JA3 (Moretta et al., 1985), we first measured the surface expression levels of CD11a and CD3ε by flow cytometry. Surface expression levels of CD11a on both cell lines were comparable (Figure 1A and Supplementary Figure 1A). Both lines displayed comparable levels of surface CD3ε and showed the presence of a CD3ε^low population that was more abundant among JA3 cells (Supplementary Figure 1B).

To assess the bull’s eye pattern of the mature IS architecture, cells were plated on SLB containing an agonistic anti-CD3ε (UCHT1) Fab′ fragment labeled with CF568 and the LFA-1 ligand ICAM-1 directly conjugated with Alexa Fluor-405, and analyzed by total internal reflection fluorescence microscopy (TIRFM) (Figure 1B and Supplementary Figures 2A,B). Cells were plated for 15 min, a time point at which, in primary T cells, the TCR and LFA-1 have segregated into the cSMAC and pSMAC, respectively. Slightly higher levels of CD3ε were observed at the interface of JA3 cells compared to E6.1 cells with SLB, as measured by mean fluorescence intensity (MFI) of the UCHT1 signal at the SLB (Figure 1C). This small increase was due to the smaller cell spreading area of JA3 cells (Supplementary Figure 3). Of note, similar to E6.1 cells, JA3 cells formed an ICAM-1 ring, which can only be elicited in the presence of a strong TCR signal, indicating that the presence of a more abundant population of CD3ε^low among JA3 cells does not compromise their ability to assemble immune synapses. The levels of LFA-1 measured by mean fluorescence intensity of ICAM-1 on SLBs were not significantly different between E6.1 and JA3 (Figure 1D), supporting the results obtained by flow cytometry (Figure 1A and Supplementary Figure 1A). Hence both Jurkat cell lines have similar capability of CD3 transport that underlies TCR spatial organization and signaling at the IS (Onnis and Baldari, 2019). However, by assessing the images in Figure 1B and Supplementary Figures 2A,B, JA3 cells formed more compact and tighter CD3-enriched cSMACs within the ICAM-1-enriched ring compared to E6.1 cells. This was confirmed by measuring intensity compactness to quantify the distribution of CD3ε clusters within the cSMAC of E6.1 and JA3 cells (see section “Materials and Methods” for description). We observed significant differences between the two cell lines, with CD3ε clusters showing a lower compactness in E6.1 cells compared to JA3 cells (Figure 1E).

As mentioned above, primary T cells have approximately 9 times more LFA-1 than E6.1 Jurkat cells and are known to form compact CD3ε-enriched cSMAC (Demetriou et al., 2020). To directly address whether increasing LFA-1 levels could improve cSMAC formation in E6.1 cells, we transduced these cells with lentiviruses encoding human CD11a and CD18, generating the E6.1 LFA-1^high line. E6.1 LFA-1^high cells expressed higher levels of surface LFA-1 compared to both parental E6.1 cells (∼2-fold) and JA3 cells (∼3-fold), as assessed by flow cytometry (Figure 1A and Supplementary Figure 1A). CD3ε surface levels were comparable across Jurkat cell lines (Supplementary Figure 1B).

To analyze TCR compartmentalization at the cSMAC, E6.1 LFA-1^high cells were plated on SLBs as for E6.1 and JA3, and analyzed by TIRFM (Figure 1B and Supplementary Figure 2C). As expected, forced LFA-1 expression in E6.1 cells increased the amount of ICAM-1-engaged LFA-1 on SLBs (Figure 1D). E6.1 LFA-1^high cells formed a tight and compact CD3ε-enriched cSMAC within the ICAM-1-enriched ring (Figures 1B,E and Supplementary Figure 2C), with a similar cell spreading area (Supplementary Figure 3) but an increase in the levels of CD3ε at the interface with the SLB compared to E6.1 cells (Figure 1C). Together, these data demonstrate that LFA-1 enhances the efficiency of TCR recruitment to the cSMAC and facilitates the formation of the core IS architecture.

To understand whether the increased levels of LFA-1 can enhance signaling, we measured the accumulation of tyrosine phosphoproteins at the IS formed in the SLB setting. E6.1, E6.1 LFA-1^high and JA3 cells were plated on SLBs containing an agonistic anti-CD3ε (UCHT1) Fab’ fragment labeled with CF568 and the LFA-1 ligand ICAM-1 directly conjugated with Alexa Fluor-405 for 15 min and analyzed by TIRFM (Figure 2A and Supplementary Figure 4). After fixation and permeabilization, cells were stained with directly conjugated primary antibodies against anti-CD3ζ Alexa Fluor-647, anti-phosphotyrosine (pTyr) Alexa-Fluor-488, and phalloidin Alexa Fluor-405 to visualize the underlying actin cytoskeleton (Figure 2A and Supplementary Figure 4). Again, JA3 cells had smaller spreading areas (Figure 2B) with levels of CD3ε and CD3ζ accumulation comparable to E6.1 and E6.1 LFA-1^high cells, while accumulation was higher in E6.1 LFA-1^high cells compared to E6.1 cells (Figures 2C,D). Quantification of the TIRFM images revealed higher pTyr levels in E6.1 LFA-1^high cells compared to parental E6.1 (Figure 2E), likely due to their more compact cSMAC. PTyr signaling in E6.1 LFA-1^high cells was also more efficient compared to JA3 cells (Figure 2E). In all Jurkat lines the underlying actin cytoskeleton formed an actin ring, which is a marker of cell activation, and LFA-1 overexpression did not alter the amount of actin at the SLB contact (Figure 2F).

To further understand whether the ability of LFA-1 to influence the architecture of the IS in the SLB setting translates into enhanced signaling in cell-cell conjugates, we compared E6.1, E6.1 LFA-1^high and JA3, cells in the classical context of SEE-specific conjugates. Jurkat cells were mixed with SEE-pulsed Raji cells (used as APC) for 15 min and the accumulation of tyrosine phosphoproteins at the T-cell:APC interface was measured by confocal microscopy. All Jurkat lines showed a comparable proportion of pTyr-positive conjugates (Figure 3A and Supplementary Figure 5). Unexpectedly, as opposed to the SLB setting, the accumulation of tyrosine phosphoproteins at the synaptic membrane upon activation was lower in E6.1 LFA-1^high cells compared to E6.1 cells, both when calculating the ratio of the membrane pTyr signal versus the average of three similarly sized regions of the non-synaptic membrane, and when calculating the ratio of the membrane pTyr signal versus the total cellular pTyr signal (Figures 3B,C). This suggests that in the more complex setting of cell-cell conjugates other integrins or costimulatory receptors contribute to the accumulation of tyrosine phosphoproteins at the IS. Remarkably, as opposed to parental E6.1 cells, E6.1 LFA-1^high cells displayed a vesicular pTyr pool that localized just beneath the synaptic membrane in the majority of conjugates and was present also in the absence of SEE (Figure 3A and Supplementary Figure 5). The pTyr⁺ pool was not observed in JA3 cells, which displayed pTyr accumulation at the synaptic membrane comparable to E6.1 cells (Figure 3A and Supplementary Figures 5,6A). Of note, when the signal of the constitutive intracellular pTyr pool was subtracted from the total cellular pTyr signal, the accumulation of tyrosine phosphoproteins at the synaptic membrane of E6.1 LFA-1^high cells was not significantly different from E6.1 of JA3 cells (Figure 3D and Supplementary Videos 1–3). Interestingly, the vesicular pTyr⁺ pool unique to E6.1 LFA-1^high cells co-localized with the intracellular pool of CD3ζ (Figure 3E, Supplementary Figures 6A–C, and Supplementary Videos 1–3).

To better visualize the accumulation of tyrosine phosphoproteins at the IS of E6.1, E6.1 LFA-1^high and JA3 cells, we performed 3D confocal imaging on SLBs. Visualization of all three cell lines by their corresponding orthogonal views revealed the presence of the internal pTyr pool only in E6.1 LFA-1^high cells (Figure 3F and Supplementary Videos 4–6). Together, these results suggest that the redistribution of tyrosine phosphoproteins into a synaptic and subsynaptic pool in E6.1 LFA-1^high cells is a consequence of forced LFA-1 expression.

To explore the outcome of the peculiar subcellular compartmentalization of tyrosine phosphoproteins in E6.1 LFA-1^high cells we carried out an immunoblot analysis on E6.1 and E6.1 LFA-1^high cells plated for 5 min or 20 min on glass-immobilized ICAM-1 in the presence or absence of anti-CD3ε mAb (clone OKT3). Interestingly, E6.1 LFA-1^high cells, either unstimulated or plated on ICAM-1 alone, had high basal levels of both p-ZAP-70 and p-Erk when compared to both E6.1 and JA3 cells (Figures 4A–C and Supplementary Figure 7). Signaling was enhanced in the presence of anti-CD3ε mAb at 5 min and further enhanced at 20 min in all cell lines (Figures 4A–C). Activated E6.1 LFA-1^high cells displayed significantly higher levels of p-ZAP-70 and p-Erk compared to E6.1 cells, suggesting that forced LFA-1 expression improves early signaling (Figures 4A–C; blue vs. green dot histograms). Interestingly, activated JA3 cells (red dot histograms) displayed lower levels of p-ZAP-70 and p-Erk in response to stimulation compared to the other cell lines, but also had the lowest levels of basal signaling (Figures 4A–C), making them the best responders.

To further investigate the potential impact of forced LFA-1 expression on early signaling in E6.1 cells, we measured Ca²⁺ mobilization from intracellular stores. Cells were loaded with the fluorescent Ca²⁺ indicator Fura-2/AM and Ca²⁺ flux was measured by fluorimetry either under resting conditions or following TCR cross-linking in Ca²⁺-free medium. The kinetics of intracellular Ca²⁺ mobilization was comparable between the two E6.1 cell lines, however total intracellular Ca²⁺ released over time was higher E6.1 LFA-1^high cells (Figure 4D). Total intracellular Ca²⁺ released over time was also higher in JA3 cells, but Ca²⁺ was released faster, reached a higher peak and returned rapidly to baseline compared to the E6.1 lines (Figure 4D, histogram), again highlighting JA3 cells as good responders.

To assess how these differences in early signaling influence the downstream biological response, we carried out a flow cytometric analysis of the activation marker CD69 on the three Jurkat lines activated under the same conditions for 16 h. CD69 expression in the two E6.1 lines showed a bimodal distribution, with a CD69^low and a CD69^high population, the former being larger in E6.1 LFA-1^high cells (Figures 5A,B). The mean fluorescence intensity of CD69 in the CD69^high population was however comparable between E6.1 and E6.1 LFA-1^high cells (Figure 5C), indicating that fewer E6.1 LFA-1^high cells responded to the stimulation compared to E6.1 cells, but that these cells were equally efficient in expressing CD69. Activated JA3 cells displayed a homogeneous distribution of CD69, and both the percentage of CD69⁺ cells and the mean fluorescence intensity of CD69 were lower compared to both E6.1 and E6.1 LFA-1^high cells (Figures 5A–C). Of note, unstimulated JA3 cells had a very low basal frequency of CD69⁺ cells compared to the two E6.1 lines, consistent with the low levels of basal signaling (Figure 5B). No difference was observed when cells were stimulated pharmacologically using a combination of the phorbol ester PMA and the calcium ionophore A23187, which bypass TCR signaling (Figure 5D), indicating that the intracellular signaling machinery downstream of the TCR is functional in all cell lines.

We extended the study to a later biological readout of T cell activation, namely IL-2 expression, which has more stringent requirements compared to CD69 (Testi et al., 1989). The frequency of IL-2 expressing cells activation was measured by intracellular staining flow cytometry. The frequency of IL-2⁺ cells was higher among E6.1 LFA-1^high cells compared to E6.1 cells, with the lowest frequency among JA3 cells (Figure 5E), suggesting a correlation between the levels of p-ZAP-70/p-Erk achieved in response to early signaling and the expression of IL-2.