Increasing LFA-1 Expression Enhances Immune Synapse Architecture and T Cell Receptor Signaling in Jurkat E6.1 Cells

The Jurkat E6.1 clone has been extensively used as a powerful tool for the genetic and biochemical dissection of the TCR signaling pathway. More recently, these cells have been exploited in imaging studies to identify key players in immunological synapse (IS) assembly in superantigen-specific conjugates and to track the dynamics of signaling molecules on glass surfaces coated with activating anti-CD3 antibodies. By comparison, Jurkat cells have been used only scantily for imaging on supported lipid bilayers (SLBs) incorporating laterally mobile TCR and integrin ligands, which allow to study synaptic rearrangements of surface molecules and the fine architecture of the mature IS, likely due to limitations in the assembly of immune synapses with well-defined architecture. Here we have explored whether upregulating the low levels of endogenous LFA-1 expression on Jurkat E6.1 cells through transduction with CD11a- and CD18-encoding lentiviruses can improve IS architecture. We show that, while forced LFA-1 expression did not affect TCR recruitment to the IS, E6.1 LFA-1high cells assembled better structured synapses, with a tighter distribution of signaling-competent TCRs at the center of the IS. LFA-1 upregulation enhanced protein phosphotyrosine signaling on SLBs but not at the IS formed in conjugates with SEE-pulsed APCs, and led to the constitutive formation of an intracellular phosphotyrosine pool co-localizing with endosomal CD3ζ. This was paralleled by an increase in the levels of p-ZAP-70 and p-Erk both under basal conditions and following activation, and in enhanced Ca2+ mobilization from intracellular stores. The enhancement in early signaling E6.1 LFA-1high cells did not affect expression of the early activation marker CD69 but led to an increase in IL-2 expression. Our results highlight a new role for LFA-1 in the core architecture of the IS that can be exploited to study the spatiotemporal redistribution of surface receptors on SLBs, thereby extending the potential of E6.1 cells and their derivatives for fine-scale imaging studies.

The Jurkat E6.1 clone has been extensively used as a powerful tool for the genetic and biochemical dissection of the TCR signaling pathway. More recently, these cells have been exploited in imaging studies to identify key players in immunological synapse (IS) assembly in superantigen-specific conjugates and to track the dynamics of signaling molecules on glass surfaces coated with activating anti-CD3 antibodies. By comparison, Jurkat cells have been used only scantily for imaging on supported lipid bilayers (SLBs) incorporating laterally mobile TCR and integrin ligands, which allow to study synaptic rearrangements of surface molecules and the fine architecture of the mature IS, likely due to limitations in the assembly of immune synapses with well-defined architecture. Here we have explored whether upregulating the low levels of endogenous LFA-1 expression on Jurkat E6.1 cells through transduction with CD11a-and CD18-encoding lentiviruses can improve IS architecture. We show that, while forced LFA-1 expression did not affect TCR recruitment to the IS, E6.1 LFA-1 high cells assembled better structured synapses, with a tighter distribution of signaling-competent TCRs at the center of the IS. LFA-1 upregulation enhanced protein phosphotyrosine signaling on SLBs but not at the IS formed in conjugates with SEE-pulsed APCs, and led to the constitutive formation of an intracellular phosphotyrosine pool co-localizing with endosomal CD3ζ. This was paralleled by an increase in the levels of p-ZAP-70 and p-Erk both under basal conditions and following activation, and in enhanced Ca 2+ mobilization from intracellular stores. The enhancement in early signaling E6.1 LFA-1 high cells did not affect expression of the early activation marker CD69 but led to an increase in IL-2 expression. Our results highlight a new role for LFA-1 in the core architecture of the IS that can be exploited to study the spatiotemporal redistribution of surface receptors on SLBs, thereby extending INTRODUCTION T cell immunological synapses (IS) are specialized cellcell junctions between T cells and antigen presenting cells (APC) that are stable, demarcated by adhesion molecules and mediate vectoral cell-cell communication through a synaptic cleft (Dustin and Colman, 2002). Kupfer described the classical architecture of T-cell activation by APCs organized into supramolecular activation clusters (SMACs) with a ring of adhesive LFA-1-ICAM1 interactions forming the peripheral (p)SMAC and a central cluster of TCR in the central (c)SMAC (Monks et al., 1998), which together define the IS (Norcross et al., 1984;Dustin and Springer, 1998). The dynamic formation of ISs were first observed with supported lipid bilayers (SLB) presenting laterally mobile ICAM-1 and pMHC complexes, which enable formation of SMACs through a T cell autonomous process (Grakoui et al., 1999). Not only does LFA-1 mediate key interactions in the pSMAC, but the higher level of LFA-1 expression on memory versus naïve CD8 T cells is associated with more stable IS (Mayya et al., 2018), although this correlation has not been further tested for causality. In addition to lateral movement of LFA-1 and TCR in the plane of the plasma membrane, vesicular trafficking also plays a key role in IS formation (Onnis and Baldari, 2019;Mastrogiovanni et al., 2020). Early IS formation studies with cellular or SLB-based antigen presentation required use of primary T cells from transgenic mice. The use of readily available cell lines such as Jurkat would be advantageous for a number of reasons.
The acute T cell leukemia-derived Jurkat cell line has represented a robust tool to study T cell signaling, allowing for the biochemical and genetic identification of key players in the TCR signaling cascade (Abraham and Weiss, 2004). Jurkat cells activated by the Raji B lymphoblastoid cell line and staphylococcal superantigen E (SEE) have been useful, but do not provide fine resolution of IS structures (Blanchard et al., 2002). Jurkat cells have also been instrumental in reconstructing the dynamics of signaling molecules following TCR triggering when plated on glass surfaces coated with activating anti-CD3 antibodies through live imaging of transfected fluorescent reporters (Bunnell et al., 2001(Bunnell et al., , 2002, which has been further refined with speckle microscopy (Kaizuka et al., 2007) and super-resolution methods (Fritzsche et al., 2017). This system led to the discovery that an F-actin ring is formed as T cells spread on surfaces with central clearance of F-actin (Bunnell et al., 2001). This central F-actin clearance with the formation of gaps in the mesh is a defining characteristic of the secretory domain that is critical for effector function (Ritter et al., 2015(Ritter et al., , 2017. A major intrinsic limitation of this approach is that, since the activating antibodies are immobile, the cells are unable to reorganize surface receptors and integrins to form the characteristic SMAC-based architecture of an IS and the method is only used to model early events in T-cell activation. The general strategy to efficiently incorporate laterally mobile antibodies into SLBs (Carrasco et al., 2004) made it possible to present laterally mobile anti-CD3 and ICAM1 to generate a Jurkat based IS model, however, in this setting TCR microclusters interspersed with LFA-1 clusters (Kaizuka et al., 2007(Kaizuka et al., , 2009Murugesan et al., 2016;Chen et al., 2017), which are instead better separated with a more compact central TCR cluster in primary CD4 + T cells (Grakoui et al., 1999;Varma et al., 2006;Kumari et al., 2015).
Here, we have sought to improve IS architecture using an engineered Jurkat subline. Several clones have been derived from the original Jurkat line ( Table 1). The most widely used to dissect TCR signaling is clone E6.1 (Weiss et al., 1984), from which subclones lacking individual TCR/CD3 components, integrin subunits or signaling molecules, such as Lck, ZAP-70, LAT, SLP-76, PLCγ, or CARMA-1, have been derived by mutagenesis (Abraham and Weiss, 2004). The basis for the less defined architecture of the IS formed by Jurkat E6.1 cells is not clear, but could include low levels of the integrin LFA-1 (CD11a/CD18) or associated co-factors such as talin and paxillin (Harburger and Calderwood, 2009). This limits the suitability of Jurkat E6.1 cells to track the dynamics of protein reorganization during IS assembly using SLB-based live imaging. Here we investigate whether increasing LFA-1 expression in Jurkat E6.1 cells can improve the architecture of ISs in the SLB setting. The results show that forcing increased LFA-1 surface expression by lentiviral transduction enhanced TCR segregation to the center of the cSMAC and local phosphotyrosine signaling. Forced LFA-1 expression did not enhance either the efficiency of TCR or tyrosine phosphoprotein accumulation at the IS formed by E6.1 cells in the setting of SEE-specific conjugates, but improved their ability to signal in response to TCR engagement, leading to increased IL-2 expression. Additionally, forced LFA-1 expression enhanced basal TCR signaling and accumulation of tyrosine phosphoproteins at CD3ζ + endosomes. The results highlight a new role for LFA-1 in the core architecture of the IS formed by E6.1 cells that can be exploited to study the spatiotemporal redistribution of surface receptors on SLBs, thereby extending the potential of E6.1 cells and their derivatives for the study of T cell autonomous IS formation.
Cells were cultured in RPMI 1640 medium (Life Technologies, #31870074) supplemented with 10% (vol/vol) FBS, 2 mM Lglutamine and 50 U/ml of Penicillin-Streptomycin at a max density of 1.5 × 10 6 /ml. 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.
For all experiments unstained cells and isotype controls were performed for background correction and gating.

Conjugate Formation
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 antirabbit Alexa-Fluor-488-and anti-mouse Alexa-Fluor-555-labeled secondary antibodies (ThermoFisher Scientific, #A11008 and #A211422, respectively).

Fluorescence Microscopy
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 zsteps. The orthogonal views and 3D reconstruction of images were performed using ImageJ (NIH).
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 (

Statistics
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.

RESULTS
Forced LFA-1 Expression in E6.1 Cells Improves TCR Segregation to the cSMAC Center 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).
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.

Increasing LFA-1 Expression in E6.1 Cells Improves Phosphotyrosine Signaling at the IS Formed on Activating SLBs but Not in SEE-Specific Cell Conjugates
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 . Horizontal lines and error bars represent mean ± SD (black line) and median (red line). Gray boxes represent 25-75 percentile. Data are from minimum of 120 cells from three independent experiments; each dot represents a cell; each color represents an independent experiment. One-way analysis of variance (ANOVA) with Tukey's post hoc test. Only significant differences are shown. * p < 0.05; * * p < 0.01; * * * * p < 0.0001.  Representative images are shown. The histogram shows the percentage (mean ± SD) of conjugates with pTyr staining at the T-cell:APC contact (right; unpaired Student's t-test; n > 40 conjugates/sample from three independent experiments). (B) Quantification of the relative mean fluorescence intensity (MFI) of anti-pTyr at the IS membrane versus the mean of anti-pTyr MFIs measured at three different membrane regions of the same size outside of the T-cell:APC contact (mean ± SD; Kruskal-Wallis test; n ≥ 20 conjugates/sample from three independent experiments). Graph boxes represent 10-90 percentile and the mean is shown as "+." (C) Quantification of the relative mean fluorescence intensity (MFI) of anti-pTyr at the IS membrane versus the MFI of the whole T cell (total) (mean ± SD; Kruskal-Wallis test; n ≥ 20 conjugates/sample from three independent experiments). Graph boxes represent 10-90 percentile and the mean is shown as "+." (D) Quantification of the relative mean fluorescence intensity (MFI) of anti-pTyr at the IS membrane versus the MFI of the whole T cell excluding the intracellular pTyr pool in E6.1 LFA-1 high cells (total*) (mean ± SD; Kruskal-Wallis test; n ≥ 20 conjugates/sample from three independent experiments). Graph boxes represent (Continued)  Figure 5B and 3D reconstructions of representative z-stacks in videos 1-3). The histogram shows the quantification (mean ± SD) of the co-localization of intracellular pTyr + pool and endosomal CD3ζ (eCD3) in E6.1 LFA-1 high cells (Manders' coefficient) (mean ± SD; Kruskal-Wallis test; n ≥ 20 conjugates/sample from three independent experiments). Graph boxes represent 10-90 percentile and the mean is shown as "+." Scale bar, 5 µm. Only significant differences are shown. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (F) Representative xy and orthogonal views from 3D confocal images of E6.1, E6.1 LFA-1 high or JA3 Jurkat cells interacting with activating [ICAM1 + anti-CD3ε UCHT1-CF568 Fab' (red)] SLB for 15 min (3D reconstructions of representative z-stacks are shown in Supplementary Videos 4-6). Cells were permeabilized and stained with directly conjugated primary antibodies against anti-phosphotyrosine [pTyr-AF488 (green)] and the cell actin cytoskeleton was labeled with phalloidin-AF405 (blue). Scale bar, 5 µm.
( 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.

Increasing LFA-1 Expression in E6.1 Cells Enhances TCR Signaling and IL-2 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 glassimmobilized 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 Ca 2+ mobilization from intracellular stores. Cells were loaded with the fluorescent Ca 2+ indicator Fura-2/AM and Ca 2+ flux was measured by fluorimetry either under resting conditions or following TCR cross-linking in Ca 2+ -free medium. The kinetics of intracellular Ca 2+ mobilization was comparable between the two E6.1 cell lines, however total intracellular Ca 2+ released over time was higher E6.1 LFA-1 high cells (Figure 4D). Total intracellular Ca 2+ released over time was also higher in JA3 cells, but Ca 2+ 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 for 5 or 20 min. Anti-ZAP-70 and anti-Erk2 antibodies were used as respective loading controls. Representative blots are shown. The migration of molecular mass markers is indicated. (C) Quantification of the relative levels of p-ZAP-70 and p-Erk1/2, normalized to the respective loading control (mean ± SD; paired Student's t-test; n = 4). Only significant differences are shown. *p < 0.05; **p < 0.01. (D) [Ca 2+ ] i mobilization in Fura-2/AM-loaded Jurkat cells. Cells were stimulated with anti-CD3ε mAb, clone OKT3 followed by anti-mouse IgG in Ca 2+ -free buffer to evaluate Ca 2+ release followed by re-addition of 1.7 mM Ca 2+ to evoke Ca 2+ influx (left); a magnification of anti-CD3ε mAb (clone OKT3) + anti-mouse IgG-induced Ca 2+ release is shown on the right. The histogram shows AUC values to quantify anti-CD3ε mAb-induced Ca 2+ release (mean ± SD; paired Student's t-test; n = 5). Only significant differences are shown. *p < 0.05; **p < 0.01.
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 on immobilized anti-CD3ε, clone OKT3 and anti-CD28 mAb. A cocktail of brefeldin A and monensin was added in the last 1 h of culture. Cells were permeabilized and stained with anti-IL-2 mAb and the percentage (%) of IL-2 + cells was quantified by flow cytometry. Only significant differences are shown. **p < 0.01; ***p < 0.001; ****p < 0.0001. 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.

DISCUSSION
Similar to all immortalized cell lines, Jurkat cells do not fully recapitulate the features of their primary CD4 + counterparts. The differences must be kept in mind when extrapolating concepts from the results obtained using this T cell model, and validation in primary T cells is mandatory. For example, loss-of-function mutations in the genes encoding the lipid phosphatases PTEN and SHIP-1 make Jurkat E6.1 cells unsuitable for studies on PI3 kinase signaling (Shan et al., 2000;Freeburn et al., 2002;Gioia et al., 2018). Additionally, because the cognate peptide-MHC ligand of the Jurkat TCR is unknown, activation must be carried out using surrogate ligands, namely agonistic anti-CD3 antibodies or SEE (Kappler et al., 1989). The signaling pathways triggered by these agonists reproduce closely, but not completely, the pathway triggered by peptide-MHC (Zamoyska, 2006). Nonetheless, Jurkat cells remain a robust tool easily amenable to genetic manipulation and biochemical studies that is extensively used to provide working hypotheses FIGURE 6 | Comparative performance of E6.1, E6.1 LFA-1 high and JA3 cells in IS architecture, TCR signaling and T-cell activation. Schematic representation of experimental settings used for testing the performance of the three Jurkat lines. The symbol " " indicates the best responder between E6.1 and E6.1 LFA-1 high cells. The symbol " " indicates experimental settings in which JA3 cells are good responders. The figure was generated with BioRender.com.
to test ex vivo on primary T cells or in vivo in genetically engineered mice.
Supported lipid bilayers have become established as a versatile tool to visualize T cell autonomous aspects of IS dynamics at nanometer to micrometer scales (Dustin, 2010). The SLB mimics an APC in many aspects, but the T cell alone drives the nanometer to micrometer scale process of microcluster and SMAC formation. Incorporating into the basic setting of a TCR ligand (pMHC or anti-CD3 Fab'), an integrin ligand (e.g., ICAM-1) and other molecules that bind costimulatory or co-inhibitory receptors has brought to light subdomain organization of the classic SMACs characterized by the segregation of specific receptors, as recently shown for CD2 and CD28 (Demetriou et al., 2020). While Jurkat cells have been widely exploited for studies of IS assembly in the setting of SEE-specific cell-cell conjugates, to date their use for imaging studies on SLBs has been limited. However, the results have demonstrated that, notwithstanding the less compact distribution of TCR clusters at the cSMAC and subtle differences in the actin cytoskeleton architecture compared to quiescent primary CD4 + T cells (Murugesan et al., 2016;Colin-York et al., 2019), this model can be exploited as a convenient toolkit to analyze the dynamics of protein segregation during IS formation (Kaizuka et al., 2007(Kaizuka et al., , 2009. Our data show that, by compensating for the low expression of LFA-1 in the most extensively used Jurkat T cell clone E6.1, it is possible to enhance the ability of these cells to form wellstructured immune synapses. In particular, while both E6.1 cells and their LFA-1 high counterparts effectively segregate the TCR, LFA-1 and F-actin in the respective SMACs, LFA-1 enhances the compartmentalization of the TCR to the cSMAC. This finding recapitulates in human T cells the finding that LFA-1 is dispensable for cSMAC formation in OVA-specific mouse CD4 + cells (Graf et al., 2007). Hence engineered LFA-1 high Jurkat E6.1 cells are a valuable model to study the redistribution of signaling molecules during IS assembly in the context of LFA-1 signaling and LFA-1-mediated adhesion using the SLB setting. The improvement in IS architecture associated with increased LFA-1 expression in E6.1 may impact also effector function requiring directed secretion of soluble factors (Huse et al., 2006) or extracellular vesicle budding (Choudhuri et al., 2014). Similarly, expression of CD40L in E6.1 cells could be studied not only in terms of transcription and surface expression, but also in terms of directed budding of TCR and CD40L positive synaptic ectosomes in the cSMAC (Saliba et al., 2019).
Surprisingly, while increasing LFA-1 expression in E6.1 cells improved their performance in the SLB system, neither conjugate formation nor pTyr signaling was enhanced in E6.1 LFA-1 high cells stimulated with SEE-pulsed APCs. This suggests that E6.1 cells may exploit other integrins or accessory surface receptors for TCR co-stimulation to compensate for the low levels of LFA-1, and indeed we found that the efficiency and extent of tyrosine phosphoprotein accumulation in E6.1 cells, but not in E6.1 LFA-1 high cells, relies in part on CD2 (unpublished results). Intriguingly, E6.1 LFA-1 high cells show a peculiar pattern of tyrosine phosphoprotein distribution at the IS, with a lower accumulation at the synaptic membrane compared to E6.1 cells concomitant with the presence of an intracellular pool that colocalizes with endosomal CD3ζ. This pool was present also in conjugates formed in the absence of SEE, albeit under these conditions it was not polarized at the T-cell:APC contact. This observation suggests that E6.1 LFA-1 high cells may have some constitutive activation due to enhanced TCR tonic signaling. This is supported by the enhanced basal activation of ZAP-70 and Erk observed in these cells. A basal endosomal accumulation of signalingcompetent tyrosine-phosphorylated CD3ζ in Jurkat cells has been previously reported (Yudushkin and Vale, 2010). Although no information of LFA-1 expression was included in this study, we speculate that the Jurkat clone used may have been LFA-1 high .
Interestingly, when comparing specific steps of the TCR signaling cascade in E6.1 and E6.1 LFA-1 high cells, forced LFA-1 expression showed an enhancing effect on the signaling readouts used, with an increase in the levels of p-ZAP-70 and p-Erk in response to activation and an increase in intracellular Ca 2+ mobilization. This correlates with a higher expression of a late activation marker, IL-2, while expression of CD69 among responder cells was comparable. We can speculate that the signals elicited by the TCR in this setting are sufficient to activate maximal CD69 expression in E6.1 cells independently of the levels of surface LFA-1. This may not apply to IL-2 expression, which has more stringent requirements compared to CD69 (Testi et al., 1989). It is noteworthy that E6.1 LFA-1 high cells show a clear bimodal pattern of CD69 expression, with a substantial increase in the CD69-negative population. We could hypothesize that the high basal ZAP-70 and Erk activation in E6.1 LFA-1 high cells makes them partially anergic, such that not all cells are able to respond.
Interestingly, our results show that the JA3 clone, which was derived from the same Jurkat line as E6.1 cells (Moretta et al., 1985), has unique features compared to both E6.1 and E6.1 LFA-1 high cells. Despite expressing LFA-1 at levels comparable to E6.1 cells, JA3 cells form well-structured synapses displaying a tight segregation of the TCR to the cSMAC. Differences can also be detected in early signaling, with lower levels of p-ZAP-70 and p-Erk in response to activation compared to the two E6.1 lines but also very low basal signaling, which makes them better responders. Additionally, the kinetics of their Ca 2+ response is faster. Nevertheless, similar to the E6.1 lines, the levels of CD69 and IL-2 expression in activated JA3 cells appear to correlate with the amount of activated p-ZAP-70 and p-Erk, making them the weakest CD69 and IL-2 expressers among the three Jurkat lines analyzed. These differences may be accounted for in part by the specific constellation of mutations accumulated in the selection process of the E6.1 and JA3 clones.

CONCLUSION
Our data extend the exploitability of Jurkat E6.1 cells by providing a strategy to make them more amenable to imaging IS formation in the SLB setting. This would potentially allow reduction of animal use and further reduce the need for primary T cells in studies on IS architecture and its abnormalities in disease. The improvement in IS architecture of E6.1 cells resulting from forced LFA-1 expression entails, however, an enhancement in basal signaling. JA3 cells, which have the "cleanest" background, could complement E6.1 cells for signaling studies to fully exploit the Jurkat paradigm of T cell signaling (Figure 6).

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

AUTHOR CONTRIBUTIONS
All the authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. CC, SB, EBC, JHF, AG, CDB, SLF, and JB performed the experimental work. CC and SB prepared the figures. SV, DP, MMD'E, and LM contributed essential reagents.

FUNDING
This work was carried out with the support of AIRC (IG-2017 -ID 20148) and Ministero dell'Istruzione, dell'Università e della Ricerca (Grant PRIN bando 2017 -2017FS5SHL) to CTB. SB and EBC are supported by ERC AdG 670930 to MLD. MLD is supported by the Kennedy Trust for Rheumatology Research. MLD and CTB are supported by ERC Synergy Grant 951329.