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

Abstract

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 Ca²⁺ 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 the potential of E6.1 cells and their derivatives for fine-scale imaging studies.

Keywords: Jurkat cell; LFA-1; TCR signaling; cSMAC; supported lipid bilayer (SLB).

FIGURE 1

Increasing LFA-1 expression in E6.1 cells improves TCR segregation to the cSMAC center. (A) Flow cytometric analysis of surface expression of LFA-1 (CD11a) on JA3, E6.1 and E6.1 LFA-1^high Jurkat cells. Representative FACS plots of LFA-1 are shown. The histogram shows GMFI of LFA-1 (mean ± SD). (B) Representative TIRFM images of E6.1, E6.1 LFA-1^high or JA3 Jurkat cells interacting with activating [ICAM1-AF405 (blue) + anti-CD3ε UCHT1-CF568 Fab’ (yellow)] SLBs for 15 min. IRM, interference reflection microscopy; BF, bright field. Scale bar, 5 μm. (C,D) Quantification of the corresponding mean fluorescent intensity (MFI) of anti-CD3ε (C) and ICAM-1 (D) from the different Jurkat lines as in (B). (E) Quantification of the CD3ε compactness at the cSMAC from the experiment in (B). 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.

FIGURE 2

Increasing LFA-1 expression in E6.1 cells improves phosphotyrosine signaling at the cSMAC. (A) Representative TIRFM images of E6.1, E6.1 LFA-1^high or JA3 Jurkat cells interacting with activating [ICAM1 + anti-CD3ε UCHT1-CF568 Fab’ (yellow)] SLB for 15 min. Cells were permeabilized and stained with directly conjugated primary antibodies against anti-CD3ζ-AF647 (red), anti-phosphotyrosine [pTyr-AF488 (green)]; the cell actin cytoskeleton was labeled with phalloidin-AF405 (blue). IRM, interference reflection microscopy; BF, bright field. Scale bar, 5 μm. (B) Quantification of cell spreading area of different Jurkat cell lines on activating SLB as in (A). Corresponding mean fluorescent intensity (MFI) of anti-CD3ε (C), anti-CD3ζ (D), anti-pTyr (E) and actin (F) from different Jurkat cell lines as in (A). Horizontal lines and error bars represent mean ± SD (black line) and median (red line). Gray box represents 25–75 percentile. Data are from minimum of 130 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.

FIGURE 3

Increasing LFA-1 expression in E6.1 cells does not improve phosphotyrosine signaling at the IS in conjugates with SEE-loaded APCs. (A) Immunofluorescence analysis of tyrosine phosphoproteins (pTyr) in 15-min conjugates of E6.1, E6.1 LFA-1^high or JA3 Jurkat cells and SEE-pulsed Raji cells (APC). 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 10–90 percentile and the mean is shown as “+.” (E) Immunofluorescence analysis of 15-min conjugates of E6.1, E6.1 LFA-1^high or JA3 cells and Raji cells (APC) in the presence or absence of SEE. Cells were co-stained with anti-pTyr and anti-CD3ζ antibodies. Representative images of E6.1 LFA-1^high cells are shown (representative xy images of E6.1 and JA3 cells are shown in Supplementary Figure 5A, orthogonal views from 3D confocal images are shown in Supplementary 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.

FIGURE 4

Increasing LFA-1 expression in E6.1 cells affects early signaling events. (A,B) Immunoblot analysis with anti-p-ZAP-70 (A) or anti-p-Erk1/2 (B) antibodies of lysates from E6.1, E6.1 LFA-1^high or JA3 Jurkat cells plated on glass-immobilized ICAM-1 in the presence or absence of anti-CD3ε mAb, clone OKT3 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) [Ca²⁺]_i mobilization in Fura-2/AM-loaded Jurkat cells. Cells were stimulated with anti-CD3ε mAb, clone OKT3 followed by anti-mouse IgG in Ca²⁺-free buffer to evaluate Ca²⁺ release followed by re-addition of 1.7 mM Ca²⁺ to evoke Ca²⁺ influx (left); a magnification of anti-CD3ε mAb (clone OKT3) + anti-mouse IgG-induced Ca²⁺ release is shown on the right. The histogram shows AUC values to quantify anti-CD3ε mAb-induced Ca²⁺ release (mean ± SD; paired Student’s t-test; n = 5). Only significant differences are shown. *p < 0.05; **p < 0.01.

FIGURE 5

Increasing LFA-1 expression in E6.1 cells improves IL-2 expression. (A) Representative FACS profiles of CD69 staining in E6.1, E6.1 LFA-1^high or JA3 Jurkat cells either unstimulated or plated on glass-immobilized ICAM-1 and anti-CD3ε mAb, clone OKT3 for 16 h. (B,C) Histograms showing the percentage (%) of CD69⁺ cells (B) and CD69 MFI (C) among cells activated as in (A) (mean ± SD; One-Way ANOVA test; n ≥ 3). (D) Histogram showing the percentage (%) of CD69⁺ cells among JA3, E6.1 and E6.1 LFA-1^high Jurkat cells stimulated for 16 h with a combination of 100 ng/ml PMA and 500 ng/ml A23187 (mean ± SD; One-Way ANOVA test; n = 3). (E) Intracellular staining flow cytometry of E6.1, E6.1 LFA-1^high or JA3 Jurkat cells stimulated for 6 h 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.

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.