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-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.

Synapse topology and downmodulation events determine the functional outcome of anti-CD19 T cell-redirecting strategies

“…For this purpose, we have used the Jurkat human T cell line, which has been widely used to study T cell activation, signaling, and IS assembly. 36 Contrary to primary T cells, Jurkat cells can be easily expanded and long-term cultured after transduction without losing CAR expression or BiTE secretion, avoiding transduction-to-transduction differences and loss of transgene expression. In addition, clonal stimulation by superantigens provides a proper control that mimics the canonical IS and, together with the high transduction efficiency achieved in Jurkat cells, allows to perform experiments with a high number of T cell-target cell interactions.…”

SARS-CoV-2 Spike protein suppresses CTL-mediated killing by inhibiting immune synapse assembly

“…Our data show that SARS-CoV-2 Spike protein binding to ACE2 interferes with the assembly of a functional IS in CTLs by inhibiting the process at an early step, concomitant with ACE2 exclusion from the IS. Potential targets of the ACE2-Spike axis are integrins, such as LFA-1, which are essential for the generation of well-structured immune synapses characterized by the tight accumulation of TCR/CD3 complexes at the center of the contact area with the APC ( Cassioli et al, 2021a ). ACE2 has been reported to constitutively interact with β1 and α5 integrins, promoting cell adhesion and modulating downstream signaling ( Clarke et al, 2012 ; Lin et al, 2004 ).…”

High efficiency sorting and outgrowth for single-cell cloning of mammalian cell lines

“…We tested suspension CHO-ES cells to compare against adherent CHO-K1 cells. Lastly, we also tested Jurkat cells which were isolated in 1977 and have become an in vitro system useful to study T-cell biology and MCF-7 which is a breast cancer cell line that was established in 1973 and has been used to study estrogen responses, hormone resistance, and testing of potential cancer therapeutics such as tamoxifen (Cassioli et al 2021 ; Lee et al 2015 ). Additionally, MCF-7 has been used to investigate the importance of the interaction between cancer cells, angiogenesis, cellular metabolism, and respiration (Comşa et al 2015 ).…”