Clustering of T Cell Ligands on Artificial APC Membranes Influences T Cell Activation and Protein Kinase C θ Translocation to the T Cell Plasma Membrane

Abstract

T cell activation is associated with active clustering of relevant molecules in membrane microdomains defined as the supramolecular activation cluster. The contact area between these regions on the surface of T cells and APC is defined as the immunological synapse. It has been recently shown that preclustering of MHC-peptide complexes in membrane microdomains on the APC surface affects the efficiency of immune synapse formation and the related T cell activation. Disruption of such clusters may reduce the efficiency of stimulation. We describe here an entirely artificial system for Ag-specific, ex vivo stimulation of human polyclonal T cells (artificial APC (aAPC)). aAPC are based on artificial membrane bilayers containing discrete membrane microdomains encompassing T cell ligands (i.e., appropriate MHC-peptide complexes in association with costimulatory molecules). We show here that preclustering of T cell ligands triggered a degree of T cell activation significantly higher than the one achieved when we used either soluble tetramers or aAPC in which MHC-peptide complexes were uniformly distributed within artificial bilayer membranes. This increased efficiency in stimulation was mirrored by increased translocation from the cytoplasm to the membrane of protein kinase θ, a T cell signaling molecule that colocalizes with the TCR within the supramolecular activation cluster, thus indicating efficient engagement of T cell activation pathways. Engineered aAPC may have immediate application for basic and clinical immunology studies pertaining to modulation of T cells ex vivo.

The effect of artificial antigen-presenting cells with preclustered anti-CD28/-CD3/-LFA-1 monoclonal antibodies on the induction of ex vivo expansion of functional human antitumor T cells

“…In order to prepare artificial APC, we used the approach described by Albani et al for the identification of rare antigen-specific T cells in autoimmune disorders [34][35][36] with the following modifications. Our artificial APC consisted of a scaffold made of GM1-enriched liposomes, which anchor microdomains where mouse anti-human CD3 (clone UCHT1, isotype IgG1k; BD PharMingen, San Diego, CA, USA), CD28 (cloneCD28.2, isotype IgG1k; BD PharMingen) and LFA1 (clone 38, isotype IgG2a; Biodesign, Saco, Maine, USA) triggering mAb had been preclustered ( Figure 1A).…”

“…To this end, biotinylated anti-CD3, anti-CD28 and anti-LFA1 mAb were combined with biotinylated cholera toxin B (Sigma-Aldrich Inc.) in a 3:1 molar ratio for 5 min at room temperature, as previously described. 34 Subsequently, neutravidin (Pierce Biotechnology, Rockford, IL, USA) was added at a ratio of 1 mol per four biotinylated moieties. After 15 min of incubation at room temperature, the ganglioside GM1-enriched liposomes (kindly provided by Dompè Biotec Spa, L'Aquila, Italy) were added.…”

“…Recent evidence indicated that preclustering of MHC-peptide complexes in membrane microdomains on the APC surface affects the efficiency of immune synapse formation and the related T-cell activation. 32,33 Building on these data, we modified the liposome-based artificial APC recently described by Albani et al, 34,35 which contained class II HLA molecule-peptide complexes associated with co-stimulatory molecules on the liposome rafts. In our artificial APC, the HLA-peptide complex was replaced by anti-CD3 mAb, and we added anti-LFA-1 mAb to allow an efficient artificial APC-T-cell interaction.…”

Establishment and Characterization of a Cell Based Artificial Antigen-Presenting Cell for Expansion and Activation of CD8+ T Cells Ex Vivo

“…The bead-based systems also have some drawbacks, including high cost of the beads, the labor-intensive process of removing the beads from the culture before infusion, and the inability of these beads to expand CD8 + T cells (21)(22)(23)(24). The artificial liposomes and exsomes show some potential for T-cell expansion, but the high cost, long-time procedure and biosafety should be considered for clinical therapy (25,26).…”

From Tango to Quadrilla

“…62,63 In agreement, clustered pMHC molecules inserted in planar lipid bilayers exert enhanced T-cell activation as compared with uniformly-distributed MHC ligands. 64 A single-molecule-imaging analysis revealed that microclusters are relatively static, although their components are in a highly-dynamic state, moving in and out of the cluster. 65 TCR microclusters move independently toward the center of the IS and fuse to form large clusters: the interconnected large clusters become then immobilized at the center of the IS, and form the TCR-cSMAC.…”