Functional CTL Capture Membrane Fragments from Tumors

Armed T cells acquire membrane fragments from APCs with which they establish an IS. Initially, it was shown that T cells acquire pMHC from APCs [54]. This process occurs in an MHC-restricted antigen-specific manner and is an early and rapid event taking place at the IS. Within minutes of T cell-APC interaction, pMHC are taken up and internalized through endocytosis [55]. This process, expressed at the cellular level by the incorporation of membrane fragments from target into effector cell outer cytoplasmic membrane, was demonstrated using fluorescently labeled pMHCs [55, 56]. Furthermore, non-specific tagging of APC membranes using biotinylation or fluorochrome dye was sufficient to label cognate T cells (Machlenkin et al., in press). TCR signaling by its antigen ligand was found to be the prime requirement for membrane capture [57]. Trogocytosis, a term coined by Joly and Hudrisier [58], describes transfer of membrane fragments from target to reactive immune cells. Based on this phenomenon, the TRAP (T cell recognition by antigen presenting cells) method was established and proved reliable to quantify antigen-specific CTL frequencies [59]. As TRAP implies, membrane capture was documented with APCs as the target cell from which T cells acquire membranes [57]. The role of tumor cells as a source of pMHC was not explored in this context, although it is clear that pMHC complexes can be obtained from tumor cells and presented unchanged on immune cells, a phenomenon named "cross dressing" and demonstrated with dendritic cells and tumors [60].

In a recent study we postulated that activated human T cells will generate an IS-like interaction with tumor cells similarly to that described with dendritic cells [61] and that as a consequence membrane fragments will be acquired by T cells. Using fluorochrome-tagged melanoma cells or peptide-loaded T2 cells, we showed that antigen-specific CD8+ T cells incorporate membrane fragments from tumor cells and fuse them with their outer cytoplasmic membrane (Fig. 8.1). T cells that captured labeled membranes were detectable by flow cytometry and sorted by FACS

Fig. 8.1 (A) Confocal microscopy of a membrane capturing MART127 35-reactive T cells. Right: T cell showing patches of tumor membrane (red, Dil) fused with T cell membrane (green, CD8-FITC); (B) T cell wrapped with target cell membrane suggesting adhesion and rolling of effector over target

cap+CD8+ T2/specific peptide cap+CD8+ T2/irrelevant peptide cap-CD8+ T2/specific peptide cap-CD8+ T2/irrelevant peptide cap+CD8+ T2/specific peptide cap+CD8+ T2/irrelevant peptide cap-CD8+ T2/specific peptide cap-CD8+ T2/irrelevant peptide

Effector:Target Ratio

Fig. 8.2 Membrane capturing CD8+ T cells show increased cytotoxicity against peptide pulsed targets and autologous melanoma cells. Cytotoxic activity of membrane capturing (cap+) or non-capturing (cap-) T cells against peptide-pulsed targets and melanoma cell lines. (A) gp100154-162-reactive A2-restricted CD8+ T cells were co-incubated with Dil-labeled 624mel melanoma cells (A2+ /gp100+) and sorted by FACS into cap+ and cap- populations of CD8+ T cells. CTL assays were done with [35S]-labeled specific (gp100154-162)- or irrelevant (HIV-derived) peptide-pulsed T2 cells as targets. (B) Melanoma patient derived CD8+ T cells were co-incubated with biotinylated autologous melanoma cells, followed by separation into cap+ and cap- fractions by streptavidin-coated magnetic beads. CTL assays were done with [35S]-labeled autologous and HLA-mismatched 624mel melanomas, as specific and non-specific targets or magnetic beads. Only a fraction of the T cells of a given clone were membrane capture capable. Cytotoxic ability was exclusively conferred to membrane capturing CD8+ T cells, whereas non-capturing CD8+ lymphocytes possessed reduced killing capacity (Fig. 8.2 A, B). Ex vivo expansion of membrane capturing T cells did not eliminate their functional cytotoxicity, implying that capturing capacity is an inherent trait of these cells. The therapeutic relevance of membrane capturing T cells was verified in a murine model of human melanoma. Tumor progression of melanoma transplanted into nude mice was significantly inhibited by membrane capturing T cells but not by non-capturing T cells of the same antigen specificity.

So far, the phenomenon of membrane capture was exploited as a new assay for antigen-specific T cell quantification and isolation. The use of tumor cells to identify antigen-specific functionally cytotoxic T cells is of paramount importance, because CTLs are selected not only by virtue of their antigen specificity, but by their capacity to damage tumor cells bearing naturally expressed antigens.

The biologic significance of membrane capture by tumor-reactive T cells is not clear. By acquiring pMHCs and other membrane-bound determinants from a target, T cells cross-dress themselves to become secondary APC (T-APC). The outcome can be manifold. T-APC may enhance antigen presentation and prolong T cell activation: membrane capturing CD4+ T cells, lacking co-stimulation, were as efficient as CD40 + B-APC for triggering epitope-specific CD8+ T cells [62]. Captured pMHC-TCR complexes undergo recycling and repeatedly generate TCR signaling and activation. Alternatively, Ag-presenting T cells expose themselves to fratricide killing by T cells of the same TCR specificity [54]. This process requires a functional perforin pathway [63], and could represent a form of activation-induced cell death.

Membrane capture by CTL from tumor cells suggests that an immune synapse of a kind occurs in the tumor microenvironment as part of the antitumor response. Live cell video microscopy of T cell:tumor interactions will be essential to illustrate the course of events that take place in the tumor microenvironment.

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