Antigen Recognition Induces Phosphatidylserine Exposure on the Cell Surface of Human CD8+ T Cells.

In eukaryotic cells the phospholipid phosphatidylserine (PS) is restricted to the inner plasma membrane leaflet. This lipid asymmetry which is maintained by the concerted action of phospholipid transport proteins is mainly lost during apoptosis. Here, we demonstrate that primary human CD8+ cytotoxic T lymphocytes (CTL) expose PS upon T-cell receptor (TCR)-mediated antigen recognition: antigen-specific CTL, recognizing the HLA-A2 binding Melan-A peptide, demonstrated a marked exposure of PS as determined by annV-FITC staining, a highly specific PS-binding protein, after 4 h of stimulation with Melan-A-loaded antigen presenting cells (APC). PS exposure was Ag-specific (>80% annV-positive T cells) but control peptide (gp100)-pulsed and unpulsed APC also induced a slight background PS exposure on the CTL. To follow more precisely the fate of annV-positive CTL after antigen-specific stimulation, we labeled Melan-A-specific CTL with the membrane dye PKH-26 and isolated all annV+ PKH-26+ CTL 4 h after stimulation with peptide-pulsed APC cells by cell sorting. Results demonstrate that the annV-positive T-cell population is heterogenous: while the annV-high T-cell population retained the annV-high phenotype and consisted of apoptotic T cells, the annV-intermediate(int) T-cell population revealed a constant decrease of annV binding and became annV-negative at 54 to 72 h after stimulation. Using three independent assays for apoptosis we found that annV-int T cells are propidium iodide negative, do not exhibit DNA strand brakes and contain no active caspase 3. In contrast, annV-int CTL revealed a strongly activated phenotype indicated by an upregulation of CD69 expression and downregulation of the TCRαβ chain expression. Fluorescence microscopic analysis demonstrated that PS is distributed inhomogenously over the plasma membrane and concentrated in membrane lipid raft domains at the immunological synapse. By studying the activity of PS transport proteins using a fluorescence-labeled PS analogue, we determined a constitutive outward transfer of PS molecules in Melan-A-specific CTL. The constitutive PS outward transport was not further accelerated after antigen re-stimulation. In sharp contrast, the inward transporting flippase was strongly inhibited in stimulated CTL resulting in an accumulation of PS molecules on the cell surface. Shielding of exposed PS by annexinV protein during antigen recognition diminished cytokine secretion, activation and cell-cell clustering of antigen-specific CTL. In summary, our data demonstrate for the first time that externalized PS on antigen-stimulated CTL is linked to T-cell activation and probably involved in cell-cell contact formation at the immunological synapse.