Lack of Negative Feedback Loops of CTLA-4 and PD-L1 as Key Mechanisms for Post-Acute T Cell Activation Until 3 Months After Ischemic Stroke

Post-stroke T lymphocytes exert differential effects both at the infarction site and systemically; these are pro-inflammatory cascades, subsequent T cell infiltration into the brain, and persisting interaction of infiltrating T lymphocytes (more specifically a subset of CD4- CD8-double-negative T cells (DNTs)). Moreover, brain-resident microglia cells exacerbate the parenchymal damage. The acute peripheral immune response is characterized by lymphopenia and persisting activation of circulating T lymphocytes. In the temporal course, it is supposed that anti-inflammatory mechanisms in general and especially the anti-inflammatory M2 phenotype of microglia cells help to recover the functionality of brain parenchyma. We assessed the long-term temporal course of peripheral blood T cell activation post stroke as well as the interaction of pan-T cells or DNTs with microglia cells ex vivo. T cell subpopulations (CD4⁺, CD8⁺, DNT) and their activation (CD25, CD57, CTLA-4 (CD152) and PD-L1 (CD274)) were analysed in peripheral blood from stroke patients and controls by flow cytometry. Pan-T cells and DNTs were isolated by magnetic beads from stroke patients' blood 3 days (t₁), 1 month (t₂), and 3 months (t₃) post stroke or once from controls, and they were incubated with or without healthy donor human microglia cells ex vivo. Upon interaction, T cell (HLA-DR) and microglia activation (HLA-DR, CX3CR1, CD32, CD86, CD206 and CD209) was assessed by flow cytometry. Moreover, IL-1β in cell culture supernatants was quantified by ELISA. Post-stroke CD4⁺ and CD8⁺ T cell activation (CD25) persisted for 1 month and declined only after 3 months. Inhibitory activation markers CTLA-4 and PD-L1 were upregulated only 3 months after stroke. Co-culture of pan-T cells and microglia had little effect on microglial activation but a strong effect on T cell activation (HLA-DR). Co-culture of DNTs with microglia cells inhibited the M2 phenotype of microglia. Stroke acutely leads to a strong activation (CD25 upregulation) of both CD4⁺ and CD8⁺ T cells. This activation persisted in the subacute post-stroke phase and declined at 3 months post stroke, being accompanied by upregulation of PD-L1 and CTLA-4. Both might be involved in terminating chronic T cell activation. DNTs might influence microglia through CX3CR1 and inhibit an M2 state ex vivo, which might contribute to cerebral inflammation post stroke.