Regulating polyphosphate to target T cell-mediated thrombosis

• Applicant: Reiner Karl Walter Mailer, Ph.D.
• Subject Area: Immunology ; Anatomy and Physiology ; Hematology, Oncology ; Clinical Immunology and Allergology ; Toxicology, Laboratory Medicine
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 470698011

Exaggerated T cell stimulation drives autoimmunity, chronic infections and inflammatory diseases that are associated with increased thrombotic risk. Thrombus formation is initiated through activation of factor XII (FXII) by the linear biopolymer polyphosphate (polyP). I recently showed that the phosphate transporter XPR1 regulates pro-coagulant polyP in platelets. We aim to reveal polyP/XPR1-mediated functions in T cells with implications for thrombosis. For the first aim we will decipher the role of polyP for T cell responses and determine the contribution of T cell polyP in thrombosis. In preliminary studies we found that stimulated CD4+ T cells accumulate polyP and initiate polyP-dependent FXII activation. Applying confocal laser scanning microscopy, imaging flow cytometry and chromogenic assays, we will identify subcellular localisation and FXII-activating potential of polyP from CD4+ T cell subsets. To analyse extra- and intracellular functions of polyP in T cells we will use soluble and membrane-permeable variants of recombinant polyphosphatase that specifically degrades polyP. Preliminary studies indicate that systemic T cell stimulation strongly increases thrombus size in vivo. We will perform venous thrombosis models and coagulation assays to demonstrate the impact of stimulated T cells on thrombosis. In a translational approach, human lymphoma cells and FXII-deficient plasma will be used to confirm the FXII-dependent effect of T cell polyP for thrombotic diseases. For the second aim we focus on XPR1 regulation of T cell-driven thromboinflammation. Using our previously established pharmacologic XPR1 inhibitors and a novel mouse strain with T cell-specific XPR1 deficiency, we will assess the role of XPR1 for T cell polyP and immune responses. Our preliminary results showed that XPR1 deficiency increases CD4+ T cell proliferation via elevated T cell antigen receptor (TCR) signalling. To define XPR1-dependent mechanisms, we will perform gene expression analysis and confirm findings on the protein level. We will compare polyP levels in CD4+ T cells that express or lack XPR1 and analyse XPR1-dependent T cell polyP in thrombosis models. Preliminary studies suggested that conditional gene deletion of Xpr1 in CD4+ T cells promotes hyperreactivity. We will use pharmacologic inhibition and membrane-permeable protein transduction for interference with XPR1 activity in vitro. To analyse the impact of XPR1 modulation on CD4+ T cells we will perform T cell proliferation assays and examine cytokine profiles. Additionally we will crossbreed T cell-specific XPR1 deficient mice with mice expressing a transgenic TCR that recognises the male antigen H-Y. This new strain will allow us to compare antigen-specific CD4+ T cell responses in vivo through flow cytometry analysis of activation-induced markers in cells that express or lack XPR1. Taken together we aim to reveal the contribution of CD4+ T cells for thrombotic diseases regulated by the polyP/XPR1-axis.

• DFG Programme: Research Grants