Regulation of CD4+ T-cell Responses through Modulation of intracellular Polyphosphate

Abstract

Polyphosphate (polyP) is a biopolymer consisting of up to several hundred linked phosphate residues and can be found in every cell type. PolyP has been associated with multiple cellular functions that are well defined in bacteria and yeasts, whereas its significance in mammalian cells remains largely unknown. However, pro thrombotic and pro inflammatory functions have been ascribed to extracellular polyP released from platelets, basophils and mast cells. In this study, intracellular polyP from CD4+ T cells was characterised and the role of polyP for T cell stimulation and differentiation was investigated. PolyP was shown to accumulate in stimulated CD4+ T cells in a glycolysis dependent manner and in response to T cell differentiation in vitro, where pro inflammatory CD4+ T cell populations display more polyP than anti inflammatory, regulatory T (Treg) cells. To modulate polyP in primary CD4+ T cells, a recombinant cell penetrating peptide polyphosphatase (CPP Ppx1) fusion protein was produced, that allows for the efficient protein transduction into viable T cells. Degradation of polyP with CPP Ppx1 resulted in calcium mobilisation and signalling in treated CD4+ T cells that increased activation marker expression and proliferation, analysed by both flow cytometry and chromatin accessibility assays. Moreover, polyP digestion by CPP Ppx1 led to enhanced glycolysis and oxidative phosphorylation, resulting in an elevated overall ATP production. Furthermore, expression of the Treg transcription factor Foxp3 was induced in CD4+ T cells with CPP Ppx1 mediated polyP reduction. However, Foxp3 expression in response to CPP Ppx1 treatment did not facilitate immunosuppressive Treg functions, which could be attributed to transient Foxp3 expression, caused by insufficient demethylation of the Foxp3 promotor, and a constant production of elevated interleukin 2 levels. In summary, this study demonstrated that polyP is differentially regulated in distinct T cell subsets and that polyP inhibits T cell activation by binding calcium. Experimental degradation of intracellular polyP results in a Treg like phenotype in vitro but did not confer suppressive functions. Thus, the regulation of T cell polyP might be a useful tool for the modulation of CD4+ T cell responses in future applications.