Functional and proteomic insight into modulation of Foxp3+ regulatory T cells by leukemic extracellular vesicles

Julian Swatler1, Laura Turos1, Dominik Cysewski2, Marta Brewinska-Olchowik1, Ewa Kozlowska3, Paulina Pilanc4, Katarzyna Piwocka1

1Laboratory of Cytometry, Nencki Institute of Experimental Biology, Warsaw, Poland, 2Mass Spectrometry Laboratory, Institute of Biochemistry and Biophysics, Warsaw, Poland, 3Department of Immunology, University of Warsaw, Warsaw, Poland, 4Laboratory of Molecular Neurobiology, Nencki Institute of Experimental Biology, Warsaw, Poland

Background: Extracellular vesicles (EVs) have been recently shown to be potent modulators of immune response, though little is known about their role in biology of Foxp3+ regulatory T cells (Tregs). EVs have also been implicated in progression of BCR-ABL-expressing chronic myeloid leukemia (CML), but not directly in regulation of immunosuppression. Our first studies have demonstrated modulation of activity of thymic regulatory T cells (tTregs) by leukemic EVs (Swatler et al., Eur J Immunol, 2020). Here, we use human ex vivo and mouse in vivo systems, as well as proteomics, to further study direct mechanisms by which leukemic EVs influence biology of Tregs.

Methods: Leukemic extracellular vesicles were isolated from conditioned media of leukemic K562 cells by differential ultracentrifugation and validated using electron microscopy and nanoparticle tracking. Human CD4+ CD25hi CD127lo Tregs and CD4+ CD25- conventional T cells were FACS-sorted and used for ex vivo assays, further analysed by multicolor flow cytometry. Protein composition of EVs was analysed using mass spectrometry. For in vivo studies, control and Rab27a knock-out (releasing significantly less EVs) 32D BCR-ABL+ GFP+ cells were used.

Results: We observed time- and dose-dependent uptake of fluorescently(CFSE)-labelled EVs by different subsets of human T cells. Treatment with leukemic EVs amplified suppressive phenotype of human sorted Tregs by upregulation of CD39, CTLA-4 and Foxp3 expression. Moreover, leukemic EVs have led to potent induction (~35% vs. ~10% in controls) of induced regulatory T cells. Increase in amount of Tregs has also been observed in spleens and bone marrow of mice injected with CML cells and bearing leukemia-like disease. Proteomic studies have revealed over 1000 proteins in K562-derived extracellular vesicles, including almost 200 proteins previously unidentified in any EVs (according to ExoCarta database). Functional analysis of proteomics data has revealed variety of proteins with immunomodulatory function, including molecules modulating T cell receptor-, TNF- and NFκB-signaling, but not TGF-β-signaling. Data from proteomic studies are under further investigation, especially in terms of modulated pathways in Treg (analysis of phospho-proteins by flow cytometry).

Conclusions: Altogether, leukemic EVs potentiate suppressive phenotype and induction of regulatory T cells in chronic myeloid leukemia, potentially contributing to immunosuppression in this disease. Regulation by EVs may enable CML cells to promote immunosuppression outside their bone marrow niche and facilitate progression to blast crisis.

Supported by grants: National Science Centre: 2013/10/E/NZ3/00673 (KP), 2018/29/N/NZ3/01754 (JS) and Foundation for Polish Science TEAM TECH Core Facility Plus/2017-2/2 (POIR.04.04.00-00-23C2/17-00) (KP).