PB0727 - The induction of Tregs by aPC depends on metabolic reprogramming and reduced αKG levels

Monday, July 11, 2022

6:30 PM – 7:30 PM

Location: Exhibition

Presentation Format: In-Person

Presenting Author(s)

Dheerendra Gupta, Msc

PhD student
Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University hospital Leipzig, Leipzig, Germany.
Leipzig, Sachsen, Germany

Co-Author(s)

AE

Ahmed Elwakiel

PhD student
Institute for Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, Leipzig University
Leipzig, Sachsen, Germany
SR

Satish Ranjan

Post Doc
Institute for Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, Leipzig University
Leipzig, Sachsen, Germany
MP

ManishKumar Pandey

PhD student
Institute for Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, Leipzig University
Leipzig, Sachsen, Germany
SK

Shruthi Krishnan

Post Doc
Institute of Experimental Internal Medicine, Otto von Guericke University
Magdeburg, Sachsen-Anhalt, Germany
RH

Reinhard Henschler

Professor
Institute for Transfusion Medicine, Leipzig University
Leipzig, Sachsen, Germany
SK

Shrey Kohli, PhD

Group leader
Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University hospital Leipzig, Leipzig, Germany
Leipzig, Sachsen, Germany
Berend Isermann, PhD MD

Institute director
Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University hospital Leipzig, Leipzig, Germany
Leipzig, Sachsen, Germany

Background: Activated protein C (aPC) is a coagulation protease known to regulate cellular signaling in innate as well as adaptive immunity. We and others have previously shown that aPC ameliorates graft versus host disease (GvHD). aPC induced regulatory T cells (Tregs) from T-effector (Teff) cells. Interestingly, cellular metabolism is known to regulate T-cell plasticity. Accordingly, we hypothesized that the coagulation protease aPC regulates T cell metabolism and plasticity.

Aims: We aim to investigate whether aPC induces Tregs via metabolic reprogramming of Teff cells.

Methods: Activation of T cells was determined by in vitro proliferation using the MLR (mixed lymphocyte reaction model: stimulation of Teff cells with AgPC, ratio 1:3) or plate-bound anti-CD3 and CD28 stimulation. Teff and Treg cell markers were measured by FACS. 5-methyl cytosine(5mC) pattern was analyzed by dot blot and FACS. Oxygen consumption rate (OCR) was measured by Seahorse and TCA metabolites by LC/MS. Steady-state T-cell phenotyping was done by analyses of T-cells from APChigh and WT mice. GvHD was induced in BALB/c mice by transplanting 2x106 T cells with 5-6x106 bone marrow cells from C57BL/6 mice.

Results: aPC treatment suppressed the global 5mC methylation in Tregs. Furthermore, induction of Tregs from Teff cells was associated with Foxp3 promoter demethylation and increased Foxp3 expression in vitro suggesting that T-cell plasticity is associated with epigenetic changes. These epigenetic changes were associated with metabolic reprogramming in T-cells, reflected by reduced OCR and alteration in TCA cycle metabolites upon aPC treatment. In particular α-ketoglutarate (αKG) was reduced in aPC-pretreated Teff cells, which was associated with reduced expression of the glutamine transporter Snat1 and Asct2 and reduced glutamate levels. These effects of aPC were reversed by addition of α-KG and glutamine resulting in increased proliferation and decreased Foxp3 expression.

Conclusion(s): These data demonstrate that aPC metabolically re-programs T-cells and induces epigenetic changes, favoring a Treg phenotype.