Human in vitro-induced regulatory T cells display Dlgh1dependent and PKC-θ restrained suppressive activity

Alexandra Zanin-Zhorov; Sudha Kumari; Keli L Hippen; Sarah C Merkel; Margaret L MacMillan; Bruce R Blazar; Michael L Dustin

doi:10.1038/s41598-017-04053-5

Human in vitro-induced regulatory T cells display Dlgh1dependent and PKC-θ restrained suppressive activity

Sci Rep. 2017 Jun 26;7(1):4258. doi: 10.1038/s41598-017-04053-5.

Authors

Alexandra Zanin-Zhorov^{1

2}, Sudha Kumari^{3

4}, Keli L Hippen⁵, Sarah C Merkel⁵, Margaret L MacMillan⁵, Bruce R Blazar⁵, Michael L Dustin^{6

7}

Affiliations

¹ Molecular Pathogenesis Program, Skirball Institute of Biomolecular Medicine, Department of Pathology, New York University School of Medicine, New York, NY, 10016, USA. Alexandra.Zanin-Zhorov@kadmon.com.
² Kadmon Corporation, LLC, New York, NY, 10016, USA. Alexandra.Zanin-Zhorov@kadmon.com.
³ Molecular Pathogenesis Program, Skirball Institute of Biomolecular Medicine, Department of Pathology, New York University School of Medicine, New York, NY, 10016, USA.
⁴ Koch institute of Integrative Cancer Research, MIT, Cambridge, MA-02139, USA.
⁵ University of Minnesota Cancer Center and Department of Pediatrics, Division of Blood and Marrow Transplantation, Minneapolis, MN, 55455, USA.
⁶ Molecular Pathogenesis Program, Skirball Institute of Biomolecular Medicine, Department of Pathology, New York University School of Medicine, New York, NY, 10016, USA. michael.dustin@kennedy.ox.ac.uk.
⁷ Kennedy Institute of Rheumatology, University of Oxford, Oxford, OX3 7FY, UK. michael.dustin@kennedy.ox.ac.uk.

Abstract

In vitro induced human regulatory T cells (iTregs) have demonstrated in vivo therapeutic utility, but pathways regulating their function have not been elucidated. Here, we report that human iTregs generated in vitro from naïve cord blood cells preferentially recruit Disc large homolog 1 (Dlgh1) and exclude protein kinase C (PKC)-θ from immunological synapses formed on supported lipid bilayers with laterally mobile ICAM-1 and anti-CD3 mAb. Also, iTregs display elevated Dlgh1 overall and Dlgh1-dependent p38 phosphorylation, higher levels of phosphatase and tensin homolog (PTEN), and diminished Akt phosphorylation. Pharmacological interruption of PKC-θ increases and Dlgh1 silencing decreases the ability of iTregs to suppress interferon-γ production by CD4⁺CD25^- effector T cells (Teff). Comparison with expanded cord blood-derived CD4⁺CD25^hi tTreg and expanded Teffs from the same donors indicate that iTreg are intermediate between expanded CD4⁺CD25^hi tTregs and Teffs, whereas modulation of suppressive activities by PKC-θ and Dlgh1 signaling pathways are shared.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Adaptor Proteins, Signal Transducing / genetics*
Adaptor Proteins, Signal Transducing / immunology
CD4 Antigens / genetics
Cell Differentiation / genetics
Discs Large Homolog 1 Protein
Fetal Blood / cytology
Fetal Blood / metabolism
Humans
Immunological Synapses / genetics*
Immunological Synapses / metabolism
Intercellular Adhesion Molecule-1 / genetics
Interleukin-2 Receptor alpha Subunit / genetics
Lipid Bilayers / immunology
Lipid Bilayers / metabolism
Lymphocyte Activation
Membrane Proteins / genetics*
Membrane Proteins / immunology
Phosphorylation
Protein Kinase C-theta / genetics*
Protein Kinase C-theta / immunology
T-Lymphocytes, Regulatory / cytology
T-Lymphocytes, Regulatory / immunology
T-Lymphocytes, Regulatory / metabolism*
p38 Mitogen-Activated Protein Kinases / genetics

Substances

Adaptor Proteins, Signal Transducing
CD4 Antigens
DLG1 protein, human
Discs Large Homolog 1 Protein
Interleukin-2 Receptor alpha Subunit
Lipid Bilayers
Membrane Proteins
Intercellular Adhesion Molecule-1
Protein Kinase C-theta
p38 Mitogen-Activated Protein Kinases

Abstract

Publication types

MeSH terms

Substances

Grants and funding