Demethylation of the RORC2 and IL17A in human CD4+ T lymphocytes defines Th17 origin of nonclassic Th1 cells.
Th17-derived Th1 lymphocytes, termed nonclassic, differ from classic Th1 cells because of the presence of retinoic acid orphan receptor (ROR)C2 and the surface expression of CD161 and CCR6. We demonstrate in this article that nonclassic Th1 cells, like Th17 cells, have a marked RORC2 and IL17A demethylation, whereas classic Th1 cells exhibit a complete methylation of these genes. The analysis of RORC2 DNA methylation in the CD4(+)CD161(+) and CD4(+)CD161(-) naive Th subsets from umbilical cord blood surprisingly revealed comparable hypermethylation levels. PCR analysis at the single-cell level revealed that RORC2 mRNA was expressed by none of the CD4(+)CD161(-) and present only in a minority of CD4(+)CD161(+) naive Th cells. These findings provide two important novel observations on the physiology of human Th17 cells: 1) they confirm at the epigenetic level the origin of nonclassic Th1 cells from Th17 cells, also identifying in the RORC2 and IL17A methylation status a novel tool for their distinction from classic Th1 cells, and 2) they demonstrate that RORC2-expressing cells are only a minority in the subset of CD4(+)CD161(+) naive Th cells, which are known to contain all Th17 cell precursors.
Department of Experimental and Clinical Medicine, University of Florence, Florence 50134, Italy; DENOTHE Center, University of Florence, Florence 50134, Italy; Department of Clinical and Experimental Medicine, Second University of Naples, Naples 80131, Italy; Center for Biomolecular Studies Supporting Human Health, Second University of Naples, Naples 80131, Italy;
Alessio
A
Mazzoni
Department of Experimental and Clinical Medicine, University of Florence, Florence 50134, Italy; DENOTHE Center, University of Florence, Florence 50134, Italy; Regenerative Medicine Unit, Careggi University Hospital, Florence 50134, Italy;
Veronica
V
Santarlasci
Department of Experimental and Clinical Medicine, University of Florence, Florence 50134, Italy; DENOTHE Center, University of Florence, Florence 50134, Italy; Regenerative Medicine Unit, Careggi University Hospital, Florence 50134, Italy;
Laura
L
Maggi
Department of Experimental and Clinical Medicine, University of Florence, Florence 50134, Italy; DENOTHE Center, University of Florence, Florence 50134, Italy;
Manuela
M
Capone
Department of Experimental and Clinical Medicine, University of Florence, Florence 50134, Italy; DENOTHE Center, University of Florence, Florence 50134, Italy;
Maria Caterina
MC
Rossi
Department of Experimental and Clinical Medicine, University of Florence, Florence 50134, Italy; DENOTHE Center, University of Florence, Florence 50134, Italy;
Valentina
V
Querci
Department of Clinical and Experimental Medicine, Second University of Naples, Naples 80131, Italy; Center for Biomolecular Studies Supporting Human Health, Second University of Naples, Naples 80131, Italy;
Raffaele
R
De Palma
Cell Biology Group, German Rheumatism Research Center Berlin, 10117 Berlin, Germany;
Hyun-Dong
HD
Chang
Regenerative Immunology and Aging, Berlin-Brandenburg Center for Regenerative Therapies, Charité University Medicine Berlin, 13353 Berlin, Germany;
Andreas
A
Thiel
Anna Meyer Children's Hospital and University of Florence, Florence 50134, Italy; and.
Rolando
R
Cimaz
Department of Experimental and Clinical Medicine, University of Florence, Florence 50134, Italy; DENOTHE Center, University of Florence, Florence 50134, Italy; Immunology and Cellular Therapy Unit, Careggi University Hospital, Florence 50134, Italy.
Francesco
F
Liotta
Department of Experimental and Clinical Medicine, University of Florence, Florence 50134, Italy; DENOTHE Center, University of Florence, Florence 50134, Italy; Immunology and Cellular Therapy Unit, Careggi University Hospital, Florence 50134, Italy.
Lorenzo
L
Cosmi
Department of Experimental and Clinical Medicine, University of Florence, Florence 50134, Italy; DENOTHE Center, University of Florence, Florence 50134, Italy; Immunology and Cellular Therapy Unit, Careggi University Hospital, Florence 50134, Italy.
Enrico
E
Maggi
Cell Biology Group, German Rheumatism Research Center Berlin, 10117 Berlin, Germany;
Andreas
A
Radbruch
Department of Experimental and Clinical Medicine, University of Florence, Florence 50134, Italy; DENOTHE Center, University of Florence, Florence 50134, Italy;
Sergio
S
Romagnani
Cell Biology Group, German Rheumatism Research Center Berlin, 10117 Berlin, Germany;
Jun
J
Dong
Department of Experimental and Clinical Medicine, University of Florence, Florence 50134, Italy; DENOTHE Center, University of Florence, Florence 50134, Italy; Regenerative Medicine Unit, Careggi University Hospital, Florence 50134, Italy; francesco.annunziato@unifi.it.
Francesco
F
Annunziato
D015704:CD4 Antigens / Q000378:metabolism; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology* / Q000378:metabolism*; D016207:Cytokines / Q000096:biosynthesis; D019175:DNA Methylation; D005786:Gene Expression Regulation; D006801:Humans; D016130:Immunophenotyping; D007371:Interferon-gamma / Q000235:genetics; D020381:Interleukin-17 / Q000235:genetics*; D008954:Models, Biological; D055651:NK Cell Lectin-Like Receptor Subfamily B / Q000378:metabolism; D057125:Nuclear Receptor Subfamily 2, Group C, Member 2 / Q000235:genetics*; D010641:Phenotype; D054504:Precursor Cells, T-Lymphoid / Q000378:metabolism; D011401:Promoter Regions, Genetic; D020825:T-Box Domain Proteins / Q000235:genetics / Q000378:metabolism; D016176:T-Lymphocyte Subsets / Q000276:immunology* / Q000378:metabolism*; D018417:Th1 Cells / Q000276:immunology / Q000378:metabolism; D058504:Th17 Cells / Q000276:immunology / Q000378:metabolism; D000098815:T-bet Transcription Factor
D015704:CD4 Antigens; D016207:Cytokines; D020381:Interleukin-17; D055651:NK Cell Lectin-Like Receptor Subfamily B; D057125:Nuclear Receptor Subfamily 2, Group C, Member 2; D020825:T-Box Domain Proteins; D000098815:T-bet Transcription Factor; D007371:Interferon-gamma