Generation of highly effective and stable murine alloreactive Treg cells by combined anti-CD4 mAb, TGF-β, and RA treatment.
The transfer of alloreactive regulatory T (aTreg) cells into transplant recipients represents an attractive treatment option to improve long-term graft acceptance. We recently described a protocol for the generation of aTreg cells in mice using a nondepleting anti-CD4 antibody (aCD4). Here, we investigated whether adding TGF-β and retinoic acid (RA) or rapamycin (Rapa) can further improve aTreg-cell generation and function. Murine CD4(+) T cells were cultured with allogeneic B cells in the presence of aCD4 alone, aCD4+TGF-β+RA or aCD4+Rapa. Addition of TGF-β+RA or Rapa resulted in an increase of CD25(+)Foxp3(+)-expressing T cells. Expression of CD40L and production of IFN-γ and IL-17 was abolished in aCD4+TGF-β+RA aTreg cells. Additionally, aCD4+TGF-β+RA aTreg cells showed the highest level of Helios and Neuropilin-1 co-expression. Although CD25(+)Foxp3(+) cells from all culture conditions displayed complete demethylation of the Treg-specific demethylated region, aCD4+TGF-β+RA Treg cells showed the most stable Foxp3 expression upon restimulation. Consequently, aCD4+TGF-β+RA aTreg cells suppressed effector T-cell differentiation more effectively in comparison to aTreg cells harvested from all other cultures, and furthermore inhibited acute graft versus host disease and especially skin transplant rejection. Thus, addition of TGF-β+RA seems to be superior over Rapa in stabilising the phenotype and functional capacity of aTreg cells.
Institute of Medical Immunology, Charité - Universitätsmedizin, Berlin, Germany.
Ulrike
U
Schliesser
Martin
M
Chopra
Andreas
A
Beilhack
Christine
C
Appelt
Simone
S
Vogel
Julia
J
Schumann
Ivo
I
Panov
Katrin
K
Vogt
Stephan
S
Schlickeiser
Sven
S
Olek
Kathryn
K
Wood
Christine
C
Brandt
Hans-Dieter
HD
Volk
Birgit
B
Sawitzki
D000208:Acute Disease; D064591:Allografts; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D058846:Antibodies, Monoclonal, Murine-Derived / Q000276:immunology / Q000494:pharmacology*; D001402:B-Lymphocytes / Q000276:immunology / Q000473:pathology; D015704:CD4 Antigens / Q000235:genetics / Q000276:immunology*; D023201:CD40 Ligand / Q000235:genetics / Q000276:immunology; D002454:Cell Differentiation / Q000187:drug effects / Q000276:immunology; D002478:Cells, Cultured; D018920:Coculture Techniques; D051858:Forkhead Transcription Factors / Q000235:genetics / Q000276:immunology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000276:immunology; D006086:Graft vs Host Disease / Q000235:genetics / Q000276:immunology / Q000473:pathology; D053645:Interleukin-2 Receptor alpha Subunit / Q000235:genetics / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D018345:Mice, Knockout; D020123:Sirolimus / Q000494:pharmacology*; D016038:Skin Transplantation; D050378:T-Lymphocytes, Regulatory / Q000276:immunology* / Q000473:pathology; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D014212:Tretinoin / Q000494:pharmacology*
D000903:Antibiotics, Antineoplastic; D058846:Antibodies, Monoclonal, Murine-Derived; D015704:CD4 Antigens; D051858:Forkhead Transcription Factors; C498833:Foxp3 protein, mouse; C508018:Il2ra protein, mouse; D053645:Interleukin-2 Receptor alpha Subunit; D016212:Transforming Growth Factor beta; D023201:CD40 Ligand; D014212:Tretinoin; D020123:Sirolimus
Alloreactive regulatory T cells; Effector T cells; Nondepleting anti‐CD4 monoclonal antibody; Retinoic acid; TGF‐β