title: Anti-CD4-mediated selection of Treg in vitro - in vitro suppression does not predict in vivo capacity to prevent graft rejection.
abstract: Regulatory T cells (Treg) have been shown to play a role in the prevention of autoimmune diseases and transplant rejection. Based on an established protocol known to generate alloantigen reactive Treg in vivo, we have developed a strategy for the in vitro selection of Treg. Stimulation of unfractionated CD4(+) T cells from naive CBA.Ca (H2(k)) mice with C57BL/10 (H2(b)) splenocytes in the presence of an anti-CD4 antibody, YTS 177, resulted in the selection of Treg able to inhibit proliferation of naive T cells. In vivo, the cells were able to prevent rejection of 80% C57BL/10 skin grafts when co-transferred to CBA.Rag(-/-) mice together with naive CD45RB(high)CD4(+) cells. Purification of CD62L(+)CD25(+)CD4(+) cells from the cultures enriched for cells with regulatory activity; as now 100% survival of C57BL/10 skin grafts was achieved. Furthermore, differentiation of Treg could be also achieved when using purified CD25(-)CD4(+) naive T cells as a starting population. Interestingly, further in vitro expansion resulted in a partial loss of CD4(+) cells expressing both CD62L and CD25 and abrogation of their regulatory activity in vivo. This study shows that alloantigen stimulation in the presence of anti-CD4 in vitro provides a simple and effective strategy to generate alloreactive Treg.
authors.affiliation: Transplantation Research Immunology Group, Nuffield Department of Surgery, University of Oxford, John Radcliffe Hospital, Oxford, UK.
authors.forename: Vanessa
authors.initials: V
authors.lastname: Oliveira
authors.forename: Birgit
authors.initials: B
authors.lastname: Sawitzki
authors.forename: Stephanie
authors.initials: S
authors.lastname: Chapman
authors.forename: Christine
authors.initials: C
authors.lastname: Appelt
authors.forename: Inga
authors.initials: I
authors.lastname: Gebuhr
authors.forename: Joanna
authors.initials: J
authors.lastname: Wieckiewicz
authors.forename: Elaine
authors.initials: E
authors.lastname: Long
authors.forename: Kathryn J
authors.initials: KJ
authors.lastname: Wood
mesh_terms: D019264:Adoptive Transfer; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology*; D000938:Antigen-Presenting Cells / Q000276:immunology; D015704:CD4 Antigens / Q000276:immunology*; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology / Q000637:transplantation; D049109:Cell Proliferation / Q000187:drug effects; D018920:Coculture Techniques; D051858:Forkhead Transcription Factors / Q000032:analysis; D006084:Graft Rejection / Q000276:immunology / Q000517:prevention & control*; D018398:Homeodomain Proteins / Q000235:genetics / Q000502:physiology; D007165:Immunosuppression Therapy / Q000379:methods; D053645:Interleukin-2 Receptor alpha Subunit / Q000032:analysis; D019041:L-Selectin / Q000032:analysis; D008213:Lymphocyte Activation / Q000187:drug effects / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D018345:Mice, Knockout; D008822:Mice, Transgenic; D054400:Receptors, CCR7 / Q000032:analysis; D016038:Skin Transplantation / Q000276:immunology; D013154:Spleen / Q000166:cytology / Q000276:immunology; D050378:T-Lymphocytes, Regulatory / Q000276:immunology* / Q000637:transplantation
chemical_list: D000911:Antibodies, Monoclonal; D015704:CD4 Antigens; C515371:Ccr7 protein, mouse; D051858:Forkhead Transcription Factors; C498833:Foxp3 protein, mouse; D018398:Homeodomain Proteins; D053645:Interleukin-2 Receptor alpha Subunit; D054400:Receptors, CCR7; D019041:L-Selectin; C064658:RAG-1 protein