title: Expanded nonhuman primate tregs exhibit a unique gene expression signature and potently downregulate alloimmune responses.
abstract: We have established two complementary strategies for purifying naturally occurring regulatory T cells (Tregs) from rhesus macaques in quantities that would be sufficient for use as an in vivo cellular therapeutic. The first strategy identified Tregs based on their being CD4+/CD25(bright). The second incorporated CD127, and purified Tregs based on their expression of CD4 and CD25 and their low expression of CD127. Using these purification strategies, we were able to purify as many as 1x10(6) Tregs from 120 cc of peripheral blood. Cultures of these cells with anti-CD3, anti-CD28 and IL-2 over 21 days yielded as much as a 450-fold expansion, ultimately producing as many as 4.7x10(8) Tregs. Expanded Treg cultures potently inhibited alloimmune proliferation as measured by a carboxyfluorescein succinimidyl ester- mixed lymphocyte reaction (CFSE-MLR) assay even at a 1:100 ratio with responder T cells. Furthermore, both responder-specific and third-party Tregs downregulated alloproliferation similarly. Both freshly isolated and cultured Tregs had gene expression signatures distinguishable from concurrently isolated bulk CD4+ T-cell populations, as measured by singleplex reverse transcriptase-polymerase chain reaction (RT-PCR) and gene array. Moreover, an overlapping yet distinct gene expression signature seen in freshly isolated compared to expanded Tregs identifies a subset of Treg genes likely to be functionally significant.
authors.affiliation: The Emory Transplant Center, Department of Surgery, Emory University School of Medicine, Atlanta, GA, USA.
authors.forename: A
authors.initials: A
authors.lastname: Anderson
authors.forename: C L
authors.initials: CL
authors.lastname: Martens
authors.forename: R
authors.initials: R
authors.lastname: Hendrix
authors.forename: L L
authors.initials: LL
authors.lastname: Stempora
authors.forename: W P
authors.initials: WP
authors.lastname: Miller
authors.forename: K
authors.initials: K
authors.lastname: Hamby
authors.forename: M
authors.initials: M
authors.lastname: Russell
authors.forename: E
authors.initials: E
authors.lastname: Strobert
authors.forename: B R
authors.initials: BR
authors.lastname: Blazar
authors.forename: T C
authors.initials: TC
authors.lastname: Pearson
authors.forename: C P
authors.initials: CP
authors.lastname: Larsen
authors.forename: L S
authors.initials: LS
authors.lastname: Kean
mesh_terms: D000818:Animals; D018106:CD28 Antigens / Q000096:biosynthesis; D017252:CD3 Complex / Q000096:biosynthesis; D015704:CD4 Antigens / Q000096:biosynthesis; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D016130:Immunophenotyping; D007376:Interleukin-2 / Q000378:metabolism; D053645:Interleukin-2 Receptor alpha Subunit / Q000096:biosynthesis; D053699:Interleukin-7 Receptor alpha Subunit / Q000096:biosynthesis; D008253:Macaca mulatta; D008954:Models, Biological; D020411:Oligonucleotide Array Sequence Analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050378:T-Lymphocytes, Regulatory / Q000276:immunology*
chemical_list: D018106:CD28 Antigens; D017252:CD3 Complex; D015704:CD4 Antigens; D007376:Interleukin-2; D053645:Interleukin-2 Receptor alpha Subunit; D053699:Interleukin-7 Receptor alpha Subunit
references.citation: Hepatobiliary Pancreat Dis Int. 2007 Aug;6(4):348-57
references.pmid: 17690028
references.citation: J Immunol Methods. 2007 Sep 30;326(1-2):10-21
references.pmid: 17689553
references.citation: Immunity. 2007 Nov;27(5):786-800
references.pmid: 18024188
references.citation: Semin Immunol. 2006 Apr;18(2):78-88
references.pmid: 16458015
references.citation: J Immunol. 2006 Mar 1;176(5):3028-36
references.pmid: 16493061
references.citation: Nat Immunol. 2008 Mar;9(3):239-44
references.pmid: 18285775
references.citation: J Immunol. 2001 Jan 15;166(2):973-81
references.pmid: 11145675
references.citation: Nat Immunol. 2002 Jan;3(1):33-41
references.pmid: 11740498
references.citation: Methods. 2001 Dec;25(4):402-8
references.pmid: 11846609
references.citation: Immunity. 2002 Feb;16(2):311-23
references.pmid: 11869690
references.citation: J Exp Med. 2002 Mar 4;195(5):657-64
references.pmid: 11877489
references.citation: Biostatistics. 2003 Apr;4(2):249-64
references.pmid: 12925520
references.citation: Nat Med. 2003 Sep;9(9):1144-50
references.pmid: 12925844
references.citation: J Neuroimmunol. 2004 Apr;149(1-2):110-20
references.pmid: 15020071
references.citation: Nat Rev Immunol. 2004 Feb;4(2):123-32
references.pmid: 15040585
references.citation: J Immunol. 2004 May 1;172(9):5753-64
references.pmid: 15100322
references.citation: Am J Transplant. 2004 Aug;4(8):1289-95
references.pmid: 15268730
references.citation: J Exp Med. 1977 Mar 1;145(3):508-22
references.pmid: 233899
references.citation: J Neuroimmunol. 1996 Jan;64(1):29-36
references.pmid: 8598387
references.citation: J Immunol. 1998 Nov 15;161(10):5236-44
references.pmid: 9820495
references.citation: Nature. 1998 Dec 17;396(6712):695-9
references.pmid: 9872320
references.citation: Am J Physiol Endocrinol Metab. 2004 Dec;287(6):E1166-70
references.pmid: 15226100
references.citation: Blood. 2005 Jan 15;105(2):750-8
references.pmid: 15374887
references.citation: Clin Immunol. 2005 Apr;115(1):3-9
references.pmid: 15870014
references.citation: J Immunol. 2005 Jun 15;174(12):7573-83
references.pmid: 15944257
references.citation: J Clin Invest. 2005 Jul;115(7):1896-902
references.pmid: 15951837
references.citation: Blood. 2005 Nov 1;106(9):3068-73
references.pmid: 16020508
references.citation: J Exp Med. 2005 Nov 21;202(10):1387-97
references.pmid: 16301745
references.citation: Exp Hematol. 2006 Jan;34(1):66-71
references.pmid: 16413392
references.citation: Transplantation. 2006 Jan 15;81(1):1-6
references.pmid: 16421467
references.citation: Semin Immunol. 2006 Apr;18(2):103-10
references.pmid: 16458533
references.citation: Am J Transplant. 2006 May;6(5 Pt 1):884-93
references.pmid: 16611324
references.citation: Exp Hematol. 2006 Jun;34(6):802-8
references.pmid: 16728286
references.citation: J Exp Med. 2006 Jul 10;203(7):1701-11
references.pmid: 16818678
references.citation: Transplantation. 2006 Jul 15;82(1):23-9
references.pmid: 16861937
references.citation: Blood. 2006 Aug 15;108(4):1291-7
references.pmid: 16627754
references.citation: Am J Transplant. 2006 Sep;6(9):1989-90
references.pmid: 16869804
references.citation: J Leukoc Biol. 2006 Oct;80(4):939-52
references.pmid: 17005908
references.citation: Transpl Immunol. 2006 Dec;17(1):7-10
references.pmid: 17157205
references.citation: J Immunol Methods. 2007 Jan 30;319(1-2):41-52
references.pmid: 17173927
references.citation: Am J Transplant. 2007 Feb;7(2):320-35
references.pmid: 17241112
references.citation: Immunology. 2007 May;121(1):129-39
references.pmid: 17425604
references.citation: Am J Transplant. 2007 Jun;7(6):1457-63
references.pmid: 17511675
references.citation: Nat Rev Immunol. 2007 Aug;7(8):650-4
references.pmid: 17653127