title: Ex-vivo expanded baboon CD4+ CD25 Hi Treg cells suppress baboon anti-pig T and B cell immune response.
abstract: BACKGROUND
  CD4(+) CD25(+) FoxP3(+) regulatory T (Treg) cells play an important role in regulating immune responses. A very small number of Treg cells are present in peripheral blood and lymphoid organs, but due to their ability to suppress the immune response, they have a high potential for immunotherapy in clinics. Successful ex-vivo expansion of naturally occurring CD4(+) CD25(+) T cells has been achieved after TCR stimulation in the presence of T cell growth factors. In this study, we evaluated the role of these Treg cells in suppressing proliferative response of baboon T and B cells to pig xenoantigens.
  
  
  METHODS
  Naturally occurring baboon CD4(+) CD25(+) regulatory T cells (nTreg) were sorted from peripheral blood and expanded in the presence of either anti-CD3/CD28 beads or irradiated pig peripheral blood mononuclear cells with IL-2. Treg cells were also enriched directly from CD4(+) T cells cultured in the presence of rapamycin (0.1-10 nm). Mixed lymphocyte culture and polyclonal B cell stimulation with ex-vivo Treg cells were performed to assess the function of ex-vivo expanded Treg cells.
  
  
  RESULTS
  The nTreg cells were expanded to more than 200-fold in 4 weeks and retained all the nTreg cell phenotypic characteristics, including high levels of FoxP3 expression. 2-fold increase in enrichment of CD4(+) CD25(+) FoxP3(+) Treg cells from CD4(+) cells was observed with rapamycin compared to cultures without rapamycin. The ex-vivo expanded Treg cells obtained from both methods were able to suppress the baboon anti-porcine xenogeneic T and B cell immune response in-vitro efficiently (more than 90% suppression at 1:1 ratio of T regulatory cells: T effector cells), and their suppression potential was retained even at 1:256 ratio. However, freshly isolated nTreg cells had only 70% suppression at 1:1 ratio, and their suppressive ability was reduced to ≤ 50% at 1:16 ratio. Furthermore, we have found that ex-vivo expanded Treg can also suppress the proliferation of B cells after polyclonal stimulation. Forty to 50 percent reduction in B cell proliferation was observed when ex-vivo expanded Treg cells were added to the culture at a 1:1 ratio. The addition of CD4(+) CD25(Neg) cells however induced vigorous proliferation.
  
  
  CONCLUSIONS
  Ex-vivo expanded CD4(+) CD25(+) FoxP3(+) Treg cells can be used to efficiently suppress xenogeneic immune responses by inhibiting T and B cell proliferation. These ex-vivo expanded Treg cells may also be used with other immunosuppressive agents to overcome xenograft rejection in preclinical xenotransplantation models.
authors.affiliation: Cardiothoracic Surgery Research Program, NHLBI, NIH, Bethesda, MD 20892, USA.
authors.forename: Avneesh K
authors.initials: AK
authors.lastname: Singh
authors.forename: Caleb N
authors.initials: CN
authors.lastname: Seavey
authors.forename: Keith A
authors.initials: KA
authors.lastname: Horvath
authors.forename: Muhammad M
authors.initials: MM
authors.lastname: Mohiuddin
mesh_terms: D000818:Animals; D015478:Antigens, Heterophile / Q000008:administration & dosage*; D001402:B-Lymphocytes / Q000166:cytology / Q000276:immunology*; D049109:Cell Proliferation; D051858:Forkhead Transcription Factors / Q000235:genetics / Q000378:metabolism; D006084:Graft Rejection / Q000517:prevention & control; D007108:Immune Tolerance; D007113:Immunity, Innate; D016130:Immunophenotyping; D007166:Immunosuppressive Agents / Q000494:pharmacology; D066298:In Vitro Techniques; D008213:Lymphocyte Activation; D010215:Papio / Q000276:immunology*; D020123:Sirolimus / Q000494:pharmacology; D034421:Sus scrofa / Q000276:immunology*; D013552:Swine; D013556:Swine, Miniature / Q000276:immunology*; D013601:T-Lymphocytes / Q000276:immunology*; D050378:T-Lymphocytes, Regulatory / Q000145:classification / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism; D014183:Transplantation, Heterologous / Q000276:immunology
chemical_list: D015478:Antigens, Heterophile; D051858:Forkhead Transcription Factors; D007166:Immunosuppressive Agents; D020123:Sirolimus