Ex-vivo expanded baboon CD4+ CD25Hi Treg cells suppress baboon anti-pig T and B cell immune response

• Published in: Xenotransplantation (Københaven) 2012-03, Vol.19 (2), p.102-111
• Main Authors: Singh, Avneesh K., Seavey, Caleb N., Horvath, Keith A., Mohiuddin, Muhammad M.
• Format: Article
• Language: English
• Subjects: CD25 antigen; CD28 antigen; CD4 antigen; Cell culture; Cell proliferation; Effector cells; Foxp3 protein; Immunoregulation; Immunosuppressive agents; Immunotherapy; Interleukin 2; Lymphocytes B; Lymphocytes T; Papio; Peripheral blood mononuclear cells; Rapamycin; T regulatory cells; T-cell receptor; Treg; xenoantigens; xenograft; Xenografts
• ISSN: 0908-665X; 1399-3089
• DOI: 10.1111/j.1399-3089.2012.00697.x

Singh AK, Seavey CN, Horvath KA, Mohiuddin MM. Ex‐vivo expanded baboon CD4+ CD25Hi Treg cells suppress baboon anti‐pig T and B cell immune response. Xenotransplantation 2012; 19: 102–111. © 2012 John Wiley & Sons A/S. : 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+ CD25Neg cells however induced vigorous proliferation. Conclusion:  Ex‐vivo expanded CD4+ CD25+ Fox