Ex-vivo expanded baboon CD4+ CD25 Hi Treg cells suppress baboon anti-pig T and B cell immune response
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Updated: May 23, 2026
Transduction and Expansion of Primary T Cells in Nine Days with Maintenance of Central Memory Phenotype
Published on: March 18, 2020
Avneesh K Singh1, Caleb N Seavey, Keith A Horvath
1Cardiothoracic Surgery Research Program, NHLBI, NIH, Bethesda, MD 20892, USA.
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.
Conclusion:
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.
