Background/Purpose: Adoptive transfer of regulatory T cells (Treg) is a promising strategy for the treatment of human autoimmune diseases. Most currently tested approaches focus on the polyclonal expansion of human thymus-derived Treg (tTreg), and are limited by potentially harmful and efficacy-limiting co-expansion of effector T cells (Teff). We hypothesized that the ex vivo isolation and massive expansion of CD4⁺/CD25^high/CD127^low/FoxP3⁺/Helios⁺ tTreg clones results in maximal purity of the generated cellular product, preserving its suppressor function stability over several re-expansion cycles while yielding cell numbers sufficient for preclinical and clinical testing.

Methods: To validate our approach for a human rheumatic disease in which antigen-specific T effector responses have been implicated in target organ damage (uveitis), we chose to identify and expand Behcet’s disease patient-derived tTreg clones. CD4⁺/CD25^high/CD127^low T cells were isolated by magnetic bead separation, subsequently cloned in a limiting dilution approach and massively expanded in at least four re-expansion cycles. Suppression was tested in suppression assays of CD3/CD28-stimulated effector T cells at repeated expansion cycles over at least 8 weeks. Clonality was demonstrated by Vβ staining.

Results: Cloning efficiency was 2-5/100 seeded cells. Treg clones were homogeneously CD4⁺/CD25^high/FoxP3⁺/Helios⁺ and stained positive for one unique TCR Vβ chain. Clones maintained around 80% suppression at 1:4 Treg:Teff ratios over as long as 11 weeks in culture and at least 4 re-expansion cycles. Proliferation was in the 10⁸ to 10⁹-fold range at day 50. When compared to tTreg clones generated from healthy human donors there were no significant differences in suppressive capacity, suppressor function stability, or phenotype.

Conclusion: Our results suggest that tTreg are not intrinsically dysfunctional in Behcet’s disease, and they deliver proof of principle that the massive monoclonal expansion of antigen-specific human Treg clones from patients with a rheumatic disease is possible and yields a functionally stable, ultra-pure cellular product for pre-clinical and clinical testing.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/massive-ex-vivo-expansion-of-functionally-stable-behcets-patient-derived-regulatory-t-cell-clones/