Regulation of Transplant Arteriosclerosis by In Vitro Expanded Human Regulatory T Cells

Objectives: Transplant arteriosclerosis is a major limitation to long-term allograft survival. It is characterized by a fibroproliferative neointimal thickening, which is driven by alloantigen-reactivity. Regulatory T cells (Tregs) are a subpopulation of leukocytes with a highly anti-inflammatory character. They are classified into natural occurring and induced Tregs, based on their origin. Due to low frequencies of those populations in peripheral blood, ex vivo cell expansion is essential for further in vivo applications.

The aim of this study was to examine the effect of in vitro expanded Tregs on transplant arteriosclerosis using an allogenic humanized mouse model.

Methods: PBMC were isolated from leukapheresis products of healthy donors. Cells were separated into CD4⁺CD25⁺CD127^lo and CD4⁺CD25^- cells by FACS and expanded in vitro by addition of IL-2 and CD3/CD28 antibody-coated beads. After verification of phenotype and in vitro suppressive capacity, T cells were used for in vivo application. To assess in vivo regulatory function of the expanded Tregs in a transplant arteriosclerosis model, PBMC -/+ expanded Tregs were injected into immunodeficient NOD.rag(−/−)γc(−/−) mice and blood samples were drawn on day 14 and 28 for immunophenotyping. Mice were sacrificed on day 28 and allogeneic human artery grafts were harvested for histopathological examination.

Results: In vitro stimulation of the respective Treg population led to a ~600-fold increase in cell numbers, while maintaining a strong FoxP3 expression. To enhance purity of the naturally occurring Tregs, CD8+ depletion had to be performed after expansion. Both Treg types suppressed the proliferation of naïve CD4+CD25- effector T cells in vitro by >80% at a 1:1 ratio. In vivo application of the expanded Tregs lead to reductions of luminal occlusion, intimal hyperplasia and cell infiltration in the human artery graft. In these experiments, induced Tregs indicated a higher immunosuppressive potential than naturally occurring Tregs, which interestingly caused media hyperplasia.

Conclusion: In vitro expansion of Tregs allows for the production of high numbers of highly immunosuppressive regulatory T cells for in vivo applications. These expanded polyspecific Tregs are functional in vivo and could enable adoptive cell therapies in clinical transplantation.