Efficient Induction and Expansion Of CD8+CD28+Foxp3+ Regulatory T Cells By TGF-beta1 and Rapamycin
Authors: Fan Huahua , Huo Xiaona , Sun Juan , Yang Yiming , Li Xiao 
Published: October 2019

Abstract

Background
Although extensive studies have focused on the CD4+CD25+Foxp3+Tregs in recent years, CD8+Tregs have also been reported to play important roles in maintenance of immune tolerance. Adoptive transfer of CD8+Tregs in rodents can prevent or treat autoimmune diseases, allograft rejection or graft-versus-host disease.

Objective
Several approaches for induction of Ag-specific CD8+Tregs have been reported, but there is currently no reliable protocol for the ex vivo induction and large-scale expansion of human polyclonal CD8+CD28+Foxp3+Tregs. Our research was designed to investigate an effective method to induce and expand polyclonal CD8+CD28+Foxp3+Tregs in vitro on a large scale to meet the clinical demand.

Methods
CD8+ T lymphocytes were isolated from adult peripheral blood with immunomagnetic beads and cultured for a week using anti-CD3/CD28 antibody-coated beads and IL-2 in combination with TGF-beta1 and rapamycin. Expanded CD8+Tregs were re-stimulated with fresh anti-CD3/CD28 antibody-coated beads and cytokines for other cycles. The phenotype of CD8+Tregs was analyzed by flow cytometry. In the suppression assay, CFSE progressive dilution was used as readout of responder cells proliferation. For in vivo experiments, the mouse models of collagen-induced arthritis (CIA) were established. 2x10E6 ex vivo induced and expanded human CD8+CD28+Foxp3+Tregs were transferred into CIA recipient mouse with the onset of RA. Clinical and histopathologic scores, cytokine and the secretion of anti-type-two collagen antidody in serum were analyzed.

Results
A large number of CD8+CD28+Foxp3+Tregs could be efficiently induced and expanded from CD8+ T lymphocytes in polyclonal stimulation culture system added TGF-beta1 and rapamycin. By repeated stimulation of CD8+ T cells for 4 weeks, we could generate larger than 1x10E10 CD8+CD28+Foxp3+Tregs without loss of their suppressive function from every 1x10E6 CD8+ T cells, which can typically be isolated from 5-10ml of peripheral blood. The expanded CD8+Tregs expressed high level of Foxp3, CD28, CD25, PD-1, CD103, CD62L, CCR7, CTLA4, CD39 and CD73, secreted small amount of IL-2, IFN-gamma, IL-10 and TGF-beta, did not secret IL-17A, displayed a partially anergic phenotype, and could inhibit the proliferation of autologous or allogeneic CD4 effective T lymphocytes activated by anti-CD3/CD28 antibody. We found that the suppression of CD8+CD28+Foxp3+Tregs in proliferation of effective T lymphocytes was mainly dependent on cell contact but not dependent on their cytotoxicity. CD8+CD28+Foxp3+Tregs did not secret IL17A or secret small amount of IFN-gamma in the presence of inflammatory cytokines such as IL-1beta and IL-6 or IL-21 and IL-23. Furthermore, CD8+CD28+Foxp3+Tregs are a kind of induced regulatory T cells, because their Treg-cell-specific demethylated region (TSDR) has a methylation status. CD8+CD28+Foxp3+Tregs treatment could significantly alleviate the severity of arthritis, and reduce the cartilage destruction of the joints of CIA mice and the level of total anti-type-two collagen IgG antibody in serum as well.

Conclusion
we have developed a simple method using TGF-beta1 and rapamycin to induce and expand highly efficient human polyclonal CD8+CD28+Foxp3+Tregs with suppressive capacities from CD8+ T cells on a large scale. This procedure of CD8+Tregs expansion we used fits with the currently available clinical grade isolation tools with the objective of facilitating easy translation into clinical practice. This approach may facilitate the clinical application of CD8+Treg-based immunotherapy in transplantation and autoimmune diseases.

Disclosures:
No relevant conflicts of interest to declare.