Large-Scale Generation of Human Allospecific Induced Tregs With Functional Stability for Use in Immunotherapy in Transplantation

Abstract

Regulatory T cells play an important role in the control of autoimmune diseases and maintenance of tolerance. In the context of transplantation, regulatory T cells (Tregs) have been proposed as new therapeutic tools that may induce allospecific tolerance toward the graft, avoiding the side effects induced by generalized immunosuppressors. Although most clinical trials are based on the use of thymic Tregs in adoptive therapy, some reports suggest the potential use of in vitro induced Tregs (iTregs), based on their functional stability under inflammatory conditions, indicating an advantage in a setting of allograft rejection. The aim of this work was to generate and expand large numbers of allospecific Tregs that maintain stable suppressive function in the presence of pro-inflammatory cytokines. Dendritic cells were derived from monocytes isolated from healthy donors and were co-cultured with CTV-labeled naïve T cells from unrelated individuals, in the presence of TGF-β1, IL-2, and retinoic acid. After 7 days of co-culture, proliferating CD4 + CD25 ++ CTV − cells (allospecific iTregs) were sorted and polyclonally expanded for 6 weeks in the presence of TGF-β1, IL-2, and rapamycin. After 6 weeks of polyclonal activation, iTregs were expanded 230,000 times, giving rise to 4,600 million allospecific iTregs. Allospecific iTregs were able to specifically suppress the proliferation of autologous CD4 + and CD8 + T cells in response to the allo-MoDCs used for iTreg generation, but not to third-party allo-MoDCs. Importantly, 88.5% of the expanded cells were CD4 + CD25 + FOXP3 + , expressed high levels of CCR4 and CXCR3, and maintained their phenotype and suppressive function in the presence of TNF-α and IL-6. Finally, analysis of the methylation status of the FOXP3 TSDR locus demonstrated a 40% demethylation in the purified allospecific iTreg, prior to the polyclonal expansion. Interestingly, the phenotype and suppressive activity of expanded allospecific iTregs were maintained after 6 weeks of expansion, despite an increase in the methylation status of the FOXP3 TSDR. In conclusion, this is the first report that demonstrates a large-scale generation of allospecific iTregs that preserve a stable phenotype and suppressor function in the presence of pro-inflammatory cytokines and pave the way for adoptive cell therapy with iTregs in transplanted patients.

Highly Purified Alloantigen-Specific Tregs From Healthy and Chronic Kidney Disease Patients Can Be Long-Term Expanded, Maintaining a Suppressive Phenotype and Function in the Presence of Inflammatory Cytokines

“…Our study is in line with a previous report showing the effect of rapamycin on Treg stability through preventing the production of pro-inflammatory cytokines in expanded Tregs and inhibiting the conversion of Tregs toward an inflammatory phenotype (60). In addition, the expansion of allospecific nTregs in the presence of IL-2 alone was able to maintain >80% FOXP3 + (Figure S9D), while both the induction and the maintenance of FOXP3 in expanded allospecific iTregs (22) and polyclonal iTregs (61) were shown to be highly dependent on the presence of TGF-b and rapamycin in the in cultures.…”

“…With this protocol, we achieved an expansion from 1,800-to 2,300-fold allo Tregs after 4 weeks of culture (Figure 2A), while previous studies reported an expansion ranging from 8-to 780-fold for 12-42 days of culture [revised in (19)]. These results are similar to those obtained in the expansion of allospecific iTregs using a slightly modified protocol, favoring the enrichment of FOXP3 + iTregs (from 60% to >90% of FOXP3 + cells) (22). Moreover, expanded allo Tregs displayed an increase in the expressions of CD25 and CTLA-4, in correlation with the increase of FOXP3, which directly upregulated the transcription of both molecules by binding to the Il2ra and Ctla4 loci (42).…”

“…Live proliferating CD4 + CD25 + CTV − Tregs (allospecific Tregs) (Figure S1B) were sorted using a MoFlo XDP cell sorter, collected in RPMI medium with 20% FBS, and cultured for 3 days in expansion medium supplemented with IL-2 (50 U/ml) plus 10% human AB serum. Then, the allospecific Tregs were polyclonally expanded using a modified protocol described previously (21,22). Briefly, the allospecific Tregs (2.5 × 10 4 cells/well) were stimulated with anti-CD3/anti-CD28 beads (bead/Treg ratio of 1:2) for 4 days in expansion medium with 10% human AB serum, IL-2 (300 U/ml), TGF-b (2.5 ng/ml), and rapamycin (100 nM).…”

“…In addition, studies have shown that long-term expansion of Tregs results in the loss of FOXP3 and may convert to potentially inflammatory T cells (20,21). Recently, our group has applied a protocol that allows the generation of a large number of functionally stable allogeneic induced Tregs (iTregs) after long-term polyclonal expansion (22). Finally, another important issue recently addressed to optimize the function of infused Tregs for the induction of effective tolerance toward the allograft is the homing capabilities of the infused Tregs (23).…”

Antigen Specific Regulatory T Cells in Kidney Transplantation and Other Tolerance Settings

“…iTregs induced with TGF-b in the presence of all-trans retinoic acid (ATRA) and rapamycin demonstrated robust suppressive function in vitro but not in vivo, in the humanized GVHD mouse model (121). Interestingly, allospecific iTregs that were induced with TGF-b1, IL-2, and ATRA in the presence of allogeneic monocyte-derived dendritic cells, can specifically suppress donor allo-responses but not third-party allo-responses, and maintain suppressive function in the presence of pro-inflammatory cytokines, despite methylation of the FOXP3 TSDR (122).…”