Abstract
Recent clinical experience has demonstrated that adoptive regulatory T (Treg) cell therapy is a safe and feasible strategy to suppress immunopathology via induction of host tolerance to allo- and autoantigens. However, clinical trials continue to be compromised due to an inability to manufacture a sufficient Treg cell dose. Multipotent adult progenitor cells (MAPCⓇ) promote Treg cell differentiation in vitro, suggesting they may be repurposed to enhance ex vivo expansion of Tregs for adoptive cellular therapy. Here, we use a Good Manufacturing Practice (GMP) compatible Treg expansion platform to demonstrate that MAPC cell-co-cultured Tregs (MulTreg) exhibit a log-fold increase in yield across two independent cohorts, reducing time to target dose by an average of 30%. Enhanced expansion is coupled to a distinct Treg cell-intrinsic transcriptional program characterized by elevated expression of replication-related genes (CDK1, PLK1, CDC20), downregulation of progenitor and lymph node-homing molecules (LEF1 CCR7, SELL) and induction of intestinal and inflammatory tissue migratory markers (ITGA4, CXCR1) consistent with expression of a gut homing (CCR7lo β7hi) phenotype. Importantly, we find that MulTreg are more readily expanded from patients with autoimmune disease compared to matched Treg lines, suggesting clinical utility in gut and/or T helper type1 (Th1)-driven pathology associated with autoimmunity or transplantation. Relative to expanded Tregs, MulTreg retain equivalent and robust purity, FoxP3 Treg-Specific Demethylated Region (TSDR) demethylation, nominal effector cytokine production and potent suppression of Th1-driven antigen specific and polyclonal responses in vitro and xeno Graft vs Host Disease (xGvHD) in vivo. These data support the use of MAPC cell co-culture in adoptive Treg therapy platforms as a means to rescue expansion failure and reduce the time required to manufacture a stable, potently suppressive product.
Highlights
Regulatory T cells (Tregs) are pivotal regulators of immune responses, maintaining self-tolerance, homeostasis, and controlling excessive immune activation through a spectrum of cell-mediated and soluble mechanisms
To establish Treg lines, CD127lo CD25hi CD4+ lymphocytes were plated at 5x104/well of round bottom 96-well plates (Corning) in 200ml 0.2mM filtered (Sartorius, Terumo) complete media consisting of X-VIVO 15 media (Lonza) containing 100mg/ml Penicillin-streptomycin, 100mg/ ml amphotericin B, 125ng/ml Rapamycin (Rapamune, Pfizer), 600 IU/ml IL-2 (Proleukin, Chiron), and 5% heat inactivated human AB serum (Sigma Aldrich) and incubated at 37°C, 5% CO2
CD4+ CD14CD127lo CD25hi live Tregs were sorted from freshly isolated peripheral blood mononuclear cells (PBMC) of healthy volunteers (n=10) and stimulated 2:1 with anti-CD3/CD28 coated beads and maintained in the presence of 600IU/ml IL-2 and 125ng/ml rapamycin for 10 days, after which cells were replated with fresh anti-CD3/CD28 coated beads (Figures 1A and S1A, B)
Summary
Regulatory T cells (Tregs) are pivotal regulators of immune responses, maintaining self-tolerance, homeostasis, and controlling excessive immune activation through a spectrum of cell-mediated and soluble mechanisms. Tregs play a key role in the prevention of autoimmune diseases, allergies, infection-induced organ pathology, transplant rejection and GvHD. Based on encouraging results in pre-clinical models, adoptive transfer of ex vivo expanded Tregs is seen as a promising therapeutic strategy to restore immune balance and promote tolerance in individuals undergoing hematopoietic stem cell- and solid organ transplantation or suffering from autoimmune diseases such as Crohn’s disease (CD) and type 1 diabetes (T1D) [2]. Early phase clinical trials have demonstrated that adoptive Treg cell therapy is safe and feasible [3]. Many clinical trials have been compromised due to manufacturing challenges, primarily in the isolation of pure Tregs and ex vivo expansion to produce sufficient cell yields for a clinical dose [3,4,5]
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