Three different methods allow large-scale expansion of clinically relevant quantities of immunosuppressive T cells.

Three research teams are reporting alternative methods for generating clinically meaningful quantities of regulatory T cells (Treg) that could potentially be used to prevent the immune systems of organ transplant recipients from rejecting donor tissues. In vivo tests showed that administering Tregs specific to donor antigens effectively prevented the attack of allogeneic skin grafts and artery transplants in mice.

In one of the studies Tregs prevented GvHD (graft versus host disease) lethality in immune-deficient mice receiving infusions of human T cells. All three research papers are published in Science Translational Medicine.

Although immunosuppressive drugs can effectively manage acute rejection, many transplant recipients will eventually succumb to immune-mediated chronic allograft rejection, or the toxicities and side effects associated with long-term immunosuppression, notes a U.K. team led by Giovanni Lombardi, M.D., at the MRC Centre for Transplantation, King’s College. Unfortunately, once chronic rejection has occurred, the only option is to carry out a repeat transplantation.

One potential alternative would be to exploit the body’s subset of naturally occurring immunosuppressive Tregs to block the immune system’s attack on transplanted organs and tissues. Tregs are a functionally and phenotypically distinct subset of lymphocytes that constitute about 1–5% of CD4+ T cells and in humans are characterized by constitutive expression of the transcription factor FoxP3 (Forkhead box P3).

It is technically possible to collect and enrich Tregs from a prospective transplant recipient, expand them ex vivo, and then reintroduce them into the patient after transplantation to alter the in vivo balance of T effector cells (Teffs) and Tregs. However, Dr. Lombardi’s team points out that administering ex vivo-expanded polyclonal Tregs could feasibly result in pan-immunosuppressive effects that cause the same problems as drug-based immunosuppression.

A far better alternative is to generate selective Tregs that only repress the immune system’s attack on cells and tissues displaying donor antigens. Current methods for the ex vivo expansion of human donor-specific Tregs require repetitive stimulation of polyclonal Tregs with antigen-presenting cells (APCs) to increase the proportion of Tregs with alloantigen specificity. Technically this is a hassle, the MRC researchers point out, and limited both by the logistical challenge of expanding such a small cell population, and the potential persistence of nonspecific Tregs.

Dr. Lombardi’s team has now identified CD69 and CD71 as cell surface markers that are co-expressed by Tregs activated by allogeneic antigens. This feature allowed them to use flow cytometry to sort and select the rare cells expressing these markers. In vitro tests showed that CD69+CD71+ Tregs enriched after activation with donor dendritic cells were able to mediate potent and specific suppression of autologous Teff proliferation following subsequent antigen challenge at Treg/Teff ratios as low as 1:100.

Importantly for potential clinical applications, sorted alloantigen-activated Tregs could be readily expanded in vitro in the presence of high doses of exogenous interleukin-2 (IL-2) without the need for further restimulation. Encouragingly, the antigen-specific Treg lines maintained the expression of Treg phenotypic markers after 4–6 weeks of culture.

The team then tested their Treg cells in a humanized xenograft mouse model of allogeneic CD4-mediated allograft injury. Teffs in combination with either polyclonal Tregs (Pc-Tregs) or antigen-specific Tregs (AgS-Tregs) were administered to the animals at a ratio of 5:1.

When the skin grafts were subsequently evaluated the researchers found that while PC-Tregs did help  prevent tissue damage induced by Teff-triggered immune responses, grafts in the Ags-Treg-treated animals were completely protected and retained histology comparable to healthy skin. The research is published in a paper titled “Human Regulatory T Cells with Alloantigen Specificity Are More Potent Inhibitors of Alloimmune Skin Graft Damage than Polyclonal Regulatory T Cells.”

The method for antigen-specific Treg enrichment developed by a team at the John Radcliffe Hospital’s Transplantation Research Immunology Group, is based on a different approach. Andrew Bushell, M.D., and colleagues stimulated mouse CD4+ T cells by culturing them with immature allogeneic dendritic cells in the presence of the phosphodiesterase 3 (PDE) inhibitor cilostamide to enrich for allogen-specific Foxp3+ T cells. Without further manipulation or selection, the resulting population delayed skin allograft rejection in mouse models.

The team subsequently found the approach was just as effective at generating human Foxp3-expressing cells, which inhibited T-cell proliferation in a standard in vitro mixed lymphocyte assay. They tested these human cilostamide-induced Tregs in a mouse model of human artery transplantation.

While the grafts in untreated animals demonstrated significant immune-related damage, those in the cilostamide-treated T-cell recipients were protected. The authors conclude that their findings “establish a method for the ex vivo generation of graft-reactive, functional mouse and human Tregs that uses a clinically approved agent, making pharmacological PDE inhibition a potential strategy for Treg-based therapies.” Dr. Bushell’s team’s work is described in a paper titled “Functional Regulatory T Cells Produced by Inhibiting Cyclic Nucleotide Phosphodiesterase Type 3 Prevent Allograft Rejection.”

A team led by Bruce R. Blazer, M.D., at the University of Minnesota Cancer Center’s division of bone marrow transplantation, meanwhile, has built on its previous work demonstrating that natural Tregs could be purified from umbilical cord blood (UCB) and expanded ex vivo using anti-CD3/CD28 mAb-coated microbeads and IL-2. A prior clinical safety trial demonstrated that these cells retained their Foxp3+ phenotype and could be tracked in blood for up to 14 days.

While the cells were well tolerated and there was also an indication that treatment reduced the incidence of graft versus host diseases in recipients, the maximum cell dose was limited by insufficient and variable Treg expansion rates. In contrast to umbilical cord blood, peripheral blood (PB) harbors larger numbers of Treg cells, and would also provide a source of autologous cells.

Unfortunately, Dr. Blazer’s group notes,  “cell sorting is a challenging GMP procedure, and overall nTreg yield from PB obtained with this isolation and expansion approach is not greatly increased over that from UCB.” Moreover, while restimulation of BP-derived cells increased total expansion about 1000-fold, cultures frequently lost Foxp3 expression and suppressive function.

The team has now developed a method, using GMP reagents and protocols, for purifying and expanding polyclonal natural Treg cells from PB. The process involves purifying cells from leukapheresis products via a two-step protocol using GMP antibody-coated magnetic beads, in which CD4+ cells are enriched by depleting cells expressing CD8, CD14, and CD19, followed by positive selection of CD25high cells, and repeated stimulation with cell-based artificial antigen presenting cells (aAPCs) to generate Foxp3+ Tregs.

The team then tested cells generated by this method which had been expanded 50 million-fold in a xenogeneic model of GvHD in which nTregs were co-transferred at a 1:1 ratio with allogeneic peripheral blood mononuclear cellss. Although the treated animals did eventually succumb to GvHD, the transferred nTregs increased median survival time and reduced weight loss.

“The degree of nTreg expansion reported here could lead to the widespread application of nTreg cellular therapy for GvHD and graft rejection through the creation of an off-the-shelf therapy using nTreg banks generated from human leukocyte antigen (HLA)–typed donors with known safety and potency records,” the authors conclude.

“The massive expansion observed with repetitive polyclonal stimulation should also allow relatively rare, autoantigen-specific nTreg clones to be expanded to treat autoimmune diseases. Ultimately, this strategy could be applied to expansion of antigen-specific nTregs.”

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