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Ex Vivo Fucosylation of Third-party Human Tregs Enhances Anti-GVHD Potency In Vivo

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Graft versus host disease (GVHD) is a rare disease, given that this disease can only develop after a stem cell transplant is performed, and only a small fraction of people undergo stem cell transplants. However, amongst those who do undergo stem cell transplants, the incidence of GVHD is high (35-50%). Thus, global scientists are working to find a way to treat GVHD.

This research shows that adoptive therapy with regulatory T cells (Tregs) to prevent GVHD would benefit from a strategy to improve homing to the sites of inflammation. In a xenogenic GVHD mouse model, fucosylated Tregs showed prolonged periods of in vivo persistence and improved survival. These preclinical data indicate that fucosylated human Tregs is an effective strategy for prevention of GVHD and, as such, warrants consideration for future clinical trials.

GVHD is a condition that might occur after an allogeneic transplant. In GVHD, the donated bone marrow or peripheral blood stem cells view the recipient's body as foreign, and the donated cells/bone marrow attack the body. It is commonly associated with bone marrow transplants and stem cell transplants and between 20% to 80% of people who have a transplant can be at risk of developing the condition.

The disease can be classified as acute GVHD and chronic GVHD, and both types can be dangerous and may result in death.

A research team from the University of Texas MD Anderson Cancer Center found that fucosylated Tregs can improve in vivo persistence, decrease GVHD, and increase overall survival in a xenogenic GVHD mouse model.

The whole research process follows the steps below:

1. CB (cord blood) Treg isolation and expansion: CB Treg expansion was performed with CD3/28 coexpressing Dynabeads.

2. Fucosylation of expanded Tregs: ex vivo expanded Tregs were harvested and incubated in fucosylation solution and resuspended in phosphate-buffered saline. Fucosylation was characterized by the presence of sLeX residues. A portion of the cells was removed pre- and postfucosylation for flow staining with CLA, CD4, CD127, and CD25 antibodies.

3. Mixed lymphocyte reaction: Allogeneic mixed lymphocyte reaction was performed using T-effector cells from PB mononuclear cells isolated from 2 unrelated donors. CB Tregs (fucosylated or untreated) were added in different ratios. Cellular proliferation was measured by incorporation of 3 H-thymidine. Results were measured using a cell harvester and a liquid scintillation counter.

4. Xenogenic GVHD mouse model: sublethally irradiated NSG mice received an intravenous injection of 107 human PBMCs and were followed every other day for weight loss, GVHD score, and survival.

5. Selectin binding assay: functional group analysis of E-, P-, L-selectin binding was made by incubating the Tregs with Fc Chimera of recombinant human E-, P-, or L-selectin. The selectin-bound cells were washed with phosphate-buffered saline and then incubated with DyLight 649 anti-human immunoglobulin G, Fc-g fragment. The cell population interacted with E-, P-, or L-selectin was then analyzed by flow cytometry.

6. Noninvasive bioluminescence imaging: ex vivo expanded Tregs were labeled with a retroviral vector that expressed Firefly luciferase and enhanced green fluorescent protein. Imaging was performed by injection of luciferin, followed by noninvasive whole-animal bioluminescence on an IVIS Spectrum imager.

This research demonstrates in this xenogeneic GVHD model that ex vivo fucosylation of expanded human CB Tregs can increase their persistence and anti-GVHD potency, which may allow more patients to benefit from the adoptive therapy with third-party CB-derived Tregs. The research team also indicates that whether these benefits will be realized in patients receiving fucosylated Tregs will be addressed in a future phase I clinical trial.

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