Ex vivo fucosylation of third-party human regulatory T cells enhances anti–graft-versus-host disease potency in vivo

The optimal way to translate the adoptive therapy with regulatory T cells (Tregs) in preventing graft versus host disease (GVHD) in the clinic remains unclear because of their small number, constituting only 1% to 5% of adult peripheral blood (PB) or umbilical cord blood (CB).^1-9  In a phase 1/2 clinical trial, large donor-to-donor variability in Treg populations was responsible for their inability to reach a clinically meaningful dose in 20% of patients and suggests that current ex vivo expansion strategies are not sufficient for broad application of this approach.²  Novel approaches to increase Treg potency are needed

Tregs bind to endothelial E- and P-selectins for trafficking to the sites of inflammation.¹⁰  E-selectin levels have been shown to be increased in patients with extensive GVHD.¹¹  Incubation of CB hematopoietic progenitor cells with the enzyme fucosyltransferase-VI results in the addition of fucose to their cell surface to form a tetrasaccharide, sialyl Lewis X (sLeX),¹²  the moiety found on P-selectin ligand.¹³  Such treatment is effective in increasing their homing and engraftment in nonobese-diabetic/severe combined immunodeficiency interleukin (IL)-2Rγ^null (NSG) mice¹⁴  and is currently being evaluated in the clinical setting.¹⁵  We hypothesized that ex vivo fucosylation would also improve Treg homing and enhance their anti-GVHD potency.

As shown previously,¹⁶  CB Treg expansion was performed with CD3/28 coexpressing Dynabeads (ClinExVivo CD3/CD28; Invitrogen Dynal AS, Oslo, Norway) in the presence of IL-2 (CHIRON Corporation, Emeryville, CA).

Ex vivo expanded Tregs were harvested and incubated in fucosylation solution (1/25 dilution of FTVI in 1 mM GDP Fucose, phosphate-buffered saline 1% human serum albumin) (Targazyme Inc, Carlsbad, California) for 30 minutes at room temperature and resuspended in phosphate-buffered saline. Fucosylation was characterized by the presence of sLeX residues, as assessed by flow cytometry with antibody HECA-452 (BD Biosciences, San Jose, CA), raised against cutaneous lymphocyte antigen (CLA), shown to be sLeX³⁰. A portion of the cells were removed pre- and postfucosylation for flow staining with CLA, CD4, CD127, and CD25 antibodies.

Allogeneic mixed lymphocyte reaction (alloMLR) was performed using T-effector cells (Teffs) from PB mononuclear cells (PBMCs) isolated from 2 unrelated donors.¹⁷  CB Tregs (fucosylated or untreated) were added in the following Teffs:Tregs ratios: 1:1, 4:1, 8:1, 10:1, 40:1, 80:1, and 100:1. Cellular proliferation was measured by incorporation of ³H-thymidine. Results were measured using cell harvester (PerkinElmer, Waltham, MA) and a liquid scintillation counter (Packard Meriden, Prospect, CT). Results are expressed in counts per minute.

Sublethally irradiated (320 cGY) NSG mice (Jackson Laboratory, Bar Harbor, ME) received intravenous injection of 10⁷ human PBMCs¹⁶  and were followed every other day for weight loss, GVHD score,¹⁸  and survival. Mice were sacrificed for moribund features, as per institutional policy. Survival was measured by Kaplan-Meier method. The GVHD score and weights were compared using the paired analysis of variance test. Protocols were approved by the Institutional Animal Care and Use Committee and the Institutional Review Board.

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 (R&D Systems, Minneapolis, MN). The selectin-bound cells were washed with phosphate-buffered saline and then incubated with DyLight 649 anti-human immunoglobulin G, Fc-γ fragment (Jackson ImmunoResearch, West Grove, PA). The cell population interacted with E-, P-, or L-selectin was then analyzed by flow cytometry.

Ex vivo expanded Tregs were labeled with a retroviral vector that expressed Firefly luciferase and enhanced green fluorescent protein (eGFP-FFLuc). Imaging was performed by injection of luciferin (20 mg/mL, 100 μL subcutaneously), followed by noninvasive whole-animal bioluminescence on an IVIS Spectrum imager, as shown before.¹⁶ 

Ex vivo fucosylation of the expanded CB Tregs was performed on day 14. A portion of the cells was removed pre- and postfucosylation for flow staining with CLA, CD4, CD127, and CD25 antibodies. Successful fucosylation of CD34⁺ cells was performed as a positive control, where a relatively high level of baseline CLA positivity was observed in activated CB CD34⁺ cells (47% ± 7%) (Figure 1A). In contrast, the baseline CLA-positivity of expanded CB Tregs was low, with average levels of 8% ± 1.3%. On incubation with FTVI enzyme, the fucosylation levels of the expanded CB Tregs increased from 8% ± 1.3% to 66% ± 4.5% (n = 5; P < .00001) (Figure 1B). Varying degrees of fucosylation were seen on testing other cell types (supplemental Figure 1, available on the Blood Web site).

We next evaluated the effect of fucosylation on the selectin homing pathways used by T cells; namely, E-, P- and L-selectins. As compared with untreated Tregs, fucosylation of CB Tregs led to their increased binding ability to E-selectin compared with untreated controls (1.5% ± 0.5% vs 38% ± 0%; P = .0001) (Figure 1C-D). The binding ability of untreated vs fucosylated Tregs for P-selectin ligand was 77% ± 3% vs 91.5% ± 0.5% (P = .04). In contrast, no significant effects were observed for L-selectin ligand expression (37% ± 14% vs 32.5% ± 7.5%; P = .8), respectively. It is possible that the homing properties of Tregs permit superior survival as a result of local environmental effects as to where Tregs reside.

To quantify their suppressive function, we showed that in an alloMLR, both untreated and fucosylated Tregs exerted similar levels of suppression at different Treg ratios, demonstrating no significant advantage of fucosylation on their in vitro function (Figure 1E).

To study their in vivo persistence, ex vivo expanded Tregs were labeled with a retroviral vector that expressed Firefly luciferase and eGFP (eGFP-FFLuc), with a transduction efficiency of 20%, followed by division of the CB Tregs into 2 fractions, where 1 fraction was fucosylated and the other untreated. Both fractions were injected at 1 × 10⁷ cells into sublethally irradiated NSG mice. The next day, mice were injected with PBMCs at 1 × 10⁷ cells. Tregs were from an HLA-A2^neg CB source and PBMCs were from an HLA-A2^pos PB source. As shown in Figure 2A, the average bioluminescent radiance in the fucosylated Treg recipients was significantly higher than in the untreated Treg recipients on days 0 and 10 postinfusion (Figure 2B). The higher frequency of fucosylated vs untreated Tregs on day 10 was predictive of observed lower PB frequencies of GVHD-inducing CD45⁺HLA-A2⁺ PBMCs on day 31 posttransplant (14% vs 88%, respectively; P < .00001) (Figure 2C).

According to preclinical data, a 1:1 ratio of Treg:conventional T-cell is optimal for Tregs to effectively suppress an allogeneic reaction.^3-5  We hypothesized that the greater persistence of fucosylated vs untreated Tregs would lead to superior GVHD protection by increasing the Treg:conventional T-cell ratio in vivo. We tested 10- to 1000-fold lower ratios of Tregs to PBMCs. Sublethally irradiated NSG mice were given fucosylated Tregs or untreated Tregs at a cell dose of 1 × 10⁶ on day −1, followed by a 10-fold higher PBMC cell dose of 1 × 10⁷ on day 0. Fucosylated Treg recipients regained their pretransplant body weight, in contrast to significant weight loss observed in the untreated Treg recipients (Figure 2D). Furthermore, an improved clinical GVHD score¹⁸  (5.7 vs 1.2; Figure 2E) was noted. Most important, at day 30, fucosylated Treg recipients had improved overall survival (70%) when compared with untreated Tregs (23%) and PBMCs alone (0%; P < .0001; Figure 2F). In preliminary experiments, improved survival was associated with fucosylated Tregs at 1 × 10⁵ cell dose when comparing untreated Tregs and PBMCs alone (supplemental data; Figure 2). No significant survival difference was noted in the fucosylated Tregs recipients at a 1 × 10⁴ cell dose (data not shown).

If, as we demonstrate in this xenogeneic GVHD model, ex vivo fucosylation of expanded human CB Tregs can increase their persistence and anti-GVHD potency, this may allow more patients to benefit from the adoptive therapy with third-party CB-derived Tregs. Such benefits of ex vivo fucosylation may also be extended to Tregs derived from other donor products. Furthermore, by empowering the Tregs by fucosylation, we propose to potentially reduce GVHD as a result of improved homing and trafficking. Whether these benefits will be realized in patients receiving fucosylated Tregs will be addressed in a future phase 1 clinical trial.

This work has been supported by the Anderson Cancer Center Start Up Funds and Lee Clark Award grants R01 HL11879, P01 CA065493, CA142106, and AI056299.

Contribution: S.P., X.L., and A.N. designed research, conducted experiments, analyzed data, and participated in manuscript writing; and N.S., H.Y., E.Y., K.R., I.M., P.Z.-M., L.M., S.W., B.R.B., and E.J.S. designed research, analyzed data, and participated in manuscript writing.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Simrit Parmar, University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Unit 423, Houston, TX 77030; e-mail: sparmar@mdanderson.org.