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. 2015 Feb 26;125(9):1502-6.
doi: 10.1182/blood-2014-10-603449. Epub 2014 Nov 26.

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

Simrit Parmar  1 , Xiaoying Liu  1 , Amer Najjar  2 , Nina Shah  1 , Hong Yang  1 , Eric Yvon  1 , Katy Rezvani  1 , Ian McNiece  1 , Patrick Zweidler-McKay  3 , Leonard Miller  4 , Steve Wolpe  4 , Bruce R Blazar  5 , Elizabeth J Shpall  1
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Ex vivo fucosylation of third-party human regulatory T cells enhances anti-graft-versus-host disease potency in vivo (VSports app下载)

Simrit Parmar (V体育官网入口) et al. Blood. .

"V体育2025版" Abstract

Adoptive therapy with regulatory T cells (Tregs) to prevent graft-versus-host disease (GVHD) would benefit from a strategy to improve homing to the sites of inflammation. We hypothesized that adding fucose to human Tregs, forming the Sialyl Lewis X moiety on P-selectin glycoprotein ligand-1, would improve their trafficking pattern. The selectin pathway recruiter, α-1,3-fucosyltransferase-VI enzyme, significantly increased Treg surface fucosylation (66% vs 8%). In a xenogenic GVHD mouse model, fucosylated Tregs showed prolonged periods of in vivo persistence. When given at a lower dose compared with the untreated Tregs, the murine recipients of fucosylated Tregs maintained weight, had ameliorated clinical GVHD, and improved survival (70% vs 30%; P < . 0001). 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. VSports手机版.

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Figures

Figure 1
Figure 1
FTVI treatment increases Treg fucosylation. Fucosylation is characterized by the presence of sLeX residues, as assessed by flow cytometry, with antibody HECA-452 (BD Biosciences, San Jose, CA), raised against CLA, shown to be sLeX30. The left side of each contour plot shows the isotype control, and the right side the results of staining (percentage CLA-positive cells). (A) Positive control. CB CD34+ cells show an endogenous rate of 56.5% CLA+ cells that increases to 100% on treatment with FTVI enzyme. (B) Expanded Tregs. Untreated ex vivo-expanded Tregs show minimal endogenous fucosylation of 6.6% that increases to 65.5% on treatment with FTVI. (C) Fucosylation of Tregs leads to increased ability to bind E-selectin ligand. Expanded Tregs were treated with FTVI enzyme for 30 minutes, followed by biotin CLA–streptavidin phycoerythrin. Unlabeled selectins (E, P, L) with APC anti-Fc were used to study selectin binding ability in untreated Tregs (left) or fucosylated Tregs (right). Significant increase in the binding ability of fucosylated Tregs to E-selectin ligand was demonstrated (41% vs 2%) (upper). A high level of endogenous expression of P-selectin ligand was seen in untreated Tregs (81.7%) that increased on fucosylation (92.9%) (lower). No change was seen in the total L-selectin ligand expression (middle). (D) Increased E-selectin binding in fucosylated Tregs. Average values from 3 independent experiments show that the extent of fucosylation is related to the binding ability of E-, P-, or L- selectin. (E) Fucosylated Tregs suppress immune response in alloMLR similar to untreated Tregs. No differences in the degree of suppression of alloMLR was identified at the varying ratio of T-effector (Teff) and Treg (untreated or fucosylated). The x-axis denotes the varying ratio of Teff:Treg. Y-axis denotes counts per minute (mean ± SEM; n = 3).
Figure 2
Figure 2
Fucosylated Tregs prevent xenogenic GVHD. (A) Longer in vivo persistence of fucosylated Tregs. Equal numbers (107 cells) of eGFP-FFLuc transduced fucosylated or untreated Tregs were injected in the NSG mice on day −1, followed by 107 PBMCs on day 0. Whole-animal luciferase imaging was performed on days −1, 0, 3, 4, 5, 7, and 1016. Continued persistence of fucosylated Tregs was demonstrated as compared with untreated Tregs. (B) On the quantification of the bioluminescence, significantly higher signal was seen on day 10 (P = .04). (C) A higher frequency of circulating PBMCs (CD45+HLA-A2pos) was seen on day 31 in the untreated Tregs cohort compared with the fucosylated Treg recipient. Fucosylated (FT) Treg cells prevent xenogeneic GVHD. Sublethally irradiated NSG mice received Treg cells or FT-Treg cells at a cell dose of 1 × 106 on day −1, followed by tail vein injection of PBMCs at a cell dose of 1 × 107 on day 0. Mice were monitored every other day for weight, GVHD score, and survival. Ten mice in each group from 3 different experiments were analyzed. (D). Weight. Fucosylated Treg recipients were able to maintain weight compared with Treg recipients and PBMC-only mice (P = .03). (E) Improved GVHD score was observed in the fucosylated Treg recipients (P = .009). (F) Significant improvement in overall survival was seen in fucosylated Treg recipients compared with untreated Tregs at 1 × 106 cell dose (P < .0001).
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References

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