Abstract
Introduction
Immunotherapy using cytotoxic T lymphocytes (CTL) has shown efficacy in the management of leukemia. However the efficacy of CTL, whether they are engineered and adoptively transferred or administered as part of allogeneic stem cell transplantation, must be balanced by their off-target toxicities, which at times can be lethal. Fucosylation, which is mediated by fucosyl transferases, is a process by which fucose sugar groups are added to cell surface receptors. Fucosylated T cells have been shown to preferentially home to inflamed tissues, including bone marrow. In view of recent data showing that fucosylation with fucosyltransferase (FT)-VI facilitates homing of regulatory T cells (T-regs) to inflamed tissues and cord blood engraftment into the bone marrow, we hypothesized that fucosylation could enhance the efficacy of CTL that target leukemia antigens. In this study, we tested whether ex vivo fucosylation of CTL that target the HLA-A2 restricted leukemia peptides, CG1 (derived from cathepsin G) and PR1 (derived from neutrophil elastase and proteinase 3), with the novel enzyme FT-VII enhances their migration and anti-leukemia functions.
Experimental design
CG1- and PR1-CTL were generated using standard methodologies. Fucosylation was achieved by incubating T cells with FTVII enzyme and GDP fucose (Targazyme). To study migration, fucosylated and non-fucosylated CTL were passed through chambers coated with a HUVEC barrier and migrated CTL were detected using cell fluorescence. To examine CTL surface markers, cells were stained for standard co-stimulatory and adhesion molecules and were analyzed using flow cytometry. Calcein AM cytotoxicity assays were used to determine the effects of fucosylation on CTL killing of target cells. In vitro effects of fucosylation on leukemia-CTL specificity was accomplished using standard CFU assays. For in vivo assessment of fucosylation on activity of CTL, NSG mice were engrafted with U937-A2 human acute myeloid leukemia (AML) cells or primary AML and were treated with intravenous injections of 5.0 x 105 fucosylated or non-fucosylated CTL. Mice were followed twice weekly and were sacrificed for bone marrow and tissue analysis at prespecified time points or when they became moribund.
Results
Fucosylated CG1-CTL and PR1-CTL showed approximately 2-fold higher migration through the HUVEC cell barrier compared to non-fucosylated CTL. Analysis of T cell surface expression of chemokine/adhesion molecules showed an approximately a 5-fold increase in CD49d and CD195, and a 50% increase in CXCR1 and CXCR3 following fucosylation. Fucosylation enhanced the cytotoxicity of leukemia specific-CTL against primary HLA-A2+ leukemia and HLA-A2+ U937 cells at increasing effector to target ratios. For primary patient AML, we show enhanced leukemia killing by fucosylated-PR1-CTL in comparison with non-fucosylated-PR1-CTL at the 20:1 effector to target (E:T) ratio (25-fold higher killing ) and the 10:1 E:T ratio (4-fold higher killing). Similar results were seen using the U937-A2 AML cell line favoring fucosylated-CG1-CTL: 20-fold higher killing at 20:1 E:T ratio and a 9-fold higher killing at the 10:1 E:T ratio. In vitro CFU assays using HLA-A2+ healthy donor bone marrow showed no change in the specificity of the antigen specific CTL following fucosylation. Specifically we show 283 and 295 colonies in the fucosylated and non-fucosylated CG1-CTL groups, respectively (P >0.05). These were also compared to irrelevant peptide HIV-CTL, which demonstrated 286 and 269 CFUs in the fucosylated and non-fucosylated HIV-CTL groups, respectively (P >0.05). In vivo experiments using CG1-CTL against primary AML showed 5-fold higher killing of AML by fucosylated CTL vs. non-fucosylated CTL. Similar results were also seen using U937-A2 AML targets.
Conclusion
Fucosylation with FT-VII enhances the efficacy of leukemia-targeting CTL against primary human AML and AML cell lines. These data demonstrate a novel approach to enhance the efficacy of antigen specific CTL that could be used in adoptive cellular immunotherapy approaches for leukemia.
Disclosures
No relevant conflicts of interest to declare.
Reduced regulatory T cells (Treg) in bone marrow preferentially associate with the expansion of cytotoxic T lymphocytes in low risk
MDS
patients
