Agonistic CD200R1 DNA Aptamers Are Potent Immunosuppressants That Prolong Allogeneic Skin Graft Survival

Abstract

CD200R1 expressed on the surface of myeloid and lymphoid cells delivers immune inhibitory signals to modulate inflammation when engaged with its ligand CD200. Signalling through CD200/CD200R1 has been implicated in a number of immune-related diseases including allergy, infection, cancer and transplantation, as well as several autoimmune disorders including arthritis, systemic lupus erythematosus, and multiple sclerosis. We report the development and characterization of DNA aptamers, which bind to murine CD200R1 and act as potent signalling molecules in the absence of exogenous CD200. These agonistic aptamers suppress cytotoxic T-lymphocyte induction in 5-day allogeneic mixed leukocyte culture and induce rapid phosphorylation of the CD200R1 cytoplasmic tail thereby initiating immune inhibitory signalling. PEGylated conjugates of these aptamers show significant in vivo immunosuppression and enhance survival of allogeneic skin grafts as effectively as soluble CD200Fc. As DNA aptamers exhibit inherent advantages over conventional protein-based therapeutics including low immunogenicity, ease of synthesis, low cost, and long shelf life, such CD200R1 agonistic aptamers may emerge as useful and safe nonsteroidal anti-inflammatory therapeutic agents.

Figure 1

DNA aptamers selected to bind to the extracellular domain of murine CD200R1 suppress cytotoxic T lymphocytes (CTL) induction in primary allo-MLC. (a) Table of selected aptamer sequences identified after 15 iterative SELEX rounds. Full-length aptamers consist of an internal variable region of 25nt, flanked by two constant regions of 25nt each. A control aptamer (cApt) composed of a randomly selected variable sequence and the same constant regions present in the library was used throughout this study. (b) DNA aptamers were added to 5-day allo-MLC with 2.5 × 10⁵ C57BL/6 responder cells incubated with an equal number of irradiated BALB/c stimulator cells. Induction of CTLs was monitored by the release of ⁵¹Cr from P815 mastocytoma target cells at a 25:1 effector-to-target ratio. Soluble CD200Fc was used as a positive control. Each bar represents the mean percent of CTL-specific lysis ± SEM from three replicate wells. Aptamers were added at 975 nmol/l (closed bars), 325 nmol/l (open bars), or 110 nmol/l (hatched bars). *P < 0.01 when compared to cApt or no treatment.

Figure 2

Truncation of CD200R1 aptamers M49 and M52. (a) Table of aptamer sequences truncated based on secondary structure predictions. Shown is aptamer name, sequence, and length in nucleotides (nt). The constant primer regions on each sequence are highlighted in red. (b) Secondary structure predictions derived from mfold were used to guide truncation of M49 and M52. (c) Truncated aptamers were added to 5-day allo-MLC and suppression of CTL induction assayed as described. Truncated sequences M52-T1 and M49-T1 suppress CTL induction with comparable efficacy to that of the full-length aptamers. Each bar represents the mean percent CTL specific lysis ± SEM from three replicate wells and is representative of two independent experiments (*P < 0.01, NS, not significant).

Figure 3

PEG-conjugated M49 and M52 function act as CD200R1 agonists. (a) PEGylated and non-PEGylated aptamers (325 nmol/l, based on nucleic acid content) were added to 5-day allo-MLC and suppression of cytotoxic T lymphocytes (CTL) induction assayed. PEG-M49 and PEG-M52 significantly suppress CTL induction. Each bar represents the mean percent CTL specific lysis ± SEM from three replicate wells. *P < 0.01 relative to no treatment; NS, not significant. (b) HEK-293 cells (grey) or HEK-293 transfectants expressing CD200R1 on the cell surface (white) were stained with a fluorescein isothiocyanate-labelled anti-CD200R1 antibody and analyzed by flow cytometry to confirm expression. (c) HEK293 cells expressing CD200R1 were incubated with the indicated concentrations of PEG-M49, PEG-M52, or PEG-cApt to monitor aptamer-induced phosphorylation of the CD200R1 cytoplasmic tail. The phosphorylated form of CD200R1 was detected by western blot using an antibody specific to phosphorylated-CD200R1 cytoplasmic tail. Supernatant from CD200-expressing cells was used as a positive control. Both PEG-M49 and PEG-M52 induce CD200R1 intracellular phosphorylation.

Figure 4

PEG-M49 and PEG-M52 function in vivo. (a) Experimental outline of in vivo experiment. BALB/c skin allografts (day 0) were transplanted onto C57BL/6 mice. These animals were subsequently treated over a 12-day period with tail vein injections of either 650 pmol PEG-M49, PEG-M52, PEG-cApt, or 325 pmol of CD200Fc at 72-hour intervals in combination with a low dose of rapamycin (0.5 mg/kg, 36 hours, i.p.). (b) After 14 days, mice were sacrificed and their splenocytes used as responder cells in ex vivo allo-MLC assays. Cultures were subsequently treated in vitro with further addition of aptamers or CD200Fc in culture (open bars) or left as is (closed bars). The in vivo administration of PEG-M49 and PEG-M52 was sufficient to suppress CTL induction without further addition of aptamer in culture (*P < 0.01). Each bar represents the mean percent of CTL specific lysis ± SEM from three replicate wells and data shown is representative of two independent experiments.

Figure 5

PEG-M49 and PEG-M52 prolong the survival of transplanted murine skin grafts. (a) Experimental outline of in vivo experiment. C57BL/6 mice (n = 6) received BALB/c skin allografts on day 0 and were treated every 3 days over 15 days with phosphate-buffered saline (PBS) (closed triangles) or 650 pmol of either PEG-cApt (open triangles), PEG-M49 (open circles), PEG-M52 (closed squares), or 325 pmol of CD200Fc (closed circles) in combination with low-dose rapamycin (0.5 mg/kg, 36 hours, i.p.). (b) Treatment with PEG-M49 and PEG-M52 significantly extended graft survival (P < 0.05, Mann–Whitney U-test) relative to control. Arrow represents time of last injection. Data shown is representative of two independent experiments.