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GPIIb/IIIa peptide immunotherapy in HLA-transgenic mice
or for the control antigen ovalbumin, were adapted from a pub- lished method.38 Microtiter plates (Nunc, Fisher Scientific, Loughborough, UK) were coated with either purified GPIIb/IIIa or ovalbumin, incubated with plasma samples diluted 1:20 in phos- phate-buffered saline, probed with anti-mouse IgG conjugated to alkaline phosphatase (Invitrogen, Fisher Scientific), and developed with p-nitrophenyl phosphate substrate (Sigma). The absorbance was measured at 405 nm.
Statistical analyses
Statistical tests were performed using SigmaPlot (SyStat Software).
Results
Seven peptides (Table 1) containing GPIIIa epitopes that are commonly recognized by Th cells from patients with ITP and are, therefore, candidates for inclusion in an immunotherapeutic product7 were screened for their effects on immunity to GPIIb/IIIa in HLA-DR15 transgenic mice. The genetically modified strain was used, not to
ensure responses to immunization with human GPIIb/IIIa, but in order to recapitulate in vivo the presentation of par- ticular GPIIIa peptides by human MHC class II molecules. GPIIIa peptides containing the T-cell epitopes that had been mapped in ITP patients would not necessarily be pre- sented by murine class II molecules to modify responses of wild-type mice to GPIIb/IIIa, and any effects would not be of direct relevance to human disease. The administra- tion of the peptides, given in soluble form via the subcu- taneous route in the absence of adjuvant signals, was designed to favor induction of regulatory versus effector responses.28,29,34,35
Stimulation of antibody responses to glycoprotein IIb/IIIa
It was first necessary to confirm that HLA-DR15 trans- genic mice could mount an antibody response to GPIIb/IIIa after immunization with the purified full-length antigen, and that no such response was elicited by of any of the seven candidate peptides considered as putative suppressive treatment. Purified GPIIb/IIIa or individual GPIIIa peptides were administered to mice, with IgG anti-
A
B
Figure 2. Treatment of HLA-DR15 transgenic mice with a combination of glycoprotein IIIa peptides containing Th epitopes inhibits anti- body responses to immunization with purified glycoprotein IIb/IIIa, but not the control antigen ovalbumin. Levels of plasma IgG antibodies reactive to (A) the antigen purified GPIIb/IIIa or (B) ovalbumin were measured by enzyme-linked immunosorbent assay in serial plasma samples from mice only immunized with GPIIb/IIIa or ovalbumin (immunized only); or pre-treated with a mixture of GPIIb/IIIa peptides 2 (aa6-20) and 82 (aa711-725) before immunization (preven- tion); or given the mixture of peptides after immunization (inhibition). Arrows indicate timing of peptide treatment (P2&82) and immuniza- tions with GPIIb/IIIa (GPIIIa) or ovabumin (OVA). Lines connect serial data points from individual mice: (A) immunized only (n=14), prevention (n=9), inhibition (n=8), tolerized (n=3); (B) immu-
nized only (n=3),
***P<0.0005, **P<0.005, *P<0.05, paired t- test, ns indicates not significant.
prevention (n=3).
haematologica | 2019; 104(5)
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