Page 232 - 2019_05-HaematologicaMondo-web

L.S. Hall et al.
bodies reactive to purified GPIIb/IIIa subsequently meas- ured by ELISA (Figure 1A). It can be seen that only purified GPIIb/IIIa, and none of the peptides, elicited a significant antibody response.
Prevention of antibody responses to glycoprotein IIb/IIIa by individual peptides
The seven peptides were next tested individually for their ability to prevent the IgG antibody response to GPIIb/IIIa immunization. Different groups of mice were each given one of the peptides, or left untreated, before immunization, and the development of anti-GPIIb/IIIa plasma antibodies compared (Figure 1B). None of the pep- tides consistently blocked the development of antibody, although there were non-significant trends for lower responses after delivery of peptides 2 (aa6-20), 70 (aa591- 605) or 82 (aa711-725).
Prevention and inhibition of antibody responses to glycoprotein IIb/IIIa by a peptide combination
It has been demonstrated that peptides used in combi- nation can be more effective than individual sequences in preventing or reversing allo- or auto-antibody produc- tion.32,34,42,43 Peptides 2 (aa6-20) and 82 (aa711-725) were selected to be tested in combination here, since epitopes they contain were previously identified as the most immunodominant,7 capable of eliciting Th responses in 65% of ITP patients tested, and they had also each demonstrated some, albeit variable, inhibition of antibody generation in the individual peptide experiments. It was determined whether prior treatment with an equimolar mixture of peptides 2 (aa6-20) and 82 (aa711-725) prevent- ed induction of the antibody response to immunization with purified GPIIb/IIIa. Given that the antibody levels are measured as relative optical density (OD) values in
A
Figure 3. Treatment of HLA-DR15 transgenic mice with glycoprotein IIIa peptides containing Th epitopes modulates splenocyte proliferative responses in vitro. Splenocytes were obtained from mice that were untreated controls (non- immunized); immunized with GPIIb/IIIa only (immunized only); pre-treated with a mixture of GPIIb/IIIa peptides 2 (aa6-20) and 82 (aa711- 725) before GPIIb/IIIa immunization (preven- tion); given the mixture of peptides after GPIIb/IIIa immunization (inhibition); or given the mixture of peptides only (tolerized). The panels show proliferation of splenocytes in response to stimulation in vitro with (A) purified GPIIb/IIIa, (B) peptide 2 or (C) peptide 82. Data points rep- resent results from individual mice: (A) non- immunized (n=10), immunized only (n=9, pre- vention (n=9), inhibition (n=12), tolerized (n=3); (B and C) non-immunized (n=7), immunized only (n=6), prevention (n=6), inhibition (n=5). *P<0.05, **P<0.001, t-test. CPM: counts per minute.
BC
1078
haematologica | 2019; 104(5)