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GPIIb/IIIa peptide immunotherapy in HLA-transgenic mice
ELISA, and not absolute concentrations, the results were designed to be interpreted as longitudinal studies of mice in each group. In contrast to control mice that received only the immunization, no significant antibody response against GPIIb/IIIa could be detected in animals pre-treated with the peptide combination (Figure 2A, left and middle panels). Extending the time after GPIIb/IIIa immunization
by a further 14 days in another group of pre-treated mice (n=3) saw no increase in antibody levels, consistent with a sustained loss, rather than delay, in responsiveness (mean ± standard deviation OD: 2.92±0.09 at day 42 versus 3.03±0.1 at day 56, n=3).
Having demonstrated that, unlike either individual pep- tide, pre-treatment with the combination of peptides 2
A
B
C
Figure 4. Treatment of HLA-DR15 transgenic mice with glycoprotein IIIa peptides containing Th epitopes modulates splenocyte populations with a CD4+CD25+FoxP3+ Treg phenotype and suppressive function. 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 (prevention); given the mixture of peptides after GPIIb/IIIa immunization (inhibition). The cells were left as unstimulated controls (none; no stimulus), or stimulated in vitro with either purified GPIIb/IIIa (GPIIIa; GPIIIa stimulation) or a mixture of all seven GPIIIa peptides (Pmix; Pmix stimulation). (A) Representative examples of flow cytometric analy- ses of CD25 and FoxP3 expression after gating on CD4+ cells. (B) Proportions of CD25+FoxP3+ cells within the splenic CD4+ populations from all the mice. Data points represent results from individual mice (non-immunized) (n=7), immunized only (n=6), prevention (n=6), inhibition (n=5). (C) The effect of depleting CD25+CD4+ cells (CD25-) on the ability of splenocytes from mice in the prevention group (n=3) to make proliferative responses when stimulated with GPIIb/IIIa. **P<0.005, t- test. CPM: counts per minute.
haematologica | 2019; 104(5)
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