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L.S. Hall et al.
Figure 5. Treatment of HLA-DR15 transgenic mice with glycoprotein IIIa peptides containing Th epitopes does not modulate splenocyte interleukin-10 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 (prevention); or given the mixture of peptides after GPIIb/IIIa immu- nization (inhibition). The cells were left as unstimulated controls (no stimulation; left panel), or stimulated in vitro with either purified GPIIb/IIIa (GPIIIa stimulation; middle panel) or the mixture of GPIIIa peptides (Pmix stimulation; right panel) and the production of interleukin-10 in culture measured by enzyme-linked immunosor- bent assay. Data points represent results from individual mice (non-immunized) (n=9), immunized only (n=9), prevention (n=9), inhibition n=8). In each panel, there are no significant differences in interleukin-10 production between the unimmunized control group and any treated groups (t-test).
(aa6-20) and 82 (aa711-725) could prevent anti-GPIIb/IIIa antibody generation, we next determined whether the mixture could also suppress responses that had already been induced, by administering the peptide product after immunization with GPIIb/IIIa (Figure 2A, right panel). As expected, a significant response to immunization was detectable by the time of the peptide administration, but there was a slight, non-significant fall in mean antibody levels over the next 14 days after treatment (mean ± stan- dard deviation OD: 0.55±0.1 at peptide treatment on day 28 versus 0.54±0.1 on day 42, n=8; P=0.8). By contrast, mean antibody levels continued to rise, modestly but sig- nificantly, in the group of control mice that received only immunization (Figure 2A, left panel) (mean ± standard deviation OD: 0.96±0.53 on day 28 versus 1.1±0.54 on day 42, n=14; P<0.05). Thus, the peptide combination blunted, but did not significantly reverse, ongoing responses to GPIIb/IIIa.
The suppressive effect of treatment of transgenic mice with the peptide combination was specific to GPIIb/IIIa responses, since there was no effect on their ability to make antibody responses to immunization with the unre- lated control antigen ovalbumin (Figure 2B).
Prevention and inhibition of T-cell responses
to glycoprotein IIb/IIIa by the peptide combination
Class-switched antibody responses classically depend on antigen-specific Th cells,19,20 which peptide immunotherapy is intended to target and render unre- sponsive.28-31 To determine the effect of treatment with the combination of GPIIIa peptides 2 (aa6-20) and 82 (aa711- 725) on Th effector function, we obtained splenocytes from control unimmunized or GPIIb/IIIa-immunized mice, or from animals that had received the peptides
either before or after immunization, and compared their ability to proliferate in vitro when stimulated with purified GPIIb/IIIa (Figure 3A). As expected, splenocytes from mice that been immunized with GPIIb/IIIa proliferated in response to the antigen, whereas there were no responses in control, unimmunized animals. Flow cytometric analy- ses demonstrated that T cells with the CD4+ helper phe- notype predominated in the proliferating cultures (>70% in all cases, n=6). Proliferation against GPIIb/IIIa was sig- nificantly reduced when immunized mice had been pre- treated with the combination of peptides 2 (aa6-20) and 82 (aa711-725), and there was also a non-significant trend for responses to be inhibited when mice were treated with the peptides after immunization.
Splenocyte responsiveness to the individual GPIIIa pep- tides 2 (aa6-20) or 82 (aa711-725) was also assayed (Figure 3B). Peptide 2 (aa6-20) elicited no consistent proliferative responses in any group, but peptide 82 (aa711-725) demonstrated significant stimulatory ability in both groups of animals that had received the peptide combina- tion in addition to immunization.
Induction of regulatory T cells by the peptide combination
Having demonstrated that treatment with a combina- tion of GPIIIa peptides can suppress both antibody and effector T-cell responses to GPIIb/IIIa immunization, we determined whether this was associated with induction of Treg cells. The proportions of cells exhibiting the CD25+FoxP3+ Treg phenotype within the CD4+ splenocyte population were compared in control, unimmunized and GPIIb/IIIa-immunized mice, and in animals that had received the peptide mixture either before or after immu- nization. Treg cells were enumerated both when spleno-
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haematologica | 2019; 104(5)