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L.S. Hall et al.
spleens after specific peptide therapy. Treg cells can sup- press antibody responses not only by blocking Th activity, but also by direct effects on B cells.40 Unlike some other examples of successful peptide therapy,28-31,44 suppression in our model was not associated with IL-10 responses, suggesting it is mediated by the CD4+CD25+FoxP3+ Treg we observed, which are classically cytokine independent, rather than by other cells such as the T regulatory 1 (Tr1) type. One notable feature of our study is that, while both immunization of mice with purified GPIIb/IIIa, and treat- ment with the peptide combination, induced Treg pheno- type cells, the populations differed in specificity. This dif- ference is demonstrated by the ability of the splenic Treg phenotype population induced in immunized mice to respond to GPIIb/IIIa, but not the peptides, and suggests an explanation for the success of immunization, and the ability of splenocytes to proliferate, in the face of such a population. Thus, the cells with the Treg phenotype induced as part of the response to GPIIb/IIIa are unable to block immunization, at least within the timeframe of our experiments, but the peptides tip the balance to suppres- sion by recruiting more effective, or additional, Treg cells not induced by the antigen alone. It is recognized that antigens and peptides can induce both effector and regula- tory cells, and that an evolving balance between these populations, particularly inter-conversion of Teff and Treg, determines the functional outcome.21,22 Our approach to peptide therapy demonstrated high efficacy in preventing antibody and T-cell responses to GPIIb/IIIa when the pep- tides were given prior to immunization, and although developing responses were blunted when mice were treat- ed after immunization, antibody levels did not fall. This may reflect a relatively slow decay of established respons- es in the face of regulation, which, if necessary, could be augmented by additional peptide doses,43 since our regi- men for combination peptide therapy was a single subcu- taneous injection, for easy translation to clinical practice. Alternatively, peptide therapy could also be combined with existing, more rapidly acting approaches, such as use
of the biologic rituximab to target B cells directly, while peptide-induced Treg cells block longer-term recruitment to the immune response. Solubility is a key feature in the efficient manufacture of pure peptides, and in their ability to induce regulation.28,29,34,35 Although peptide 2 (aa6-20) needed no modification to meet this criterion, we extend- ed the sequence of peptide 82 (aa711-725) with an argi- nine-lysine wrapper in order to ensure adequate solubility, so that the combined peptides were appropriate for fur- ther product development.
The current results add to the body of work indicating that the manipulation of Treg cells offers an effective strat- egy for ameliorating ITP and other autoimmune antibody- mediated diseases,16-22,48 and the data demonstrate that this goal could be achieved by peptide immunotherapy. The ability of a combination of peptides to suppress antibody and T-cell responses to GPIIb/IIIa, and to induce Treg, in our pre-clinical model provides further justification for human clinical trials of this approach in ITP. Differences between species inevitably present a risk that positive results from murine studies do not translate to human patients, but we have reduced this by testing mice with partially humanized immune systems, and by asking highly defined questions as to the ability of GPIIIa pep- tides presented in vivo by human MHC class II molecules to induce suppression. A product containing GPIIIa pep- tides 2 (aa6-20) and 82 (aa711-725) therefore represents the basis for development, with the initial indication being those ITP patients in whom conventional immunosup- pressive treatments have failed.
Funding
The study was funded by grants from the Medical Research Council (UK) Confidence in Concept, the Scottish National Blood Transfusion Service and the Wellcome Trust (UK).
Acknowledgments
We would like to acknowledge the assistance of Iain Fraser Cytometry Centre at the University of Aberdeen.
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