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GPIIb/IIIa peptide immunotherapy in HLA-transgenic mice
cytes had received no further stimulus in vitro, and after incubation with either purified GPIIb/IIIa, or the peptide mix. Figure 4A illustrates examples of contour plots obtained from individual animals from each group, and Figure 4B summarizes all the data. Without stimulation in culture, the proportions of cells with the Treg phenotype were low, typically <5% of CD4+ cells, irrespectively of whether the mice had been immunized or received pep- tide. However, after in vitro stimulation with purified GPIIb/IIIa the Treg population increased, typically to >10% of CD4+ cells, in all the immunized groups, both peptide-treated and -untreated, reaching proportions of Treg that were significantly higher than those seen in non- immunized controls. When stimulated in vitro with the peptide mix, increases in Treg were seen in mice that had received peptide either before or after immunization, but not in animals given only the immunization. Overall, the observed inhibition of antibody and Th effector function by peptide therapy is therefore associated with recruit- ment of cells with a Treg phenotype, which differ in responsiveness from those induced by the purified full- length antigen. This is consistent with induction of sup- pression to GPIIb/IIIa by peptide-specific Treg. It was con- firmed that Treg cells contribute to the suppressive effect in peptide-treated mice since splenocyte proliferative responsiveness to GPIIb/IIIa was restored by depletion of the CD25+ T-cell population that contains the Treg cells (Figure 4C).
Production of the regulatory cytokine IL-10 is not neces- sarily associated with the classic CD25+FoxP3+ Treg popu- lation, but represents another mechanism by which effec- tor responses can be downregulated following peptide immunotherapy.19-23,28,29,44 When we measured production of IL-10 by splenocytes (Figure 5), there were no differ- ences between levels of the cytokine in cultures from mice that had been non-immunized, immunized, or given immunization and peptides, although across all groups there was a trend for IL-10 production to be higher when splenocytes were stimulated with the peptide mix com- pared to unstimulated or GPIIb/IIIa-stimulated cultures. Therefore, in this model of peptide therapy, there is little evidence for IL-10 having a role in suppression.
Discussion
As part of a strategy to develop and evaluate specific peptide therapy for ITP, this study determined the ability of sequences containing immunodominant helper epi- topes to suppress responses to the major human platelet autoantigen GPIIb/IIIa by humanized mice expressing HLA-DR15. None of seven GPIIIa peptides, when tested individually, was able reliably to inhibit antibody respons- es by the mice to immunization with purified GPIIb/IIIa, but the combination of peptides 2 (aa6-20) and 82 (aa711- 725) did suppress the development of antibodies and pro- liferative effector T-cell responses. These inhibitory effects were associated with induction of a specific population of Treg cells, and raise the prospect that the peptide combi- nation may be effective in ameliorating ITP in patients.
Previous studies in other diseases have suggested that combinations of peptides are markedly more effective than individual sequences in suppressing pathogenic immune responses in murine models,32,34,42,43 and several of these combinations have progressed into the clinic, with
promising results recently reported from human trials. These include phase I trials of a combination of three HLA-DQ2-restricted immunodominant peptides in the ImmusanT product Nexvax2 to treat celiac disease,45 phase IIa trials of a mixture of four myelin peptides in the Apitope product ATX-MS-1467 in patients with multiple sclerosis (ClinicalTrials.gov Identifier: NCT01973491), and a phase I trial in type 1 diabetes of the product MultiPepT1De containing proinsulin sequences.46 Combinations of peptides may be more effective than individual sequences in triggering therapeutic suppression for a number of reasons. First, different class II molecules vary in their peptide binding preferences,47 and so mix- tures of peptides can maximize the likelihood of effective presentation to CD4+ T cells in HLA-disparate patient populations.29-31 However, this does not account for the superior efficacy of the combination of peptides 2 (aa6-20) and 82 (aa711-725) versus the individual sequences in the current murine model, in which only HLA-DR15 is avail- able. Here, a second explanation, that additional peptides interact with a wider pool of T-cell specificities and are therefore more likely to tip the balance of the response towards regulation, is more relevant.29-31,34 In vivo studies such as these provide the opportunity to catalogue the complex immune properties that therapeutic peptides can exhibit alone or in combination. Individual peptides can induce different balances between effector and regulatory T cells,21,22 yet can synergize in suppression, as illustrated here by the ability of peptide 2 (aa6-20), but not 82 (aa711- 725), to stimulate the proliferation of splenocytes, when mice need to be given both peptides to inhibit GPIIb/IIIa responses. The combination of peptides 2 (aa6-20) and 82 (aa711-725) is attractive as a candidate product to treat ITP, not only because of the current demonstration of effi- cacy in a pre-clinical model, but also because the sequences appear promiscuous in their ability to be pre- sented by different HLA-DR molecules, and are together recognized by T cells from the majority of patients.7 The finding that the inhibitory effects of the peptides are lim- ited to responses to GPIIb/IIIa, and not control antigen, demonstrates specificity of suppression and suggests that immunity to infection would not be compromised by treatment.
In many animal models of immune-mediated disease, and clinical investigations, the efficacy of peptide immunotherapy is associated with suppression of effector T cells and induction of Treg populations.29-32,43-46 The approach is attractive for development as a novel treat- ment for ITP since the disease is associated with an impairment of CD4+CD25+FoxP3+ Treg cells, with their frequency being reduced in the circulation, spleen and bone marrow.2,16-18 Dysregulation of Treg cells is also restored in line with platelet counts after treatment with dexamethasone, rituximab or thrombopoietin.2,4 Analysis of a SCID murine model of ITP, which is induced by adop- tive transfer of splenocytes from CD61-/- mice that have been immunized with CD61+ platelets, further demon- strated a dysregulation of CD4+CD25+FoxP3+ Treg cells.48 These mice were successfully treated with intravenous immunoglobulins, which normalized both platelet and Treg counts.48 The suppression of antibody responses to GPIIb/IIIa in HLA-DR transgenic mice reported here now raises the prospect that Treg can be boosted therapeutical- ly in ITP, with populations of antigen-responsive cells with a CD4+CD25+FoxP3+ Treg phenotype induced in the
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