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GPIIb/IIIa peptide immunotherapy in HLA-transgenic mice
cell cytotoxicity has also been demonstrated against platelets and megakaryocytes.10,11 Class switching and somatic mutation of the autoantibodies12 indicate that their production is T-dependent, in line with the finding that autoreactive CD4+ helper T (Th) cells specific for the corre- sponding platelet antigens are activated in the disease.7,13-15 Evidence has accumulated that the loss of peripheral toler- ance in ITP reflects an imbalance between regulatory and effector CD4+ T-cell activity, with, in particular, impair- ment of the suppressive function of the CD25+FoxP3+ reg- ulatory T (Treg) subset.2,16-18 ITP therefore fits the paradigm emerging in other autoantibody-mediated diseases, in which pathogenic responses are helper-dependent and arise and/or are sustained due to inadequate Treg suppres- sion.19-21
The current first-line treatment for ITP, steroids, inhibits autoantibody production and platelet degradation by inducing generalized immunosuppression.23,24 However, the numerous side effects of this treatment include infec- tion and cardiovascular disease, which are more frequent causes of death than bleeding in ITP patients.25 Second-line treatments, for instance rituximab to deplete B cells, immunoglobulin infusions or splenectomy, are also non- specific and typically further intensify immunosuppres- sion. Third-line thrombopoietic agents are expensive, and although they can induce lasting remission in some patients26 and induce immune tolerance to platelet autoantigens in a murine model of ITP,27 these effects are not fully understood, and longer term side effects have yet to be fully reported.23,24 In contrast to previous untargeted approaches, antigen-specific immunotherapy offers the prospect of rebalancing pathogenic CD4+ Th and Treg responses, without compromising the rest of the immune system.28-32 Precedents in models of other immune-mediat- ed diseases demonstrate that this can be achieved by appropriate administration of short, synthetic peptides containing CD4+ T-cell epitopes from the respective target antigens, with peptide products being tested in clinical tri- als for the treatment of a number of conditions, including type I diabetes, multiple sclerosis and rheumatoid arthri- tis.28-32 The approach induces immune regulation, which, in addition to suppressing inflammatory T-cell responses, also has the potential to inhibit helper-dependent antibody production, as demonstrated by the success of peptide therapy in blocking IgG responses to the RhD blood group protein in a humanized murine model.33,34 Once peptides that span major T-cell epitopes have been mapped, key
requirements for effective immunotherapy include deliv- ery of these sequences in soluble form, in the absence of any adjuvant material.28,29,34,35 Despite an initial focus on mucosal administration of peptides, a variety of routes are now known to be effective, including subcutaneous.29,31,34
The fine specificity of autoreactive Th cells in ITP has been characterized with a view to developing specific pep- tide therapy that restores helper tolerance and avoids long- term immunosuppressive steroid treatment or splenecto- my.7 When a panel of 15-mer peptides spanning the entire length of GPIIIa was screened for helper epitopes, particu- lar sequences were found to elicit recall responses by CD4+ T cells from patients with ITP. Despite variation between different cases, seven GPIIIa peptides (aa6-20, aa331-345, aa361-375, aa421-435, aa591-605, aa661-675 and aa711- 725) were commonly recognized, with one or more elicit- ing responses by T cells from 84% of patients. Of these peptides, two predominated (aa6-20 and aa711-725), with either or both being stimulatory in 65% of patients.
The aim of the current work, and the next step in devel- oping specific immunotherapy for ITP patients, was to test and compare the efficacy of the seven dominant GPIIIa peptides, particularly aa6-20 and aa711-725, in suppressing responses to GPIIb/IIIa in a pre-clinical animal model. In order to replicate restriction and peptide-binding prefer- ences of the human major histocompatibility complex (MHC), the model used was a humanized transgenic mouse expressing HLA-DR rather than murine class II mol- ecules. A strain transgenic for HLA-DR15 was chosen for these proof-of-principle studies, since it is an established model for investigating responses restricted by human class II,33,34 and, although there is no consensus as to whether certain class II alleles predispose to, or protect against, ITP,36,37 DRB1*1501 was well represented in our cohort of patients7 and is a common allele in many popu- lations. We demonstrate that sequences aa6-20 and aa711- 725 in combination are effective in suppressing Th and IgG antibody responses to immunization of the mice with GPIIIa, and that this effect is associated with induction of a Treg population.
Methods
Purification of glycoprotein IIb/IIIa
GPIIb/IIIa was purified as previously reported,38 with minor modifications. In brief, apheresed platelets (provided by the
Table 1. GPIIIa immunodominant peptides. Peptides, previously reported to contain Th epitopes that are immunodominant in patients with immune thrombocytopenia,7 were manufactured to their wild-type sequence or extended using an arginine-lysine wrapper to improve manufac- turability and solubility.
Peptide nomenclature
P2
P44
P47
P53
P70
P77
GPIIIa sequence
TTRGVSSCQQCLAVS-acid
KRGVLSMDSSNVLQLIVRK-acid
DLPEELSLSFNATCL-acid
FKDSLIVQVTFDCDC-acid
PGSYGDTCEKCPTCP-acid
DDCVVRFQYYEDSSG-acid
KRALLIWKLLITIHDRKRK-acid *Modified by inclusion of an RK wrapper – added in bold.
Amino acids
aa6-20
aa331-345 aa361-375 aa421-435 aa591-605 aa661-675 aa711-725
Modified to improve manufacture
No
Yes* No No No No Yes*
P82
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