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The following abstract was selected for presentation at the American Transplant Congress in a plenary session:

Ex Vivo Expansion of Human Antigen-Specific Natural Tregs Using IgG Fc Derived Peptides (Tregitopes)

Francesca D’Addio, Olaf Boenisch, Toshiko Watanabe, William Martin, Annie De Groot, Nader Najafian

Transplantation Research Center-Brigham & Women’s Hospital and Children’s Hospital; Epivax Inc. and University of Rhode Island

A set of natural T regulatory epitopes in IgG that induce tolerance to immunogenic proteins has been recently identified. These Tregitopes can bind to MHC class II molecules and specifically induce natural regulatory T cells (Tregs) and, when co-administered with an antigen, lead to expansion of antigen-specific Tregs. Using a HLA class II Tregitope with high affinity to HLA 0401/0701 (Tr289, 9-mer peptides) derived from the Fc fragment of human IgG, we set out to explore the feasibility of expansion of antigen-specific nTregs and their ability to modulate allo-immune responses in a mixed lymphocyte reaction.

We isolated human PBMC from responders with either a class II HLA Tr289-binding or HLA Tr289-non binding phenotype. We incubated the cells with alloantigen and human Tr289/control peptide (10mcg/ml) for 5 days (d). Responder cells were labeled with CFSE and samples were taken on d3 and d5 for flow and luminex analysis (primary MLR). After 5d, cells were washed, incubated with fresh alloantigen for 48 hours and analyzed in a similar fashion (secondary MLR). CD4+Foxp3+ (Tregs) were stained for HLA specific tetramer to assess antigen-specificity.

Tregs expanded from d0 to d3 and more impressively to d5 with Tr289 (15 vs. 6% control) in a HLA restricted manner (control with non binding HLA: 5 and 6% respectively). There was increased IL-10 production in HLA Tr289-binding responders when cells were treated with Tr289 both in primary (4552 vs. 305 pg/ml p<0.03) and secondary MLR (193 vs. 115 pg/ml, p<0.04). In the secondary MLR, Tr289-treated CD4+ cells derived from HLA Tr289-binding responders proliferated less (CD4+CFSE+ 65 vs 51% control peptide, p=0.009) and produced lower amounts of pro-inflammatory cytokines (IL-6, IFN-g, TNF-alfa) whereas no difference was detected in proliferation and cytokine production in Tr289-non binders (70 vs. 71.5% with and without Tr289 respectively). Staining for HLA specific tetramer showed an increase of Tregs after restimulation in Tr289-treated cells (2.1 vs. 0.7%) consistent with HLA binding affinity of the Tregitope.

This is the first report to establish the role of Tregitopes in regulating human allo-immune responses by inducing antigen-specific Tregs ex vivo. This provides the rationale to develop new strategies to harness the potential of Tregs to modulate immune response in transplantation.

Keywords: Epitopes; HLA matching; Immunoglobulins (Ig); Tolerance
Wednesday, May 5, 2010 8:45 AM
Plenary Session IV (8:30 AM-10:00 AM)
Room: Hall F