Characterization of MHC class II-restricted T-cell receptors for T-cell therapy of HBV infection

Abstract

Background & Aim Hepatitis B virus (HBV) infection remains a severe health problem with current treatment options being unable to achieve viral clearance. Since virus eradication is known to be accompanied by a strong T-cell response in patients with resolved infection, adoptive T-cell therapy represents a promising therapeutic approach. We recently demonstrated that CD8+ T cells grafted with T-cell receptors (TCR) restricted by MHC class I have the potential to cure HBV infection in vitro and in vivo. Nevertheless, also CD4+ T cells are known to play an important role in resolving HBV infection and may therefore potentially benefit T-cell therapy. We thus identified, cloned and characterized MHC class II-restricted TCRs from HBV-specific CD4+ T cells. Methods, Results & Conclusion HBV peptides were used to stimulate PBMC from donors with cleared HBV infection. HBV-specific CD4+ T cells secreting TNF-α were sorted by flow cytometry and clonally expanded. Subsequently, variable sequences of TCR α- and β-chains were identified, codon-optimized and cloned into a retroviral vector with murine constant domains. After retroviral transduction, all TCRs were expressed on primary T cells and characterized in depth regarding their MHC restriction, specificity, binding affinity and functionality. A total of 23 TCRs specific for 7 different epitopes from HBV envelope, core or polymerase protein were generated. Through retroviral titration, TCR integration was limited to under 5 integrates per cell, as determined by qPCR. Transduction rates ranging from 65-85% were measured by flow cytometry, where strength of surface expression was found to be characteristic of the TCR rather than the number of integrates. Through co-culture with partially matched lymphoblastoid cell lines, 6 different MHC class II restrictions were identified, including HLA-DRB1*01, *07, *04 and *13 or HLA-DPB1*02 and *04. 17 TCRs recognized at least 3 HBV genotype variants and 12 TCRs had a high binding affinity with EC50 values in a low nanomolar range. Similarly, 12 TCRs were able to recognize endocytosed and intracellularly processed viral antigen. 20 TCRs were capable of activating CD8+ T cells, inducing granzyme B secretion in 75-100% of transduced T cells. Taken together, our set of 23 MHC class II-restricted TCRs recognizes epitopes derived from HBV core, surface and polymerase protein. These TCRs enabled both CD4+ and CD8+ T cells to detect antigen on MHC class II in a nanomolar range, resulting in antigen-specific T-cell activation.