ABSTRACT
Adoptive therapy using T cells redirected to target tumor- or infection-associated antigens is a promising strategy that has curative potential and broad applicability. In order to accelerate the screening process for suitable antigen-specific T cell receptors (TCRs), we developed a new approach circumventing conventional in vitro expansion-based strategies. Direct isolation of paired full-length TCR sequences from non-expanded antigen-specific T cells was achieved by the establishment of a highly sensitive PCR-based T cell receptor single cell analysis method (TCR-SCAN). Using MHC multimer-labeled and single cell-sorted HCMV-specific T cells we demonstrate a high efficacy (approximately 25%) and target specificity of TCR-SCAN receptor identification. In combination with MHC-multimer based pre-enrichment steps, we were able to isolate TCRs specific for the oncogenes Her2/neu and WT1 even from very small populations (original precursor frequencies of down to 0.00005% of CD3(+) T cells) without any cell culture step involved. Genetic re-expression of isolated receptors demonstrates their functionality and target specificity. We believe that this new strategy of TCR identification may provide broad access to specific TCRs for therapeutically relevant T cell epitopes.
Subject(s)
Histocompatibility Antigens/chemistry , Immunotherapy , Protein Multimerization , Receptors, Antigen, T-Cell/isolation & purification , Receptors, Antigen, T-Cell/therapeutic use , Single-Cell Analysis , Amino Acid Sequence , Animals , Antigens, Neoplasm/immunology , Cell Culture Techniques , Cytomegalovirus/immunology , Epitopes , Gene Transfer Techniques , HEK293 Cells , Histocompatibility Antigens/metabolism , Humans , Jurkat Cells , Mice , Molecular Sequence Data , Polymerase Chain Reaction , Receptors, Antigen, T-Cell/chemistry , Receptors, Antigen, T-Cell/immunology , Receptors, Antigen, T-Cell, alpha-beta , Sequence Analysis, Protein , Species Specificity , TransgenesABSTRACT
Basophils are associated with T helper 2 (Th2) cell-polarized immune responses such as allergic disorders or helminth infections. To directly address the role of basophils for type 2 immunity, we generated transgenic mice with constitutive and selective deletion of basophils. Differentiation and accumulation of Th2 cells, induction of eosinophilia, and increase in serum IgE or IgG1 induced by allergens or by infection with the helminth Nippostrongylus brasiliensis appeared to be basophil independent. Further, basophils were not required for passive IgE- or IgG1-mediated systemic anaphylaxis. However, basophils were essential for IgE-meditated chronic allergic dermatitis and for protection against secondary infection with N. brasiliensis. These results demonstrate that basophils play an important role for protective immunity against helminths and orchestrate chronic allergic inflammation, whereas primary Th2 cell responses can operate efficiently in the absence of this cell type.
