T cell receptor transduction onto regulatory T cells to treat autoimmune disease

Background \u0026 Aim Autoimmune disease is caused by failure of self-tolerance mechanisms. Central to self-tolerance is the regulatory T cell (Treg) subset, which actively suppresses pro-inflammatory self-reactivity. Tregs, like all T cells, are activated when their T cell receptor (TCR) recognises its cognate antigen in the context of peptide-MHC complexes. Using the model autoimmune disease, Goodpasture\u0027s, we have shown that a reduction in Tregs specific for the Goodpasture\u0027s autoantigen potentiates disease. Here, we show that lentiviral transduction of TCRs specific for the GP autoantigen is a feasible way of developing autoantigen-specific Treg therapy. Methods, Results \u0026 Conclusion Autoantigen-specific CD4+ T cells were isolated from Goodpasture\u0027s disease patient PBMCs by single cell flow cytometric sorting using tetramers. The single cells then underwent T cell receptor sequencing to determine the TCR alpha and TCR beta chain CDR3 sequence and Variable, Diverse and Joining gene usage. After analysing dozens of paired alpha-beta TCR sequences, a clonal population expressing the same TCR was identified in each patient (GP1-TCR and GP2-TCR). Lentiviral vectors coding for these alpha-beta TCR genes plus GFP reporter were constructed and used to transduce Jurkat cells, a human T cell line lacking endogenous TCR expression. The Jurkat cells were shown to express both GFP and GP1-TCR or GP2-TCR by flow cytometry. Co-culture of the transduced Jurkat cells with a DR15-specific B-Lymphoblastiod Cell Line (as antigen presenting cells) in the presence or absence of GP peptide resulted in an increase of Jurkat cell activation only in the group with GP peptide. This was measured by an upregulation of CD69 cell surface expression by flow cytometry and increased IL2 secretion by ELISA. This suggests GP1-TCR and GP2-TCR are specific for GP peptide. Using custom designed lentiviral constructs, GP1TCR was successfully lentivirally transduced onto primary human Tregs. Therefore, the use of single cell sequencing and enhanced lentiviral transduction of TCRs onto Tregs allow for the development of potent autoantigen specific Tregs that can be used to treat autoimmune disease.