Figure 3. Cloning of murine TCRs from CXorf61-specific T cells induced by immunization of HLA-transgenic mice and identification of HLA-A*02-restricted epitopes.

A. Detection of CXorf61-specific T cells after 5 rounds of immunization of HLA-A*0201-transgenic mice with CXorf61 encoding IVT RNA. Spleen cells from three mice were analyzed for reactivity against CXorf61 overlapping peptide pool (CXorf61 pool) or predicted HLA-A*0201-binding CXorf61-derived peptides by IFNy-ELISPOT assay. B. FACS sorting of CXorf61-specific murine CD8+ T cells from spleen cells of immunized HLA-A*0201-transgenic mice after in vitro restimulation with overlapping peptide pools. Cells were gated on CD3+/CD8+ lymphocytes. Single CD8+/CD137+ T cells were isolated C. Specificity testing of murine TCRs isolated from CD8+ T cells of CXorf61-immunized mice. CD8+ T cells of a HLA-A*0201-positive healthy donor were transfected with TCR-α/β chain RNAs and tested for recognition of K562-A*0201 transfected with CXorf61 IVT RNA or pulsed with CXorf61 peptide pool or HLA-A*02 binding peptides by IFNγ-ELISPOT. D. OKT3-activated CD8+ T cells from a healthy donor were transfected with the murine TCR#4 IVT RNA and co-cultured with autologous immature DCs transfected with CXorf61 IVT RNA or loaded with CXorf61_66–74. Specific killing was analyzed by luciferase cytotoxicity assay. Positive controls: Phorbol-12-myristate-13-acetate treated spleen cells (PMA); Staphylococcus Enterotoxin B treated cells (SEB); negative controls: spleen cells without stimulus (medium); irrelevant HLA-A*0201-restricted peptide PLAC1_31–39 (control peptide); pool of overlapping 15mer peptides representing the irrelevant antigen HIV-gag (control pool); IVT RNA encoding the irrelevant antigen TPTE (control RNA), T cells transfected without TCR RNA (CD8 mock).