Mutated FNDC3B generates a naturally immunogenic neoepitope in patient 2. (A) Twenty-six missense mutations were identified in patient 2 CLL cells, from which 37 epitopes (from 36 unique peptides; 1 peptide was predicted to bind to 2 HLA alleles) from 16 mutations were predicted to bind to patient 2’s MHC class I alleles. A total of 18 peptides from 12 mutations were experimentally confirmed to bind. Posttransplant T cells (∼4 years) from patient 2 were stimulated with autologous DCs or B cells pulsed with 3 pools of experimentally validated binding mutated peptides (18 peptides total) for 2 weeks ex vivo (supplemental Table 5). (B) Increased IFN-γ secretion was detected by ELISPOT assay in T cells stimulated with pool 1 peptides. (C) Of pool 1 peptides, increased IFN-γ secretion was detected against the mut-FNDC3B peptide (lower, averaged results from duplicate wells are displayed). (Upper) Predicted and experimental IC50 scores of mut- and wt-FNDC3B peptides. (D) T cells reactive to mut-FNDC3B demonstrate specificity to the mutated epitope but not the corresponding wild-type peptide (concentrations: 0.1-10 µg/mL) and are polyfunctional, secreting IFN-γ, GM-CSF, and IL-2 (Tukey post-hoc tests from 2-way analysis of variance modeling; mut vs wt). (E) (Left) Mut-FNDC3B-specific T cells are reactive in a class I-restricted manner and (right) recognize an endogenously processed and presented form of mutated FNDC3B, because they recognized HLA-A2 APCs transfected with a plasmid encoding a minigene of 300 bp encompassing the FNDC3B mutation (2-sided Welch t test) but not wild-type sequences beyond the background of nontransfected APCs alone. (Top right) Western blot analysis confirming expression of minigenes encoding mut- and wt-FNDC3B. (F) Patient 2 CD8+ T cells specifically recognizing mut-FNDC3B (5 μg/mL) demonstrate antitumor responses in an IFN-γ ELISPOT assay (2-sided Welch t test). (G) Specificity of T cells recognizing the mut-FNDC3B epitope detected by HLA-A2+/mut−FNDC3B tetramer-positive T cells in patient 2.