Fig. 1: Cloning of specific cytotoxic T lymphocytes (CTLs) from a patient with anti-PIT-1 hypophysitis (Case 1).

a Design of overlapping peptides (OPs) for covering PIT-1. The amino acid sequence translated from the open reading frame of PIT-1 was used to design OPs. b Schematic of peptide pool (PP) exposure and isolation of reactive CTLs from peripheral blood mononuclear cells (PBMCs). CD4⁺CD25⁺ depleted PBMCs were prepared on day 0. PP21-30 was exposed to the CD4⁺CD25⁺ depleted PBMCs every 14 days. After four repeated exposures, the reactivity of CTLs was confirmed based on 4-1BB expression upon re-exposure to PP21-30. c Isolation of PP21-30 reactive CTLs on the basis of the Vβ-subtype. The 4-1BB expression was evaluated 24 h after PP21-30 exposure. Anti-CD3 antibody was used as positive control. All data were gated on live cells. Gating on Vβ7.1⁺ was added only for the rightmost panel. d Determination of reactive OPs from PP21-30. The 4-1BB⁺ proportion on Vβ7.1⁺ CTLs was evaluated 24 h after each OP exposure, and the ratio of 4-1BB⁺ cells on Vβ7.1⁺ CTLs is shown; the original flow cytometry panels appear in Supplementary Fig. 1e. OP30 is indicated with an arrow, and its amino acid sequence is provided. Source data are provided as a Source Data file. e TCR repertoire analysis of OP30-reactive Vβ7.1⁺ CTLs. Next-generation sequencing (NGS) was applied for the analysis. Source data are provided as a Source Data file. f Flow cytometry analysis for OP30-reactivity of PB-CTLs with TCR-expressing vector transduction. The lentivirus vectors shown in Supplementary Fig. 2f were used for the transduction. The 4-1BB expression was evaluated 24 h after OP30 exposure. The anti-CD3 antibody was used as positive control. Representative data from five independent experiments are shown. All data were gated on CD8⁺CD45⁺ live cells.