a, Thawed PBMCs from week 16 collected from patient 7 revealed 60% of the live PBMCs to be CD3+ T cells. Data are representative of results from two independent experiments. b, Thawed PBMCs from patient 7 at week 16 were tested ex vivo by ELISPOT, in which 2 × 105 PBMCs were added per well and exposed overnight to 10 μg ml−1 of peptides covering ARHGAP35MUT (ASP34 or ASP35 peptides) compared to negative control (OVA peptide). Positive control, CEF peptides. Experiment was performed once in triplicate wells. c, Results of ex vivo ELISPOT (n = 3 biologically independent samples). Together, these results indicate that the frequencies of ASP35- and ASP34-reactive T cells were 39 and 29 (after subtracting background) per 360,000 T cells, respectively. Detection of ASP35-reactive T cells (F10) and ASP34-reactive T cells (H02) in brain at relapse was 1 each among 277 single intracranial T cells (Fig. 4c). The rate of T cells that recognize immunizing neoantigens is highly enriched in the brain compared to the periphery, P = 0.030 for ASP35 and P = 0.023 for ASP34, two-sided Poisson test.