(A) Primary data of an exemplary IFN-γ ELISPOT assay (CRC patient 1056) with T cell fractions before and after depletion of CD4⁺CD25⁺ Tregs. Mean ± SEM; horizontal lines indicate samples containing (black bars) or lacking (white bars) Tregs; *P < 0.05 (2-tailed Student’s t test). (B) Cumulative total IFN-γ spots from Treg-depleted T cells for control antigen (IgG) in responding and nonresponding patients and responding and nonresponding TAAs. Mean ± SEM; ***P < 0.0001 (2-tailed Student’s t test); IgG, n = 21–28; TAAs, n = 50–106. (C) Data represent the percentage change in tested individuals that exerted significant T cell reactivity against the tested TAAs after depletion of Tregs relative to the response rates before Treg depletion. *P < 0.05 (Fisher’s exact test); n = 10–34. (D) Comparison of TAA reactivity in CRC patients before and after depletion of Tregs. Data represent the percentage of tests where significant T cell reactivity against a TAA was measured. *P < 0.05 (Fisher’s exact test); n = 246–254. (E) Frequencies of TAA-specific Tmems before and after Treg depletion in individual CRC patients and healthy donors. Mean ± SEM; **P < 0.01; ***P < 0.001 (2-tailed Student’s t test); n = 209–269. (F) TAAs were classified into Treg-dependent Tmem responses (>10% change in response rate) or Treg-independent Tmem responses (<10% change in response rate). Mean ± SEM; asterisks indicate significant difference between the groups indicated by horizontal line; **P < 0.01. Telo, telomerase. (G) Patients were tested for the presence of HLA-A*0201 and grouped into an HLA-A*0201–positive and an HLA-A*0201–negative cohort. Depicted are the proportions of CRC patients for both groups among all individuals of the respective group tested that exerted significant T cell reactivity against tested polypeptides. **P < 0.01; NS, P > 0.05 (Fisher’s exact test); n = 93–189.