Understanding CD8 + T‐cell responses toward the native and alternate HLA‐A∗02:01‐restricted WT1 epitope

The Wilms’ tumor 1 (WT1) antigen is expressed in solid and hematological malignancies, but not healthy tissues, making it a promising target for cancer immunotherapies. Immunodominant WT1 epitopes, the native HLA‐A2/WT1 126‐134 ( R MFPNAPYL) (HLA‐A2/RMFPNAPYL epitope (WT1A)) and its modified variant Y MFPNAPYL (HLA‐A2/YMFPNAPYL epitope (WT1B)), can induce WT1‐specific CD8 + T cells, although WT1B is more stably bound to HLA‐A∗02:01. Here, to further determine the benefits of those two targets, we assessed the naive precursor frequencies; immunogenicity and cross‐reactivity of CD8 + T cells directed toward these two WT1 epitopes. Ex vivo naive WT1A‐ and WT1B‐specific CD8 + T cells were detected in healthy HLA‐A∗02:01 + individuals with comparable precursor frequencies (1 in 10 5 –10 6 ) to other naive CD8 + T‐cell pools (for example, A2/HIV‐Gag 77‐85 ), but as expected, ~100 × lower than those found in memory populations (influenza, A2/M1 58‐66 ; EBV, A2/BMLF1 280‐288 ). Importantly, only WT1A‐specific naive precursors were detected in HLA‐A2.1 mice. To further assess the immunogenicity and recruitment of CD8 + T cells responding to WT1A and WT1B, we immunized HLA‐A2.1 mice with either peptide. WT1A immunization elicited numerically higher CD8 + T‐cell responses to the native tumor epitope following re‐stimulation, although both regimens produced functionally similar responses toward WT1A via cytokine analysis and CD107a expression. Interestingly, however, WT1B immunization generated cross‐reactive CD8 + T‐cell responses to WT1A and could be further expanded by WT1A peptide revealing two distinct populations of single‐ and cross‐reactive WT1A + CD8 + T cells with unique T‐cell receptor‐αβ gene signatures. Therefore, although both epitopes are immunogenic, the clinical benefits of WT1B vaccination remains debatable and perhaps both peptides may have separate clinical benefits as treatment targets.