Ribosome profiling shows variable sensitivity to detect open reading frames for conventional and different types of cryptic T cell antigens.

T cell-based immunotherapies targeting antigens on tumor cells have shown efficacy as anti-cancer treatments. While neoantigens are created by somatic mutations acquired during tumorigenesis, allogeneic stem cell transplantation as treatment for hematological malignancies exploits minor histocompatibility antigens encoded by genetic differences between patients and donors. Screening methods to predict neoantigens and minor histocompatibility antigens typically consider only conventional antigens created by nonsynonymous mutations or polymorphisms coding for amino acid changes in canonical open reading frames (ORFs). However, unconventional ORFs encoding peptides outside the known human proteome also provide an important source of cryptic antigens targeted in anti-tumor responses. Here, we used the recently expanded repertoire of human leukocyte antigen (HLA) class I-restricted minor histocompatibility antigens identified in patients treated with allogeneic stem cell transplantation by a method unbiased regarding the type of antigen to explore the sensitivity of ribosome profiling to detect ORFs for different types of T cell antigens. Ribosome profiling showed high sensitivity to detect upstream ORFs for cryptic antigens similar to canonical ORFs for conventional antigens, while cryptic antigens in out-of-frame ORFs and ORFs in long non-coding RNAs were largely missed. In conclusion, ribosome profiling shows variable sensitivity to detect ORFs for canonical and different types of cryptic T cell antigens.