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

Area of Science:

• Immunology
• Genomics
• Proteomics

Background:

• T cell-based immunotherapies are vital anti-cancer treatments targeting tumor antigens.
• Conventional screening methods for neoantigens and minor histocompatibility antigens focus on canonical open reading frames (ORFs).
• Unconventional ORFs encoding peptides outside the known proteome represent a significant source of cryptic antigens for anti-tumor responses.

Purpose of the Study:

• To evaluate the sensitivity of ribosome profiling in detecting various types of T cell antigen ORFs.
• To compare the detection capabilities of ribosome profiling for canonical versus cryptic antigens.

Main Methods:

• Utilized a recently expanded repertoire of human leukocyte antigen (HLA) class I-restricted minor histocompatibility antigens from allogeneic stem cell transplantation patients.
• Employed a ribosome profiling method unbiased by antigen type to identify ORFs.
• Analyzed the sensitivity of ribosome profiling for detecting canonical ORFs, upstream ORFs, out-of-frame ORFs, and ORFs in long non-coding RNAs.

Main Results:

• Ribosome profiling demonstrated high sensitivity in detecting upstream ORFs, similar to canonical ORFs for conventional antigens.
• Detection of cryptic antigens within out-of-frame ORFs and ORFs in long non-coding RNAs was largely unsuccessful.
• Significant variability in ribosome profiling sensitivity was observed across different ORF types.

Conclusions:

• Ribosome profiling is a sensitive tool for identifying certain types of T cell cryptic antigens, particularly those originating from upstream ORFs.
• The method's effectiveness is limited for cryptic antigens encoded by out-of-frame ORFs and long non-coding RNAs.
• Further optimization is needed to enhance the detection of all cryptic T cell antigen sources using ribosome profiling.