An unexplored angle: T cell antigen discoveries reveal a marginal contribution of proteasome splicing to the immunogenic MHC class I antigen pool

Abstract

In the current era of T cell-based immunotherapies, it is crucial to understand which types of MHC-presented T cell antigens are produced by tumor cells. In addition to linear peptide antigens, chimeric peptides are generated through proteasome-catalyzed peptide splicing (PCPS). Whether such spliced peptides are abundantly presented by MHC is highly disputed because of disagreement in computational analyses of mass spectrometry data of MHC-eluted peptides. Moreover, such mass spectrometric analyses cannot elucidate how much spliced peptides contribute to the pool of immunogenic antigens. In this Perspective, we explain the significance of knowing the contribution of spliced peptides for accurate analyses of peptidomes on one hand, and to serve as a potential source of targetable tumor antigens on the other hand. Toward a strategy for mass spectrometry independent estimation of the contribution of PCPS to the immunopeptidome, we first reviewed methodologies to identify MHC-presented spliced peptide antigens expressed by tumors. Data from these identifications allowed us to compile three independent datasets containing 103, 74, and 83 confirmed T cell antigens from cancer patients. Only 3.9%, 1.4%, and between 0% and 7.2% of these truly immunogenic antigens are produced by PCPS, therefore providing a marginal contribution to the pool of immunogenic tumor antigens. We conclude that spliced peptides will not serve as a comprehensive source to expand the number of targetable antigens for immunotherapies.

Keywords: PCPS; spliced antigens; tumor antigens.

Fig. 1.

Proteasomal activity generates both regular and spliced (antigenic) peptides. (A) The proteasome consists of one or two RPs, a cap and two rings consisting of seven α/β subunits each that form the CP. Ubiquitinated proteins or polypeptides are captured by the RP and processed by the CP into peptides. (B) Proteins are cleaved by means of nucleophilic substitution; reactive threonines attack the carbonyl carbon of scissile amino acid bonds, resulting in separate peptides bound to the β subunits. Conventional peptides are formed through hydrolysis, while spliced peptides are generated by transpeptidation of a donor peptide. (C) Six tumor-expressed cis-spliced antigens have been identified thus far, of which four are ligated in reverse order (^rev). The antigen derived from SP110 is a minor H antigen, whereas the other five are tumor-associated antigens. Three of the latter group are derived from GP100.

Fig. 2.

Schematic overview of the forward antigen identification strategies. (A) Prior to identification, T cells are isolated from a patient, and relevant tumor or minor H antigen–specific T cell clones are selected. Three main forward strategies are regularly used to identify the recognized peptide antigen (B–D). (B) Peptides are eluted from MHC molecules that are restriction elements of an antigen-specific T cell clone. Eluted peptides are fractionated high-performance LC and separately loaded on cells to measure reactivity by the antigen-specific T cell clone. The antigen-positive fraction is analyzed by MS/MS to determine the putative sequence of the recognized peptide. (C) Cells are transfected with fractions of a cDNA library and tested for their ability to activate the antigen-specific T cell clone. Antigen-positive fractions are subsequently sequenced to determine the cDNA coding for the antigen. (D) For WGAS, EBV-LCLs from the 1000 Genomes Project are phenotyped as minor H antigen positive or negative using a T cell recognition assay. The genetic variation encoding the antigen of interest can be determined by analyzing the association of the phenotypes with the publicly available genotypes of 4·10⁷ SNPs from the same cells. Former genetic linkage analyses were based on a similar principle, but were less powerful because of the smaller number of genotyped variations. (E) Final validation of the epitope for each of those strategies (B–D) is achieved by testing specific (truncated) cDNA constructs or synthetic peptides, which are selected based on MHC-binding prediction algorithms, for recognition by the T cell clone.

Fig. 3.

Spliced antigens constitute a marginal fraction of the MHC-I-presented antigen pool. (A) Methods for antigen identification and their bias toward spliced versus nonspliced antigens. (B) We evaluated the percentage of spliced peptides within the CAP database. The CAP database contains 474 tumor antigens, of which 286 are unique MHC-I restricted. Four (1.4%) of these are spliced peptides. Literature analyses revealed that 103 of the 286 antigens were identified with largely unbiased forward methods, still including four spliced peptides (3.9%). (C) The second independent dataset contains 76 molecularly characterized MHC-I-restricted minor H antigens that were identified with forward methods. One (1.4%) of these is a spliced antigen. (D) A third independent dataset consists of recently identified but unpublished minor H antigens discovered by our largely unbiased GWAS strategy using 83 different antigen-specific T cell clones. Of the eight antigens that are currently unresolved, two are, in all likelihood, encoded by cryptic transcripts. The remaining six (7.2%) antigens are potential spliced antigens or other unconventionally encoded or processed antigens. We concluded that the maximum percentage of spliced antigens in this dataset is therefore 7.2%.