The T210M Substitution in the HLA-a*02:01 gp100 Epitope Strongly Affects Overall Proteasomal Cleavage Site Usage and Antigen Processing

Abstract

MHC class I-restricted epitopes, which carry a tumor-specific mutation resulting in improved MHC binding affinity, are preferred T cell receptor targets in innovative adoptive T cell therapies. However, T cell therapy requires efficient generation of the selected epitope. How such mutations may affect proteasome-mediated antigen processing has so far not been studied. Therefore, we analyzed by in vitro experiments the effect on antigen processing and recognition of a T210M exchange, which previously had been introduced into the melanoma gp100 209 Protein degradation by the ubiquitin-proteasome system plays an important role in regulating cellular protein homeostasis. Concomitant with the degradation of proteins, the 20S proteasome, which is the catalytic core of the ubiquitin-proteasome system, generates peptides of 8 -12 amino acids in length or N-terminally extended precursor peptides that, after trimming by endoplasmic reticulum (ER) 3 resident amino peptidases (ERAPs), can bind MHC class I molecules in the ER to be presented at the cell surface to CD8 ϩ T cells for immune recognition. These antigenic peptides can be produced by the proteasome by normal peptide-bond hydrolysis or by proteasomecatalyzed peptide splicing. The latter reaction generates peptides that have a different sequence than the original antigen, and it can occur by binding fragments of a single molecule (cis proteasome-catalyzed peptide splicing) or of two distinct molecules (trans proteasome-catalyzed peptide splicing) (1-4).In vitro experiments performed with purified 20S proteasomes were shown to closely reflect the in vivo situation, making it an ideal platform to study the generation of non-spliced and spliced antigenic peptides in vitro (1,(5)(6)(7)(8)(9)(10)(11). Under ideal conditions the 20S proteasome exists in two isoforms, i.e. the standard 20S proteasome (s-proteasomes) with the active site subunits ␤1, ␤2, and ␤5 and the 20S immunoproteasomes (i-proteasomes) with the inducible active site subunits ␤1i, ␤2i, and ␤5i. Constitutive expression of true i-proteasomes appears to be restricted to a small number of mainly immune cells like B or T cells. In contrast, the expression of so-called intermediatetype proteasomes containing both standard-and immuno-active subunits appears to be more frequent. Intermediate-type proteasomes are expressed in most tumor cells and in many tissues of the human body under normal physiological nutrition and growth conditions (12). It has been recently shown that the active subunit composition of 20S proteasomes in principle does not affect the quality of proteasome-generated peptides (5,13,14). Nevertheless, proteasomal subunit composition can strongly affect cleavage site usage within a given substrate and hence the relative quantity of non-spliced or spliced peptides produced. Such quantitative differences in the generation of cleavage products can strongly affect cell surface presentation

ER‐aminopeptidase 1 determines the processing and presentation of an immunotherapy‐relevant melanoma epitope

“…As already anticipated, 20S proteasomes obtained from melanoma cell lines did not produce the minimal gp100 209‐217 epitope in detectable amounts, confirming the result of previous studies carried out with various proteasomes and conditions . Two N‐extended versions – i.e .…”

“…Proteasomes were purified from cell lines grown in the absence of inflammatory stimuli to analyze their baseline activity. Consistently with previous studies [10,12,13,35], the minimal gp100 209-217 epitope was not detectable in melanoma proteasome digestions, whereas its N-terminal extended versions could be quantified ( Fig. 2A).…”

“…They are produced in significant smaller amounts than the gp100 205‐217 peptide by 20S proteasome purified from melanoma cell lines (Fig. A) and other cell types , though. These quantitative observations suggest that the trimming of the N‐extended versions of the minimal gp100 209‐217 epitope could play a significant role in the presentation of the gp100 209‐217 epitope by melanoma cells to CTLs.…”

“…By combining the outcome of this and other studies , we can sketch how gp100 209‐217 ‐containing epitopes are presented to CTLs: in the cytosol, proteasomes produce two potential epitopes, i.e. gp100 207‐217/T210M and gp100 208‐217/T210M that share the gp100 209‐217/T210M minimal sequence I T / M DQVPFSV.…”

A large fraction of HLA class I ligands are proteasome-generated spliced peptides

“…The few pioneering studies on PCPS had already provided hints about generative 6 mechanisms. For instance, they suggested that PCPS prefers specific peptide sequences, although the limited number of spliced peptides/epitopes identified has so far precluded an analysis with sufficient statistical power (2,8,(14)(15)(16)(17)(18)(19)(20)(21). With our large pool of spliced antigenic peptides this became possible.…”