The T210M Substitution in the HLA-a*02:01 gp100 Epitope Strongly Affects Overall Proteasomal Cleavage Site Usage and Antigen Processing.
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 gp100209-217 tumor epitope to improve the HLA-A*02:01 binding and its immunogenicity. A quantitative analysis of the main steps of antigen processing shows that the T210M exchange affects proteasomal cleavage site usage within the mutgp100201-230 polypeptide, leading to the generation of an unique set of cleavage products. The T210M substitution qualitatively affects the proteasome-catalyzed generation of spliced and non-spliced peptides predicted to bind HLA-A or -B complexes. The T210M substitution also induces an enhanced production of the mutgp100209-217 epitope and its N-terminally extended peptides. The T210M exchange revealed no effect on ERAP1-mediated N-terminal trimming of the precursor peptides. However, mutant N-terminally extended peptides exhibited significantly increased HLA-A*02:01 binding affinity and elicited CD8(+) T cell stimulation in vitro similar to the wtgp100209-217 epitope. Thus, our experiments demonstrate that amino acid exchanges within an epitope can result in the generation of an altered peptide pool with new antigenic peptides and in a wider CD8(+) T cell response also towards N-terminally extended versions of the minimal epitope.
From the Institut für Biochemie, Charité-Universitätsmedizin Berlin, 10117 Berlin, Germany.
Kathrin
K
Textoris-Taube
From the Institut für Biochemie, Charité-Universitätsmedizin Berlin, 10117 Berlin, Germany.
Christin
C
Keller
Centre for Integrative Systems Biology and Bioinformatics, Department of Life Sciences, Imperial College London, SW7 2AZ, United Kingdom, and.
Juliane
http://orcid.org/0000-0003-2515-9707
J
Liepe
From the Institut für Biochemie, Charité-Universitätsmedizin Berlin, 10117 Berlin, Germany.
Petra
P
Henklein
Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, California 92037.
John
J
Sidney
Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, California 92037.
Alessandro
A
Sette
From the Institut für Biochemie, Charité-Universitätsmedizin Berlin, 10117 Berlin, Germany, p-m.kloetzel@charite.de.
Peter M
PM
Kloetzel
From the Institut für Biochemie, Charité-Universitätsmedizin Berlin, 10117 Berlin, Germany, michele.mishto@charite.de.
Michele
http://orcid.org/0000-0003-3042-2792
M
Mishto
D019943:Amino Acid Substitution; D017951:Antigen Presentation / Q000235:genetics / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D018984:Epitopes, T-Lymphocyte / Q000235:genetics / Q000276:immunology*; D015789:HLA-A2 Antigen / Q000235:genetics / Q000276:immunology*; D015235:HLA-B Antigens / Q000235:genetics / Q000276:immunology; D006801:Humans; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000276:immunology*; D058951:gp100 Melanoma Antigen / Q000235:genetics / Q000276:immunology*
D018984:Epitopes, T-Lymphocyte; C435939:HLA-A*02:01 antigen; D015789:HLA-A2 Antigen; D015235:HLA-B Antigens; D058951:gp100 Melanoma Antigen; D046988:Proteasome Endopeptidase Complex
antigen presentation; antigen processing; mutant; protein degradation; ubiquitin-dependent protease