Journal: Frontiers in Immunology

Article Title: MediMer: a versatile do-it-yourself peptide-receptive MHC class I multimer platform for tumor neoantigen-specific T cell detection

doi: 10.3389/fimmu.2023.1294565

Figure Lengend Snippet: Detection of neoepitope- and TAA-specific CD8 + T cell populations in the peripheral blood of melanoma patient using multimerized easYmers, dtSCTs and dsSCD (MediMers) in a complementary manner. (A) Identified antigen-specific CD8 + T cell populations in the patient’s peripheral blood by dual color-encoded pMHC-I multimer staining covering all six HLA alleles of a melanoma patient. In total, 148 pMHC-I multimers were generated (107 predicted neoepitopes, 24 non-mutant tumor-associated antigens (TAA) and 17 viral epitopes) on the basis of a dsSCD for HLA-C*08:02 and easYmers covering the remaining five HLA-I allotypes. EasYmer HLA-B*14:01 was used as a surrogate for the patient’s HLA-B*14:02 allotype. For the initial cell staining up to 60 dual color-encoded pMHC-I multimer pairs were used in one single pMHC-I library and detected pMHC-I multimer + populations were verified afterwards by at least two additional independent stainings with up to 10 dual color-encoded pMHC-I multimer pairs. Representative dot plots are shown in Supplementary Figure 3D . Zero values were converted to 0.0001 to allow for plotting on a log scale. (B, C) pMHC-I multimer-guided single-cell TCR repertoire and cell surface protein expression analysis. pMHC-I multimer + CD8 + T cells were cell sorted using a pMHC-I multimer library comprising uniquely DNA-barcoded and population size-dependent dual color-encoded pMHC-I multimer pairs (see Supplementary Figure 2C ) combined with a panel of 30 DNA-barcoded cell phenotyping antibodies ( Supplementary Table 4 ). Multimerized dsSCD, easYmer and dtSCT were used in combination. An experimentally obtained 10x scSeq data set is shown as tSNE plot based on surface marker expression including the pMHC-I multimer labeling of a total of 3236 individual cells represented by dots. Cell clustering based on pMHC-I multimer barcode detection (B) and cell surface expression of selected T cell memory markers (C) is shown. (D, E) Validation of cloned MAGE-A3 and NY-ESO-1-specific TCR from the 10x scSEQ data set. TRBV/TRBJ and TRAV/TRAJ subtypes and respective CDR3 sequences are displayed in the table at the bottom. (D) Antigen-specific staining of a MAGE-A3 168-176 /A*01:01-specific TCR (MM-01) and two NY-ESO-1 139-147 /HLA-C*08:02-specific TCR (MM-02 and MM-03) expressed in J76 CD8+ cells with multimerized dtSCT and dsSCD representing the cognate (teal lines) or a viral control (black lines) epitope. (E) Co-culture of TCR-expressing J76 CD8+ cells with autologous peptide-pulsed B cells. The expression of early T cell activation marker CD69 ± SD in [%] of TCR + J76 CD8+ cells after 18h co-culture in triplicates in presence of cognate (teal) or a control (black) peptide at various concentrations. Two cloned dominant TCRs (MM-04 and MM-05) derived from the OSGEP V91D /HLA-C*08:02 multimer scSEQ cluster lacked expression in J76 CD8+ cells or were not stained by corresponding the pMHC-I multimer, respectively. ND, not determined.

Article Snippet: Sorted DNA-barcoded pMHC-I multimer + CD8 + T cells of the melanoma patient were analyzed by single cell RNA sequencing (scRNA-Seq) utilizing the Chromium NEXT GEM Single Cell 5′ TCR profiling and Feature Barcode Technology v2 (dual index) reagent kit (10x Genomics), which enables the combined interrogation of cell surface protein expression including pMHC-I multimer binding (CSP), TCR (VDJ) and gene expression (GEX).

Techniques: Staining, Generated, Mutagenesis, Expressing, Marker, Labeling, Biomarker Discovery, Clone Assay, Control, Co-Culture Assay, Activation Assay, Derivative Assay