Figure 1.
Strategy to identify HLA-I-restricted MiHAs. Visual representation of the strategy that was followed to identify HLA-I–restricted MiHAs targeted in 39 patients who responded to DLI after HLA-matched alloSCT with GVL reactivity in the absence or presence of GVHD. (A) Patient samples collected after DLI were enriched for CD8+ T cells. Activated T cells were sorted based on CD137 after 2 days of in vitro stimulation with a pre-alloSCT patient sample (left), or based on HLA-DR as in vivo marker. (B) Growing T-cell clones were tested against patient EBV-LCLs and donor EBV-LCLs pulsed with peptide mixes of known MiHAs. (C) T-cell clones recognizing known antigens were subjected to a combinatorial peptide test to identify the antigen, shown here for the MiHA HA-2. (D) T-cell clones for unknown antigens that exclusively recognized patient EBV-LCL, but not peptide-pulsed donor EBV-LCLs were tested against 10 pools each containing 5 EBV-LCLs. Based on recognition patterns, T-cell clones were clustered, exemplified here by 5 clones with 2 distinct recognition patterns (blue/purple and orange). (E) From each cluster, representative T-cell clones were selected for GWAS. T-cell clones were tested against an optimized panel of 191 EBV-LCLs that were sequenced as part of the 1000 Genomes Project. HLA-restriction was determined by shared HLA alleles on EBV-LCLs that were recognized by T cells (here HLA-B∗07:02-restricted). EBV-LCLs expressing the relevant HLA-restriction allele were divided into antigen-positive and -negative groups based on IFN-γ secretion. All 11.1 million SNPs (minor allele frequency >0.01) were scanned for association with T-cell recognition patterns, here exemplified by heterozygous (orange, nucleotides CG) or homozygous (red, nucleotides CC in example) genotyping for positive EBV-LCLs and homozygous allelic variants (blue, nucleoties GG in example) in negative EBV-LCLs. (F) SNPs with strong association were investigated for encoding polymorphic peptides with predicted binding to the relevant HLA allele, and candidates (orange) and their allelic variants (blue) were tested for T-cell recognition for validation. Numbers in schematic illustration represent range of activated T cells per patients (A), total number of growing T-cell clones (B), T-cell clones for which a MiHA was identified by combinatorial peptide mixes (C), T-cell clones reactive to patient EBV-LCLs (D), T-cell clones for which GWAS performed representative of clustered T-cell clones (number in parentheses) (E) and number of identified new MiHAs (number of T-cell clones targeting these in parentheses) (F).

Strategy to identify HLA-I-restricted MiHAs. Visual representation of the strategy that was followed to identify HLA-I–restricted MiHAs targeted in 39 patients who responded to DLI after HLA-matched alloSCT with GVL reactivity in the absence or presence of GVHD. (A) Patient samples collected after DLI were enriched for CD8+ T cells. Activated T cells were sorted based on CD137 after 2 days of in vitro stimulation with a pre-alloSCT patient sample (left), or based on HLA-DR as in vivo marker. (B) Growing T-cell clones were tested against patient EBV-LCLs and donor EBV-LCLs pulsed with peptide mixes of known MiHAs. (C) T-cell clones recognizing known antigens were subjected to a combinatorial peptide test to identify the antigen, shown here for the MiHA HA-2. (D) T-cell clones for unknown antigens that exclusively recognized patient EBV-LCL, but not peptide-pulsed donor EBV-LCLs were tested against 10 pools each containing 5 EBV-LCLs. Based on recognition patterns, T-cell clones were clustered, exemplified here by 5 clones with 2 distinct recognition patterns (blue/purple and orange). (E) From each cluster, representative T-cell clones were selected for GWAS. T-cell clones were tested against an optimized panel of 191 EBV-LCLs that were sequenced as part of the 1000 Genomes Project. HLA-restriction was determined by shared HLA alleles on EBV-LCLs that were recognized by T cells (here HLA-B∗07:02-restricted). EBV-LCLs expressing the relevant HLA-restriction allele were divided into antigen-positive and -negative groups based on IFN-γ secretion. All 11.1 million SNPs (minor allele frequency >0.01) were scanned for association with T-cell recognition patterns, here exemplified by heterozygous (orange, nucleotides CG) or homozygous (red, nucleotides CC in example) genotyping for positive EBV-LCLs and homozygous allelic variants (blue, nucleoties GG in example) in negative EBV-LCLs. (F) SNPs with strong association were investigated for encoding polymorphic peptides with predicted binding to the relevant HLA allele, and candidates (orange) and their allelic variants (blue) were tested for T-cell recognition for validation. Numbers in schematic illustration represent range of activated T cells per patients (A), total number of growing T-cell clones (B), T-cell clones for which a MiHA was identified by combinatorial peptide mixes (C), T-cell clones reactive to patient EBV-LCLs (D), T-cell clones for which GWAS performed representative of clustered T-cell clones (number in parentheses) (E) and number of identified new MiHAs (number of T-cell clones targeting these in parentheses) (F).

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