Figure 3
Figure 3. Representative tetramer-guided epitope mapping results. (A) Analysis of an initial possible tetramer-positive response to F8-SNP-Pool 2238 peptides. CD4 cells from severe HA subject GIS-005-019, whose F8 gene encodes the M2238 sequence, were stimulated with irrelevant peptides (FVIII-A2 peptide pool 6), tetanus peptides (TT506-525, a known HLA-DRB1*0301–restricted epitope or TT946-965, a known HLA-DRB1*11:01–restricted epitope), or with F8 ns-SNP peptide pool 2238 peptides for 2 weeks in culture and then stained with phycoerythrin (PE)-labeled HLA-DR0301 and DR1101 tetramers loaded with the same peptides used for stimulation. Cultures stimulated with tetanus peptide showed the expected strong T-cell staining by tetramers loaded with these known T-cell epitopes. Cultures stimulated with F8 SNP-Pool 2238 peptides showed low-level, possibly nonspecific, staining by DR0301 and DR1101 tetramers loaded with these peptides. (B) Decoding the pooled-peptide results using the individual peptides comprising F8 SNP-Pool 2238. CD4 cells were cultured for 2 more days following the initial tetramer staining experiment (A) and then stained using HLA-DR1101 and DR0301 tetramers loaded with the 4 individual peptides comprising the original peptide pool. Again, low-level tetramer staining was seen using tetramers loaded with several of these individual 2238 peptides. (C) Attempts to isolate T-cell clones recognizing sequences encoded by ns-SNPs in F8. Similarly, tetramer-positive T cells from the cultures stimulated with F8 SNP-Pool 2238 (shown in panel A) were single-cell sorted and expanded with PHA for 6 weeks in an attempt to isolate FVIII-specific T-cell clones. They were then incubated with fluorescent HLA-DR0301 and DR1101 tetramers loaded with the F8 SNP-Pool 2238 peptides. Representative results are shown for 2 of these expanded cultures. The PHA expansion did not produce any T-cell clones recognizing the pooled peptides, indicating the earlier “borderline” tetramer-positive staining was likely nonspecific. These negative staining results are in distinct contrast to our earlier studies, in which T-cell responses to “sequence-mismatched” FVIII products in mild HA subjects were identified initially by tetramer staining of CD4 T cells and then unambiguously verified by isolation of T-cell clones and lines with HLA-restricted responses to the mismatched FVIII sequence.7,8,40,41 (D) Isolation of tetanus-specific T-cell clones as positive controls. T cells showing positive staining using tetramers loaded with tetanus peptides (A) were single-cell sorted onto 96-well plates and expanded with PHA for 6 weeks to isolate tetanus-specific T-cell clones, as positive controls. Representative staining results are shown for 2 of 12 and 2 of 32 T-cell clones isolated from these cultures, stained with DR0301 and DR1101 tetramers loaded with tetanus peptides TT 506-525 and TT946-965, respectively.

Representative tetramer-guided epitope mapping results. (A) Analysis of an initial possible tetramer-positive response to F8-SNP-Pool 2238 peptides. CD4 cells from severe HA subject GIS-005-019, whose F8 gene encodes the M2238 sequence, were stimulated with irrelevant peptides (FVIII-A2 peptide pool 6), tetanus peptides (TT506-525, a known HLA-DRB1*0301–restricted epitope or TT946-965, a known HLA-DRB1*11:01–restricted epitope), or with F8 ns-SNP peptide pool 2238 peptides for 2 weeks in culture and then stained with phycoerythrin (PE)-labeled HLA-DR0301 and DR1101 tetramers loaded with the same peptides used for stimulation. Cultures stimulated with tetanus peptide showed the expected strong T-cell staining by tetramers loaded with these known T-cell epitopes. Cultures stimulated with F8 SNP-Pool 2238 peptides showed low-level, possibly nonspecific, staining by DR0301 and DR1101 tetramers loaded with these peptides. (B) Decoding the pooled-peptide results using the individual peptides comprising F8 SNP-Pool 2238. CD4 cells were cultured for 2 more days following the initial tetramer staining experiment (A) and then stained using HLA-DR1101 and DR0301 tetramers loaded with the 4 individual peptides comprising the original peptide pool. Again, low-level tetramer staining was seen using tetramers loaded with several of these individual 2238 peptides. (C) Attempts to isolate T-cell clones recognizing sequences encoded by ns-SNPs in F8. Similarly, tetramer-positive T cells from the cultures stimulated with F8 SNP-Pool 2238 (shown in panel A) were single-cell sorted and expanded with PHA for 6 weeks in an attempt to isolate FVIII-specific T-cell clones. They were then incubated with fluorescent HLA-DR0301 and DR1101 tetramers loaded with the F8 SNP-Pool 2238 peptides. Representative results are shown for 2 of these expanded cultures. The PHA expansion did not produce any T-cell clones recognizing the pooled peptides, indicating the earlier “borderline” tetramer-positive staining was likely nonspecific. These negative staining results are in distinct contrast to our earlier studies, in which T-cell responses to “sequence-mismatched” FVIII products in mild HA subjects were identified initially by tetramer staining of CD4 T cells and then unambiguously verified by isolation of T-cell clones and lines with HLA-restricted responses to the mismatched FVIII sequence.7,8,40,41  (D) Isolation of tetanus-specific T-cell clones as positive controls. T cells showing positive staining using tetramers loaded with tetanus peptides (A) were single-cell sorted onto 96-well plates and expanded with PHA for 6 weeks to isolate tetanus-specific T-cell clones, as positive controls. Representative staining results are shown for 2 of 12 and 2 of 32 T-cell clones isolated from these cultures, stained with DR0301 and DR1101 tetramers loaded with tetanus peptides TT 506-525 and TT946-965, respectively.

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