Deep TCRB sequencing of tetramer-sorted cell populations from T-cell line 56A-L2
| Cells* . | Frequency (%) . | Count . | TCRBV gene† . | TCRBD gene† . | TCRBJ gene† . | Junction sequence . | Sequence identifier‡ . |
|---|---|---|---|---|---|---|---|
| Unsorted | 82.65 | 226 589 | 24-1*01 | 1 or 2§ | 2-3*01 | CATSEVTSTDTQYF | Seq07 |
| Unsorted | 11.45 | 31 403 | 27*01 | Not identified | 1-1*01 | CASSFDPLNTEAFF | Seq01 |
| Unsorted | 3.78 | 10 368 | 20-1|| | 1*01 | 2-7*01 | CSAKGQGPYEQYF | Seq08 |
| Unsorted | 0.15 | 421 | 19¶ | 2*01 or 2*02 | 1-1*01 | CASSVAGGLNTEAFF | Seq09 |
| Unsorted | 0.13 | 361 | 24-1*01 | 1*01 | 2-3*01 | CATSEVNSTDTQYV | Seq10 |
| Unsorted | 0.13 | 349 | 27*01 | 1*01 | 2-7*01 | CASSLRTGGSYYEQYF | Seq11 |
| Unsorted | 0.12 | 326 | 7-8# | 2*01 or 2*02 | 2-3*01 | CASSSAPRLTSGRTDTQYF | Seq03 |
| High | 93.60 | 56 880 | 27*01 | Not identified | 1-1*01 | CASSFDPLNTEAFF | Seq01 |
| High | 4.89 | 2971 | 24-1*01 | 1 or 2 | 2-3*01 | CATSEVTSTDTQYF | Seq07 |
| High | 0.57 | 344 | 20-1|| | 1*01 | 2-7*01 | CSAKGQGPYEQYF | Seq08 |
| High | 0.19 | 113 | 7-8# | 2*01 or 2*02 | 2-3*01 | CASSSAPRLTSGRTDTQYF | Seq03 |
| High | 0.14 | 84 | 27*01 | 1*01 | 1-1*01 | CASSFDQLNTEAFV | Seq12 |
| Medium | 77.28 | 281 766 | 24-1*01 | 1 or 2 | 2-3*01 | CATSEVTSTDTQYF | Seq07 |
| Medium | 10.67 | 38 897 | 20-1|| | 1*01 | 2-7*01 | CSAKGQGPYEQYF | Seq08 |
| Medium | 8.26 | 30 122 | 27*01 | Not identified | 1-1*01 | CASSFDPLNTEAFF | Seq01 |
| Medium | 0.77 | 2799 | 7-8# | 2*01 or 2*02 | 2-3*01 | CASSSAPRLTSGRTDTQYF | Seq03 |
| Medium | 0.11 | 419 | 24-1*01 | 1*01 | 2-3*01 | CATSEVNSTDTQYV | Seq10 |
| Low** | 94.31 | 143 552 | 24-1*01 | 1 or 2 | 2-3*01 | CATSEVTSTDTQYF | Seq07 |
| Low | 4.78 | 7276 | 20-1|| | 1*01 | 2-7*01 | CSAKGQGPYEQYF | Seq08 |
| Low | 0.26 | 403 | 27*01 | Not identified | 1-1*01 | CASSFDPLNTEAFF | Seq01 |
| Low | 0.15 | 229 | 24-1*01 | 1*01 | 2-3*01 | CATSEVNSTDTQYV | Seq10 |
| Cells* . | Frequency (%) . | Count . | TCRBV gene† . | TCRBD gene† . | TCRBJ gene† . | Junction sequence . | Sequence identifier‡ . |
|---|---|---|---|---|---|---|---|
| Unsorted | 82.65 | 226 589 | 24-1*01 | 1 or 2§ | 2-3*01 | CATSEVTSTDTQYF | Seq07 |
| Unsorted | 11.45 | 31 403 | 27*01 | Not identified | 1-1*01 | CASSFDPLNTEAFF | Seq01 |
| Unsorted | 3.78 | 10 368 | 20-1|| | 1*01 | 2-7*01 | CSAKGQGPYEQYF | Seq08 |
| Unsorted | 0.15 | 421 | 19¶ | 2*01 or 2*02 | 1-1*01 | CASSVAGGLNTEAFF | Seq09 |
| Unsorted | 0.13 | 361 | 24-1*01 | 1*01 | 2-3*01 | CATSEVNSTDTQYV | Seq10 |
| Unsorted | 0.13 | 349 | 27*01 | 1*01 | 2-7*01 | CASSLRTGGSYYEQYF | Seq11 |
| Unsorted | 0.12 | 326 | 7-8# | 2*01 or 2*02 | 2-3*01 | CASSSAPRLTSGRTDTQYF | Seq03 |
| High | 93.60 | 56 880 | 27*01 | Not identified | 1-1*01 | CASSFDPLNTEAFF | Seq01 |
| High | 4.89 | 2971 | 24-1*01 | 1 or 2 | 2-3*01 | CATSEVTSTDTQYF | Seq07 |
| High | 0.57 | 344 | 20-1|| | 1*01 | 2-7*01 | CSAKGQGPYEQYF | Seq08 |
| High | 0.19 | 113 | 7-8# | 2*01 or 2*02 | 2-3*01 | CASSSAPRLTSGRTDTQYF | Seq03 |
| High | 0.14 | 84 | 27*01 | 1*01 | 1-1*01 | CASSFDQLNTEAFV | Seq12 |
| Medium | 77.28 | 281 766 | 24-1*01 | 1 or 2 | 2-3*01 | CATSEVTSTDTQYF | Seq07 |
| Medium | 10.67 | 38 897 | 20-1|| | 1*01 | 2-7*01 | CSAKGQGPYEQYF | Seq08 |
| Medium | 8.26 | 30 122 | 27*01 | Not identified | 1-1*01 | CASSFDPLNTEAFF | Seq01 |
| Medium | 0.77 | 2799 | 7-8# | 2*01 or 2*02 | 2-3*01 | CASSSAPRLTSGRTDTQYF | Seq03 |
| Medium | 0.11 | 419 | 24-1*01 | 1*01 | 2-3*01 | CATSEVNSTDTQYV | Seq10 |
| Low** | 94.31 | 143 552 | 24-1*01 | 1 or 2 | 2-3*01 | CATSEVTSTDTQYF | Seq07 |
| Low | 4.78 | 7276 | 20-1|| | 1*01 | 2-7*01 | CSAKGQGPYEQYF | Seq08 |
| Low | 0.26 | 403 | 27*01 | Not identified | 1-1*01 | CASSFDPLNTEAFF | Seq01 |
| Low | 0.15 | 229 | 24-1*01 | 1*01 | 2-3*01 | CATSEVNSTDTQYV | Seq10 |
Cells were sorted from this polyclonal line based on their binding avidity for the DRB1*01:01-FVIII2194-2213 tetramer into high-, medium-, and low-avidity populations (P2, P3, and P4 gates, respectively, in supplemental Figure 4); 200 000 cells were collected from each population except for the smaller high-avidity population, which consisted of 112 000 cells. Genomic DNA was isolated from cells, and deep TCRB-CDR3 sequencing was performed by Adaptive Biotechnologies. Sequences were analyzed with ImmunoSEQ Analyzer version 2.0. Productive sequences with a frequency ≥0.1% are shown.
Gene names for nucleotide sequences were determined using IMGT/V-QUEST version 3.3.4.50
Sequences were given a numerical identifier for labeling purposes. Analyzed nucleotide sequences are in supplemental Table 6.
Multiple gene names result from >1 known annotated V, D, or J gene sequence matching equally well with the aligned sequence.
Sequence was identical to 20-1*01, *02, *03, *04, and *05 alleles.
Sequence was identical to 19*01, *02, and *03 alleles.
Sequence was identical to 7-8*01 and *03 alleles.
Results shown are for the low-avidity CD4 P4 gate in supplemental Figure 4. TCRB sequences identified in the low-avidity CD4-low P5 gate were similar, as the 3 most frequent sequences were identical with similar frequencies.