Enumeration of A2/Tumor Antigen Tetramer Binding Lymphocytes in Ex Vivo or Short-term Cultured LN Cells from HLA-A2+ Melanoma Patients
| Melanoma patient . | . | Type of LN . | . | PCR . | . | Exp. no. . | . | Days in culture* . | . | % CD8+ in lymphocyte gate‡ . | . | % tetramer+ in CD8+ LN cells . | ||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Melan-A . | . | Tyrosinase . | Melan-A . | . | Tyrosinase . | |||||||||||
| LAU 50 | TILN | + | + | 21.2 | 1 | 12.3 | 0.94 | 0.08 | ||||||||
| LAU 56 | TILN | − | − | 21.2 | 1 | 4.8 | 0.64 | 0 | ||||||||
| LAU 132 | TILN | + | + | 21.2 | 21 | 93 | 3.67 | 0.63 | ||||||||
| LAU 181 | NLN | + | − | 3.3 | 1 | 11.8 | 0.29 | |||||||||
| 3.3 | 1 | 11.8 | 0.23 | |||||||||||||
| 18.5 | 1 | 13.7 | 0.35 | |||||||||||||
| 18.5 | 1 | 13.8 | 0.30 | |||||||||||||
| TILN | + | + | 3.3 | 1 | 41 | 0.51 | ||||||||||
| 3.3 | 1 | 41.9 | 0.48 | |||||||||||||
| 18.5 | 1 | 41.2 | 0.46 | |||||||||||||
| 18.5 | 1 | 43.3 | 0.49 | |||||||||||||
| LAU 203 | TILN | + | + | 21.2 | 16 | 89 | 12.00 | 0.05 | ||||||||
| LAU 233 | TILN | + | + | 21.2 | 12 | 76 | 21.10 | 0.48 | ||||||||
| LAU 240 | TILN-1 | + | + | 21.2 | 1 | 15.6 | 0.82 | 0.03 | ||||||||
| TILN-2 | + | + | 21.2 | 1 | 4.3 | 0.52 | 0 | |||||||||
| LAU 253 | TILN-1 | + | + | 23.7 | 1 | 13.5 | 1.05 | 0.14 | ||||||||
| 23.7 | 1 | 14.6 | 1.21 | |||||||||||||
| TILN-2 | + | + | 23.7 | 1 | 10.2 | 0.78 | ||||||||||
| 23.7 | 1 | 10.2 | 0.81 | |||||||||||||
| LAU 267 | NLN | − | − | 4.3 | 0 | 11.2 | 0.22 | |||||||||
| 4.3 | 0 | 11.4 | 0.12 | |||||||||||||
| 18.5 | 1 | 12.6 | 0.21 | |||||||||||||
| 18.5 | 1 | 12.9 | 0.30 | |||||||||||||
| TILN-1 | + | + | 4.3 | 0 | 9.7 | 0.45 | ||||||||||
| 4.3 | 0 | 9.9 | 0.40 | |||||||||||||
| 18.5 | 1 | 10.4 | 0.45 | |||||||||||||
| 18.5 | 1 | 10.1 | 0.47 | |||||||||||||
| 1 | 10.4 | 0.62 | ||||||||||||||
| TILN-2 | + | + | 4.3 | 0 | 10.3 | 3.27 | ||||||||||
| 4.3 | 0 | 10.8 | 3.52 | |||||||||||||
| 18.5 | 1 | 11.8 | 3.53 | |||||||||||||
| 18.5 | 1 | 11.9 | 3.14 | |||||||||||||
| 18.5 | 1 | 11.8 | 3.70 | |||||||||||||
| Melanoma patient . | . | Type of LN . | . | PCR . | . | Exp. no. . | . | Days in culture* . | . | % CD8+ in lymphocyte gate‡ . | . | % tetramer+ in CD8+ LN cells . | ||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Melan-A . | . | Tyrosinase . | Melan-A . | . | Tyrosinase . | |||||||||||
| LAU 50 | TILN | + | + | 21.2 | 1 | 12.3 | 0.94 | 0.08 | ||||||||
| LAU 56 | TILN | − | − | 21.2 | 1 | 4.8 | 0.64 | 0 | ||||||||
| LAU 132 | TILN | + | + | 21.2 | 21 | 93 | 3.67 | 0.63 | ||||||||
| LAU 181 | NLN | + | − | 3.3 | 1 | 11.8 | 0.29 | |||||||||
| 3.3 | 1 | 11.8 | 0.23 | |||||||||||||
| 18.5 | 1 | 13.7 | 0.35 | |||||||||||||
| 18.5 | 1 | 13.8 | 0.30 | |||||||||||||
| TILN | + | + | 3.3 | 1 | 41 | 0.51 | ||||||||||
| 3.3 | 1 | 41.9 | 0.48 | |||||||||||||
| 18.5 | 1 | 41.2 | 0.46 | |||||||||||||
| 18.5 | 1 | 43.3 | 0.49 | |||||||||||||
| LAU 203 | TILN | + | + | 21.2 | 16 | 89 | 12.00 | 0.05 | ||||||||
| LAU 233 | TILN | + | + | 21.2 | 12 | 76 | 21.10 | 0.48 | ||||||||
| LAU 240 | TILN-1 | + | + | 21.2 | 1 | 15.6 | 0.82 | 0.03 | ||||||||
| TILN-2 | + | + | 21.2 | 1 | 4.3 | 0.52 | 0 | |||||||||
| LAU 253 | TILN-1 | + | + | 23.7 | 1 | 13.5 | 1.05 | 0.14 | ||||||||
| 23.7 | 1 | 14.6 | 1.21 | |||||||||||||
| TILN-2 | + | + | 23.7 | 1 | 10.2 | 0.78 | ||||||||||
| 23.7 | 1 | 10.2 | 0.81 | |||||||||||||
| LAU 267 | NLN | − | − | 4.3 | 0 | 11.2 | 0.22 | |||||||||
| 4.3 | 0 | 11.4 | 0.12 | |||||||||||||
| 18.5 | 1 | 12.6 | 0.21 | |||||||||||||
| 18.5 | 1 | 12.9 | 0.30 | |||||||||||||
| TILN-1 | + | + | 4.3 | 0 | 9.7 | 0.45 | ||||||||||
| 4.3 | 0 | 9.9 | 0.40 | |||||||||||||
| 18.5 | 1 | 10.4 | 0.45 | |||||||||||||
| 18.5 | 1 | 10.1 | 0.47 | |||||||||||||
| 1 | 10.4 | 0.62 | ||||||||||||||
| TILN-2 | + | + | 4.3 | 0 | 10.3 | 3.27 | ||||||||||
| 4.3 | 0 | 10.8 | 3.52 | |||||||||||||
| 18.5 | 1 | 11.8 | 3.53 | |||||||||||||
| 18.5 | 1 | 11.9 | 3.14 | |||||||||||||
| 18.5 | 1 | 11.8 | 3.70 | |||||||||||||
NLN, Macroscopically normal LN. Exp., Experiment.
LN cell suspensions were cultured in complete medium supplemented with rIL-2 and rIL-7 at 37°C for the indicated number of days. In the assays where d = 0, fresh LN cells were analyzed immediately after cell suspension preparation. In the assays where d = 1, thawed LN cells were cultured for 16–20 h before analysis.
Aliquots of TILNs or NLNs were analyzed by three-color flow cytometry after staining with anti-CD3 or anti-CD45RA, or anti-CD45RO, anti-CD8, and either A2/Melan-A or A2/tyrosinase tetramers as in Fig. 2,A. CD8+ LN cells were gated, and the percentage of tetramer+ cells was determined by setting a region as in Fig. 2 A. CD3+CD8− LNs were also analyzed on a separate gate, and the percentage of tetramer+ cells varied between 0.00 and 0.06% of gated cells, with one single exception where this percentage was 0.22% for cells of patient LAU 233 stained with the A2/ Melan-A tetramers (not shown).