Enumeration of A2/Tumor Antigen Tetramer Binding Lymphocytes in Ex Vivo LN Cells Draining Nonmelanoma Tumors
| LN no. . | . | Patient . | . | Age . | . | Carcinoma . | . | Stage . | . | Type of LN* . | . | HLA-A2‡ . | . | % CD8+§ . | . | % CD8+ tetramer+∥ . |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| yr | ||||||||||||||||
| 1 | LAU 292 | 75 | Bladder | BCGitis¶ | Right ilioobturator NLN | + | 2.2 2.7 | 0.18 0.08 | ||||||||
| 2 | LAU 293 | 46 | Breast | pT2N0Mx | Low axillary NLN | − | 9.7 7.2 | 0.18 0.13 | ||||||||
| 3 | LAU 293 | External axillary NLN | − | 9.1 8 | 0.17 0.2 | |||||||||||
| 4 | LAU 295 | 61 | Renal cell | pT2N0Mx | Right pararenal NLN | − | 5.6 6 | 0.11 0.14 | ||||||||
| 5 | LAU 296 | 75 | Lung | pT1N1Mx | Intertracheal NLN | − | 8.5 8.2 | 0.09 0.18 | ||||||||
| 6 | LAU 296 | Right paratracheal NLN | − | 6.5 7.3 | 0.11 0.11 | |||||||||||
| 7 | LAU 297 | 75 | Colon | pT4N0M1 | Left colon NLN | − | 3.5 4.6 | 0.08 0.06 | ||||||||
| 8 | LAU 297 | Paraaortic NLN | − | 5.4 5.8 | 0.04 0.04 | |||||||||||
| 9 | LAU298 | 49 | Bladder | pT4aN2 | Left TILN | + | 3.2 3.6 | 0.1 0.14 | ||||||||
| 10 | LAU 298 | Right NLN | + | 6 | 0.11 | |||||||||||
| 11 | LAU 299 | 68 | Prostate | pT3bN0 | Left ilioobturator NLN | + | 4.4 | 0.11 | ||||||||
| 12 | LAU 301 | Gastric | pT3N0Mx | Satellite NLN | + | 3.1 3.3 | 0.08 0.04 |
| LN no. . | . | Patient . | . | Age . | . | Carcinoma . | . | Stage . | . | Type of LN* . | . | HLA-A2‡ . | . | % CD8+§ . | . | % CD8+ tetramer+∥ . |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| yr | ||||||||||||||||
| 1 | LAU 292 | 75 | Bladder | BCGitis¶ | Right ilioobturator NLN | + | 2.2 2.7 | 0.18 0.08 | ||||||||
| 2 | LAU 293 | 46 | Breast | pT2N0Mx | Low axillary NLN | − | 9.7 7.2 | 0.18 0.13 | ||||||||
| 3 | LAU 293 | External axillary NLN | − | 9.1 8 | 0.17 0.2 | |||||||||||
| 4 | LAU 295 | 61 | Renal cell | pT2N0Mx | Right pararenal NLN | − | 5.6 6 | 0.11 0.14 | ||||||||
| 5 | LAU 296 | 75 | Lung | pT1N1Mx | Intertracheal NLN | − | 8.5 8.2 | 0.09 0.18 | ||||||||
| 6 | LAU 296 | Right paratracheal NLN | − | 6.5 7.3 | 0.11 0.11 | |||||||||||
| 7 | LAU 297 | 75 | Colon | pT4N0M1 | Left colon NLN | − | 3.5 4.6 | 0.08 0.06 | ||||||||
| 8 | LAU 297 | Paraaortic NLN | − | 5.4 5.8 | 0.04 0.04 | |||||||||||
| 9 | LAU298 | 49 | Bladder | pT4aN2 | Left TILN | + | 3.2 3.6 | 0.1 0.14 | ||||||||
| 10 | LAU 298 | Right NLN | + | 6 | 0.11 | |||||||||||
| 11 | LAU 299 | 68 | Prostate | pT3bN0 | Left ilioobturator NLN | + | 4.4 | 0.11 | ||||||||
| 12 | LAU 301 | Gastric | pT3N0Mx | Satellite NLN | + | 3.1 3.3 | 0.08 0.04 |
A cut-off value of 0.25 was calculated as the mean of a total of 22 determinations (0.11) of CD8+ tetramer+ LN cells from 12 LNs analyzed from nonmelanoma tumors, plus 3 SD (0.14).
Based on pathology examination of a fragment of the same LN.
An aliquot of the LN cell suspension was stained with HLA-A2–specific mAb BB7.2.
LN cell suspensions were cultured overnight in complete medium supplemented with rIL-2 and rIL-7. Aliquots of the cell suspension were analyzed by two-color flow cytometry with anti-CD8PerCP and A2/Melan-A tetramers. The percentage of CD8+ cells was calculated relative to all cells included in the live cell gate.
Live CD8+ LN cells were electronically gated, and the percentage of tetramer+ cells was calculated by setting a region similar to those used in Fig. 2 A. The determinations were generally performed in duplicate except in those LNs in which there were insufficient numbers of cells.
Subacute granulomatous cystitis after therapy with BCG. No residual tumor was found upon anatomopathological examination of resected tissues. The primary tumor had been resected 2 yr earlier by endoscopy.