Article Figures & Data
Figures
- FIGURE 1.
Assessment of functional reactivity and BV usage by A2/Melan-A tetramer-positive and -negative TILN from an A2+ melanoma patient. Short term cultured TILN from HLA-A2+ melanoma patient LAU 233 were triple stained with anti-CD8 mAbs together with anti-BV mAbs and A2/Melan-A tetramers as detailed in Materials and Methods. Frequencies of cells in the quadrants were calculated with CellQuest software. Results are shown for gated CD8bright cells. The A2/Melan-A tetramer vs CD8 dot plot is shown for gated live lymphocytes (A). Selected dot plots for BV-14 (E) and BV-17 (F) are shown. CD8bright lymphocytes from TILN were sterile sorted into A2/Melan-A tetramer+ and tetramer− populations. After overnight culture, sorted populations were tested for their cytolytic activity against 51Cr-labeled T2 cells (HLA-A2+). B, Bars represent the difference in specific lysis obtained in the presence of peptide Melan-A26–35 (EAAGIGILTV; 1 μM) minus the percent lysis on T2 cells without peptide. C, Specific cytolytic activity of tetramer+ lymphocytes in response to graded concentrations of the peptide Ag (▪; Melan-A26–35; EAAGIGILTV) or to an irrelevant antigenic peptide (□; NY-ESO-1157–165; SLLMWITQC) (29 ). D, Specific cytolytic activity of tetramer+ lymphocytes on the autologous melanoma line Me 305 (□), the HLA-A2+ Melan-A-expressing melanoma cell line Me 290 (○), the HLA-A2+ Melan-A-negative melanoma cell line NA-8 MEL (▵), or the HLA-A2− melanoma cell lines, Me 242 B1 (▾) and Me 329 M2 (♦), in the absence (open symbols) or the presence (filled symbols) of the antigenic peptide Melan-A26–35 (1 μM).
- FIGURE 2.
A2/Melan-A tetramer-guided TCR BV region analysis of TILN. TILN from HLA-A2+ melanoma patients were stained A2/Melan-A tetramers together with anti-BV and anti-CD8 mAbs as detailed in Materials and Methods. Results were analyzed with CellQuest software. After gating on CD8bright living lymphocytes A2/Melan-A tetramer vs BV dot plots were analyzed. Dot plots were divided into four quadrants as illustrated in Fig. 1, E and F, and the percentages of CD8+ A2/Melan-A tetramer+ or tetramer− cells expressing a given BV were calculated. For example in Fig. 1E the upper right quadrant contains tetramer+ cells that express BV14. These cells represent 6.5% of the total CD8+ population. Considering that total tetramer+ cells account for 22.5% of CD8+ cells in the sample, it follows that the tetramer+ BV14+ fraction represents 28.8% of total tetramer+ cells (A). A similar calculation was performed for tetramer− cells (B).
- FIGURE 3.
A2/Melan-A tetramer-guided TCR BV region analysis of TIL. TIL from HLA-A2+ melanoma patients were stained as described in Fig. 2. Percentages of BV+ A2/Melan-A tetramer+ (A) or BV+ A2/Melan-A tetramer− (B) cells were calculated with CellQuest software as detailed in Fig. 2. Results are shown for gated CD8bright cells.
- FIGURE 4.
BV usage analysis of CD8+ A2/Melan-A tetramer+ peptide stimulated PBMC from melanoma patient LAU 203. CD8+ highly enriched cells from patient LAU 203 were stimulated with peptides and autologous APC as detailed in Materials and Methods. Two parallel cultures (a and b) were simultaneously set for either Melan-A27–35 nonapeptide (A) or the Melan-A26–35 decapeptide (B). After 2 wk of culture, cells were triple stained with anti-CD8 mAb, A2/Melan-A tetramers and the listed anti-BV mAb. Numbers represent the percentage of BV+ A2/Melan-A+ cells on gated CD8bright cells calculated with CellQuest software as detailed in Fig. 2. The percentages of CD8+ A2/Melan-A tetramer+ cells contained in the cultures at the moment of the analysis were 3.5% (AAGIGILTV; a), 4.6% (AAGIGILTV; b), 4.0% (EAAGIGILTV; a), and 4.4% (EAAGIGILTV; b), respectively.
Tables
- Table I.
Clinical status of metastatic melanoma patients and enumeration of A2/Melan A tetramer binding lymphocytes in TIL(N)
Patient Codea Age (yr) Tumor Stage (AJCC)b Samplea %CD8+ in Lymphocyte Gatec %CD8+ A2/Melan-A Tetramer+ LAU 156 50 T3N0M1 Paravertebral metastasis 97 9.7 LAU 156 51 T3N0M1 Paravertebral metastasis 85.4 14.7 LAU 212 71 pTXN2cM0 Subcutaneous metastasis 92 3.9 LAU 337 25 pT3aN2M1 Soft tissue metastasis 34.2 0.3 LAU 321 65 pT2N2cM0 Subcutaneous metastasis 65.5 0.2 LAU 343 78 pT3aN2cM0 Subcutaneous metastasis 63.6 15.5 LAU 253 61 pT3aN2M0 Tumor-infiltrated lymph node 62.3 21.8 LAU 50 65 pT4aN2cM0 Tumor-infiltrated lymph node 47.5 14.5 LAU 233 75 pT4N2cM0 Tumor-infiltrated lymph node 87.4 24.4 LAU 203 66 pTxN2cM0 Tumor-infiltrated lymph node 98 4.9 LAU 314 42 pT2N0M1 Tumor-infiltrated lymph node 67.7 <0.01 LAU 198 71 pT3aN1M0 Tumor-infiltrated lymph node 52.8 <0.01 -
a All samples included in this series were collected by surgical dissection, finely minced with needles and cultured for 2–3 wk in complete medium supplemented with hrIL-2 and hrIL-7.
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b AJCC, American Joint Committee on Cancer.
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c Aliquots of TIL(N) were analyzed by two-color flow cytometry after staining with anti-CD8 and A2/Melan-A tetramers. CD8bright cells were gated, and the percentage of CD8+ A2/Melan-A tetramer+ cells was calculated with CellQuest software.
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