Article Figures & Data
Figures
- Fig. 1.
Correlation between NK cell frequency and CD16bright subpopulation in TIL. A, NK frequency (X axis) of TIL (left; n = 19) and PBMCs of patients (right; n = 13) plotted against the percentage of CD16bright NK cells (Y axis). Symbols, patients. Left, TIL with high (⧫, >20% of lymphocytes) or low (◊, <20% of lymphocytes) NK content. Right, patients with tumors of low (○) or high (•) NK content. B, CD16bright cells among gated CD3-CD56+ lymphocytes of TIL and PBMC of patients 3080 and 3074. The mAb combination was CD3, CD56, and CD16. Quadrants were adjusted according to isotype controls tested for each sample. The percentage of CD56+ cells is given in corresponding quadrants.
- Fig. 2.
Cytotoxicity of NK-PBMC from healthy donors and renal cell carcinoma patients and NK-TIL of high (high-NK-TIL) or low NK content (low-NK-TIL). A, cytotoxicity against K562 with effector-to-target cell ratio of 20:1 or 40:1 (*). NK cells were enriched to similar percentages (60-95%; circles). Three samples of the high-NK-TIL group had percentages of NK between 35% and 42% and were used without enrichment (diamonds). Horizontal bars, median values. B, redirected lysis against NKp46-loaded P815. Only enriched NK populations were used. Vertical bar, maximal value obtained with isotype (MOPC21)–loaded P815. Individual isotype values ranged between 2% and 10% in different assays. Effector-to-target cell ratio was 20:1 (black columns) or 40:1 (gray columns).
- Fig. 3.
Expression of NKp46, CD3ζ, NKG2D, DAP10, and DAP12/KARAP by PBMC and TIL. A to C, dot plots of gated CD3-CD56+ lymphocytes. Quadrants were adjusted according to isotype controls. Numbers in top right quadrants, percentage of positive cells of the CD56+CD3− population. A, PBMC-3088 (27% CD56+CD3− NK cells) and TIL-3088 (12% CD56+CD3− NK cells) stained with mAbs against CD3, CD56, and NKp46. B, PBMC-2936 (7% CD56+CD3− NK cells) and TIL-2936 (13% CD56+CD3− NK cells) stained with mAbs against CD3, CD56, and CD3ζ. C, PBMC-2928 (21% CD56+CD3− NK cells) and TIL-2928 (8% CD56+CD3− NK cells) stained with mAbs against CD3, CD56, and NKG2D. D, semiquantitative RT-PCR of PBMC-239, PBMC-2986, TIL-2986, and TIL-2936 with primers for DAP10, DAP12/KARAP, and GAPDH. Identical amounts of cDNA and PCR products were used in each case. Fragment length is presented in brackets.
- Fig. 4.
Perforin, granzyme A, and granzyme B expression of NK-PBMC and NK-TIL from renal cell carcinoma patients (A and B, gated on CD56+CD3− cells) and NK lines NKL, NK-92, YT, and 2DL2+NK (C). mAbs combinations were CD3 and CD56 and either perforin, granzyme A or granzyme B. A, percentages of perforin+ (first row), granzyme A+ (second row), or granzyme B+ (third row) NK cells. Groups are as follows: NK-PBMC of renal cell carcinoma patients (renal cell carcinoma-PBMC, black symbols), NK cells of high NK-TIL (gray symbols), or low NK-TIL (open symbols). B, perforin expression levels of NK cells of renal cell carcinoma-PBMC (top row), high NK-TIL (middle row), and low NK-TIL (bottom row). Two examples are shown for each category. Percentages of perforinhigh NK cells are quoted in each histogram. Mean fluorescence intensities for isotype controls ranged from 5 to 7 in each case (not shown). C, cytotoxin expression levels of NK lines: perforin (light gray histograms), granzyme A (dark gray), and granzyme B (dashed lines). Isotype control for perforin is shown in each case (dotted lines). Isotype controls for the other antibodies showed comparable fluorescence intensity (not shown). D, cytotoxicity of NK lines against K562 at declining effector-to-target cell ratio. Cytotoxic assays and stainings (C) were done in parallel.
Tables
- Table 1.
Clinicopathologic variables of renal cell carcinoma patients
Patient ID Sex Age* TNM grade† Tumor stage‡ Follow-up§ (mo) High NK content 3109 M 67 T1bN0M0 G2 I A-DF (12) 3091 F 31 T2N0M0 G2 I A-DF (16) 3073 M 61 T1aN0M0 G2 I A (18) 3074 F 66 T1bN0M0 G2 I A-DF (18) 2926 M 52 T1NxMx G2-3 I A (38) 2924 M 47 T1N0M0 G2 I A (39) 3094 M 47 T2N0M0 Gx I ND 2864 M 68 T3aNxM0 G2 II A-DF (45) 3093 M 68 T3bN0M0 G2 III A-PR (15) 2913 F 76 T3bN0M0 G2 III A-DF (43) 2900 F 67 T3bN0Mx G2 III A-DF (43) 0174 F 74 T3aNxMx G3 III ND 3108 M 59 T3bNxM0 G2 III ND Low NK content 3100 M 34 T2N0M0 G2 I A-DF (14) 3075 M 68 T1N0M0 G2 I A-DF (18) 2863 M 47 T1bN0M0 G2 I DOD (34) 2928 M 57 T2N0Mx G3 I A-DF (38) 2990 F 69 T1NxMx G2 I A-DF (42) 2907 M 68 T1N0Mx G2 I A-DF (42) 2901 M 83 T2NxMx G2 I A-DF (43) S306 M 68 T1bN0M0 Gx I ND S333 F 76 T1bN0Mx G2 I ND 2936 M 61 T3N0Mx G3 II/III A (36) S310 M 65 T3bNxMx G3 III ND 3086 F 74 T3bN0M0 G3 III A-DF (16) 3080 M 53 T3bN0M0 G2 III A-DF (17) 2986 F 74 T3bN0Mx G2 III A (27) 2869 F 80 T3bN2M0 G2 III A (45) 2988 M 61 T3bN2M1 G3 IV DOD (12) 3088 M 35 T4N0M0 Gx IV A-PR (15) 2916 M 73 T3bN0M1 G2 IV DOD (27) 2890 M 68 T3bN0M1 G2 IV A-PR (43) 2885 F 80 T1N0M1 G3 IV A-SD (44) NOTE: Only clear cell carcinomas were selected. Patients did not receive any treatment in addition to surgery.
Abbreviations: M, male; F, female; A, alive; A-DF; alive-disease free; A-PR, alive-progress; A-SD, alive-stable disease; DOD; dead-of-disease; ND, not determined.
↵* Age, in years, at operation; mean = 61.3y for high-NK group and 61 years for low-NK group.
↵§ Follow-up was completed until August 2005 with a median follow-up of 34 months (range: 12-45). Numbers in brackets are months of survival after operation.
- Table 2.
CD16 surface expression NK cells correlates with the frequency of NK cells in renal cell carcinoma tumors
n* %NK (range)† %CD16+ (range)‡ NK-PBMC§ 13 12.7 (2.0-26.0) 89.5 (72.4-98.7) NK-TIL∥ 22 59.3 (24.8-96.9) High NK-TIL 7 35.4 (22.0-62.1) 89.9 (82.0-96.9)¶ Low NK-TIL 14 12.2 (5.9-17.9) 43.5 (24.8-68.9)¶ ↵* Number of lymphocyte populations investigated.
↵† Mean percentage (range) of CD3−CD56+ NK cells.
↵‡ Mean percentage (range) of CD16+ cells within gated CD3−CD56+ NK cells.
↵§ PBMC from renal cell carcinoma patients.
↵∥ TIL from renal cell carcinoma tissues, grouped according to their NK cell content (high-NK-TIL, >20% NK; low-NK-TIL, <20% NK).
↵¶ P value between both groups of NK-TIL was <0.0001 (other P values are given in the text).
Volume 12, Issue 3
