Article Figures & Data
Figures
- Fig. 1.
Immunohistochemical staining of advanced human ovarian carcinomas. A, a high frequency of CD45+ leukocytes is seen after staining with anti-CD45 mAb (magnification, ×20); B, a high proportion of NKG2D+ cells is seen. In average, these represent 15% of total leukocytes (magnification, ×10); C, CD8+ cells are noted within a tumor islet (magnification, ×20). D, CD57+ NK cells are only occasionally present in advanced ovarian carcinoma (<1% of total CD45+ cells); E, double fluorescent terminal deoxynucleotidyl transferase-mediated nick end labeling/anti-CD3 immunostaining demonstrates absence of apoptosis, as detected by terminal deoxynucleotidyl transferase-mediated nick end labeling (FITC, green), in CD3+ tumor-infiltrating lymphocytes (rhodamine, red). F, Letal protein is expressed predominantly by tumor cells in ovarian carcinomas. Nuclei were counterstained with hematoxylin. These images are representative of stage III ovarian carcinomas with intratumoral T cells.
- Fig. 2.
Letal expression increases with tumor progression but is associated with better outcome. A, quantification of Letal mRNA levels by TaqMan PCR in human normal ovaries, benign tumors (n = 6), borderline tumors (n = 4), stage I (n = 9), and stage III ovarian carcinomas (n = 43). B, Letal mRNA expression analyzed by TaqMan PCR in tumor islets harboring intratumoral T cells and tumor islets lacking intratumoral T cells, isolated by laser capture microdissection. C–E, Kaplan-Meier curves for overall survival based on the presence (red) versus absence (black) of Letal mRNA (C), the presence (red) versus absence (black) of intratumoral T cells (D), suboptimal (red) versus optimal (black) debulking (E), and the presence (red) versus absence (black) of NKG2D staining in tumor islets (F). G, Kaplan-Meier curves for overall survival in patients with tumors characterized by optimal debulking plus tumor-infiltrating lymphocytes (TILs) present plus Letal present (black), TILs present plus either optimal debulking or Letal present (red), and TILs present plus any combination of debulking and Letal mRNA expression (green).
- Fig. 3.
Letal induces sustained expansion of tumor-infiltrating or tumor-associated CD28− effector lymphocytes. A, most CD8+ lymphocytes in solid tumors and ascites do not express the costimulatory molecule CD28. B, sustained expansion of sorted tumor-infiltrating or tumor-associated CD8+CD28− lymphocytes through CD3/Letal costimulation provided by Letal+ K32 cells bearing anti-CD3 monoclonal antibody. Left, tumor-infiltrating lymphocytes procured from solid tumors; right, tumor-associated lymphocytes sorted from tumor ascites. C, combined stimulation with anti-CD3 and Letal induces a marked increase in IFN-γ secretion by CD8+CD28− cells. Results are compared with a pool of supernatants from the same cells activated with K32 cells bearing anti-CD3 monoclonal antibody alone.
- Fig. 4.
Letal engagement increases the glycolytic flux and protects lymphocytes from genotoxic death. A, Letal alone or in combination with CD3 stimulation up-regulates Glut-1 as efficiently as CD3/CD28 costimulation in CD8+ lymphocytes. Shaded: lymphocytes stimulated with CD3 alone. B, Letal alone or in combination with CD3 stimulation up-regulates glucose uptake by CD8+ lymphocytes more efficiently than CD3/CD28 costimulation. C, Letal engagement protects CD8+ lymphocytes from genotoxic drugs. Peripheral CD8+ T cells were stimulated for 3 days with the indicated factors and then incubated with cisplatin. Note that results are expressed as percentage of apoptotic cells. All results are representative of at least three experiments.
- Fig. 5.
Letal engagement protects lymphocytes from Fas-dependent apoptosis. A, select ovarian carcinoma specimens exhibit intense Fas ligand staining in tumor cells by immunohistochemistry. B, down-regulation of Fas/CD95 by peripheral blood lymphocytes upon CD3/Letal engagement. Lymphocytes were stimulated for 4 days with the indicated conditions, and Fas expression was analyzed by flow cytometry. Shaded: unstimulated CD8+ cells at day 4. C, Letal stimulation induces resistance to Fas ligand-dependent apoptotic death. CD8+ lymphocytes treated with the indicated factors for 3 days were exposed to agonistic anti-Fas antibody EOS9.1 that delivers an apoptotic signal to Fas-expressing cells. More than 25% of Letal-stimulated lymphocytes resist apoptosis after 18 h. A representative analysis of three experiments is shown. Note that results are expressed as percentage of nonapoptotic cells.
Tables
- Table 1
Association with other clinicopathological variables
A. Letal Letal present (%) Letal absent (%) P Intratumoral T cells Present 46.7 75.0 0.063 Absent 53.3 25.0 NKG2Da Present 60.0 75.0 0.307 Absent 40.0 25.0 Debulking Optimal 26.7 42.9 0.295 Suboptimal 73.3 57.1 Histotype Serous, mucinous, or endometroid 53.3 71.4 0.235 Clear cell or undifferentiated 46.7 28.6 Paclitaxel Included 40.0 60.7 0.194 Not included 60.0 39.3 Response to chemotherapy Complete 73.3 78.6 0.698 Noncompleteb 26.7 21.4 B. NKG2D NKG2D presenta (%) NKG2D absenta (%) P Intratumoral T cells Present 38.5 76.7 0.016 Absent 61.5 23.3 Debulking Optimal 30.8 40.0 0.565 Suboptimal 69.2 60.0 Histotype Serous, mucinous, or endometroid 46.2 73.3 0.086 Clear cell or undifferentiated 53.8 26.7 Paclitaxel Included 61.5 50.0 0.486 Not included 38.5 50.0 Response to chemotherapy Complete 69.2 80.0 0.443 Noncompleteb 30.8 20.0 -
a In tumor islets.
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b Comprises partial response and no response.
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- Table 2
Risk ratios from multivariate Cox proportional hazards model analysis
Group Risk ratio P Good prognosis Intratumoral T cells present plus optimal debulking plus Letal present 0.01 <0.001 Moderate prognosis Intratumoral T cells present plus either Letal absent or suboptimal debulking or both 0.25 0.005 Poor prognosis Intratumoral T cells absent plus either Letal absent or suboptimal debulking or both 1.00
Volume 64, Issue 6
