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NKG2D is a Key Receptor for Recognition of Bladder Cancer Cells by IL-2-Activated NK Cells and BCG Promotes NK Cell Activation.

García-Cuesta EM, López-Cobo S, Álvarez-Maestro M, Esteso G, Romera-Cárdenas G, Rey M, Cassady-Cain RL, Linares A, Valés-Gómez A, Reyburn HT, Martínez-Piñeiro L, Valés-Gómez M - Front Immunol (2015)

Bottom Line: More recently, other immune effectors such as monocytes, natural killer (NK), and NKT cells have been suggested to play a role in this immune response.However, NK cells were activated efficiently when BCG was included in mixed lymphocyte cultures, suggesting that NK activation after mycobacteria treatment requires the collaboration of various immune cells.The total numbers of NK cells did not vary during treatment, indicating that a more detailed study of NK cell activation in the tumor site will be required to evaluate the response in each patient.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Oncology, National Centre for Biotechnology (CNB-CSIC) , Madrid , Spain.

ABSTRACT
Intravesical instillation of bacillus Calmette-Guérin (BCG) is used to treat superficial bladder cancer, either papillary tumors (after transurethral resection) or high-grade flat carcinomas (carcinoma in situ), reducing recurrence in about 70% of patients. Initially, BCG was proposed to work through an inflammatory response, mediated by phagocytic uptake of mycobacterial antigens and cytokine release. More recently, other immune effectors such as monocytes, natural killer (NK), and NKT cells have been suggested to play a role in this immune response. Here, we provide a comprehensive study of multiple bladder cancer cell lines as putative targets for immune cells and evaluated their recognition by NK cells in the presence and absence of BCG. We describe that different bladder cancer cells can express multiple activating and inhibitory ligands for NK cells. Recognition of bladder cancer cells depended mainly on NKG2D, with a contribution from NKp46. Surprisingly, exposure to BCG did not affect the immune phenotype of bladder cells nor increased NK cell recognition of purified IL-2-activated cell lines. However, NK cells were activated efficiently when BCG was included in mixed lymphocyte cultures, suggesting that NK activation after mycobacteria treatment requires the collaboration of various immune cells. We also analyzed the percentage of NK cells in peripheral blood of a cohort of bladder cancer patients treated with BCG. The total numbers of NK cells did not vary during treatment, indicating that a more detailed study of NK cell activation in the tumor site will be required to evaluate the response in each patient.

No MeSH data available.


Related in: MedlinePlus

Effect of BCG exposure on bladder cancer cell lines. (A) Bladder cancer cell lines T24, RT-112, J82 were incubated in the presence of BCG (two bacteria per bladder cell) for the indicated times and analyzed by flow cytometry for the expression of NKG2D-L and MHC. Data show the average and SD of three independent experiments. (B) Degranulation experiments. Bladder cancer cell lines T24, RT-112, J82 were incubated in the presence of BCG for the indicated times and blocked with anti-MHC-I antibody before being co-incubated with NK cells for 2 h at an E:T ratio of 1:2. Data show the average and SD of three independent experiments.
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Figure 4: Effect of BCG exposure on bladder cancer cell lines. (A) Bladder cancer cell lines T24, RT-112, J82 were incubated in the presence of BCG (two bacteria per bladder cell) for the indicated times and analyzed by flow cytometry for the expression of NKG2D-L and MHC. Data show the average and SD of three independent experiments. (B) Degranulation experiments. Bladder cancer cell lines T24, RT-112, J82 were incubated in the presence of BCG for the indicated times and blocked with anti-MHC-I antibody before being co-incubated with NK cells for 2 h at an E:T ratio of 1:2. Data show the average and SD of three independent experiments.

Mentions: After intravesical instillation of BCG in bladder cancer patients, mycobacteria first contact with the bladder epithelium and some reports indicate that urothelium could contribute to immune modulation. For example, urothelial cells have been shown to release cytokines after contact with Escherichia coli Hu734 (32) and to express toll-like receptors (33). In other experiments, exposure to Mycobacterium tuberculosis (Mtb) increased surface expression of one of the NKG2D-L (34). For these reasons, we investigated whether exposure to BCG affected the presence of NKG2D ligands on the surface of a panel of bladder cancer cell lines. Cells were initially treated with BCG for 1 and 4 days and no major changes were observed in the expression of NKG2D ligands (Figure 4A), then longer times were analyzed and, up to day 7 no differences were observed (data not shown). Since it is difficult to estimate how many bacteria encounters each bladder cell during treatment, the experiments were performed using several ratios of BCG:bladder cells (1:1–10:1) and no difference in surface NKG2D ligands was observed. Similar data were obtained with two other strains of BCG. To investigate if BCG-exposed bladder cells were also recognized by IL-2-activated NK cells through other receptor–ligand interaction, degranulation assays were performed after treating urothelial cells with BCG (Figure 4B). Surprisingly, only minor changes were observed in the recognition of these cells.


NKG2D is a Key Receptor for Recognition of Bladder Cancer Cells by IL-2-Activated NK Cells and BCG Promotes NK Cell Activation.

García-Cuesta EM, López-Cobo S, Álvarez-Maestro M, Esteso G, Romera-Cárdenas G, Rey M, Cassady-Cain RL, Linares A, Valés-Gómez A, Reyburn HT, Martínez-Piñeiro L, Valés-Gómez M - Front Immunol (2015)

Effect of BCG exposure on bladder cancer cell lines. (A) Bladder cancer cell lines T24, RT-112, J82 were incubated in the presence of BCG (two bacteria per bladder cell) for the indicated times and analyzed by flow cytometry for the expression of NKG2D-L and MHC. Data show the average and SD of three independent experiments. (B) Degranulation experiments. Bladder cancer cell lines T24, RT-112, J82 were incubated in the presence of BCG for the indicated times and blocked with anti-MHC-I antibody before being co-incubated with NK cells for 2 h at an E:T ratio of 1:2. Data show the average and SD of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4459093&req=5

Figure 4: Effect of BCG exposure on bladder cancer cell lines. (A) Bladder cancer cell lines T24, RT-112, J82 were incubated in the presence of BCG (two bacteria per bladder cell) for the indicated times and analyzed by flow cytometry for the expression of NKG2D-L and MHC. Data show the average and SD of three independent experiments. (B) Degranulation experiments. Bladder cancer cell lines T24, RT-112, J82 were incubated in the presence of BCG for the indicated times and blocked with anti-MHC-I antibody before being co-incubated with NK cells for 2 h at an E:T ratio of 1:2. Data show the average and SD of three independent experiments.
Mentions: After intravesical instillation of BCG in bladder cancer patients, mycobacteria first contact with the bladder epithelium and some reports indicate that urothelium could contribute to immune modulation. For example, urothelial cells have been shown to release cytokines after contact with Escherichia coli Hu734 (32) and to express toll-like receptors (33). In other experiments, exposure to Mycobacterium tuberculosis (Mtb) increased surface expression of one of the NKG2D-L (34). For these reasons, we investigated whether exposure to BCG affected the presence of NKG2D ligands on the surface of a panel of bladder cancer cell lines. Cells were initially treated with BCG for 1 and 4 days and no major changes were observed in the expression of NKG2D ligands (Figure 4A), then longer times were analyzed and, up to day 7 no differences were observed (data not shown). Since it is difficult to estimate how many bacteria encounters each bladder cell during treatment, the experiments were performed using several ratios of BCG:bladder cells (1:1–10:1) and no difference in surface NKG2D ligands was observed. Similar data were obtained with two other strains of BCG. To investigate if BCG-exposed bladder cells were also recognized by IL-2-activated NK cells through other receptor–ligand interaction, degranulation assays were performed after treating urothelial cells with BCG (Figure 4B). Surprisingly, only minor changes were observed in the recognition of these cells.

Bottom Line: More recently, other immune effectors such as monocytes, natural killer (NK), and NKT cells have been suggested to play a role in this immune response.However, NK cells were activated efficiently when BCG was included in mixed lymphocyte cultures, suggesting that NK activation after mycobacteria treatment requires the collaboration of various immune cells.The total numbers of NK cells did not vary during treatment, indicating that a more detailed study of NK cell activation in the tumor site will be required to evaluate the response in each patient.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Oncology, National Centre for Biotechnology (CNB-CSIC) , Madrid , Spain.

ABSTRACT
Intravesical instillation of bacillus Calmette-Guérin (BCG) is used to treat superficial bladder cancer, either papillary tumors (after transurethral resection) or high-grade flat carcinomas (carcinoma in situ), reducing recurrence in about 70% of patients. Initially, BCG was proposed to work through an inflammatory response, mediated by phagocytic uptake of mycobacterial antigens and cytokine release. More recently, other immune effectors such as monocytes, natural killer (NK), and NKT cells have been suggested to play a role in this immune response. Here, we provide a comprehensive study of multiple bladder cancer cell lines as putative targets for immune cells and evaluated their recognition by NK cells in the presence and absence of BCG. We describe that different bladder cancer cells can express multiple activating and inhibitory ligands for NK cells. Recognition of bladder cancer cells depended mainly on NKG2D, with a contribution from NKp46. Surprisingly, exposure to BCG did not affect the immune phenotype of bladder cells nor increased NK cell recognition of purified IL-2-activated cell lines. However, NK cells were activated efficiently when BCG was included in mixed lymphocyte cultures, suggesting that NK activation after mycobacteria treatment requires the collaboration of various immune cells. We also analyzed the percentage of NK cells in peripheral blood of a cohort of bladder cancer patients treated with BCG. The total numbers of NK cells did not vary during treatment, indicating that a more detailed study of NK cell activation in the tumor site will be required to evaluate the response in each patient.

No MeSH data available.


Related in: MedlinePlus