Background
Materials and Methods
Cell lines
Patienta | T cell phenotype | Expression of CCR2 (% positive cells)b | |
---|---|---|---|
# | Dukes' Disease Stage | ||
296674 | A | CD4 | 58.8 |
298884 | B | CD4/CD8 | 68 |
1003485 | B | CD4/CD8 | 48.2 |
05193 | B | CD4/CD8 | 27.4 |
Reagents
Chemokine determination by RT-PCR or ELISA
Phenotyping
T-cell migration in organotypic CRC culture (reconstruct)
Blocking of T-cell migration in reconstruct
Chemotaxis assay
Immunohistochemistry
Statistical analyses
Results
Functional characteristics of CTL007 in reconstruct
Lymphocytes | E:T | Total number of tumor cells mean ± SD/field (10 fields) | Number of apoptotic tumor cells mean ± SD/field (10 fields) | Percentage of apoptotic cells, mean ± SD/field (10 fields) |
---|---|---|---|---|
Experiment I | ||||
CTL007 | 1:1 | 80.5 ± 38.8 | 23.5 ± 8.7 | 31.7 ± 11c, d |
PHA blast | 1:1 | 136.2 ± 54.8 | 15.9 ± 6.8 | 12.1 ± 3.9c |
No lymphocytes | NA | 90.1 ± 3.5 | 7.1 ± 2.8 | 8.1 ± 3.5d |
Experiment II | ||||
CTL007 | 10:1 | 116.7 ± 19.4 | 94.8 ± 19 | 79.5 ± 6.7a, b |
PHA blast | 10:1 | 118.3 ± 23.6 | 34.7 ± 11.8 | 27.5 ± 4.7a |
No lymphocytes | NA | 121.1 ± 22.3 | 25.8 ± 10.7 | 21.3 ± 10.1b |
Phenotypic characteristics of CTL007 and WC007 CRC cells
Parameter investigateda | Cell lines (% cells positive) | |
---|---|---|
CRC WC007 | CTL007 | |
HLA Class I | 99.8 | 95.6 |
HLA Class II | < 1 | 60 |
CD4 | NA | 96 |
CD8 | NA | < 1 |
CD25 | < 1 | 30 |
CD40L | 2.1 | 13 |
CD44 | 48 | 79.7 |
CD80 (B7-1) | 85 | < 1 |
CD49a (α1 integrin) | 48.2 | 26.1 |
CD49b (α2 integrin) | 63.2 | 32.5 |
CD29 (β1 integrin) | 56.9 | 79.8 |
CD95 (FAS) | 65.1 | 95.1 |
CD95L (FASL) | 19 | < 1 |
CD54 (ICAM-1) | 84.5 | 89.3 |
CD11a (LFA-1a) | < 1 | 84.2 |
Chemokine and chemokine receptor involved in CTL007 migration toward WC007 cells
Chemokine receptors expressed by CTL007a | Chemokines | |||
---|---|---|---|---|
Chemokine receptors
|
% positive
cells
|
Known to bind
to receptor
|
Expressed by WC007
b
| |
RT-PCR | ELISA (pg/ml) | |||
CCR1 | 5.7 | CCL3 | + | < 30c |
CCL5 | - | NDd | ||
CCL7 | - | ND | ||
CCL13 | - | ND | ||
CCL15 | + | 35 | ||
CCR2 | 65 | CCL2 | + | 51.6 |
CCL7 | - | ND | ||
CCL8 | - | ND | ||
CCL13 | - | ND | ||
CCR3 | 19.4 | CCL7 | - | ND |
CCL8 | - | ND | ||
CCL11 | - | ND | ||
CCL13 | - | ND | ||
CCL24 | - | ND | ||
CCR5 | 10.1 | CCL3 | + | < 30c |
CCL4 | + | 16 | ||
CCL5 | - | ND | ||
CCR7 | 9.5 | CCL19 | + | < 20c |
CCL21 | + | < 30c | ||
CCR9 | 16.4 | CCL25 | - | ND |
CXCR1 | 16.7 | CXCL6 | - | ND |
CXCL8 | - | ND | ||
CXCR2 | 15.4 | CXCL1 | - | ND |
CXCL5 | - | ND | ||
CXCL6 | - | ND | ||
CXCL7 | - | ND | ||
CXCL8 | - | ND | ||
CXCR3 | 16.9 | CXCL9 | - | ND |
CXCL10 | - | ND | ||
CXCL11 | + | 35.6 | ||
CXCL13 | - | ND | ||
CXCR4 | 17.6 | CXCL12 | - | ND |
CXCR5 | 32.4 | CXCL13 | - | ND |
Blocking agent | Number of tumor cells/field (10 fields) | Number of apoptotic cells/field (10 fields) | % of apoptotic cells | % of tumor cell apoptosis inhibition |
---|---|---|---|---|
None | 21.8 ± 9.7 | 8.2 ± 9 | 40.5 ± 9.7b | - |
Control IgG | 21 ± 2.2 | 8.4 ± 1.1 | 40.1 ± 4.9c | - |
Anti-CCR1 Ab | 22 ± 3.4 | 10.0 ± 1.6 | 45.6 ± 5.0 | -13.7 |
Anti-CCR2 Ab | 19.6 ± 1.1 | 2.6 ± 0.5 | 13.2 ± 2.3c | 67.1 |
Anti-CCR3 Ab | 22.4 ± 2.8 | 9.2 ± 1.9 | 40.7 ± 4.2 | 1.5 |
Anti-CCR5 Ab | 21 ± 2.6 | 9.6 ± 1.8 | 45.5 ± 3.8 | -13.4 |
Anti-CCR7 Ab | 18.8 ± 2.4 | 7.2 ± 1.6 | 38.0 ± 4.1 | 5.2 |
Anti-CXCR3 Ab | 21.4 ± 1.5 | 9.2 ± 0.8 | 43.1 ± 1.8 | -7.5 |
CCL2 | 21.4 ± 4.4 | 3.6 ± 1.1 | 17.2 ± 6.1b | 57.5 |
Anti-CCL2 Ab | 17.8 ± 2.6 | 2.2 ± 0.4 | 12.7 ± 3.8c | 68.6 |