Tumor-infiltrating lymphocytes (TIL) were tested for cytotoxicity against autologous tumor cells in a study utilizing a chemically induced cancer ofthe bladder (transitional cell carcinoma),
BC
-47, in inbred ACI/N rats. From tumors grown after subcutaneous implantation of
BC
-47 in the rats TIL were separated by density gradient centrifugation and incubated in plastic dishes for separation of non-adherent from adherent cells. The non-adherent cells were further fractionated into T and
B
cells by the panning method using anti-rat F(
ab
')
2 antibody. The cell fractions were each added to
BC
-47 in culture to be assessed for antitumor effect by the crystal violet dye exclusion method and 3H-thymidine incorporation inhibition assay. Peripheral blood mononuclear cells (PBMC) were also tested as described above. TIL expressed significantly higher cytotoxicity against
BC
-47 with the mean % cytotoxicity of 56.
6
±5.
6
% and 87.5±
7
.
1
% at
E
/T ratios of 10:
1
and 20:
1
, respectively, as compared to PBMC(
9
.
9
±5.0% at
E
/T 10:
1
) (P<0.001). The adherent cells,
B
and T cell fractions showed respective % cytotoxicity of 92.
4
±2.
8
%, 57.
9
±10.
6
% and
9
.
9
±
7
.
8
% at an
E
/T ratio of 10:
1
. TIL pretreated with IFN or rIL-2 for 24or 48 hours did not exhibit any noticeably enhanced antitumor activity at an
E
/T ratio of 5:
1
. Prevention of direct contact of
BC
-47 cells and TIL by an interposed Millipore membrane (0.45μm) resulted in an unequivocal reduction of antitumor effect. This finding clearly indicates that the direct contact with
BC
-47cells and TIL is necessary for TIL to manifest their cytotoxicity against the autologous tumor cells, suggesting that TIL exhibit autologous tumor killing activity through cell-mediated cytotoxicity.
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