3.2.1 Hsp70 membrane-positive tumor cells are infiltrated by TKD stimulated PBL
The 14-mer peptide TKD did not only enhance the cytotoxic activity of NK cells against Hsp70 membrane-positive tumor cells but also induces migration of NK cells towards them. A first hint that membrane-bound Hsp70 attracts NK cells was given by the following experiment: TKD stimulated PBL were co-incubated with exponentially growing Hsp70 membrane-positive CX+ and Hsp70 membrane- negative CX- cells. After 24 h the morphology of the cells in the co-cultures and controls (tumor cells alone) were analyzed by light microscopy (Figure 14).
Figure 14: Infiltration of Hsp70 membrane-positive tumor cell clusters by TKD activated PBL.
Hsp70 membrane-positive CX+ and Hsp70 membrane-negative CX- cells (0.1 x 106 cells/well) were seeded in 96 well flat-bottom plates and grown for 48 h. Tumor cells were co-incubated either with medium or with TKD stimulated PBL for another 24 h. Light microscopical analysis is shown for tumor cells only (control, upper row) or tumor cells co-incubated with TKD activated PBL (+ PBL. lower row) at a magnification of 50 x on a Zeiss Axiovert 25 microscope. Small inserts show a 2.5-fold magnification of one representative cell cluster. The scale bar in the lower right panel indicates 100 µm.
Under control conditions both cell lines showed a similar morphology forming adherent spheroidal cell clusters (Figure 14, control). If TKD activated effector cells
were added, the Hsp70 membrane-positive CX+ cell clusters were infiltrated by PBL, but not their Hsp70 membrane-negative counterparts (Figure 14, + PBL). Cell viability of CX+ cells also appeared to be reduced. This finding was confirmed by the fact that Hsp70 membrane-positive tumor cells are more susceptible to lysis mediated by TKD stimulated NK cells, than Hsp70 membrane-negative tumor cells (Figure 13).
3.2.2 TKD stimulated PBL selectively migrate towards Hsp70 membrane- positive tumor cells and supernatants derived thereof
Additional migration assays were performed using a transwell culture system. As tumor target cells the Hsp70 membrane-positive CX+ and Hsp70 membrane- negative CX- tumor cells were used. These cells were cultured in the lower compartment for 48 h. TKD activated PBL were added to the upper compartment and after a 4 h co-incubation period specific migration was measured. As shown in Figure 15A migration of TKD activated PBL towards Hsp70 plasma membrane-positive CX+ (11%) cells was higher than migration towards their negative counterparts (5%). As a positive control the cytokine IL-15 (10 ng/ml) was used as an attractant. Comparable to data published by Allavena et al (1997) migration towards IL-15 was 15% (data not shown). It has been shown that co-incubation of IL-2 activated NK cells and K562 target cells results in effector-target cell conjugates that trigger chemokine secretion and chemoattraction of other NK cells (Nieto et al 1998). To exclude chemoattraction induced by direct cell-cell contact, cell-free supernatants derived from CX+ and CX- tumor cells on day 2 were used. As demonstrated in Figure 15B left panel, TKD activated PBL showed 12% migration towards supernatants derived from CX+ cells, whereas only 3% of the PBL migrated towards supernatants derived from CX- cells. This suggests that migration of TKD activated PBL might be induced by a soluble factor that is produced selectively by Hsp70 membrane-positive tumor cells.
RESULTS 44
Figure 15: TKD activated PBL selectively migrate towards Hsp70 membrane-positive tumor cells and supernatants derived therof. Migration was analyzed in a transwell system with two compartments separated by a polycarbonate membrane with a pore size of 5 µm. (A) In the lower compartment CX+ and CX- tumor cells (0.2 x 106 cells/well) were grown for 48 h in 600 µl of RPMI- 1640 medium supplemented with 10% heat inactivated FCS. PBL stimulated with low dose IL-2 (100 IU/ml) plus TKD (2 µg/ml) for 4 days, were labeled with sodium 51chromate (100 µCi) and transferred into theupper chamber of the transwell system. Following a 4 h co-incubation period radioactivity was determined in PBL that migrated to the lower chamber. Specific migration was calculated as described in Materials and Methods. Results are shown as the mean ± SD of at least five independent experiments; statistically significant (student's τ-test) * p < 0.05. (B) In a next set of experiments 600 µl of cell-free culture supernatant (SN) of CX+ and CX- tumor cells cultured for 48 h were administered in the lower compartment. Following a 4 h co-incubation period with IL-2 and TKD stimulated PBL (left panel) or IL-2 stimulated PBL (right panel), radioactivity was determined in the migrated cells of the lower chamber. Specific migration was calculated as described above. Results are shown as the mean ± SD of at least five independent experiments; statistically significant (student's τ-test) * p < 0.05, ** p < 0.005.
Identical results were obtained with the pancreatic tumor system Colo+/Colo- (Gastpar, Gross et al 2004, appendix iv). Similar to the CX+/CX- sublines the Hsp70 membrane-positive Colo+ (73% Hsp70 membrane-positive cells) subline and the Hsp70 membrane-negative Colo- (34% Hsp70 membrane-positive cells) subline was generated from the pancreas carcinoma cell line Colo357 (50% Hsp70 membrane- positive cells) by flow cytometric cell sorting using the Hsp70 specific antibody cmHsp70.1. In this system TKD activated PBL migrated towards Colo+ cells (10%) and their supernatants (12%) whereas no migration against Colo- cells and their supernatants was observed (6% and 6% respectively). It has to be mentioned that stimulation of PBL with low dose IL-2 and TKD is a prerequisite for the induction of the migratory capacity since PBL stimulated with IL-2 alone did not migrate at all (Figure 15B, right panel).
3.2.3 NK cells migrate and exhibit lytic activity against Hsp70 membrane- positive tumor cells
A phenotypic analysis of the migrated cells revealed that selectively CD3-/CD16/56 NK cells but not CD3+ T cells were attracted by supernatants derived from Hsp70 membrane-positive tumor cells (Figure 16).
Figure 16: NK cells migrate towards and exhibit lytic activity against Hsp70 membrane-positive
tumor target cells. A 4 h migration assay was performed using TKD stimulated PBL against
supernatants of Hsp70 membrane-positive CX+ tumor cells to determine the phenotype and the lytic activity of non-migrated and migrated cells in comparison to the initial PBL population. (A) Non migrated PBL (middle panel), and migrated cells (right panel) were harvested and analyzed by multiparameter flow cytometry using CD3-FITC and CD16/56-PE antibodies. The phenotype of each cell population was compared to the initial PBL population (left panel) that had been stimulated with TKD for 4 days. Numbers in the dot blot diagrams indicate percentage of positively stained cells for each quadrant. Data of one representative result of three independent experiments is shown. (B) Concomitantly, the cytolytic activity of the three effector cell populations (initial PBL, non-migrated PBL, and migrated PBL) was tested in a standard 4 h 51Cr release assay. CX+ (solid circles) and CX- (open circles) cells served as tumor target cells.Effector to target ratios ranged from 1/1 to 10/1. Mean values of three independent experiments ± SD is shown.
As shown in Figure 16A the cells of the original population contained 34% CD3 T cells, 11% CD3+/CD16/56+ NKT cells and 29% CD3-/CD16/56+ NK cells. The non- migrated cells showed no significant differences in the percentage of CD3+ T cells (36%) and CD3+/CD16/56+ NKT cells (7%), whereas a drastic decrease in CD3- /CD16/56+ NK cells from 29% to 17% could be observed. Concomitantly, the CD3-
RESULTS 46 /CD16/56+ increased significantly from 29% to 61% in the migrated cell population and the amount of CD3+/CD16/56- T cells was reduced from 34% to 18%. Taken together, these data indicate, that CD3-/CD16/56+ NK cells were specifically attracted by Hsp70 membrane-positive tumor cells and supernatants derived thereof. In addition, the migrated cell population showed a very strong lytic activity against Hsp70 membrane-positive CX+ target cells (solid circles) and a weaker activity against Hsp70 membrane-negative CX- target cells (open circles) (Figure 16B, right panel), whereas non-migrated cells showed a very weak lytic activity against all tumor target cells (Figure 16B, middle panel) indicating that CD3-/CD16/56+ NK cells were selectively attracted by Hsp70 membrane-positive tumor cells and specifically lyse them. Again, similar results were observed with the Colo+/Colo- system (Gastpar, Gross et al 2004, appendix iv).
Since NK cells not only migrated towards Hsp70 membrane-positive tumor cells, but also to supernatants derived thereof, a soluble factor was postulated that might be released by Hsp70 membrane-positive tumor cells. This was proven by the fact that both Hsp70 and TKD initiated migratory capacity in NK cells in a concentration- dependent (1 – 5 µg/ml), highly selective and chemokine-independent manner (Gastpar, Gross et al 2004, appendix iv). Taken together these results indicate that Hsp70 as well as TKD stimulate cytolysis, proliferation and chemotaxis in CD3- /CD16/56+ NK cells.