Colon carcinoma cells induce CXCL11-dependent migration of CXCR3-expressing cytotoxic T lymphocytes in organotypic culture

Abstract

Adoptive immunotherapy of cancer patients with cytolytic T lymphocytes (CTL) has been hampered by the inability of the CTL to home into tumors in vivo. Chemokines can attract T lymphocytes to the tumor site, as demonstrated in animal models, but the role of chemokines in T-lymphocyte trafficking toward human tumor cells is relatively unexplored. In the present study, the role of chemokines and their receptors in the migration of a colon carcinoma (CC) patient's CTL toward autologous tumor cells has been studied in a novel three-dimensional organotypic CC culture. CTL migration was mediated by chemokine receptor CXCR3 expressed by the CTL and CXCL11 chemokine secreted by the tumor cells. Excess CXCL11 or antibodies to CXCL11 or CXCR3 inhibited migration of CTL to tumor cells. T cell and tumor cell analyses for CXCR3 and CXCL11 expression, respectively, in ten additional CC samples, may suggest their involvement in other CC patients. Our studies, together with previous studies indicating angiostatic activity of CXCL11, suggest that CXCL11 may be useful as an immunotherapeutic agent for cancer patients when transduced into tumor cells or fused to tumor antigen-specific Ab.

Fig. 1

Functions of CTL020 cells in MLTC. CTL020 was generated in MLTC by stimulating PBMC of CC patient 020 with irradiated autologous WC020 CC cells. Cultures were restimulated every 2 weeks with irradiated autologous CC cells and 20 U/ml of natural IL-2. a CTL responses. CTL020 cells were stimulated for 4 days with irradiated autologous tumor cells and IL-2. CTL lysis of ⁵¹Cr-labeled autologous CC cells and control target cells [allogeneic Daudi (LAK target) and K562 (NK target)] was determined in 10 h ⁵¹Cr-release assay at various E:T ratios. b Cytokine production. CTL020 cells were incubated with various numbers of irradiated WC020 CC cells or without tumor cells in the presence of recombinant IL-2. IFN-γ and IL-4 in supernatants obtained from cultured CTL after 2 and 4 days, respectively, were measured by ELISA. All values obtained from cultures stimulated with tumor cells are significantly (P < 0.01) different from controls (CTL020 cells incubated without tumor cells)

Fig. 2

CTL020 cell migration toward autologous WC020 CC cells in reconstruct. A Schema of reconstruct. B CTL020 cell migration. The bottom layer of reconstructs contained 4.5 × 10⁴ fibroblasts in 450 μl type I collagen gel. After 24 h, CC cells (1 × 10⁵) were seeded on top of the bottom layer. After 24 h, CC cells were stained with CellTracker Blue CMAC. A separating layer of fibroblasts in collagen gel (500 μm) was then placed on top of the CC cell layer, followed by addition of a top layer containing 3 × 10⁵ pre-stained (CFDA-Green, Invitrogen) CTL020, mixed with 2.5 × 10⁴ fibroblasts and type I collagen gel (a); control cultures were prepared without lymphocytes (b). Reconstructs were harvested on day 6 (3 days after adding T cells), fixed in buffered formalin, and embedded in paraffin. Sections were photographed in the Nikon fluorescence microscope using appropriate filters. Magnification ×400 . Note the presence of CFDA-Green-labeled CTL in the tumor cell layer (a). C Induction of apoptosis in WC020 cells by CTL020—qualitative analysis. The reconstructs were harvested on day 7, fixed in buffered formalin, embedded in paraffin, and stained with H&E. Magnification ×400 . Note the presence of apoptotic tumor cells in cultures with WC020 plus CTL020 cells (a), but not in control cultures with WC020 plus PHA blasts (b), or WC020 only (c). D Induction of apoptosis in WC020 cells by CTL020—quantitative analysis. The percentage of apoptotic tumor cells was determined by counting apoptotic nuclei, based on nuclear morphology, and intact tumor cells in 20 fields of sections stained with H&E. Data represent means + SD of 20 fields. Values with the same symbol are significantly different from each other (P < 0.0001)

Fig. 3

Autologous CC cells WC020 produce CXCL11, and CTL020 cells express the corresponding receptor CXCR3. a Production of CXCL11. CTL020 cells were cultured with irradiated WC020 tumor cells in T-cell medium for 4 days. WC020 CC cells were cultured in CC medium; after 2 days of culture the medium was replaced and supernatants were collected 4 days later. FCFB/1 fibroblast cells were cultured in DMEM for 4 days. CXCL11 production in supernatants obtained from various cells was measured in ELISA. b Expression of CXCR3. Cultured cells of CTL020, FCFB/1, and WC020 were incubated with saturating concentration (5 μg/ml) of anti-CXCR3 MAB (solid line) or mouse IgG control (dotted line) in RPMI-1640 with 5% FBS for 1 h at 4°C. After washing, fluoresceinated goat anti-mouse IgG Ab was added. The expression of CXCR3 was detected by flow cytometry