Jie Mu

Enhanced Induction of Antitumor T-Cell Responses by Cytotoxic T Lymphocyte-associated Molecule-4 Blockade: The Effect Is Manifested Only at the Restricted Tumor-bearing Stages

Cytotoxic T lymphocyte-associated molecule-4 (CTLA-4), a second counterreceptor for the B7 family of costimulatory molecules, functions as a negative regulator of T-cell activation. Here, we investigated whether the blockade of the CTLA-4 function leads to enhancement of antitumor T-cell responses at various stages of tumor growth. Unfractionated spleen cells taken from CSAIM fibrosarcoma-bearing mice 1-2 weeks after CSA1M cell implantation (early tumor-bearing mice) contained tumor-primed T cells that produced interleukin 2 and IFN-gamma through collaboration with antigen-presenting cell-binding tumor antigens when cultured in vitro. However, this initial lymphokine-producing capacity decreased at later stages of tumor growth (7-10 weeks after tumor cell implantation). Anti-CTLA-4 monoclonal antibody (mAb) was added to whole-spleen cell cultures from early or late tumor-bearing mice. Spleen cells from early tumor-bearing mice exhibited enhanced production of interleukin 2 and IFN-gamma upon in vitro culture in the presence of anti-CTLA-4 mAb. However, addition of anti-CTLA-4 mAb to whole-spleen cell cultures from late tumor-bearing mice failed to display such an enhancement. Consistent with these in vitro results, the in vivo antitumor effect of anti-CTLA-4 administration was observed in a tumor-bearing stage-restricted manner; in vivo administration of anti-CTLA-4 (1 mg/mouse, three times at 1-week intervals) into early tumor-bearing mice resulted in regression of growing tumors, whereas the same treatment did not affect tumor growth when performed for late tumor-bearing mice. Similar anti-CTLA-4 effect was observed in another tumor (OV-HM ovarian carcinoma) model. These in vitro and in vivo results indicate that CTLA-4 blockade in tumor-bearing individuals enhances the capacity to generate antitumor T-cell responses, but the expression of such an enhancing effect is restricted to early stages of tumor growth.

IL-12-induced tumor regression correlates with in situ activity of IFN-gamma produced by tumor-infiltrating cells and its secondary induction of anti-tumor pathways.

Administration of recombinant interleukin‐12 (rIL‐12) into CSA1M fibrosarcoma‐bearing mice results in complete regression of growing tumors. This tumor regression is associated with massive lymphoid cell infiltration to tumor sites and is completely blocked by injection of anti‐interferon‐γ (IFN‐γ) monoclonal antibody (mAb). We investigated whether anti‐IFN‐γ mAb exerts its suppressive effect on tumor regression by blocking the IL‐12‐induced lymphoid cell migration to tumor sites or by inhibiting the secondary effects of IFN‐γ produced by infiltrating cells. Injection of anti‐IFN‐γ mAb to CSA1M‐bearing mice before IL‐12 treatment prevented the induction of tumor regression, whereas this treatment affected only marginally the infiltration of lymphoid cells to tumor masses. In accordance with this, IFN‐γ mRNA was expressed inside tumor masses by infiltrating cells after IL‐12 therapy irrespective of whether anti‐IFN‐γ mAb was injected. However, anti‐IFN‐γ mAb treatment almost completely abrogated the in situ expression of inducible nitric oxide synthase (iNOS) as well as IFN‐inducible protein‐10 (IP‐10) genes as examples of IFN‐γ‐inducible genes. Immunohistochemical analyses also revealed that the expression of iNOS protein was completely inhibited by anti‐IFN‐γ injection. These results suggest that the implementation of in situ IFN‐γ activity and its secondary induction of antitumor pathways such as iNOS and IP‐10 expression are important processes in the IL‐12‐induced tumor regression. J. Leukoc. Biol. 62: 450–457; 1997.

A Defect in Cell–to–cell Adhesion via Integrin–Fibronectin Interactions in a Highly Metastatic Tumor Cell Line

We investigated the role of integrin–fibronectin (FN) interactions in tumor cell adhesion. Two cloned tumor cell lines designated OV–LM (low–metastatic) and OV–HM (high–metastatic) were isolated from a murine ovarian carcinoma, OV2944. OV–LM and OV–HM cells exhibited high and low RGDS–sequence–dependent adhesiveness to FN, respectively. Both lines expressed comparable levels of α5 and αv integrins, which are capable of reacting with RGDS on FN. To compare the functions of these integrins between the two tumor lines, the signaling mechanism following FN stimulation was examined. Significant levels of phosphorylation of focal adhesion kinase(FAK)were detected in bothOV–LM and OV–HM cells before FN stimulation. Whereas the level of FAK phosphorylation was appreciably enhanced in OV–LM cells stimulated with FN, stimulation of OV–HM cells with FN induced a reduction in the FAK phosphorylation in association with a significant decrease in theamount of FAK protein in the soluble compartment of cell lysates. A difference in the deposition of FN on the cell surfacewas also observed between the two types of tumor lines; OV–HM cells had an appreciably smaller amount of FN than OV–LM. Consistent with the functional abnormality of the integrin–FAK system and the smaller amount of FN on OV–HM, this clone exhibited a reduced cell–cell adhesion in the in vitro cell aggregation assay. Namely, OV–LM cells displayed a time–dependent increase in the formation of cell aggregates, whereas most OV–HM cells remained single. The formation of aggregates by OV–LM cells was inhibited by addition of RGDS peptide. These results indicate that the highly metastatic clone, OV–HM, exhibits a decreased capacity of cell–cell adhesion mediated by integrin–FN interactions and suggest that this defect is mainly due to the dysfunction of integrins/FAK rather than a decrease in the amount of integrins expressed on tumor cells.