Nonredundant roles of antibody, cytokines, and perforin in the eradication of established Her-2/neu carcinomas

Abstract

Since the mechanisms by which specific immunity destroys Her-2/neu carcinoma cells are highly undetermined, these were assessed in BALB/c mice vaccinated with plasmids encoding extracellular and transmembrane domains of the protein product (p185(neu)) of the rat Her-2/neu oncogene shot into the skin by gene gun. Vaccinated mice rejected a lethal challenge of TUBO carcinoma cells expressing p185(neu). Depletion of CD4 T cells during immunization abolished the protection, while depletion of CD8 cells during the effector phase halved it, and depletion of polymorphonuclear granulocytes abolished all protection. By contrast, Ig mu-chain gene KO mice, as well as Fcgamma receptor I/III, beta-2 microglobulin, CD1, monocyte chemoattractant protein 1 (MCP1), IFN-gamma, and perforin gene KO mice were protected. Only mice with both IFN-gamma and perforin gene KOs were not protected. Although immunization also cured all BALB/c mice bearing established TUBO carcinomas, it did not cure any of the perforin KO or perforin and IFN-gamma KO mice. Few mice were cured that had knockouts of the gene for Ig mu-chain, Fcgamma receptor I/III, IFN-gamma, or beta-2 microglobulin. Moreover, vaccination cured half of the CD1 and the majority of the MCP1 KO mice. The eradication of established p185(neu) carcinomas involves distinct mechanisms, each endowed with a different curative potential.

Figure 1

Cytotoxic and antibody response to p185^neu of WT BALB/c mice shot at 2 week intervals with p185 plasmids. Spleens and sera were collected 2 weeks after the last immunization. The upper panel shows cytotoxicity of splenocytes evaluated in a 48-hour [³H]thymidine release assay against TUBO (squares, H-2^d, p185^neu positive), F1F-neu (triangles, H-2^d, p185^neu positive), N202.1A (filled circles, H-2^q, p185^neu positive); and F1F (open circles, H-2^d, p185^neu negative) targets. No significant cytotoxicity was found in mice shot with empty plasmids (data not shown). The lower panel shows the specific binding potential titer of anti-p185^neu Ab in a pool of sera from WT BALB/c, BALB-IFNγKO, and BALB-μIgKO mice shot with empty (white bars) or p185 (black bars) plasmids.

Figure 2

Ability of gene gun vaccination to cure established TUBO carcinomas. The left panels show growth and regression of 2-mm (a), 4-mm (b) or 6-mm (c) mean diameter carcinomas after two p185 plasmid shots (arrows). No modulation of tumor growth was observed after shots with empty plasmids (data not shown). The right panels show histological stages of the tumors. When the first shot was performed, the 2-mm carcinomas (d) consisted of well-defined neoplastic lobules surrounded by a delicate stroma. The 4-mm carcinomas (e) consisted of an established mass invading the subcutaneous fibroadipose tissue and almost completely occupying the microscope field. The 6-mm carcinomas (f) could no longer be fully contained within the microscope field. These carcinomas show an expansive and invasive growth pattern. Magnification, ×25.

Figure 3

Morphological events associated with the eradication of the 2-mm mean diameter TUBO carcinomas in immunized WT BALB/c mice. Twenty days after the first shot with empty vector, a large, solid carcinoma (a) with a delicate stroma and without appreciable collagen deposition (c) formed by round, monomorphous, r-p185^neu-expressing neoplastic cells (e) was evident. Immunohistochemistry showed that the tumor mass was moderately infiltrated by PMNs (g) and characterized by a very slight production of the fibrogenic cytokine TGF-β1 (i). At the same time, mice shot with p185 plasmids displayed small neoplastic cell aggregates (b), with an evident downmodulation of p185^neu expression on the cell membranes (f), surrounded by a massive collagen deposition and devastating fibrotic reaction evidenced as blue-green staining by the trichrome method (d). A marked infiltration by PMN (h) and expression of TGF-β1 (j) were evident among and inside the neoplastic cell aggregates. Magnification, ×630.

Figure 4

Mechanism of r-p185 carcinoma eradication. When the tumor was 2 mm in diameter, various KO mice were shot with p185 plasmid. Although all WT BALB/c mice were cured (20 of 20 cured at day 200) (Figure 2a), vaccination did not cure any BALB-pfpKO mice (0 of 7 cured, P < 0.0001 versus WT BALB/c) (f) or BALB-IFNγ-pfpKO mice (0 of 7 cured, P < 0.0001) (c). Vaccination did not cure 89% of BALB-μIgKO mice (2 of 18 cured, P < 0.0001) (a), 88% of BALB-FcγRI/IIIKO mice (1 of 8 cured, P < 0.001) (b), 86% of BALB-IFNγKO mice (3 of 21 cured, P < 0.0001) (e), and 78% of BALB-β2mKO mice (2 of 9 cured, P < 0.001) (g). Vaccination cured 54% of BALB-CD1KO mice (6 of 11 cured, P < 0.006) (h) and 80% of BALB-MCP1KO mice (12 of 15 cured, P < 0.07) (d). Arrows show the days of the shots, and the gray area shows the growth and the rejection of the tumor in WT BALB/c mice bearing 2-mm tumors.