Human T lymphocyte responses against lung cancer induced by recombinant truncated mouse EGFR

Abstract

The induction of active cellular responses against EGFR should be a promising approach for the treatment of those receptor-positive tumors. However, the immunity against EGFR is presumably difficult to elicit by vaccine based on self or syngeneic EGFR due to the immune tolerance acquired during the development in immune system. We proposed a model to break immune tolerance against self-EGFR through an altered immunogen source based on xenogeneic homologous EGFR. We have previously shown human EGFR as a xenoantigen could induce specific immune responses in mouse and cross-react with mouse EGFR, and resulted in therapeutic benefits for EGFR-positive mouse tumor. Here, we show a recombinant form of extracellular domain of mouse EGFR, in the presence of DCs, could activate human peripheral T cells to proliferate, secret IFN-gamma, the induced responses could cross-react with human EGFR and kill autologous EGFR-positive lung cancer cells which could be blocked by anti-CD8 and anti-MHC class I antibody. There is no detectable cytotoxical activity against lung tissue, liver tissue and kidney tissue derived from paracancerous normal tissue. These observations suggest that antitumor immunity induced by the truncated mouse EGFR may be provoked in a cross-reaction between mouse EGFR and self-EGFR, and may provide insight into treatment of EGFR-positive tumors through induction of the autoimmune responses against EGFR.

Fig. 1.

Induction of proliferative T cells response. T cells were primed with reMER (recombinant form of extracellular domain of mouse EGFR)-pulsed DCs (reMER-DC), reHER (recombinant form of extracellular domain of human EGFR)-pulsed DCs (reHER-DC), ovalbumin-pulsed DCs (ovalbumin-DC), PBS-treated DCs (PBS-DC), PBS alone or proteins alone as described in Methods. These cells were subjected ³H-thymidine incorporation assay. The representative experiments were shown from 4 cases

Fig. 2.

Activation of IFN-γ-producing T cells. T cells were primed with reMER-DC, reHER-DC, ovalbumin-DC, PBS-DC, DCs alone or PBS alone as described in Methods, and subjected to ELISPOT assay for detection IFN-γ-producing T cells. The representative experiments were shown from 4 cases

Fig. 3.

Induction of cross-reactive T cell responses. reMER-DC primed autologous T cells were restimulated with reHER-DC, ovalbumin-DC, PBS-DC, DC or PBS alone, and then subjected with ³H-thymidine incorporation assay and IFN-γ-specific ELISPOT assay. The representative experiments were shown from 4 cases

Fig. 4.

Induction of CTLs against autologous lung cancer cells. (a) Cytotoxic killing activity, primed T cells were tested for cytotoxicity against autologous lung cancer cells in a 4-h ⁵¹Cr release assay and results were expressed as % cytotoxicity at an E:T ratio of 50:1. The data shown are from patient#1, 2, 5 and 10. (b) CD8-dependent MHC class I-restricted CTL response. Activated T cells from CTL detected cases were blocked by anti-CD4, anti-CD8, anti-MHC class I, anti-MHC class II and control Igs and then subjected to ⁵¹Cr release assay. The results were expressed as % inhibition. (c) CTL response against autologous but not allogeneic lung cancer cells. Primed T cells from CTL detected cases were tested for cytotoxicity against autologous and allogeneic lung cancer cells, autologous monocytes, or A549 human lung cancer cell lines in ⁵¹Cr release assay

Fig. 5.

Expression of EGFR on target cells. (a) Lysates of the collected tumor cells were separated SDS-PAGE, electroblotted onto PVDF membrane, and probed with anti-EGFR for detection expression of EGFR. To confirm protein loading, membranes were stripped, and reprobed with anti-actin. (b) Cells derived from paracancerous tissue of lung cancer, liver cancer and kidney cancer were subjected Western Blot as described above. Lane 1 and 2 represented paracancerous lung tissue derived from 2 patients; lane 3, 4 and 5 represented paracancerous liver tissue derived from 3 patients; lane 6 represented paracancerous kidney tissue derived from 1 patient