Restoration of MHC-I on Tumor Cells by Fhit Transfection Promotes Immune Rejection and Acts as an Individualized Immunotherapeutic Vaccine

Abstract

The capacity of cytotoxic-T lymphocytes to recognize and destroy tumor cells depends on the surface expression by tumor cells of MHC class I molecules loaded with tumor antigen peptides. Loss of MHC-I expression is the most frequent mechanism by which tumor cells evade the immune response. The restoration of MHC-I expression in cancer cells is crucial to enhance their immune destruction, especially in response to cancer immunotherapy. Using mouse models, we recovered MHC-I expression in the MHC-I negative tumor cell lines and analyzed their oncological and immunological profile. Fhit gene transfection induces the restoration of MHC-I expression in highly oncogenic MHC-I-negative murine tumor cell lines and genes of the IFN-γ transduction signal pathway are involved. Fhit-transfected tumor cells proved highly immunogenic, being rejected by a T lymphocyte-mediated immune response. Strikingly, this immune rejection was more frequent in females than in males. The immune response generated protected hosts against the tumor growth of non-transfected cells and against other tumor cells in our murine tumor model. Finally, we also observed a direct correlation between FHIT expression and HLA-I surface expression in human breast tumors. Recovery of Fhit expression on MHC class I negative tumor cells may be a useful immunotherapeutic strategy and may even act as an individualized immunotherapeutic vaccine.

Keywords: Fhit; MHC-I restoration; antitumor immunity; cytotoxic T lymphocytes; immune profile; immunotherapy; vaccine.

Figure 1

Changes produced in B11 tumor cells by the Fhit gene transfection. (A) MHC class I surface expression of B11 and TB11-Fhit tumor cell lines in baseline conditions: H-2 K^d (gray line), H-2 D^d (dotted line), and H-2 L^d (black line). B11 is negative in baseline conditions; TB11-Fhit recovered surface expression of all three H-2 molecules. Data from one experiment are depicted; (B) Transcription levels of Fhit, H-2 class I heavy chain, β₂microglobulin, and several APM components detected by real-time RT-PCR; (C) Transcriptional expression of IFN-γ signal transduction pathway genes in B11 and TB11-Fhit tumor cell lines. Data were normalized using β-actin and GAPDH as housekeeping genes. Only genes with changes in their expression are depicted. Data for B11 are set at 1. Values are depicted as means ± SD of three independent experiments performed in quadruplicate. * p < 0.05. A two-tailed Student’s t-test was used for statistical analysis.

Figure 2

In vitro changes produced in B9 tumor cells by the Fhit gene transfection. (A) Microscopic image of B9 and TB9-Fhit tumor cell lines. Both tumor cell lines show very similar cellular morphology; (B) In vitro proliferation index of B9 and TB9-Fhit tumor cell lines. TB9-Fhit shows a lower proliferation index. The proliferation index was calculated: final cell number/initial cell number. Values are depicted as means ± SD of three independent experiments performed; (C) In vitro migration and invasion assays compare B9 and TB9-Fhit tumor cells. TB9-Fhit shows lower migratory and invasive potential. Values are depicted as means ± SD of three independent experiments; (D) Transcriptional expression of cell cycle genes in B9 and TB9-Fhit tumor cell lines. Data were normalized using β-actin and GAPDH as housekeeping genes. Only genes with changes in their expression are depicted. Data for B9 are set at 1. Values are depicted as means ± SD of three independent experiments performed in quadruplicate. * p < 0.05. A two-tailed Student’s t-test was used for statistical analysis. Scale bar, 100 μm.

Figure 3

In vivo oncogenicity of untransfected and Fhit-transfected tumor cells in immunocompetent and immunodepleted mice. (A) In vivo tumor growth curves (n = 10 mice per group) of B9 and TB9-Fhit tumor cells (cell dose 6.25 × 10⁵) in female/male immunocompetent mice. TB9-Fhit was rejected in 100% of female mice and 50% of male mice. Fisher’s exact test showed that tumor rejection significantly differed between male and female mice. Assays were repeated twice; (B) In vivo tumor growth curves (n = 10 mice per group) of TB9-Fhit tumor cells (cell dose 6.25 × 10⁵) in female nude mice. Identical results were found in male nude mice and in CD8+ T lymphocyte-immunodepleted male/female immunocompetent mice. TB9-Fhit tumor cells grew in all animals. Assays were repeated twice.

Figure 4

The cytokine/chemokine profile in untransfected and Fhit-transfected tumor cells and in mice inoculated with Fhit-transfected tumor cells. (A) The cytokine/chemokine profile secreted by Fhit-transfected versus non-transfected tumor cells. Fhit transfection of tumor cells modified the pattern of cytokine/chemokine secretions; (B) The cytokine/chemokine profile in plasma of female/male mice inoculated with Fhit-transfected tumor cells versus control mice. Results show chemokine/cytokine levels at 42 (42d) and 56 (56d) days post-inoculation of tumor cells. The male mice with primary tumor are depicted as 56d-t. Only cytokines/chemokines presented changes in their expression are depicted. Differences were found between female and male mice and between male mice with and without a primary tumor. Values are depicted as means ± SD of two independent experiments performed in duplicate. * p < 0.05. A two-tailed Student’s t-test or ANOVA test, followed by Tukey’s post-hoc test, was used for statistical analysis.

Figure 5

Immunogenicity and immunoprotection of female mice inoculated with Fhit-transfected B9/B11 tumor cells. (A) TB9- and TB11-Fhit immunoprotected the hosts against B9 and B11 tumor cells. The animals (n = 10 mice per group) were previously inoculated with Fhit-transfected tumor cells and then, 60 days later, inoculated with non-transfected tumor cells; B9 and B11 tumor cells were immune rejected in all mice; (B) TB9- and TB11-Fhit immunoprotected the hosts against other tumor cell clones of the GR9 tumor system and even against GR9 bulk tumor cells; (C) TB9- and TB11-Fhit did not immunoprotect the hosts against 4T1 breast carcinoma cells or CT26 colon carcinoma cells. The assays were repeated twice.

Figure 6

Expression of FHIT and MHC-I in human breast carcinoma cells analyzed by immunohistochemistry. (A) Representative tumor with intense FHIT and HLA-I expressions; (B) Representative tumor with moderate FHIT and HLA-I expressions; (C) Representative tumor with weak FHIT and HLA-I expressions. (D) Representative tumor with negative FHIT and HLA-I expressions. The same tumor cells were analyzed in each case for FHIT and HLA-I expression. Magnification 200×.