Expression of co-stimulator 4-1BB molecule in hepatocellular carcinoma and adjacent non-tumor liver tissue, and its possible role in tumor immunity

Abstract

Aim: To investigate the expression of 4-1BB molecule in hepatocellular carcinoma (HCC) and its adjacent tissues.

Methods: Reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the gene expression of 4-1BB in hepatocarcinoma and its adjacent tissues, and peripheral blood mononuclear cells (PBMCs) from both HCC and health control groups. Flow cytometry was used to analyse the phenotypes of T cell subsets from the blood of HCC patients and healthy volunteers, and further to determine whether 4-1BB molecules were also expressed on the surface of CD4+ and CD8+ T cells. The localization of 4-1BB proteins on tumor infiltrating T cells was determined by direct immunofluorescence cytochemical staining and detected by confocal microscopy.

Results: 4-1BB mRNA, which was not detectable in normal liver, was found in 19 liver tissues adjacent to tumor edge (<1.0 cm). Low expression of 4-1BB mRNA was shown in 8 tumor tissues and 6 liver tissues located within 1 to 5 cm away from tumor edge. In PBMCs, 4-1BB mRNA was almost not detected. Percentage of CD4+, CD8+ and CD3+/CD25+ T cells, as well as ratio of CD4 to CD8 revealed no difference between groups (P>0.05, respectively), while a significant lower percentage of CD3+ T cell was found in HCC group as compared to healthy control group (P<0.05). However, 4-1BB molecules were almost not found on the surface of CD4+ and CD8+ T cells in HCC and healthy control group. Double-staining of 4-1BB+/CD4+ and 4-1BB+/CD8+ immunofluorescence on tumor infiltrating T cells was detected in 13 liver tissues adjacent to tumor edge (<1.0 cm) by confocal microscopy.

Conclusion: Although HCC may escape from immune attack by weak immunogenicity or downregulated expression of MHC-1 molecules on the tumor cell surface, tumor infiltrating T cells can be activated via other costimulatory signal pathways to exert a limited antitumor effect on local microenvironment. The present study also implicates that modulating 4-1BB/4-1BBL costimulatory pathway may be an effective immunotherapy strategy to augment the host response.

Figure 1

Representative RT-PCR results of 4-1BB mRNA ex-pression in normal liver, and HCC tissues as well as its adja-cent tissues. (M, GeneRulerTM 100 bp DNA Ladder Plus; N, normal liver; H, HCC tissues; P, adjacent tissues to HCC (<1 cm), P’: liver tissues located within 1 to 5 cm away from tumor edge)

Figure 2

Representative RT-PCR results of 4-1BB mRNA ex-pression in PBMCs (M,GeneRuler^TM 100 bp DNA Ladder Plus; N, healthy control; H, HCC group).

Figure 3

Comparison of 4-1BB molecules on CD4⁺ or CD8⁺ PBMCs between healthy controls and HCC patients.

Figure 4

Confocal laser micrographs of TILs in HCC adjacent tissues processed for the immunocytochemical detection of 4-1BB and CD4 or CD8 molecules. (A and D, FITC immunofluorescence for CD4 and CD8 molecules respectively; B and E, PE immunof-luorescence for 4-1BB molecules; C and F, automatically synthetic micrographs by computer soft for A and B, and D and E, respectively).