Effector T lymphocyte subsets in human pancreatic cancer: detection of CD8+CD18+ cells and CD8+CD103+ cells by multi-epitope imaging

Abstract

Pancreatic cancer is characterized by an increasing incidence and an extremely poor prognosis. It is resistant to most of the conventional treatment modalities. Histomorphologically, it presents with a strong desmoplastic reaction around cancer cells, and lymphocytes are typically localized as aggregates in the fibrotic interstitial tissue. Using the method of multi-epitope imaging with fluorochrome-tagged specific MoAbs which allows the simultaneous localization and characterization of T cells in tissues, we studied phenotypes and distribution of tumour-infiltrating lymphocytes (TIL) in pancreatic cancer. CD3+ T cells comprised up to 90% of the tumour-infiltrating cells which were either CD4+ or CD8+, most of them being memory cells (CD45RO+). In decreasing order of frequency, T lymphocytes carried the markers for CD45RO, CD18, CD103 and TCR gammadelta. Very few natural killer cells (CD56+) were observed. Twenty percent of CD8+ were labelled with CD103. These CD8+CD103+ T cells, analogous to the gut intraepithelial lymphocytes (IEL), were found in the fibrous interstitial tissue. Furthermore, an inverse correlation was found between the expression of CD18, the beta2-integrin, which mediates adhesion of activated lymphocytes, and CD45RO in the CD8+ subset of TIL (P = 0.046). In conclusion, phenotyping of T lymphocytes in pancreatic cancer raises the possibility that pancreatic cancer cells develop several strategies to escape the T cell-induced cytolysis by (i) the aggregation of cytotoxic CD8+CD103+ T cells in the fibrous tissue distant from the tumour cells, and (ii) the presence of CD18-bearing cells which lack the expression of the activation marker CD45RO.

Fig. 1

Fluorescent immunohistochemistry. (A) Localization of CD4⁺ and CD8⁺ T cells in the fibrous interstitial tissue in pancreatic cancer by immunofluorescence. CD4⁺ and CD8⁺ cells are seen in clusters; there is no distinct pattern noticeable in the distribution of the two T cell subsets. This image was produced by the superimposition of the grey scale fluorescent images obtained for binding with the antibodies against CD4 and CD8, respectively, on the phase contrast image. Each image was assigned a separate colour channel (red, CD4; green, CD8) and the phase contrast image was ascribed the colour blue. (B) Image acquired by superimposition of the fluorescent images obtained on binding with the CD8 (red) and TCR γδ⁺ (green) antibodies. White arrows indicate CD8⁺ TCR γδ⁺ cells. Due to the exact superimposition of the two images, the areas of overlap appear white.

Fig. 2

Multi-epitope-imaging. The phase contrast image gives the orientation of the lymphocytes. All images depict the same high-power field, i.e. the same lymphocytes as seen labelled with different T cell markers. The panels show the grey scale fluorescent images of a section of pancreatic cancer.

Fig. 3

Multi-epitope-imaging. (A) Superimposition of the fluorescent images obtained on binding with the CD8 (red) and CD103 (green) antibodies. White arrows indicate CD8⁺ CD103⁺ cells. (B) Superimposition of the fluorescent images obtained on binding with the CD8 (red) and CD18 (green) antibodies. The white arrows indicate CD8⁺ CD18⁺ cells.