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. 2014 Jul;8(5):982-97.
doi: 10.1016/j.molonc.2014.03.001. Epub 2014 Apr 2.

L1CAM promotes enrichment of immunosuppressive T cells in human pancreatic cancer correlating with malignant progression (V体育安卓版)

Evelin Grage-Griebenow  1 , Elfi Jerg  1 , Artur Gorys  1 , Daniel Wicklein  2 , Daniela Wesch  3 , Sandra Freitag-Wolf  4 , Lisa Goebel  1 , Ilka Vogel  5 , Thomas Becker  6 , Michael Ebsen  7 , Christoph Röcken  8 , Peter Altevogt  9 , Udo Schumacher  2 , Heiner Schäfer  10 , Susanne Sebens  11
Affiliations

L1CAM promotes enrichment of immunosuppressive T cells in human pancreatic cancer correlating with malignant progression

"V体育安卓版" Evelin Grage-Griebenow et al. Mol Oncol. 2014 Jul.

Abstract

Regulatory T cell (T-reg) enrichment in the tumor microenvironment is regarded as an important mechanism of tumor immune escape. Hence, the presence of T-regs in highly malignant pancreatic ductal adenocarcinoma (PDAC) is correlated with short survival. Likewise, the adhesion molecule L1CAM is upregulated during PDAC progression in the pancreatic ductal epithelium also being associated with poor prognosis. To investigate whether L1CAM contributes to enrichment of T-regs in PDAC, human CD4(+)CD25(+)CD127(-)CD49d(-) T-regs and CD4(+)CD25(-) T-effector cells (T-effs) were isolated by magnetic bead separation from blood of healthy donors VSports手机版. Their phenotype and functional behavior were analyzed in dependence on human premalignant (H6c7) or malignant (Panc1) pancreatic ductal epithelial cells, either exhibiting or lacking L1CAM expression. T cells derived from blood and tumors of PDAC patients were analyzed by flow cytometry and findings were correlated with clinical parameters. Predominantly T-regs but not T-effs showed an increased migration on L1CAM expressing H6c7 and Panc1 cells. Whereas proliferation of T-regs did not change in the presence of L1CAM, T-effs proliferated less, exhibited a decreased CD25 expression and an increased expression of CD69. Moreover, these T-effs exhibited a regulatory phenotype as they inhibited proliferation of autologous T cells. Accordingly, CD4(+)CD25(-)CD69(+) T cells were highly abundant in PDAC tissues compared to blood being associated with nodal invasion and higher grading in PDAC patients. Overall, these data point to an important role of L1CAM in the enrichment of immunosuppressive T cells in particular of a CD4(+)CD25(-)CD69(+)-phenotype in PDAC providing a novel mechanism of tumor immune escape which contributes to tumor progression. .

Keywords: CD171; Immune evasion; Malignant progression; Pancreatic adenocarcinoma; Regulatory T cells; Tumor stroma V体育安卓版. .

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Figures

Figure 1
Figure 1
The prevalence of CD4+CD25+CD127−CD49d− T‐regs in blood and pancreatic tissues of CP and PDAC patients compared to healthy donors. PBMCs from blood of healthy donors, CP and PDAC patients as well as cells migrated out of pancreatic tissues of CP and PDAC patients were stained and analyzed by flow cytometry for expression of CD4, CD25, CD49d and CD127. Data are presented as (A) % CD4+ cells in all lymphocytes, (B) % CD25+, (C) % CD25− and (D) % CD25+ CD127− CD49d− cells in CD4+ T cells. Bars indicate the means. Statistical significance is indicated by the respective p‐values. Red marks indicate data from blood‐derived cells, black marks from tissue‐derived cells.
Figure 2
Figure 2
L1CAM predominantly increases cell migration of T‐regs. T‐regs and T‐effs were isolated by negative selection. A) T‐regs and T‐effs were stained and analyzed by flow cytometry for expression of CD4, CD25, CD69, CD49d, CD127, CD45RO and FoxP3. Data are presented as dot plots from one representative isolation. B) T‐effs and T‐regs were cultured at a ratio of 50:1. Proliferation of T cells was determined either by 3H‐thymidine assay after 5 days or Ki‐67 staining and subsequent flow cytometry after 3 days. Data from one representative experiment are presented as % proliferating T‐effs. C + D) T‐regs‐ and T‐effs were labeled with CFSE and seeded on transwells inserts covered with C) H6c7‐mock and H6c7‐L1CAM cells or D) Panc1L1low and Panc1L1high cells, respectively. After 24 h, migrated T cells were determined by measuring the fluorescence in the lower compartments. Data are presented as % migrated cells with migration of T‐regs on HPDE‐mock and Panc1L1low cells set as 100%. Means ± SD from 3 to 5 experiments are shown. Statistical significance is indicated by the respective p‐values. Photographs show representative L1CAM stainings of C) H6c7‐mock and H6c7‐L1CAM cells as well as D) Panc1L1low and Panc1L1high cells demonstrating the different L1CAM expression level.
Figure 2
Figure 2
(Continued)
Figure 3
Figure 3
L1CAM alters the phenotype of T‐effs. T‐regs and T‐effs were cocultured for 72 h with H6c7‐mock and H6c7‐L1CAM cells or Panc1L1low and Panc1L1high cells, respectively, in the absence (none) or presence of T cell activation/expansion beads (+beads). Then, both T cell populations were stained and analyzed by flow cytometry for expression of (A) CD49d/CD127 and (B) CD25. Data are presented as histograms from one representative experiment out of 11. Empty curves indicate staining with the isotype control, filled curves with the specific antibody. Values in brackets indicate median fluorescence intensity (left value: isotype control, right value: specific antibody). Main changes are depicted in the red frame and highlighted with red, bold letters.
Figure 4
Figure 4
L1CAM reduces proliferation but increases CD69 expression in T‐effs. T‐regs (A + B) and T‐effs (A–E) were cocultured for 72 h with T cell activation/expansion beads and in the presence of (A,C,D) H6c7‐mock and H6c7‐L1CAM cells or (B + E) Panc1L1low and Panc1L1high cells, respectively. A + B) T‐regs and T‐effs were stained and analyzed by flow cytometry for expression of Ki‐67. C) T‐effs were analyzed for the expression of Ki‐67 and supernatants of the respective cocultures for the presence of IL‐2 and IFN‐γ. One representative experiment is shown. D + E) T‐effs were stained and analyzed by flow cytometry for expression of CD69. Data are presented as histograms from one representative experiment out of 11 (A,B,D,E). Values in brackets indicate median fluorescence intensity of isotype control (empty curves) and of specific antibody (filled curves).
Figure 4
Figure 4
(Continued)
Figure 5
Figure 5
In the presence of L1CAM T‐effs acquire an immunosuppressive phenotype. T‐effs were cocultured for 72 h with T cell activation/expansion beads and in the presence of (A) H6c7‐mock and H6c7‐L1CAM cells or (B) Panc1L1low and Panc1L1high cells, respectively. Then, autologous T‐effs (T‐effsa) that had been cultured in parallel, were cultured together with one of the cocultured T‐eff populations at a ratio of 75:1 and in the presence of T cell activation/expansion beads for further 72 h. T cells were stained and analyzed by flow cytometry for expression of Ki‐67. Data are presented as mean fluorescence intensity (MFI) over control. Means ± SD from 5 to 6 experiments are shown. Statistical significance is indicated by the respective p‐values. C) Representative histograms from Ki‐67 stainings of T‐effsa cocultured with T‐effs from cocultures with H6c7‐L1CAM cells without depletion of CD25+ T cells (T‐effs), with depletion of CD25+ cells (CD25− Teffs). As control, T‐effsa were cocultured under similar conditions with enriched CD25+ T cells (CD25+ T‐effs) or T‐regs that had been cocultured with H6c7‐L1CAM cells before. D) After coculture with H6c7‐mock or H6c7‐L1CAM cells, T‐effs were stained for CD25, CD69 and membrane‐bound TGF‐β1. Representative histograms from TGF‐β1 stainings in CD25−CD69+ T‐effs are shown. E) Levels of IL‐2, IL‐12p40 and SDF‐1α were determined in supernatants from T‐effsa cultured either alone or together with T‐effs that had been cocultured with H6c7‐L1CAM cells before. Data from one representative experiment are shown.
Figure 5
Figure 5
(Continued)
Figure 6
Figure 6
A high prevalence of CD4+CD25−CD69+ T cells in tissues of PDAC patients correlates with malignant progression. PBMCs from blood of healthy donors, CP and PDAC patients as well as cells migrated out of pancreatic tissues of CP and PDAC patients were stained and analyzed as described. Data are presented as (A) % CD69+, (B) % CD25+CD69+ and (C) % CD25−CD69+ cells in CD4+ T cells. D) Representative dot plots from CD69 and CD25 staining of PBMCs from blood of healthy donors, CP and PDAC patients as well as of cells migrated out of pancreatic tissues of CP and PDAC patients. Correlation of (E) % CD69+, (F) % CD25+CD69+ and (G) % CD25−CD69+ cells in CD4+ T cells in PDAC tissues with the indicated clinical parameters that are status of lymphangioinvasion (L), haemangioinvasion (V), nodal invasion (N) and grading (G1/G2 = well/moderately differentiated; G3/G4 = poorly/undifferentiated). Bars indicate the means. Statistical significance is indicated by the respective p‐values.
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