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In vivo imaging of cytotoxic T cell infiltration and elimination of a solid tumor.

Boissonnas A, Fetler L, Zeelenberg IS, Hugues S, Amigorena S - J. Exp. Med. (2007)

Bottom Line: We use a combination of two-photon intravital microscopy and immunofluorescence on ordered sequential sections to analyze the infiltration and destruction of solid tumors by CTLs.We show that in the periphery of a thymoma growing subcutaneously, activated CTLs migrate with high instantaneous velocities.CTLs migrating along blood vessels preferentially adopt an elongated morphology.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale U653, Immunité et Cancer, Pavillon Pasteur, Institut Curie, F-75245 Paris Cedex 05, France.

ABSTRACT
Although the immune system evolved to fight infections, it may also attack and destroy solid tumors. In most cases, tumor rejection is initiated by CD8(+) cytotoxic T lymphocytes (CTLs), which infiltrate solid tumors, recognize tumor antigens, and kill tumor cells. We use a combination of two-photon intravital microscopy and immunofluorescence on ordered sequential sections to analyze the infiltration and destruction of solid tumors by CTLs. We show that in the periphery of a thymoma growing subcutaneously, activated CTLs migrate with high instantaneous velocities. The CTLs arrest in close contact to tumor cells expressing their cognate antigen. In regions where most tumor cells are dead, CTLs resume migration, sometimes following collagen fibers or blood vessels. CTLs migrating along blood vessels preferentially adopt an elongated morphology. CTLs also infiltrate tumors in depth, but only when the tumor cells express the cognate CTL antigen. In tumors that do not express the cognate antigen, CTL infiltration is restricted to peripheral regions, and lymphocytes neither stop moving nor kill tumor cells. Antigen expression by tumor cells therefore determines both CTL motility within the tumor and profound tumor infiltration.

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Dynamics of CTL interactions with tumor cells. TPLSM images of OT1-CFP cells (A, blue; B, magenta) within EG7-GFP tumors (green) during early phase (day 4) and late phase (day 5) of tumor rejection. Collagen fibers (blue) are imaged by SHG. Examples of typical migratory paths (red) are shown. Bars: (A) 27 μm; (B) 39 μm. (C) Scatter plots of T cell mean velocity, confinement ratio, and arrest coefficients of all cells analyzed. Data from three to four independent experiments were pooled. (Alive-Early, n = 20; Dead-Late, n = 121; Alive-Late, n = 77). ***, P < 0.001. (D) TPLSM images (90 × 90 μm) representing typical migratory paths (red) for each class of interaction as described in Material and methods (Stable, black; Confined, dark gray; Serial, light gray; Fleet, white). Bar, 28 μm. (E) Histograms representing the relative fraction of the different classes of interaction in areas displaying alive or dead tumor cells during the early (day 4) and the late phase (day 5) of tumor regression.
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fig4: Dynamics of CTL interactions with tumor cells. TPLSM images of OT1-CFP cells (A, blue; B, magenta) within EG7-GFP tumors (green) during early phase (day 4) and late phase (day 5) of tumor rejection. Collagen fibers (blue) are imaged by SHG. Examples of typical migratory paths (red) are shown. Bars: (A) 27 μm; (B) 39 μm. (C) Scatter plots of T cell mean velocity, confinement ratio, and arrest coefficients of all cells analyzed. Data from three to four independent experiments were pooled. (Alive-Early, n = 20; Dead-Late, n = 121; Alive-Late, n = 77). ***, P < 0.001. (D) TPLSM images (90 × 90 μm) representing typical migratory paths (red) for each class of interaction as described in Material and methods (Stable, black; Confined, dark gray; Serial, light gray; Fleet, white). Bar, 28 μm. (E) Histograms representing the relative fraction of the different classes of interaction in areas displaying alive or dead tumor cells during the early (day 4) and the late phase (day 5) of tumor regression.

Mentions: The change in the dynamics of OT1 cell motility in the EG7 tumors between the early and late phases could therefore correspond to the arrests of OT1 cells during tumor cell killing. To investigate this possibility, we analyzed the dynamics of OT1 cells expressing cyan fluorescent protein (OT1-CFP) in tumors expressing GFP during the early or late rejection phases (Videos S5 and S6, available at http://www.jem.org/cgi/content/full/jem.20061890/DC1). As shown in Fig. 4 A and Video S5, during the early phase the majority of the arrested OT1-CFP cells are in contact with living EG7-GFP tumor cells. During the late phase, OT1-CFP cells seem to resume motility in regions where the tumor cells are dead (Fig. 4 B and Video S6). We therefore compared the dynamic of the OT1 cells in regions where the tumor cells are alive or dead. As shown in Fig. 4 C, the OT1-CFP cells displayed lower mean velocity (1.1 ± 0.4 μm/min) and confinement ratios (0.15 ± 0.05) and higher arrest coefficients (82 ± 11%) in regions where the tumor cells are alive during early phase than in regions where the tumor cells are dead during late phase (mean velocity, 6.4 ± 2.5 μm/min; confinement ratio, 0.7 ± 0.2; and arrest coefficient, 17 ± 18%). During the late phase, we eventually found regions of the tumors where at least parts of the EG7-GFP cells are still alive. OT1-CFP cells in these regions only partially resumed motility compared with regions where most of the tumor cells were dead (Fig. 4 C).


In vivo imaging of cytotoxic T cell infiltration and elimination of a solid tumor.

Boissonnas A, Fetler L, Zeelenberg IS, Hugues S, Amigorena S - J. Exp. Med. (2007)

Dynamics of CTL interactions with tumor cells. TPLSM images of OT1-CFP cells (A, blue; B, magenta) within EG7-GFP tumors (green) during early phase (day 4) and late phase (day 5) of tumor rejection. Collagen fibers (blue) are imaged by SHG. Examples of typical migratory paths (red) are shown. Bars: (A) 27 μm; (B) 39 μm. (C) Scatter plots of T cell mean velocity, confinement ratio, and arrest coefficients of all cells analyzed. Data from three to four independent experiments were pooled. (Alive-Early, n = 20; Dead-Late, n = 121; Alive-Late, n = 77). ***, P < 0.001. (D) TPLSM images (90 × 90 μm) representing typical migratory paths (red) for each class of interaction as described in Material and methods (Stable, black; Confined, dark gray; Serial, light gray; Fleet, white). Bar, 28 μm. (E) Histograms representing the relative fraction of the different classes of interaction in areas displaying alive or dead tumor cells during the early (day 4) and the late phase (day 5) of tumor regression.
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fig4: Dynamics of CTL interactions with tumor cells. TPLSM images of OT1-CFP cells (A, blue; B, magenta) within EG7-GFP tumors (green) during early phase (day 4) and late phase (day 5) of tumor rejection. Collagen fibers (blue) are imaged by SHG. Examples of typical migratory paths (red) are shown. Bars: (A) 27 μm; (B) 39 μm. (C) Scatter plots of T cell mean velocity, confinement ratio, and arrest coefficients of all cells analyzed. Data from three to four independent experiments were pooled. (Alive-Early, n = 20; Dead-Late, n = 121; Alive-Late, n = 77). ***, P < 0.001. (D) TPLSM images (90 × 90 μm) representing typical migratory paths (red) for each class of interaction as described in Material and methods (Stable, black; Confined, dark gray; Serial, light gray; Fleet, white). Bar, 28 μm. (E) Histograms representing the relative fraction of the different classes of interaction in areas displaying alive or dead tumor cells during the early (day 4) and the late phase (day 5) of tumor regression.
Mentions: The change in the dynamics of OT1 cell motility in the EG7 tumors between the early and late phases could therefore correspond to the arrests of OT1 cells during tumor cell killing. To investigate this possibility, we analyzed the dynamics of OT1 cells expressing cyan fluorescent protein (OT1-CFP) in tumors expressing GFP during the early or late rejection phases (Videos S5 and S6, available at http://www.jem.org/cgi/content/full/jem.20061890/DC1). As shown in Fig. 4 A and Video S5, during the early phase the majority of the arrested OT1-CFP cells are in contact with living EG7-GFP tumor cells. During the late phase, OT1-CFP cells seem to resume motility in regions where the tumor cells are dead (Fig. 4 B and Video S6). We therefore compared the dynamic of the OT1 cells in regions where the tumor cells are alive or dead. As shown in Fig. 4 C, the OT1-CFP cells displayed lower mean velocity (1.1 ± 0.4 μm/min) and confinement ratios (0.15 ± 0.05) and higher arrest coefficients (82 ± 11%) in regions where the tumor cells are alive during early phase than in regions where the tumor cells are dead during late phase (mean velocity, 6.4 ± 2.5 μm/min; confinement ratio, 0.7 ± 0.2; and arrest coefficient, 17 ± 18%). During the late phase, we eventually found regions of the tumors where at least parts of the EG7-GFP cells are still alive. OT1-CFP cells in these regions only partially resumed motility compared with regions where most of the tumor cells were dead (Fig. 4 C).

Bottom Line: We use a combination of two-photon intravital microscopy and immunofluorescence on ordered sequential sections to analyze the infiltration and destruction of solid tumors by CTLs.We show that in the periphery of a thymoma growing subcutaneously, activated CTLs migrate with high instantaneous velocities.CTLs migrating along blood vessels preferentially adopt an elongated morphology.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale U653, Immunité et Cancer, Pavillon Pasteur, Institut Curie, F-75245 Paris Cedex 05, France.

ABSTRACT
Although the immune system evolved to fight infections, it may also attack and destroy solid tumors. In most cases, tumor rejection is initiated by CD8(+) cytotoxic T lymphocytes (CTLs), which infiltrate solid tumors, recognize tumor antigens, and kill tumor cells. We use a combination of two-photon intravital microscopy and immunofluorescence on ordered sequential sections to analyze the infiltration and destruction of solid tumors by CTLs. We show that in the periphery of a thymoma growing subcutaneously, activated CTLs migrate with high instantaneous velocities. The CTLs arrest in close contact to tumor cells expressing their cognate antigen. In regions where most tumor cells are dead, CTLs resume migration, sometimes following collagen fibers or blood vessels. CTLs migrating along blood vessels preferentially adopt an elongated morphology. CTLs also infiltrate tumors in depth, but only when the tumor cells express the cognate CTL antigen. In tumors that do not express the cognate antigen, CTL infiltration is restricted to peripheral regions, and lymphocytes neither stop moving nor kill tumor cells. Antigen expression by tumor cells therefore determines both CTL motility within the tumor and profound tumor infiltration.

Show MeSH
Related in: MedlinePlus