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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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Distinct migratory patterns of T cells within tumors. (A) TPLSM images of OT1-GFP cells (green) within an EG7 tumor during the late phase of tumor rejection (day 5). Vessels (red) are labeled by 70 kD rhodamine-dextran. Several representative migration tracks of OT1-GFP cells are shown for cells either in close contact or not with blood vessels (green); individual tracks are plotted after normalization of their initial positions (bottom). Bar, 120 μm. (B) Scatter plot of the mean velocities of OT1 cells either in contact or not with blood vessels. ns, not significant. (C) Representative cell shapes of cells in close contact (right) or not (left) with the blood vessels (45 × 45 μm). Cells at distance or in close contact with blood vessels were tracked (mean = 10 min in both conditions), and the means of their elongation index during their migratory path were plotted (bottom). ***, P < 0.001. Bar, 28 μm. (D) Top graph shows the elongation index along time of representative cells migrating at distance (black) or in close contact (red) with blood vessels. Straight lines indicate the mean value of the elongation index for cells in close contact (1.9, red) or at distance (1.6, black) from blood vessels. Bottom graph shows the elongation index along time of a representative cell that migrates first within the tumor matrix (black) and thereafter in close contact with a blood vessel (red). Straight line indicates the mean value of its elongation index (1.9, black).
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fig5: Distinct migratory patterns of T cells within tumors. (A) TPLSM images of OT1-GFP cells (green) within an EG7 tumor during the late phase of tumor rejection (day 5). Vessels (red) are labeled by 70 kD rhodamine-dextran. Several representative migration tracks of OT1-GFP cells are shown for cells either in close contact or not with blood vessels (green); individual tracks are plotted after normalization of their initial positions (bottom). Bar, 120 μm. (B) Scatter plot of the mean velocities of OT1 cells either in contact or not with blood vessels. ns, not significant. (C) Representative cell shapes of cells in close contact (right) or not (left) with the blood vessels (45 × 45 μm). Cells at distance or in close contact with blood vessels were tracked (mean = 10 min in both conditions), and the means of their elongation index during their migratory path were plotted (bottom). ***, P < 0.001. Bar, 28 μm. (D) Top graph shows the elongation index along time of representative cells migrating at distance (black) or in close contact (red) with blood vessels. Straight lines indicate the mean value of the elongation index for cells in close contact (1.9, red) or at distance (1.6, black) from blood vessels. Bottom graph shows the elongation index along time of a representative cell that migrates first within the tumor matrix (black) and thereafter in close contact with a blood vessel (red). Straight line indicates the mean value of its elongation index (1.9, black).

Mentions: To characterize and quantify the cells migrating along blood vessels in more detail, we took advantage of certain regions of the tumor periphery that displayed a highly organized blood vessel pattern orientated along one major axis (Fig. 5 A). In these areas, numerous CTLs maintained close contact with vessels during migration (Video S11, available at http://www.jem.org/cgi/content/full/jem.20061890/DC1) for relatively long distances (114 ± 63 μm, n = 17). The CTLs adopted an anisotropic migration pattern along the axis of the blood vessels as indicated by a higher correlation coefficient (no-contact, r = 0.31 and contact, r = 0.73; P < 0.001) (Fig. 5 A, bottom). The ability of several cells to migrate from a blood vessel to another confirmed that the CTLs are probably not in the lumen of the vessels, but either in the tissue or within the “sheath” around the vessels. Migration along blood vessels proceeds with a mean velocity similar to that of the CTLs migrating at distance of visible blood vessels (8.2 ± 2.4 μm/min and 8.6 ± 2.8 μm/min, respectively; Fig. 5 B).


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)

Distinct migratory patterns of T cells within tumors. (A) TPLSM images of OT1-GFP cells (green) within an EG7 tumor during the late phase of tumor rejection (day 5). Vessels (red) are labeled by 70 kD rhodamine-dextran. Several representative migration tracks of OT1-GFP cells are shown for cells either in close contact or not with blood vessels (green); individual tracks are plotted after normalization of their initial positions (bottom). Bar, 120 μm. (B) Scatter plot of the mean velocities of OT1 cells either in contact or not with blood vessels. ns, not significant. (C) Representative cell shapes of cells in close contact (right) or not (left) with the blood vessels (45 × 45 μm). Cells at distance or in close contact with blood vessels were tracked (mean = 10 min in both conditions), and the means of their elongation index during their migratory path were plotted (bottom). ***, P < 0.001. Bar, 28 μm. (D) Top graph shows the elongation index along time of representative cells migrating at distance (black) or in close contact (red) with blood vessels. Straight lines indicate the mean value of the elongation index for cells in close contact (1.9, red) or at distance (1.6, black) from blood vessels. Bottom graph shows the elongation index along time of a representative cell that migrates first within the tumor matrix (black) and thereafter in close contact with a blood vessel (red). Straight line indicates the mean value of its elongation index (1.9, black).
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Related In: Results  -  Collection

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fig5: Distinct migratory patterns of T cells within tumors. (A) TPLSM images of OT1-GFP cells (green) within an EG7 tumor during the late phase of tumor rejection (day 5). Vessels (red) are labeled by 70 kD rhodamine-dextran. Several representative migration tracks of OT1-GFP cells are shown for cells either in close contact or not with blood vessels (green); individual tracks are plotted after normalization of their initial positions (bottom). Bar, 120 μm. (B) Scatter plot of the mean velocities of OT1 cells either in contact or not with blood vessels. ns, not significant. (C) Representative cell shapes of cells in close contact (right) or not (left) with the blood vessels (45 × 45 μm). Cells at distance or in close contact with blood vessels were tracked (mean = 10 min in both conditions), and the means of their elongation index during their migratory path were plotted (bottom). ***, P < 0.001. Bar, 28 μm. (D) Top graph shows the elongation index along time of representative cells migrating at distance (black) or in close contact (red) with blood vessels. Straight lines indicate the mean value of the elongation index for cells in close contact (1.9, red) or at distance (1.6, black) from blood vessels. Bottom graph shows the elongation index along time of a representative cell that migrates first within the tumor matrix (black) and thereafter in close contact with a blood vessel (red). Straight line indicates the mean value of its elongation index (1.9, black).
Mentions: To characterize and quantify the cells migrating along blood vessels in more detail, we took advantage of certain regions of the tumor periphery that displayed a highly organized blood vessel pattern orientated along one major axis (Fig. 5 A). In these areas, numerous CTLs maintained close contact with vessels during migration (Video S11, available at http://www.jem.org/cgi/content/full/jem.20061890/DC1) for relatively long distances (114 ± 63 μm, n = 17). The CTLs adopted an anisotropic migration pattern along the axis of the blood vessels as indicated by a higher correlation coefficient (no-contact, r = 0.31 and contact, r = 0.73; P < 0.001) (Fig. 5 A, bottom). The ability of several cells to migrate from a blood vessel to another confirmed that the CTLs are probably not in the lumen of the vessels, but either in the tissue or within the “sheath” around the vessels. Migration along blood vessels proceeds with a mean velocity similar to that of the CTLs migrating at distance of visible blood vessels (8.2 ± 2.4 μm/min and 8.6 ± 2.8 μm/min, respectively; Fig. 5 B).

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