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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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CTLs motility during tumor infiltration and clearance. (A) TPLSM images (260 × 260 μm) of OT1-GFP cells (green) within tumors (EG7 and EL4) of anesthetized mice at early (day 3) and late (day 5) time points after adoptive transfer. Vascular vessels are labeled by 70 kD rhodamine-dextran (red); typical migratory path (white) examples are shown. Bar, 47 μm. (B) Overlay of 50 individual T cell tracks plotted after aligning their starting positions. (C) Scatter plots of T cell mean velocity, confinement ratio, and arrest coefficients of all cells analyzed. Data from two to four independent experiments were pooled (day 3: EG7, n = 675; EL4, n = 275; day 5: EG7, n = 1249; EL4, n = 126). ns, not significant. ***, P < 0.001.
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fig2: CTLs motility during tumor infiltration and clearance. (A) TPLSM images (260 × 260 μm) of OT1-GFP cells (green) within tumors (EG7 and EL4) of anesthetized mice at early (day 3) and late (day 5) time points after adoptive transfer. Vascular vessels are labeled by 70 kD rhodamine-dextran (red); typical migratory path (white) examples are shown. Bar, 47 μm. (B) Overlay of 50 individual T cell tracks plotted after aligning their starting positions. (C) Scatter plots of T cell mean velocity, confinement ratio, and arrest coefficients of all cells analyzed. Data from two to four independent experiments were pooled (day 3: EG7, n = 675; EL4, n = 275; day 5: EG7, n = 1249; EL4, n = 126). ns, not significant. ***, P < 0.001.

Mentions: To investigate the role of antigen recognition in the motility of antigen-specific CTLs within tumors, we next performed intravital time-lapse two-photon imaging. 8–10 d after s.c. injection of the tumors, OT1-GFP cells were adoptively transferred into tumor-bearing mice. At days 3–4 (early phase of rejection) or 5–6 (late phase of rejection) after adoptive transfer of OT1-GFP cells, the mice were anesthetized and injected i.v. with fluorescent dextran to visualize blood vessels. As shown in Videos S1 and S2 (available at http://www.jem.org/cgi/content/full/jem.20061890/DC1), during the early phase of rejection OT1-GFP moved actively in EL4 tumors but had reduced motility in EG7 tumors. During the late phase of rejection, OT1-GFP cells moved actively in both tumors (Videos S3 and S4, available at http://www.jem.org/cgi/content/full/jem.20061890/DC1). Individual cell trajectories were tracked (representative examples are shown in Fig. 2, A and B), and the mean velocity, arrest coefficient (the proportion of time every individual remains arrested), and confinement ratio (the ratio of the distance between the initial and the final positions of each cell to the total distance covered by that cell) were determined (Fig. 2 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)

CTLs motility during tumor infiltration and clearance. (A) TPLSM images (260 × 260 μm) of OT1-GFP cells (green) within tumors (EG7 and EL4) of anesthetized mice at early (day 3) and late (day 5) time points after adoptive transfer. Vascular vessels are labeled by 70 kD rhodamine-dextran (red); typical migratory path (white) examples are shown. Bar, 47 μm. (B) Overlay of 50 individual T cell tracks plotted after aligning their starting positions. (C) Scatter plots of T cell mean velocity, confinement ratio, and arrest coefficients of all cells analyzed. Data from two to four independent experiments were pooled (day 3: EG7, n = 675; EL4, n = 275; day 5: EG7, n = 1249; EL4, n = 126). ns, not significant. ***, P < 0.001.
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fig2: CTLs motility during tumor infiltration and clearance. (A) TPLSM images (260 × 260 μm) of OT1-GFP cells (green) within tumors (EG7 and EL4) of anesthetized mice at early (day 3) and late (day 5) time points after adoptive transfer. Vascular vessels are labeled by 70 kD rhodamine-dextran (red); typical migratory path (white) examples are shown. Bar, 47 μm. (B) Overlay of 50 individual T cell tracks plotted after aligning their starting positions. (C) Scatter plots of T cell mean velocity, confinement ratio, and arrest coefficients of all cells analyzed. Data from two to four independent experiments were pooled (day 3: EG7, n = 675; EL4, n = 275; day 5: EG7, n = 1249; EL4, n = 126). ns, not significant. ***, P < 0.001.
Mentions: To investigate the role of antigen recognition in the motility of antigen-specific CTLs within tumors, we next performed intravital time-lapse two-photon imaging. 8–10 d after s.c. injection of the tumors, OT1-GFP cells were adoptively transferred into tumor-bearing mice. At days 3–4 (early phase of rejection) or 5–6 (late phase of rejection) after adoptive transfer of OT1-GFP cells, the mice were anesthetized and injected i.v. with fluorescent dextran to visualize blood vessels. As shown in Videos S1 and S2 (available at http://www.jem.org/cgi/content/full/jem.20061890/DC1), during the early phase of rejection OT1-GFP moved actively in EL4 tumors but had reduced motility in EG7 tumors. During the late phase of rejection, OT1-GFP cells moved actively in both tumors (Videos S3 and S4, available at http://www.jem.org/cgi/content/full/jem.20061890/DC1). Individual cell trajectories were tracked (representative examples are shown in Fig. 2, A and B), and the mean velocity, arrest coefficient (the proportion of time every individual remains arrested), and confinement ratio (the ratio of the distance between the initial and the final positions of each cell to the total distance covered by that cell) were determined (Fig. 2 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