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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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Activation of specific T cells in antigen-expressing tumors. C57Bl6 mice were injected s.c. with five 105 EG7 and EL4 tumor cells in either flank. 8–10 d later, the mice were adoptively transferred with 107 purified naive CD8+ cells from OT1-GFP mice. (A) Evolution of EG7 (▪) and EL4 (□) tumor size after adoptive transfer. (mean ± SD, n = 5 independent experiments). Early phase (day 3–4) and late phase (day 5–6) were defined in Material and methods. (B) Density of OT1-GFP cells within EG7 and EL4 at early (day 3) and late (day 6) phase of tumor rejection were calculated from tumor section by counting the number of OT1-GFP cells per mm2. (C) Expression of CD69 (hatch marked) and CD62L (gray) activation markers in GFP+ cells as analyzed by flow cytometry (dLN, draining LN) (mean ± SD, n = 3–6). (D) IFN-γ production of GFP+ cells in EG7 and EL4 tumors after 3 h of treatment with Brefeldin A without antigen restimulation (mean ± SD, n = 5). *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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fig1: Activation of specific T cells in antigen-expressing tumors. C57Bl6 mice were injected s.c. with five 105 EG7 and EL4 tumor cells in either flank. 8–10 d later, the mice were adoptively transferred with 107 purified naive CD8+ cells from OT1-GFP mice. (A) Evolution of EG7 (▪) and EL4 (□) tumor size after adoptive transfer. (mean ± SD, n = 5 independent experiments). Early phase (day 3–4) and late phase (day 5–6) were defined in Material and methods. (B) Density of OT1-GFP cells within EG7 and EL4 at early (day 3) and late (day 6) phase of tumor rejection were calculated from tumor section by counting the number of OT1-GFP cells per mm2. (C) Expression of CD69 (hatch marked) and CD62L (gray) activation markers in GFP+ cells as analyzed by flow cytometry (dLN, draining LN) (mean ± SD, n = 3–6). (D) IFN-γ production of GFP+ cells in EG7 and EL4 tumors after 3 h of treatment with Brefeldin A without antigen restimulation (mean ± SD, n = 5). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Mentions: To analyze the role of antigen recognition in tumor infiltration and in CTL motility within tumors, we inoculated individual C57BL/6 mice s.c. with two tumors: the EL4 thymoma in one flank and the EG7 (OVA-expressing EL4 cells) in the other flank. Naive GFP-expressing, OVA-specific, TCR-transgenic OT1 cells (OT1-GFP) were adoptively transferred to the mice when the EL4 and EG7 tumors were 500–1,000 mm3 (around day 10). OT1-GFP cells induced the complete rejection of EG7, but not of EL4 tumors, within 6–8 d (Fig. 1 A). After the adoptive transfer of OT1-GFP cells, the tumors continued to grow for 3 d. After 3–4 d, the tumor stops growing before the size actually starts decreasing at days 5–6. At days 7–8, the tumors continued to shrink and disappeared completely. We defined two periods for the analysis of the active rejection process: the “early” rejection phase corresponds to days 3–4 after adoptive transfer when the tumor stops growing; the “late” rejection phase corresponds to days 5–6 when the size of the tumors starts to decrease, but before complete rejection (which occurs at days 7–8).


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)

Activation of specific T cells in antigen-expressing tumors. C57Bl6 mice were injected s.c. with five 105 EG7 and EL4 tumor cells in either flank. 8–10 d later, the mice were adoptively transferred with 107 purified naive CD8+ cells from OT1-GFP mice. (A) Evolution of EG7 (▪) and EL4 (□) tumor size after adoptive transfer. (mean ± SD, n = 5 independent experiments). Early phase (day 3–4) and late phase (day 5–6) were defined in Material and methods. (B) Density of OT1-GFP cells within EG7 and EL4 at early (day 3) and late (day 6) phase of tumor rejection were calculated from tumor section by counting the number of OT1-GFP cells per mm2. (C) Expression of CD69 (hatch marked) and CD62L (gray) activation markers in GFP+ cells as analyzed by flow cytometry (dLN, draining LN) (mean ± SD, n = 3–6). (D) IFN-γ production of GFP+ cells in EG7 and EL4 tumors after 3 h of treatment with Brefeldin A without antigen restimulation (mean ± SD, n = 5). *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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Related In: Results  -  Collection

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fig1: Activation of specific T cells in antigen-expressing tumors. C57Bl6 mice were injected s.c. with five 105 EG7 and EL4 tumor cells in either flank. 8–10 d later, the mice were adoptively transferred with 107 purified naive CD8+ cells from OT1-GFP mice. (A) Evolution of EG7 (▪) and EL4 (□) tumor size after adoptive transfer. (mean ± SD, n = 5 independent experiments). Early phase (day 3–4) and late phase (day 5–6) were defined in Material and methods. (B) Density of OT1-GFP cells within EG7 and EL4 at early (day 3) and late (day 6) phase of tumor rejection were calculated from tumor section by counting the number of OT1-GFP cells per mm2. (C) Expression of CD69 (hatch marked) and CD62L (gray) activation markers in GFP+ cells as analyzed by flow cytometry (dLN, draining LN) (mean ± SD, n = 3–6). (D) IFN-γ production of GFP+ cells in EG7 and EL4 tumors after 3 h of treatment with Brefeldin A without antigen restimulation (mean ± SD, n = 5). *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Mentions: To analyze the role of antigen recognition in tumor infiltration and in CTL motility within tumors, we inoculated individual C57BL/6 mice s.c. with two tumors: the EL4 thymoma in one flank and the EG7 (OVA-expressing EL4 cells) in the other flank. Naive GFP-expressing, OVA-specific, TCR-transgenic OT1 cells (OT1-GFP) were adoptively transferred to the mice when the EL4 and EG7 tumors were 500–1,000 mm3 (around day 10). OT1-GFP cells induced the complete rejection of EG7, but not of EL4 tumors, within 6–8 d (Fig. 1 A). After the adoptive transfer of OT1-GFP cells, the tumors continued to grow for 3 d. After 3–4 d, the tumor stops growing before the size actually starts decreasing at days 5–6. At days 7–8, the tumors continued to shrink and disappeared completely. We defined two periods for the analysis of the active rejection process: the “early” rejection phase corresponds to days 3–4 after adoptive transfer when the tumor stops growing; the “late” rejection phase corresponds to days 5–6 when the size of the tumors starts to decrease, but before complete rejection (which occurs at days 7–8).

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