Figure 1.
Acute lung inflammation induced by antigen-specific CD8+ T cells cultured with low dose IL-2 resolves.
CC10-OVA. Thy1.2 transgenic mice or controls (B6.Thy1.2) were injected i.v. with the indicated numbers of activated OT-I. Thy1.1 T cells expanded with either high dose IL-2 (25U/ml) or low dose IL-2 (12.5U/ml). (A) Survival was dependent on the number of OT-I transferred and the dose of IL-2 given during culture. (B) The percentage of neutrophils (gated by size and Gr1Hi), CD3+Thy1.2+ and OT-I. Thy1.1+ T cells were measured in the BAL. (C) The numbers of cells in the lungs were measured on Day 4 after transfer of OT-I T cells. (D) Lung histology 4 days after transfer demonstrates peribronchial and perivascular mononuclear infiltrates in CC10-OVA mice compared to B6 mice; 7 µm sections were stained with H and E, magnification, 10X. Unpaired t tests were used to determine significance between controls (B6) and CC10-OVA mice, *p<0.05, **p<0.01, ***p<0.0001.
Figure 2.
Bystander lymphocytes are required to resolve lung injury mediated by OT-I.
CC10-OVA.RAG-/- or B6.RAG-/- mice were given 1x105 OT-I activated in vitro with low dose IL-2 on Day 0. On Day -2, some animals received 5x107 lymph node (LN) and spleen cells from B6 wild-type mice. Mice were sacrificed and lungs and BAL were analyzed at different time-points as indicated. (A) Experimental plan is outlined. (B) CC10-OVA.RAG-/- mice that received only OT-I T cells died within 7 days (No sp > CC10-OVA.RAG-/-). CC10-OVA.RAG-/- mice that received LN/spleen cells 2 days prior to transfer of OT-I survived (Sp > CC10-OVA.RAG-/-). Control mice were not affected (No Sp > B6.RAG-/-). (C) Lung tissue was harvested from CC10-OVA.RAG-/- given LN/spleen cells prior to OT-I transfer and B6.RAG-/- mice on days 4 and 30 after transfer of OT-I T cells. Sections (7 µm) were stained with H and E. Original magnification: 10X. (D) BAL from CC10-OVA.RAG-/- mice given LN/spleen cells prior to OT-I transfer and controls was harvested and analyzed on days 4, 6, and 10 after adoptive transfer of 1x105 OT-I T cells. Results are the mean ± SEM, CC10-OVA. RAG-/- mice (n=3), B6.RAG-/- (n=3) at each time-point.
Figure 3.
CD4+ T cells are required to prevent lethal lung injury.
CC10-OVA. RAG-/- mice were injected with LN/spleen cells or CD4 depleted LN/spleen Thy1.2 cells 2 days prior to i.v. injection of activated 1x105 OT-I. Thy1.1 T cells. (A) Survival was dependent on CD4+ T cells. (B) Day 4 after OT-I transfer, frequency of BAL cells was measured in B6.RAG-/- compared to CC10.OVA.RAG-/- with or without CD4 depletion of LN/spleen cells.
Figure 4.
Neutrophils are not required for lethal lung injury.
CC10-OVA.RAG-/- mice were injected with anti-Ly6G antibody or isotype control antibody i.p. two days prior to transfer of OT-I T cells and on days 3 and 5 after OT-I transfer. (A) Anti-Ly6G antibody (dotted line) specifically depleted neutrophils (Gr1Hi) cells in both the lung and spleen as demonstrated by the absence of the Gr1Hi peak by histogram compared to isotype control treated mice (solid line). Gray histogram is negative control for Gr1 staining. (B) No difference in mortality between anti-Ly6G treated animals and isotype control treated animals was found.
Figure 5.
ICOS+ Tregs are increased in lungs of mice with CD8-mediated acute lung injury.
Lung cells were harvested and analyzed by flow cytometry on day 4 after adoptive transfer of 1x105 OT-I T cells (low dose IL-2). (A) Frequency of CD4+ Foxp3+ Tregs in the lungs. (B) Frequency of ICOS + CD25+ CD4+Tregs in the lung; data are representative of three independent experiments. (C) Representative FACS plot from lungs of control or CC10.OVA mice. (D) Left panel is ICOS expression on CD4+Foxp3+ T cells from lungs of B6 (dotted line) and B6.CC10-OVA (solid line). Right panel is ICOS expression on CD4+Foxp3+ Tregs from lungs of B6.RAG-/- (dotted line) and B6.CC10-OVA.RAG-/- mice (solid line).
Figure 6.
ICOS-/- lymphocytes do not protect from CD8-mediated lung injury in mice.
CC10-OVA.RAG-/- mice were injected with wild-type LN/spleen cells, ICOS deficient LN/spleen cells, or ICOS deficient LN/spleen cells co-transferred with 0.5x106 or 1x106 ICOS+/+ Tregs 2 days prior to i.v. injection of activated 1x105 OT-I T cells. (A) Survival (B) Total lung cells, percentage and absolute numbers of CD4+, CD4+Foxp3+ T cells. (C) Percentage and absolute number of OT-I. Thy1.1+ cells and CD8+ Thy1.2+ T cells. (D) Percentage and absolute number of OT.I. Thy1.1+ T cells and CD8+Thy1.2+ T cells expressing IFNγ or TNFα in the lungs at Day 4 were measured.
Figure 7.
ICOS-/- Tregs have defects in IL-10 production and proliferation.
CD4+CD25+ Treg were isolated from C57Bl/6 ICOS+/+ and ICOS-/- mice and stimulated for 72 hours in vitro with anti-CD3 and IL-2 (A and B). A) Regulatory cytokine expression was measured by qRT-PCR. (B) IL-10 in the culture supernatants was measured by ELISA. C and D: single-cell suspensions of spleen and lymph node cells from C57Bl/6 ICOS+/+ and ICOS-/- mice were CFSE labeled, cultured for 72 hours in the presence of anti-CD3, and analyzed by flow cytometry. C. Percentage of CD4+Foxp3+ T cells that were CFSELo was measured. D. Survival of CD4+Foxp3+ T cells from ICOS+/+ and ICOS-/- mice was determined using a fixable live/dead dye (*p < 0.05, **p<0.01 – unpaired Student’s t test).
Figure 8.
ICOS-/- Tregs protect from lethal lung injury.
CC10-OVA.RAG-/- mice were injected with CD4-depleted wild-type LN/spleen cells in combination with 1x106 ICOS+/+CD4+CD25+ or ICOS-/-CD4+CD25+ Tregs 2 days prior to i.v. injection of activated 1x105 OT-I T cells. Survival was compared.