0 10 3 10 4 10 5 15 0 10 3 10 4 10 5 40 CFSE Control Immunized 93 4.1 100 80 60 40 20 0 % of undivided cells# of divided cells Control Immunized Control Immunized 10 7 10 6 10 5 10 4 10 3 Relative cell # T IM Naive Transferred cells 100 80 60 40 20 0 % of IFN- + TNF- + cells p=0.0021 % of XCL1 + cells 0.15 0.1 0.05 0 Transferred F5 CD8 T cellsEndogenous CD8 T cells 10 20 30 50 40 0 % of XCL1 + cells Peptide: + + - - + + - - T IM F5 recipie nt mice Naive F5 recipient mice T IM F5 recipie nt mice Naive F5 recipient mice p=0.0061 NS Open Resource 1 T IM cells proliferate in response to a viral challenge. CFSE-labelled T IM F5 CD8 T cells were transferred into syngeneic hosts that were subsequently immunized with VV- NP. Three days after viral infection the proliferation of F5 CD8 T cells from the spleens of recipient mice was analyzed by FACS. CFSE profiles as well as the mean±SD of the percentages of undivided and of the number of divided CD8 + F5TCR + cells in control and immunized animals are shown. One representative experiment out of two is shown. Open Resource 2 Secondary T IM - derived memory CD8 T cells contain a higher proportion of IFN- + /TNF- + cells than naive-derived primary memory CD8 T cells. Naive or T IM F5 CD8 T cells were transferred into congenic hosts that were subsequently immunized with VV-NP. Six weeks after immunization, the spleen CD8 T cells from both types of recipients were re-stimulated by the antigenic peptide in vitro for 4 hours in the presence of monensin and the ability of transferred cells to co-produce IFN- and TNF- was assessed by FACS analysis. Mean±SD from 3 pooled independent experiments. Two-tailed unpaired t- test. Open Resource 3 Secondary anti-inﬂuenza memory CD8 T cells generated from F5 T IM -transferred cells have an advantage in XCL1 production. Naive or T IM F5 CD8 T cells were transferred into congenic hosts that were subsequently immunized intra-nasally with a H1N1 inﬂuenza virus, engineered to express the NP68 epitope. Six weeks after immunization, the spleen CD8 T cells from both types of recipients were re-stimulated or not by the antigenic peptide in vitro for 4 hours in the presence of monensin and the ability of transferred (F5) and endogenous cells to secrete XCL1 was assessed by FACS analysis. The mean±SD of the percentage of XCL1 + cells among transferred (left panel) or endogenous (right panel) CD8 T cells from T -

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Immunized. Control. 10 7. 100. % of IFN- g + TNF- a + cells. 80. 10 6. 40. p=0.0021. 15. 60. 50. 10 5. 93. 4.1. 40. 40. Relative cell #. 10 4. 20. 30. 10 3. 0. 20. 10. 0 10 3 10 4 10 5. 0 10 3 10 4 10 5. 0. -. -. +. +. - PowerPoint PPT Presentation

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0 103 104 105

CFSE

Control Immunized

93 4.1

100

% of undivided cells # of divided cells

Control Immunized Control Immunized

107

106

105

104

103

Rela

tive

cell

TIM Naive

Transferred cells

100

% of IFN-+TNF-+ cells

p=0.0021

% o

f XCL

1+ cel

0.15

0.1

0.05

Transferred F5 CD8 T cells Endogenous CD8 T cells

% o

f XCL

1+ cel

Peptide: ++- - ++- -

TIM F5 recipient

mice

Naive F5 recipient

mice

TIM F5 recipient

mice

Naive F5 recipient

mice

p=0.0061 NS

Open Resource 1 TIM cells proliferate in response to a viral challenge. CFSE-labelled TIM F5 CD8 T cells were transferred into syngeneic hosts that were subsequently immunized with VV-NP. Three days after viral infection the proliferation of F5 CD8 T cells from the spleens of recipient mice was analyzed by FACS. CFSE profiles as well as the mean±SD of the percentages of undivided and of the number of divided CD8+F5TCR+ cells in control and immunized animals are shown. One representative experiment out of two is shown.

Open Resource 2 Secondary TIM-derived memory CD8 T cells contain a higher proportion of IFN-+/TNF-+ cells than naive-derived primary memory CD8 T cells. Naive or TIM F5 CD8 T cells were transferred into congenic hosts that were subsequently immunized with VV-NP. Six weeks after immunization, the spleen CD8 T cells from both types of recipients were re-stimulated by the antigenic peptide in vitro for 4 hours in the presence of monensin and the ability of transferred cells to co-produce IFN- and TNF- was assessed by FACS analysis. Mean±SD from 3 pooled independent experiments. Two-tailed unpaired t-test.

Open Resource 3 Secondary anti-influenza memory CD8 T cells generated from F5 TIM-transferred cells have an advantage in XCL1 production. Naive or TIM F5 CD8 T cells were transferred into congenic hosts that were subsequently immunized intra-nasally with a H1N1 influenza virus, engineered to express the NP68 epitope. Six weeks after immunization, the spleen CD8 T cells from both types of recipients were re-stimulated or not by the antigenic peptide in vitro for 4 hours in the presence of monensin and the ability of transferred (F5) and endogenous cells to secrete XCL1 was assessed by FACS analysis. The mean±SD of the percentage of XCL1+ cells among transferred (left panel) or endogenous (right panel) CD8 T cells from TIM- and naive-recipients are shown. One Representative of two independent experiments. Two-tailed unpaired t-test.