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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. -. -. +. +.

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  1. Immunized Control 107 100 % of IFN-g+TNF-a+ cells 80 106 40 p=0.0021 15 60 50 105 93 4.1 40 40 Relative cell # 104 20 30 103 0 20 10 0 103 104 105 0 103 104 105 0 - - + + - - + + CFSE TIM F5 recipient mice TIM F5 recipient mice Naive F5 recipient mice Naive F5 recipient mice % of undivided cells # of divided cells 100 Open Resource 2 Secondary TIM-derived memory CD8 T cells contain a higher proportion of IFN-g+/TNF-a+ 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-g and TNF-a was assessed by FACS analysis. Mean±SD from 3 pooled independent experiments. Two-tailed unpaired t-test. 80 60 40 20 0 Control Immunized Control Immunized Naive TIM Transferred cells 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. Transferred F5 CD8 T cells Endogenous CD8 T cells p=0.0061 NS 0.15 0.1 % of XCL1+ cells % of XCL1+ cells 0.05 0 Peptide: 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.

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