0 103 104 105

15

0 103 104 105

40

CFSE

Control Immunized

93 4.1

100

80

60

40

20

0

% of undivided cells # of divided cells

Control Immunized Control Immunized

107

106

105

104

103

Rela

tive

cell

#

TIM Naive

Transferred cells

100

80

60

40

20

0

% of IFN-+TNF-+ cells

p=0.0021

% o

f XCL

1+ cel

ls

0.15

0.1

0.05

0

Transferred F5 CD8 T cells Endogenous CD8 T cells

10

20

30

50

40

0

% o

f XCL

1+ cel

ls

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.