(A) Mice that received a small number (5×10⁵) of P14 TCR-tg Stat3^+/+ (filled square) and Stat3^−/− (open circle) cells were infected with LCMV. P14 CD8⁺ T cells in spleen were enumerated at day 8, 40 and 80 p.i. Numbers of total P14, KLRG1^lo IL-7R^hi and KLRG1^hi IL-7R^lo subsets were plotted in line graphs. (B) Dot plots show the expression of KLRG1 and IL-7R in P14 CD8⁺ T cells at day 8 and 80 p.i. (C) Histograms show the expression of CD27, CD122, Granzyme B and BCL-2, and percentage of CD62L⁺ and IL-2⁺ in P14 Stat3^+/+ (shaded histogram) and Stat3^−/− (dashed line histogram) CD8⁺ T cells at day 80 p.i. MFI or percentage of “positive” cells shown in the upper left corner.

(A) WT and Il21−/− mice were infected with LCMV and either treated with αIL-10 mAb or mock injected with PBS for 25 days. (A) KLRG1 and IL-7R expression on D^bGP_33–41 tetramer⁺ CD8⁺ T cells was examined at days 25 p.i. (B) The numbers of total D^bGP_33–41 tetramer⁺ (open bars) and KLRG1^lo IL-7R^hi (black bars) CD8⁺ T cells at day 25 p.i. are shown in the stacked bar graphs. Data shown are representative of two independent experiments (Statistical analyses show the comparison between KLRG1^lo IL-7R^hi groups, * denotes p<0.05 and n.s. denotes “not significant”).

(A) Histograms show the expression of BCL-6 in LCMV-specific IL-7R^hi (black line) and IL-7R^lo (shaded) subsets at day 8 and 40 p.i. based on intracellular staining and flow cytometry. (B) Cd8α ^−/− bone marrow was mixed with either Bcl6^−/− or Bcl6^+/+ bone marrow (at a 90:10 ratio) and used to reconstitute irradiated Cd8⁺α ^−/− mice that were then infected with LCMV two months later. D^bNP_396–404 tetramer⁺ CD8⁺ T cells were examined for expression of KLRG1 and IL-7R at days 8 and 60 p.i. (C) Numbers of total (open) or KLRG1^lo IL-7R^hi (solid) D^bNP_396–404⁺ memory CD8⁺ in spleen at day 60 were plotted in the bar graphs. Data shown are representative of two independent experiments (** denotes to p<0.01).

(A) Stat3^+/+ (filled square) and Stat3^−/− (open circle) mice were infected with LCMV, and D^bGP_33–41-specific CD8⁺ T cells in spleen were enumerated at day 8, 40 and 80 p.i. (B) Bar graphs show viral titers in the serum at day 8 p.i. LOD denotes the level of detection. Serum from naïve mice infected with CL13 served as a positive control. (C) Dot plots show expression of KLRG1 and IL-7R in D^bGP_33–41 tetramer⁺ CD8⁺ T cells at day 8 and 80 p.i. (D) Numbers of KLRG1^lo IL-7R^hi and KLRG1^hi IL-7R^lo subsets from (A and B) were plotted in line graphs. (E) Histograms show the expression level of CD27, CD122, Granzyme B and BCL-2, and percentage of CD62L⁺ and IL-2⁺ in D^bGP_33–41 tetramer⁺ Stat3^+/+ (shaded histogram) and Stat3^−/− (dashed line histogram) CD8⁺ T cells at day 80 p.i. (F) Dot plots show the IFNγ and TNFα expression in GP_33–41 peptide stimulated Stat3^+/+ and Stat3^−/− splenocytes at day 80 p.i. Data shown are representative of at least three independent experiments per time point. MFI or percentage of “positive” cells (mean±SEM) shown in the upper left or right corner throughout the whole manuscript.

(A) LCMV infected mice were given BrdU in drinking water from days 30 to 40 p.i., BrdU incorporation (±SEM) in Stat3^+/+ (solid line) and Stat3^−/− (dashed line) memory CD8⁺ T cell and isotype control (shaded) is shown in histograms. (B and C) Equal numbers of Stat3^+/+ and Stat3^−/− LCMV-specific memory CD8⁺ T cells were adoptively transferred into naïve hosts that were subsequently infected with LCMV Cl13. A control group of “Naive” mice that did not receive donor memory CD8⁺ T cells are also shown. Bar graphs show the numbers of secondary effector CD8⁺ T cells (B) and serum viral titers (C) in the recipient mice at day 6 p.i. Data shown are representative of three independent experiments (** denotes p<0.01).

(A and B) Histogram plots showing the expression of Eomes, T-bet and BCL-6 in D^bGP_33–41 tetramer⁺ Stat3^+/+ (shaded) and Stat3^−/− (dashed line) CD8⁺ T cells at days 8 (A) and 40 (B) post infection. (C) Western blot shows the amount of Blimp-1 and Actin (loading control) in P14 CD8⁺ T cells isolated at days 8 and 40 p.i. by FACS. Numbers below the graph indicate the normalized abundance of Blimp-1 measured by densitometry.

(A) Western blot shows the amount of SOCS3 and GRP94 (loading control) in KLRG1^hi IL-7R^lo (TE) or KLRG1^lo IL-7R^hi (MPC) P14 CD8⁺ T cells isolated by FACS 8 days p.i. Numbers below the graph indicate the normalized amount of SOCS3 measured by densitometry. (B) Histogram plots showing the expression of SOCS3 in D^bGP_33–41 tetramer⁺ Stat3^+/+ (shaded) and Stat3^−/− (dashed line) CD8⁺ T cells at day 8 and 40 p.i. (C) SOCS3 expression in P14 Stat3^+/+ or Stat3^−/− LCMV-specific day 6 effector CD8⁺ T cells treated in vitro with IL-10 and IL-21 for 8 hrs (black line) or left untreated (shaded). (D) Activated P14 CD8⁺ T cells were transduced with retroviruses (RV) containing MigR1 empty vector or MigR1-SOCS3, stimulated with IL-12 (black line) or left untreated (dashed line), and then the amount of pSTAT4₆₉₃ was measured by flow cytometry. (E) Histogram plot (left) shows the levels of pSTAT4₆₉₃ in day 7 P14 Stat3^+/+ (shaded) and Stat3^−/− (dashed line) effector CD8⁺ T cells after IL-12 (10ng ml⁻¹) stimulation. Grey line shows untreated control cells. Line graph (right) shows MFI of pSTAT4₆₉₃ (normalized to unstimulated controls) in day 7 P14 Stat3^+/+ (squares) and Stat3^−/− (open circles) CD8⁺ T cells stimulated over a range of IL-12 concentrations. (F) Plots show the amount of pSTAT4₆₉₃ directly ex vivo in P14 Stat3^+/+ (shaded) and Stat3^−/− (dashed line) LCMV-specific memory CD8⁺ T cells one day after infection with Listeria monocytogenes. (G) Activated P14 CD8⁺ T cells were transduced with control RV (MigR1) or a SOCS3 shRNAi RV and adoptively transferred into C57BL/6 mice that were subsequently infected with LCMV. Contour plots show expression of KLRG1 and IL-7R in the RV transduced cells at day 30 p.i. Data shown are representative of at least three independent experiments.