(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) 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) 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) 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 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) 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) 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.