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. 2011 Nov 23;35(5):792-805.
doi: 10.1016/j.immuni.2011.09.017.

An interleukin-21-interleukin-10-STAT3 pathway is critical for functional maturation of memory CD8+ T cells

Weiguo Cui  1 , Ying Liu, Jason S Weinstein, Joseph Craft, Susan M Kaech
Affiliations

An interleukin-21-interleukin-10-STAT3 pathway is critical for functional maturation of memory CD8+ T cells

Weiguo Cui et al. Immunity. .

Abstract

Memory CD8(+) T cells are critical for long-term immunity, but the genetic pathways governing their formation remain poorly defined. This study shows that the IL-10-IL-21-STAT3 pathway is critical for memory CD8(+) T cell development after acute LCMV infection. In the absence of either interleukin-10 (IL-10) and IL-21 or STAT3, virus-specific CD8(+) T cells retain terminal effector (TE) differentiation states and fail to mature into protective memory T cells that contain self-renewing central memory T cells. Expression of Eomes, BCL-6, Blimp-1, and SOCS3 was considerably reduced in STAT3-deficient memory CD8(+) T cells, and BCL-6- or SOCS3-deficient CD8(+) T cells also had perturbed memory cell development. Reduced SOCS3 expression rendered STAT3-deficient CD8(+) T cells hyperresponsive to IL-12, suggesting that the STAT3-SOCS3 pathway helps to insulate memory precursor cells from inflammatory cytokines that drive TE differentiation. Thus, memory CD8(+) T cell precursor maturation is an active process dependent on IL-10-IL-21-STAT3 signaling.

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Figures

Figure 1
Figure 1. STAT3-deficient memory CD8+ T cells sustain a terminally differentiated effector phenotype
(A) Stat3+/+ (filled square) and Stat3−/− (open circle) mice were infected with LCMV, and DbGP33–41-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 DbGP33–41 tetramer+ CD8+ T cells at day 8 and 80 p.i. (D) Numbers of KLRG1lo IL-7Rhi and KLRG1hi IL-7Rlo 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 DbGP33–41 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 GP33–41 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.
Figure 2
Figure 2. STAT3 acts in a CD8+ T cell autonomous manner to generate a long-lived pool of mature memory CD8+ T cells
(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, KLRG1lo IL-7Rhi and KLRG1hi IL-7Rlo 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.
Figure 3
Figure 3. STAT3 is required for memory CD8+ T cell self-renewal and protective immunity
(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).
Figure 4
Figure 4. Both IL-10 and IL-21 promote memory CD8+ T cell differentiation
(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 DbGP33–41 tetramer+ CD8+ T cells was examined at days 25 p.i. (B) The numbers of total DbGP33–41 tetramer+ (open bars) and KLRG1lo IL-7Rhi (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 KLRG1lo IL-7Rhi groups, * denotes p<0.05 and n.s. denotes “not significant”).
Figure 5
Figure 5. Stat3−/− CD8+ T cells have reduced expression of Eomes and BCL-6
(A and B) Histogram plots showing the expression of Eomes, T-bet and BCL-6 in DbGP33–41 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.
Figure 6
Figure 6. BCL-6 is important for maintaining KLRG1lo IL-7Rhi memory CD8+ T cells following infection
(A) Histograms show the expression of BCL-6 in LCMV-specific IL-7Rhi (black line) and IL-7Rlo (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. DbNP396–404 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 KLRG1lo IL-7Rhi (solid) DbNP396–404+ 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).
Figure 7
Figure 7. STAT3-dependent SOCS3 expression suppresses IL-12 signaling and enhances memory CD8+ T cell differentiation
(A) Western blot shows the amount of SOCS3 and GRP94 (loading control) in KLRG1hi IL-7Rlo (TE) or KLRG1lo IL-7Rhi (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 DbGP33–41 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.
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