ThPOK derepression is required for robust CD8 T cell responses to viral infection

Abstract

In the thymus, the transcription factor ThPOK is essential for the development of the CD4 helper T cell lineage, whereas active repression of ThPOK is critical for the development of the CD8 cytotoxic T cell lineage. ThPOK gene silencing is thought to be irreversible in peripheral CD8 T cells. We noticed that ThPOK repression is readily abrogated upon in vitro TCR stimulation of peripheral CD8 T cells. This observation prompted us to investigate a role for ThPOK in the CD8 T cell response to an acute viral infection. We observed that a functional deficiency of ThPOK does not affect CD8 T cell differentiation into effector T cells and the long-term persistence of Ag-specific memory T cells. However, in the absence of functional ThPOK, clonal expansion is significantly less in both primary and secondary CD8 T cell responses. Long-lived, Ag-specific CD8 T cells with a functional deficiency in ThPOK fail to produce high amounts of IL-2 and also fail to express high levels of granzyme B upon rechallenge. Our data reveal an unexpected role for ThPOK in CD8 T cells in promoting expansion and boosting the response to antigenic challenge.

FIGURE 1

Activation of peripheral CD8 T cells leads to ThPOK up-regulation. A, Splenic CD8 T cells derived from ThPOK^wt/gfp (open histograms) or ThPOK^wt/wt (shaded histograms) mice were exposed to the indicated concentrations of anti-CD3 and a fixed concentration of soluble anti-CD28 (1 μg/ml). After 2 days the cultures were removed from anti-CD3-coated plates and cultured in the presence of rIL-2 (10 U/ml). At 2, 4, and 6 days of culture the level of ThPOK/GFP expression was assessed by flow cytometry. The numbers inserted in the histograms refer to the GFP MFI. B, MFI of GFP expression vs time in culture is plotted. Results are representative of three independent experiments.

FIGURE 2

Foreign Ag-specific effector and memory T cells up-regulate ThPOK expression during an infection. Left panels, ThPOK^wt/gfp and ThPOK^wt/wt mice were infected with LCMV Armstrong. T cells specific for the LCMV-derived GP₃₃ epitope in the spleen were identified using D^b/GP₃₃ tetramer and anti-CD8 staining at 6, 8, and >100 days postinfection and 5 days after rechallenge of immune mice (>100 days after the primary infection) with recombinant L. monocytogenes expressing GP₃₃. The numbers indicate the percentage of tetramer-positive cells among CD8 T cells in ThPOK^wt/gfp mice. Right panels, ThPOK/GFP expression in D^b/GP₃₃ tetramer-positive CD8 T cells was determined by flow cytometry. Shaded histograms represent ThPOK^wt/wt tetramer⁺ cells. Numbers indicate the GFP MFI for ThPOK^wt/gfp cells. Results are representative of three independent experiments.

FIGURE 3

Reduced accumulation of effector P14 T cells in the absence of functional ThPOK. A, CD44^lowCD4⁻ThPOK^hd/hd or ThPOK^wt/wt P14 cells (Thy1.1) (10⁴) were transferred into B6 hosts that were subsequently immunized with LCMV. At 8 days postinfection, the fraction of both types of P14 T cells and their absolute numbers in spleen and liver were determined. The results are representative of eight independent experiments for spleen and two independent experiments for liver. B, Host mice were injected with 1 mg of BrdU i.p. (open histograms) or left untreated (shaded histograms) at 6 days postinfection. Six hours later, cells harvested from the spleen were analyzed for BrdU incorporation. The numbers in the histograms are the percentages of BrdU⁺ cells among P14 cells. C, At the indicated time points the levels of Bcl-2 expression (open histograms) in ThPOK^hd/hd P14 or ThPOK^wt/wt P14 cells were determined by flow cytometry. Shaded histograms represent isotype controls and numbers refer to the MFI of Bcl-2.

FIGURE 4

Normal differentiation of effector T cells in the absence of functional ThPOK. A, Splenocytes harvested at day 8 from mice that received either ThPOK^hd/hd or ThPOK^wt/wt P14 cells plus LCMV were stimulated in vitro with the GP₃₃ peptide for 5 h. The percentages of P14 T cells producing IFN-γ, TNF-α, and IL-2 after stimulation are shown. The numbers in parenthesis indicate the MFI of IFN-γ of P14 T cells. B, ThPOK^hd/hd or ThPOK^wt/wt P14 cells were analyzed for granzyme B and T-bet expression 8 days postinfection. Shaded histograms are isotype controls and numbers refer to the MFI of granzyme B or T-bet.

FIGURE 5

P14 T cells lacking functional ThPOK can form long-lived T cells that exhibit impaired ability to produce IL-2. A, The percentages and the absolute numbers of ThPOK^hd/hd and ThPOK^wt/wt P14 T cells in the spleen at >100 days postinfection with LCMV were determined by CD8, Vα2, and Thy1.1 expression. B, Fractions of both types of long-lived P14 T cells that produce IFN-γ and IL-2 in response to in vitro stimulation with the GP₃₃ peptide for 5 h.

FIGURE 6

P14 memory T cells lacking ThPOK show reduced accumulation upon rechallenge. A, Ninety to 180 days postinfection, CD19⁺ cell-depleted spleen cells containing 10⁴ ThPOK^hd/hd or ThPOK^wt/wt P14 cells were transferred into new B6 hosts, and rechallenged with LCMV. The percentages and absolute numbers of each type of P14 T cell 7 days after rechallenge were analyzed for CD8 and Thy1.1 expression. Results are representative of four independent experiments. B, B6 host mice were injected with (open histograms) or without (shaded histograms) 1 mg of BrdU i.p. 6 days after rechallenge. The percentages of BrdU incorporation in ThPOK^hd/hd or ThPOK^wt/wt P14 cells in PBL were analyzed 6 h after BrdU injection. Results are representative of two independent experiments. C, Bcl-2 expression levels (open histograms) in ThPOK^hd/hd and ThPOK^wt/wt P14 cells from the spleen before (90–180 days postinfection) or 7 days after the secondary challenge. Shaded histograms are isotype controls. The numbers in the histograms indicate MFI of Bcl-2.

FIGURE 7

Reduced expression levels of granzyme B and CD27 during the secondary response in the absence of ThPOK. A, Five days after the secondary LCMV challenge, spleen cells from rechallenged mice were stimulated in vitro with GP₃₃ peptide for 5 h. The production of IFN-γ and TNF-α in ThPOK^hd/hd or ThPOK^wt/wt P14 cells are shown. B, Granzyme B expression levels of ThPOK^hd/hd (shaded histogram) or ThPOK^wt/wt (open histogram) P14 cells in the spleen 5 days after rechallenge were analyzed. C, CD27 expression on ThPOK^hd/hd P14 (shaded histogram) or ThPOK^wt/wt P14 cells (open histogram) 7 days after rechallenge was analyzed by flow cytometry. Results are representative of two independent experiments.