Autocrine IFN-γ promotes naive CD8 T cell differentiation and synergizes with IFN-α to stimulate strong function

Abstract

Autocrine IFN-γ signaling is important for CD4 differentiation to Th1 effector cells, but it has been unclear whether it contributes to CD8 T cell differentiation. We show in this paper that naive murine CD8 T cells rapidly and transiently produce low levels of IFN-γ upon stimulation with Ag and B7-1, with production peaking at ∼8 h and declining by 24 h. The autocrine IFN-γ signals for upregulation of expression of T-bet and granzyme B and induces weak cytolytic activity and effector IFN-γ production. IFN-α acts synergistically with IFN-γ to support development of strong effector functions, whereas IL-12 induces high T-bet expression and strong function in the absence of IFN-γ signaling. Thus, IFN-γ is not only an important CD8 T cell effector cytokine, it is an autocrine/paracrine factor whose contributions to differentiation vary depending on whether the response is supported by IL-12 or type I IFN.

FIGURE 1. Naïve CD8 T cells produce IFN-γ early in response to Ag and B7-1-dependent costimulation

Spleen cells from OT-I and pmel, and lymph node cells from P14 TCR transgenic mice (all 16 weeks of age) were stained with anti-CD44 mAb and stimulated with cognate peptide for 4 hr (middle row) or 8 hr (bottom row), permeabilized and stained with anti- IFN-γ mAb as described in Materials and Methods. (A) Top panel shows OT-I cells cultured for 4 hours in the absence of cognate peptide and bottom panel shows OT-I cells stimulated with Ag for 8 hours and stained with isotype control Ab. Isotype controls were done for all samples at both 4 and 8 hr, and in all cases showed fewer than 1.5 % IFNg ⁺ events in the CD44^low/int gate. (B-D) Each column shows the CD44 expression profile (top row) for the indicated cell type, and dot plots for IFN-γ × CD44 for cells stimulated with Ag for 4 hr (middle row) and 8 hr (bottom row). For dot plots, percent of cells in each gated quadrant is shown, and the mean fluroscence intensity (MFI; geometric mean) for cells in some quadrants are shown. OT-I.IFN-γ^−/− cells examined in parallel had fewer than 1.5% IFN-γ ⁺ events in the CD44 ^low/int gate at 4 or 8 hr (not shown). In an independent experiment examining cells stimulated for 8 hr, the percent IFN-γ₊ CD44 ^low/int cells for OT-I, pmel, P14, and OT-I. IFN-γ^−/− were 43.1%, 8.6%, 27.4% and 1.0% respectively.

Figure 2. Early IFN-γ production by naïve cells is increased by B7-1-dependent costimulation but not IL-2, and is rapid and transient

(A) Purified CD44^low OT-I T cells were stimulated in vitro for 8 hr with Ag alone or Ag along with co-immobilized B7-1/Fc ligand (Ag/B7), and IFN- γ production was determined by IC staining. Results shown are mean and range for duplicate samples. Five independent experiments confirmed that Ag/B7 stimulation results in 2- to 3-fold greater numbers of IFN-γ⁺ cells than does Ag alone. (B) Purified CD44^low OT-I (black line) and OT-I.IFNγ^−/− (solid gray) T cells were stimulated with aAPC having just Ag, or Ag and B7-1 (Ag/B7) immobilized on the surface either without or with IL-2 added (2.5U/ml). IFN-γ production was determined by IC staining after 8 hours of stimulation. Percent IFN-γ+ OT-I cells is shown for the indicated gating. (C) Purified CD44 ^low OT-I cells were stimulated with Ag/B7 for varying times and IFN-γ production determined by IC staining. Shown are representative dot plots (CD44 × IFN-γ) of stimulated cells at 4 hr (top) and 8 hr (bottom). In all cases, samples stained with isotype control Ab had < 1% IFN-γ+ cells. (D) IFN-γ+ cells were determined as in (C), and are shown as the percent of total OT-I cells. Results are expressed as mean +/− SD at each time point for two independent experiments. Five independent experiments confirmed these results.

FIGURE 3. Peptide Ag dose response for early IFN-γ production

LN cells from OT-I mice (8 weeks old) were stained with anti-CD44 mAb and stimulated in vitro with aAPC prepared by immobilizing H-2K^b.DimerX and B7-1/Fc on the surface and pulsing with varying concentrations of peptide Ag prior to being washed and added to the cultures. After 8 hr of stimulation the cells were permeabilized and stained for IFN-γ. (A) CD44 expression on OT-I cells incubated in the absence of peptide Ag. (B) IFN- γ expression of OT-I cells incubated for 8 hr in the absence of Ag. (C) Representative dot plot of OT-I cells stimulated with peptide Ag (10⁻⁶ M) for 8 hr. (D) The percent of IFN-g+ cells of the total cells in the CD44low/int gate and in the CD44hi gate, using gates shown in C, at varying peptide concentrations. (E) The MFI (geometric mean) of the IFN-g+ cells in the CD44low/int and CD44hi gates. At the lowest peptide concentrations there were too few events to obtain meaningful MFI values.

Figure 4. Naïve OT-I cells from Rag^−/− mice and mice deficient for both IL-12 and Type I IFN receptors produce early IFN-γ

(A-C) Purified CD44^low OT-I.IFNγ^−/− (A) OT-I (B), and OT-I.Rag^−/− cells (C) were stimulated with aAPC having Ag and B7-I immobilized on the surface, and IFN-γ production determined by IC staining after 8 hours. Isotype control Ab staining is solid gray. Percent IFN-γ+ cells is shown for the indicated gating. Shown are representative histograms from triplicate samples. (D) CD8 T cells from OT-I cells deficient for IL-12 and Type I IFN receptors (OT-I.IL-12βR^−/−.IFNAR^−/−) were stimulated for 8 hr with aAPC having Ag and B7-I/Fc on the surface, stained with anti-CD44 mAb, and IFN-γ production determined by IC staining. The CD44 expression profile of cells at 8 hr is shown. (E) A representative dot plot (CD44 × IFN-γ) of cells stimulated with aAPC that were not pulsed with peptide Ag, i.e. a no Ag control. (F) A representative dot plot of cells stimulated with aAPC that had been pulsed with Ag. In an independent experiment, 13% of the CD44^low/int cells produced IFN-γ. The peptide dose response for the OT-I.IL-12βR^−/−.IFNAR^−/− cells was essential the same as for wild type OT-I (not shown).

FIGURE 5. IFN-γ signals for development of weak effector functions, but is not required for IL-12-mediated development of effector functions

Naïve OT-I, OT-I. IFN-γ^−/− (OT-I.γ ko), and OT-I. IFN-γR^−/− (OT-I.γ Rko) cells were stimulated for 3 days in vitro with Ag/B7 in the absence or presence of IL-12, washed and assayed. (A) OT-I (solid lines) and OT-I.γ ko (dashed lines) were stimulated for 3 days with Ag/B7 in the absence (open symbols) or presence of IL- 12 (filled symbols) and cytolytic activity determined in a 4 hr ⁵¹Cr-release assay. Separate lines are shown for cells from independent cultures of each cell type. (B) As in A, but examining OT-I.γ Rko cells (dotted lines). For nine independent experiments, cytolytic activity of OT-I cells in the absence of IL-12 was 12% +/− 9% (SD) of the activity of cells stimulated with IL-12, while no activity was detected for OT-I.γko or OT-I.γRko cells. (C) GrzB levels were determined by IC staining and representative histograms are shown. Solid lines are OT-I cells and dashed lines are OT-I. IFN-γRko cells stimulated with Ag/B7 in the absence (top) or presence (bottom) of IL- 12. Gray lines are isotype control Ab. (D) GrzB levels are shown as mean fluorescence intensity (MFI: geometric mean) of IC staining. Additions to the 3 day cultures with Ag/B7 are shown, and included neutralizing anti- IFN-γ Ab (α-IFNγ), IFN-γ and IL-12 in various combinations. Values are means and ranges for duplicate samples.

FIGURE 6. Early IFN-γ signals for development of weak late IFN- γ effector production, but is not required for IL-12-mediated development of strong IFN-γ effector function

(A – C) OT-I, OT-I. IFN-γR^−/−, and OT-I. IFN-γ^−/− cells were stimulated for 8 hr with Ag/B7 and IFN-γ production determined by IC staining. (D – G) OT-I or OT-I. IFN-γR^−/− cells were cultured for 3 days with Ag/B7 and either no addition (D and F) or with IL-12 added (E and G), and IFN-γ production then determined by IC staining after 4 hr re-stimulation with peptide Ag. Gray lines are isotype Ab controls. Thin lines in (D) and (E) are cells that had neutralizing anti- IFN-γAb present during the culture period. Representative histograms are shown.

FIGURE 7. IFN-γ-dependent differentiation yields effector cells that can mediate tumor growth control

OT-I and OT-I. IFN-γR^−/− (OT-I.γ Rko)cells were stimulated in vitro with Ag/B7 for 3 days with no cytokine addition (None) or with IL-12 added. Cells were then washed and adoptively transferred into mice bearing subcutaneous B16.OVA tumors that had been growing for 10 days (7 mice/group), and tumor size was determined by two right-angle measurements on day 21 of tumor growth (i.e. 11 days after adoptive transfer). Results are shown as mean tumor area (mm²) +/− SD, and p values are by Student’s T test. Essentially the same results were obtained in a second independent experiment. Comparison of OT-I/IL-12 versus IFNγRko/IL-12 at 20 × 10⁵ cell input yields a p value of 0.03, and comparision at 2 × 10⁵ cell input yields a p value of 0.07.

FIGURE 8. IFN-γ and IFN-α synergize to induce development of strong effector functions

(A) OT-I (solid lines) and OT-I. IFN-γ^−/− (dashed lines) cells were stimulated for 3 days in vitro with Ag/B7 and no cytokine addition (None), or either IL-12 (1μg/ml) or IFN-α (1,000U/ml) added to the cultures. Cells were then harvested and cytolytic activity was determined in a ⁵¹Cr-release assay. (B) OT-I (black lines) and OT-I. IFN-γR^−/− (gray lines) cells were stimulated as in A and harvested at day 3. Cells were re-stimulated for 4 hours with SIINFEKL peptide and IFN-γ production then determined by IC staining (B). (C) OT-I (solid lines) and OT-I. IFN-γR^−/−(dashed lines) cells were stimulated for 3 days with Ag/B7 and varying amounts of IFN-α added to the cultures as indicated. On day 3 cells were harvested and cytolytic activity (top panel), GrzB expression (middle panel) and IFN-γ production (bottom panel) were determined. Cytolytic activity (top) is expressed as Lytic Units per 1×10⁶ cells.

FIGURE 9. IFN-γ upregulates expression of T-bet but does not affect Eomes expression

(A) OT-I and OT-I. IFN-γ^−/− cells were stimulated with Ag/B7 without added cytokine (None) or with IL-12 added. At the indicated times cells were harvested and Eomes protein expression determined by IC staining. Expression in naïve cells (left panels) was also determined. Gray lines are isotype control Ab. (B) Cells were stimulated as above with no cytokine added (None), or with IFN-γ or IL-12 added. Cells were harvested after 72 hr and T-bet protein levels determined by IC staining. Gray line is isotype control Ab, and vertical dotted lines are to aid comparisons. (C) Cells were stimulated and T-bet expression levels determined as in B at the indicated times. Results are expressed as mean fluorescence intensity (MFI: geometric mean). (D) OT-I and OT-I. IFN-γ^−/− cells were stimulated with Ag/B7 and varying concentrations of IFN-α. Cells were harvested at 72 hr and T-bet levels determined by IC staining. Results are expressed as MFI (geometric mean) of T-bet.