Role of PKR and Type I IFNs in Viral Control during Primary and Secondary Infection

Type I interferons (IFNs) are known to mediate viral control, and also promote survival and expansion of virus-specific CD8+ T cells. However, it is unclear whether signaling cascades involved in eliciting these diverse cellular effects are also distinct. One of the best-characterized anti-viral signaling mechanisms of Type I IFNs is mediated by the IFN-inducible dsRNA activated protein kinase, PKR. Here, we have investigated the role of PKR and Type I IFNs in regulating viral clearance and CD8+ T cell response during primary and secondary viral infections. Our studies demonstrate differential requirement for PKR, in viral control versus elicitation of CD8+ T cell responses during primary infection of mice with lymphocytic choriomeningitis virus (LCMV). PKR-deficient mice mounted potent CD8+ T cell responses, but failed to effectively control LCMV. The compromised LCMV control in the absence of PKR was multifactorial, and linked to less effective CD8+ T cell-mediated viral suppression, enhanced viral replication in cells, and lower steady state expression levels of IFN-responsive genes. Moreover, we show that despite normal expansion of memory CD8+ T cells and differentiation into effectors during a secondary response, effective clearance of LCMV but not vaccinia virus required PKR activity in infected cells. In the absence of Type I IFN signaling, secondary effector CD8+ T cells were ineffective in controlling both LCMV and vaccinia virus replication in vivo. These findings provide insight into cellular pathways of Type I IFN actions, and highlight the under-appreciated importance of innate immune mechanisms of viral control during secondary infections, despite the accelerated responses of memory CD8+ T cells. Additionally, the results presented here have furthered our understanding of the immune correlates of anti-viral protective immunity, which have implications in the rational design of vaccines

FOXO3 Regulates CD8 T Cell Memory by T Cell-Intrinsic Mechanisms

CD8 T cell responses have three phases: expansion, contraction, and memory. Dynamic alterations in proliferation and apoptotic rates control CD8 T cell numbers at each phase, which in turn dictate the magnitude of CD8 T cell memory. Identification of signaling pathways that control CD8 T cell memory is incomplete. The PI3K/Akt signaling pathway controls cell growth in many cell types by modulating the activity of FOXO transcription factors. But the role of FOXOs in regulating CD8 T cell memory remains unknown. We show that phosphorylation of Akt, FOXO and mTOR in CD8 T cells occurs in a dynamic fashion in vivo during an acute viral infection. To elucidate the potentially dynamic role for FOXO3 in regulating homeostasis of activated CD8 T cells in lymphoid and non-lymphoid organs, we infected global and T cell-specific FOXO3deficient mice with Lymphocytic Choriomeningitis Virus (LCMV). We found that FOXO3 deficiency induced a marked increase in the expansion of effector CD8 T cells, preferentially in the spleen, by T cell-intrinsic mechanisms. Mechanistically, the enhanced accumulation of proliferating CD8 T cells in FOXO3-deficient mice was not attributed to an augmented rate of cell division, but instead was linked to a reduction in cellular apoptosis. These data suggested that FOXO3 might inhibit accumulation of growth factor-deprived proliferating CD8 T cells by reducing their viability. By virtue of greater accumulation of memory precursor effector cells during expansion, the numbers of memory CD8 T cells were strikingly increased in the spleens of both global and T cell-specific FOXO3-deficient mice. The augmented CD8 T cell memory wa

Loss of FOXO3 leads to a tissue-specific increase in expansion of effector CD8 T cells.

<p>(<b>A</b>) Dynamic, in vivo alterations in phosphorylation of FOXO- associated signaling proteins in LCMV-specific CD8 T cells. C57BL/6 mice were infected with LCMV, and at the indicated days PI, splenocytes were stained with anti-CD8, MHC-I tetramer (D^b/NP396 or D^b/GP33) and either the anti-P-Akt (T308), P-FOXO-1/3a (T24/T32), or P-mTOR (S2448) antibodies. As controls, these antibodies were pre-incubated with their specific antigenic peptide before adding on to the cells. Representative flow plots (left) gated on tetramer-binding CD8s from day 5 PI mice indicate specific antibody staining in relation to the peptide blocked controls. Plotted data (Corrected MFI) is expressed as the difference of observed MFI for the phospho-specific protein and peptide-blocked control (right), divided by the peptide-blocked control. Data are representative of at least three independent experiments. (<b>B</b>) +/+ and FOXO3−/− mice were infected with LCMV, and at 8 days PI, LCMV specific CD8 T cells were quantified in spleen, liver and lymph nodes by staining with anti-CD8 and MHC-I tetramers. Data are representative of 4 independent experiments with 4–6 mice/group/experiment.</p

The secondary CD8 T cell response in FOXO3 deficient mice.

<p>+/+ and FOXO3−/− mice were infected with LCMV-Armstrong and after 90 days PI, these mice were challenged with 2.5×10⁶ PFU of LCMV clone 13. (<b>A</b>) Five days after LCMV clone13 infection, mononuclear cells from spleen, liver and lymph nodes were collected and the total number of LCMV-specific CD8 T cells was determined by staining with anti-CD8 and LCMV-specific MHC-I tetramers. Data are from 4–6 mice/group; error bars represent the SEM and * indicates statistical significance at p<.05. (<b>B</b>) Splenocytes from +/+ or FOXO3−/− mice were stained with anti-CD8, MHC-I tetramers and anti-Ki67. The percentage of Ki67 positive cells amongst tetramer positive CD8 T cells was determined by flow cytometry. (<b>C</b>) Splenocytes were stimulated ex vivo with LCMV-specific peptides for 5 hours. Following stimulation, cells were stained for surface expression of CD8 and intracellular expression of IFNγ, IL-2 and TNFα. The top number in the plots on the left is the MFI of staining for IFNγ. The bottom number in the plots indicates the percentage of total splenocytes that are CD8 and IFNγ positive. The plots on the right are gated on IFNγ^+ve CD8 T cells and the numbers are the percentages of cells that produced IFNγ and IL-2, IFNγ and TNFα, or IFNγ, IL-2, and TNFα. (<b>D</b>) LCMV titers in serum and lung were determined by plaque assay using a vero cell monolayer. Each symbol represents the viral titer of an individual mouse.</p

FOXO3 regulates CD8 T cell expansion through T cell-intrinsic mechanisms.

<p>(<b>A</b>) +/+ and FOXO3L mice were infected with LCMV. At 8 days PI, mononuclear cells from spleen, liver and lymph nodes were stained with anti-CD8 and MHC-I tetramers. Data are from 3–4 independent experiments with 4–6 mice/group/experiment; error bars represent the SEM and * indicates statistical significance at p<.03. (<b>B</b>) Investigation of in vivo proliferation and apoptosis by BrdU incorporation/Ki67 staining and Annexin V staining. +/+ and FOXO3L mice were infected with LCMV and given a BrdU injection at 6 days PI and administered BrdU in drinking water between days 6–8 PI. At the end of the BrdU pulse, splenocytes were stained with anti-CD8, MHC-I tetramers and anti-BrdU. The percentage of BrdU positive cells amongst tetramer positive CD8 T cells was determined by flow cytometry (top panel). In parallel studies, mononuclear cells were stained with anti-CD8, MHC-I tetramer and anti-Ki67 (middle panel) or Annexin V (lower panel). The percentage of Ki67 or Annexin V positive cells amongst tetramer positive CD8 T cells was determined by flow cytometry. Data are from 2–3 independent experiments; Graphs represent data from 6–8 mice/group/experiment; error bars represent the SEM and * indicates statistical significance at p<.001. (<b>C</b>) At day 8 PI, splenocytes were stimulated in vitro with LCMV specific peptides for 5 hours. Following stimulation, cells were stained for surface expression of CD8 and intracellular expression of IFNγ, IL-2 and TNFα. Representative dot plots (left) are gated from total lymphocytes with the top numbers indicating the MFI for IFNγ on CD8 T cells while the bottom number in the plot indicates the percentage of total splenocytes that are CD8 and IFNγ positive. Dot plots (right) represent the percentages of IL-2 and/or TNFα producing cells among IFNγ^+ve CD8 T cells. Representative plots from 1 of 6 individual experiments are illustrated. (<b>D</b>) To deplete CD4 T cells, +/+ and FOXO3L mice were injected with 100 ug of the monoclonal antibody, GK1.5, at days 0 and 4 relative to LCMV infection. At day 8 PI, splenocytes were stained with anti-CD8 and MHC-I tetramers to determine the total number of LCMV-specific CD8 T cells. Bars represent data collected from at least 4 mice; error bars represent the SEM and * indicates statistical significance at p<.01.</p

Absence of FOXO3 does not affect the phenotype or function of effector CD8 T cells.

<p>(<b>A</b>) At 8 days after LCMV infection, splenocytes from +/+ and FOXO3−/− mice were isolated and stained for expression of CD44, CD62L, CD27 and CD122 on NP396- (top) and GP33- (bottom) specific CD8 T cells. Representative histograms in panel A are gated on CD8 and MHC-I tetramer positive populations with the numbers indicating MFI for the indicated protein in either +/+ or FOXO3−/− mice. (<b>B</b>) Total splenocytes were stained with MHC-I tetramer, anti-CD8, anti-CD127 and anti-KLRG-1, and the total number of SLECs (KLRG-1^high/CD127^low) and MPECs (CD127^high/KLRG-1^low) were quantified by flow cytometry. Data are from 4 to 6 independent experiments with 3–6 mice/group/experiment; error bars represent the SEM and * indicates statistical significance at p<.05. (<b>C</b>) On day 8 PI, Splenocytes from +/+ or FOXO3−/− mice were stimulated with LCMV epitope peptides for 5 hours directly ex-vivo. Following stimulation, cells were stained for cell surface CD8 and intracellular IFNγ, IL-2 and TNFα. Panel <b>C</b> shows cytokine production by effector CD8 T cells. Representative dot plots (left) are gated on total lymphocytes with the top number indicating observed MFI for IFNγ staining in peptide-stimulated CD8 T cells and the bottom number indicating percentage of total splenocytes that are CD8 and IFNγ positive. Dot plots (right) represent the percentage of IL-2 and/or TNFα producing cells among IFNγ^+ve CD8 T cells. (<b>D</b>) Intracellular staining for Granzyme B. The FACS histograms are gated on LCMV-specific CD8 T cells from +/+ (BLUE) and FOXO3−/− (RED) mice. The green histogram represents staining with isotype control antibodies. The data are MFIs for Granzyme B expression +/− SD.</p

Effect of FOXO3 deficiency on contraction of CD8 T cells.

<p>(<b>A</b>) +/+ and FOXO3−/− mice were infected with LCMV and at the indicated days PI, cells from spleen, liver and lymph nodes were stained with anti-CD8 and MHC-I tetramers. Data are representative of 3 to 8 independent experiments with 4–6 mice/group/experiment for each indicated time point. (<b>B</b>) +/+ and FOXO3−/− mice were infected with LCMV and given a BrdU injection at either 8 or 12 days PI and administered BrdU in drinking water between either days 8–11 or 12–15 PI. At the end of each BrdU pulse (day 12 or day 15), splenocytes were stained with anti-CD8, MHC-I tetramers and anti-BrdU. The percentage of BrdU positive cells amongst tetramer binding CD8 T cells for each pulse (8–11, or 12–15 days PI) was determined by flow cytometry. Data are the mean of at least 6 +/+ or FOXO3−/− mice/group.</p