Figure 1.
Socs4R108X/R108X mice show increased susceptibility to influenza A infection.
(A) Mice were infected i.n. with 20 pfu H1N1 PR8 virus and weight loss monitored for 20 days. Mice that lost more than 20% of their initial body weight (shown as separate lines in the graph) were considered moribund and removed from the study. (B) Kaplan-Meier survival plot of Socs4R108X/R108X and Balb/c control mice. Survival of Socs4R108X/R108X mice was significantly lower than controls (p = 0.002, Log-rank test). Combined data from two independent experiments are shown. (C) Comparison of lung viral titres on day 5 post-PR8 infection, *** indicates p value of 0.008 (t-test). (D) Socs4R108X/R108X mice showed significantly greater weight loss than Balb/c controls (p = 0.005) and slower recovery following infection with 103pfu of H3N2 X31 influenza virus. Data shown are representative of independent experiments (n = 4). (E) Socs4R108X/R108X mice display significantly higher lung viral titres than control mice following infection with 103pfu of H3N2 X31 influenza virus. Combined data from independent experiments are shown, * indicates p value <0.05. (F–G) Wild-type mice reconstituted with Socs4R108X/R108X bone marrow show increased disease severity following influenza infection. Lethally irradiated wild-type Thy1.1 mice were reconstituted with Socs4R108X/R108X Thy1.2 or WT Thy1.2 bone marrow and infected with H3N2 X31 influenza A virus. Weight loss (F) was monitored for 6 days, at which time point lungs were harvested for viral titer estimation (G). Pooled data from two independent experiments are shown.
Figure 2.
Elevated cytokine and chemokine production in lungs of Socs4R108X/R108X is associated with an increased influx of T cells.
(A) Cytokine and chemokine levels were analysed by ELISA and Bioplex in lung homogenates at day 3 post-infection with X31 virus. Mean data ± S.E.M. are shown for biological replicates (n = 4 for Balb/c, n = 5 for Socs4R108X/R108X), * indicates p<0.05, **<0.005. (B) Phenotypic analysis of lung hematopoietic subsets in Socs4R108X/R108X and Balb/c mice at day 3 post-infection. Flow cytometry analysis was performed on homogenized lungs and extracted BAL. Data plotted include combined cell numbers from lungs and BAL, * indicates p<0.05, **<0.005. (C) Expression of Socs4 mRNA in immune cells recovered from BAL and in the lungs of Balb/c mice infected with X31 virus. Mean data ± S.E.M. are shown for n = 3 biological replicates, u/inf = uninfected.
Figure 3.
Altered tissue distribution of virus-specific CD8 T cells in Socs4R108X/R108X mice.
(A) Total numbers (BAL+MLN+SPL) of virus-specific (KdNP147 positive) CD8 T cells following X31 influenza virus infection. Mean values (n = 5) are plotted, error bars represent SEM. (B) Significant differences were observed in the distribution of KdNP147 positive CD8 T cells in lungs (BAL) and spleens of Socs4R108X/R108X and Balb/c control mice following infection. *p<0.05, **<0.005, ***<0.001 (C) Percentage of GzmB positive KdNP147 positive CD8 T cells in various tissues on d10 post-infection (left hand panel); percentage of CD107a positive (middle panel) or GzmB positive (right hand panel) IFN-γ positive CD8 T cells on d10 post-infection following ex vivo stimulation with KdNP147 peptide. Mean values (n = 5) are plotted, error bars represent SD.
Figure 4.
Reduced downregulation of CD62L expression on Socs4R108X/R108X CD8 cells in following X31 influenza infection.
(A) Flow cytometric analysis showing the gating strategy and percentages of CD62Lhi and CD62Ll°CD8 T cells on day 5 post-infection with X31 influenza virus. Representative dot plots are shown from control Balb/c and Socs4R108X/R108X mice. (B) Total CD8 T cell numbers in the lymph node (MLN), lungs (BAL) and spleen (SPL) of Socs4R108X/R108X and Balb/C mice on day d5, d6 and d7 following infection with X31 influenza virus. (C) Total number and percentages of CD62Lhi and CD62Ll°CD8 cells in the MLN of Socs4R108X/R108X and Balb/C mice following X31 influenza virus infection.
Figure 5.
Defective TCR responses in Socs4R108X/R108X CD8 T cells.
CD4 or CD8 T cells were purified from Socs4R108X/R108X or wild-type spleens by negative selection, plated onto anti-CD3 coated plates in the presence of IL-2 and analysed at the indicated timepoints. (A) Q-PCR analysis showing increased expression of Socs4 mRNA in purified T cells from Balb/c mice following TCR engagement (mean±SEM, n = 3). (B) Cells were analysed by flow cytometry for the expression of the T cell activation marker, CD62L (mean±S.D.), n = cells derived from 3 mice. (C–D) Cells were labelled with Cell Trace Violet (CTV) for analysis of proliferative responses. Cell numbers per division are shown in the left hand panels; total cell numbers in the centre panels, (C) CD4 T cells, (D) CD8 T cells. Results are shown as the mean±S.E.M. using pooled data from 2 independent experiments (n = 5 mice per group per experiment). Representative histograms are plotted for each subset (day 4; right hand panels).