Fig 2: MHV68 infection induces IL-22 production across anatomical sites and immune cell populations.C57BL/6 (BL6) mice were intranasally infected with 1000 PFU of MHV68. Tissues and serum were harvested at 9 dpi and 16 dpi, corresponding to the lytic and peak latent phases of infection, respectively. (A–D) IL-22 protein levels in the lungs at 9 dpi (A) and 16 dpi (D) were determined by ELISA. (B, E) IL-22 protein levels in the spleens at 9 dpi (B) and 16 dpi (E) were quantified by ELISA. (C, F) Serum IL-22 concentrations at 9 dpi (C) and 16 dpi (F) were measured using a flow cytometry-based multiplex bead assay. (G–J) Total numbers of IL-22-producing γδ T cells (defined as TCRγδ + IL-22 + cells) (G, I), NK cells (defined as CD3-CD49 + IL-22 + cells) (H,J), NK T cells (defined as CD3 + CD49 + IL-22 + cells) (K,M), and ILC3 cells (defined as CD45 + Lineage-Thy1 + TCRβ- TCRγδ-CD127 + RORγT + IL-22 + cells) (L,N) in the spleen and peritoneal cavity, respectively, at 16 dpi. (K–N). (O–R) Total numbers of IL-22-producing virus-specific CD4⁺ (defined asCD3 + CD4 + IL-22 + cells) (O,Q) and CD8⁺ (defined as CD3 + CD8 + IL-22 + cells) (P,R) T cells in the spleen and peritoneal cavity, respectively, at 16 dpi. Virus-specific T cells were identified following ex vivo stimulation with the immunodominant MHV68 peptides gp150 (MHC class II) or ORF6 (MHC class I), followed by intracellular cytokine staining and flow cytometric analysis. Each experimental group consists of 3–4 animals and each symbol represent an individual mouse. Data are pooled from two to three independent experiments. Mean and standard error of the mean are shown. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 3: IL-22 deficiency is associated with reduced systemic BAFF levels without altering the balance between germinal center compartments.BL6 and IL-22-/- mice were infected as described in the legend to Fig 1 and analyzed at 16 days post-infection (dpi), corresponding to the peak of latency. (A, B) Total numbers of centroblasts (defined as CXCR4 + CD86-) (A) and centrocytes (defined as CXCR4-CD86+) (B) within the germinal center B-cells (defined as B220 + GL7 + CD95+) compartment were determined by flow cytometry. (C) Centroblast-to-centrocyte ratio in infected BL6 and IL-22-/- mice. (D–F) Serum concentrations of BAFF (D), CXCL12 (E), and CXCL13 (F) were quantified using a flow cytometry-based multiplex bead assay. Each symbol represents an individual mouse. Data are pooled from two to three independent experiments with 2–4 mice per group. Mean and standard error of the mean are shown. *P < 0.05.

Fig 4: IL-22 deficiency reduces virus specific and polyclonal humoral response during chronic infection.BL6 and IL-22-/- mice were either mock treated or infected as described in the legend to Fig 1. Serum was collected at 16 days post-infection (dpi; peak latency) and 42 dpi (long-term latency) to evaluate humoral immune responses. (A–D) Serum concentrations of total IgG (A), total IgM (B), MHV68-specific IgG (C), and MHV68-specific IgM (D) at 16 dpi were determined by ELISA. (E) Anti-nuclear antibody (ANA) reactivity at 16 dpi was quantified by indirect immunofluorescence using HEp-2 cell substrates. IgG fluorescein isothiocyanate (FITC)-conjugated antibody was used for detection (F) ANA staining quantification of the sera from mock- and MHV68-infected BL6 and IL-22-/- mice by corrected total cell fluorescence (CTCF) (G) Serum anti-double-stranded DNA (anti-dsDNA) IgG levels at 16 dpi were determined by ELISA. (H–K) Serum concentrations of total IgG (H), total IgM (I), MHV68-specific IgG (J), and MHV68-specific IgM (K) at 42 dpi were determined by ELISA. (L) Serum anti-dsDNA IgG levels at 42 dpi. Data are pooled from two to five independent experiments, with each symbol representing an individual mouse. Mean and standard error of the mean are shown. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 5: IL-22 is not required for CD8 + T cell functional capacity or systemic inflammatory stability.BL6 and IL-22-/- mice were infected as described in the legend to Fig 1. (A-C) Flow cytometry-based analysis of IFNγ production by viral specific splenic CD8 + T cells at 16dpi. Splenocytes were restimulated ex vivo with Orf6 MHV68 peptide, and the frequency (A, B) and absolute number (C) of IFNγ+ cells were determined by intracellular cytokine staining (ICS). (D–F) Systemic inflammatory milieu at 16 dpi. Serum levels of IFNγ (D), TNFα (E), and IL-6 (F) were measured using a flow cytometry-based multiplex bead assay. Each experimental group consists of 3–4 animals and each symbol represent an individual mouse. Data are pooled from two to three independent experiments. Mean and standard error of the mean are shown.