Absence of cross-presenting cells in the salivary gland and viral immune evasion confine cytomegalovirus immune control to effector CD4 T cells

Abstract

Horizontal transmission of cytomegaloviruses (CMV) occurs via prolonged excretion from mucosal surfaces. We used murine CMV (MCMV) infection to investigate the mechanisms of immune control in secretory organs. CD4 T cells were crucial to cease MCMV replication in the salivary gland (SG) via direct secretion of IFNγ that initiated antiviral signaling on non-hematopoietic cells. In contrast, CD4 T cell helper functions for CD8 T cells or B cells were dispensable. Despite SG-resident MCMV-specific CD8 T cells being able to produce IFNγ, the absence of MHC class I molecules on infected acinar glandular epithelial cells due to viral immune evasion, and the paucity of cross-presenting antigen presenting cells (APCs) prevented their local activation. Thus, local activation of MCMV-specific T cells is confined to the CD4 subset due to exclusive presentation of MCMV-derived antigens by MHC class II molecules on bystander APCs, resulting in IFNγ secretion interfering with viral replication in cells of non-hematopoietic origin.

Figure 1. Helper functions exerted by CD4 T cells are dispensable for MCMV control.

(A) MCMV titers in the SG in CD4^-/- (open triangles), MHCII^-/- (open circles) and B6 (closed circles) mice at different time points post infection. Dotted line shows detection limit. (B–E) M45- and M38-specific CD8 T cells were quantified by tetramer staining of pooled SG leukocytes of CD4^-/- (open triangles), MHCII^-/- (open circles) and B6 (closed circles) mice at different time points post MCMV infection. Representative results of three independent experiments are shown.

Figure 2. CD4 T cells exert direct antiviral mechanisms by secretion of IFNγ.

(A) γ-irradiated CD4^-/- were reconstituted with 50% bone marrow of CD4^-/- mice and 50% bone marrow of either CD4^-/-, IFNγ^-/-, TNFα^-/-, PKOB or B6 mice. Mice were infected with a MCMV mutant expressing the firefly luciferase under the control of the m157 promoter. 10 minutes prior to analysis, D-luciferin was injected i.p. and active virus replication was detected by in vivo bioluminescence imaging in one month infected chimeras. (B, C) 2 to 4 months post infection viral titers were determined by plaque assay. Dotted line shows detection limit. Combined results of three individual experiments are shown. Percentages of mice bearing a virus load above the detection limit (non-controllers) are indicated in B and the virus titers are shown in C. (D) IFNγR^-/- or B6 mice were γ-irradiated and reconstituted with of B6 or IFNγR^-/- bone marrow, respectively. MCMV titers in the SG were determined 8 weeks post infection. Representative results of two independent experiments are shown. (* p<0.05, ** p<0.01, *** p<0.001, 2-tailed unpaired student's t-test).

Figure 3. MCMV-specific CD8 T cells isolated from the SG secrete IFNγ.

(A) Kinetic analysis of the number of IFNγ-secreting CD8 T cells after ex vivo restimulation with the epitopes M45 (closed squares) and M38 (closed triangles) or CD4 T cells after stimulation with a lysate of MCMV infected cells (open circle) in the SG. B) Total numbers of IFNγ-producing CD8 T cells isolated from SG of wild type (black bars) or MHCII^-/- (open bars) mice after ex vivo restimulation with either M45, M38 or IE3 peptides after 2, 8 or 16 weeks post infection. Data of SGs pooled from three to four mice per group are displayed.

Figure 4. CD4 as well as CD8 T cells infiltrate the infected SG tissue and CD8 T cell infiltration is CD4 T cell independent.

SGs were isolated three weeks post infection from B6 (A and B) or MHCII^-/- (C) mice infected with a GFP-expressing MCMV mutant. Cryosections of SG were counterstained with anti-CD4 (A; red) or anti-CD8 (B and C; red) and anti-CD11c (blue). MCMV-bearing AGECs (green) were situated either distal to immune infiltrates (left column) or in proximity to infiltrates (middle columns). In some infiltrates cells with very low fluorescent intensity for GFP were present (middle column; arrow), most likely representing cells not directly infected with MCMV but instead cells that had taken up remnants of MCMV-infected cells. Dense leukocyte infiltrates were often devoid of GFP⁺ cells (right column). Confocal images were taken with 20 times magnification. Scale bar indicates 100 µm. One representative picture of a minimum of 10 is shown.

Figure 5. MHC class I and II expression is absent in intact MCMV infected cells and may be re-expressed at low levels on infected cells in proximity to leukocyte infiltrates.

Three weeks post MCMV-GFP infection, SG sections isolated from B6 mice were stained for MHC class I (A to C; blue) or MHC class II (D and E; blue) as well as with phalloidin (A to E; red), visualizing actin. MCMV-infected cells which are situated distal (A and D) or in proximity (B and E) to cell infiltrates were analyzed. Expression of MHC class I on uninfected AGECs is shown in C. Arrows point out weak MHC class I or MHC class II expression on AGECs. Confocal images were taken with 100 times magnification. Scale bar indicates 10 µm. One representative picture of a minimum of 10 is shown.

Figure 6. Cross-presenting APCs are absent in SG tissue.

(A) Spleen cells and SG resident leukocytes from naïve or 4 week MCMV infected mice were stained for I-A^b, CD11c, CD8α, CD4 or CD103 and B220. Left plots are gated on B220 negative cells and stained for I-A^b and CD11c, right plots are gated on B220⁻, MHC class II⁺, CD11c⁺ cells and stained for CD4 respectively CD103 and CD8α. Frequencies of gated cells among the parent population are indicated. B) Total numbers of total CD11⁺ I-A^b+ B220⁻ (upper diagram) or CD11⁺ I-A^b+ B220⁻ CD8α⁺ CD4⁻ (lower diagram) DCs resident in the spleen (black bar) or APCs in the SG (open bar) are shown for different time points post MCMV infection. C) DCs were isolated four weeks post MCMV infection from spleen (left column) or APCs from the SG (right column) and pulsed with VLPs cross-linked to either the LCMV-derived gp33 (upper left plots) or to gp61 (upper right plots) peptides or pulsed with the peptide (lower plots). 10⁵ naïve CFSE-labeled gp33-specific CD8 T cells (P14) or naïve CFSE-labeled gp61-specific CD4 T cells (Smarta) were added to equal numbers of splenic DCs or SG derived APCs. After 5 days of incubation, CFSE dilution was analyzed by gating on P14 or Smarta cells. One representative example of three individual experiments is shown.

Figure 7. MCMV control in the SG in presence or absence of viral immune evasion genes.

(A) B6, CD4^-/- or MHCII^-/- mice were infected with MCMV-Δm04Δm06Δm152 or its parental BAC-derived virus (MCMV wt) and SG virus titers were determined 28 days post infection. (B) B6 and CD4^-/- mice were depleted of CD8 T cells and infected with MCMV-Δm04Δm06Δm152 or its parental BAC-derived virus (MCMV wt) and SG virus titers were determined 28 days post infection. Combined data of two independent experiments is shown. (* p<0.05, ** p<0.01, *** p<0.001, 2-tailed unpaired student's t-test).

Figure 8. Virus-specific CD4 but not CD8 T cells control MCMV replication in the salivary gland.

Surface expression of MHC class I and II is effectively suppressed in infected acinar glandular epithelial cells (AGECs), thereby inhibiting direct recognition by MCMV-specific CD8 and CD4 T cells. Phagocytic APCs situated in close proximity to virus-infected cells take up MCMV antigens, either in from of defective viral particles or apoptotic bodies of infected cells, and present MCMV-derived peptide antigens on MHC class II molecules to CD4 T cells but not to CD8 T cells due to their inability to cross-present particulate antigens. Activated MCMV-specific CD4 T cells secrete IFNγ which consequently binds to its receptor on the cell surface of the infected cell as well as on adjacent AGECs. Activation of IFNγR signaling cascade on adjacent cells induces an antiviral state, rendering them resistant to MCMV replication and IFNγ signaling in infected cells leads to the termination of MCMV replication.