Figure 8. Via targeting ICOSL, m138 reduces T-cell mediated control of MCMV infection in vivo.

(A) BALB/c mice were intraperitoneally (i.p.) inoculated with 2 × 10⁶ PFU MCMV-GFP. Two days post-infection mice were sacrificed and peritoneal exudate cells were extracted and analyzed by flow cytometry for surface expression of ICOSL and MHCI, using specific mAbs against each of these molecules. Blue histograms represent the expression of uninfected (GFP-) cells and green histograms represent the expression of MCMV-infected (GFP+) cells from the same mouse. The isotype for each antibody was used as a negative control (dotted lines). The results obtained from a representative infected mouse out of two are shown. (B) BALB/c mice (n = 4/group) were mock-infected or i.p. infected with 1 × 10⁶ PFU of MCMVwt or MCMVΔm138. At 6 hpi, peritoneal exudate cells were extracted and surface expression of ICOSL and CD80 assessed by flow cytometry on the surface of DCs (CD11⁺ MHC II⁺ CD3^-CD19^- NKp46^- cells), gating on m04-positive cells when derived from MCMVwt- or MCMVΔm138-infected mice. Results are expressed as the mean +/-SD of the MFI values obtained for samples from two independent experiments. (C) Table displaying the immunomodulatory effects of the different MCMVs on the cellular targets of m138. (D) On the top, a schematic representation of the MCMVΔm138 and MCMVm138ΔIg2 in vivo infection assay is shown. C57BL/6 mice (n = 5–7/group) with or without NK, or NK, CD4⁺ and CD8⁺ T-cell depletion, as indicated, were intravenously (i.v.) inoculated with 2 × 10⁵ PFU/mouse of MCMVwt (yellow circles), MCMVΔm138 (pink triangles) or MCMVm138ΔIg2 (blue triangles). At day 14 post-infection mice were sacrificed and viral titers in salivary glands and lungs of individual mice were determined by standard plaque assays. Horizontal bars indicate the median values. The Kruskal-Wallis test was used to assess statistical differences between experimental groups. *p<0.05, **p<0.01. A representative experiment out of two performed is shown.

Figure 8—figure supplement 1. m138 contributes to immune evasion of CD4⁺ T cells in spleens and lungs of MCMV-infected mice.

On the top, a schematic representation of the adoptive transfer in vivo assay is shown. 1 × 10⁵ M25-specific transgenic CD4⁺ T cells were transferred (i.v.) into C57BL/6 SCID mice (n = 4–5/group), which were depleted of NK cells as indicated, and either mock-infected or i.p. infected with 2 × 10⁵ PFU/mouse of MCMVwt or MCMVΔm138. Seven dpi, spleens, lungs, and salivary glands were harvested, and analyzed by flow cytometry using anti-CD45.1 and anti-CD4-specific mAbs. The frequency of CD45.1⁺ CD4⁺ cells for each organ is indicated. Results are expressed as the mean +/-SEM of the percentage values obtained for samples in each group.