Cytomegalovirus encodes a positive regulator of antigen presentation

Abstract

Murine cytomegalovirus encodes three regulators of antigen presentation to antiviral CD8 T cells. According to current paradigms, all three regulators are committed to the inhibition of the presentation of antigenic peptides. Whereas m152/gp40 catalyzes the retention of peptide-loaded major histocompatibility complex (MHC) class I molecules in a cis-Golgi compartment, m06/gp48 binds stably to class I molecules and directs them into the cellular cargo-sorting pathway of lysosomal degradation. Regulator m04/gp34 also binds stably to class I molecules, but unlike m152 and m06, it does not downmodulate MHC class I cell surface expression. It has entered the literature as a direct inhibitor of T-cell recognition of the MHC-peptide complex at the cell surface. In this work, we have studied the presentation of antigenic viral peptides in cells infected with a comprehensive set of mutant viruses expressing the three regulators separately as well as in all possible combinations. The results redefine m04 as a positive regulator dedicated to the facilitation of antigen presentation. When expressed alone, it did not inhibit T-cell recognition, and when expressed in the presence of m152, it restored antigen presentation by antagonizing the inhibitory function of m152. Its intrinsic positive function, however, was antagonized and even slightly overcompensated for by the negative regulator m06. In an adoptive cell transfer model, the opposing forces of the three regulators were found to govern immune surveillance in the infected host. While negative regulators, also known as immunoevasins, are common, the existence of a positive regulator is without precedent and indicates an intriguing genetic potential of this virus to influence antigen presentation.

FIG. 1.

Regulation of MHC class I cell surface expression by vRAPs. Primary BALB/c-derived MEFs were either left uninfected (n.i., no infection) (A) or were infected with mutant mCMV-Δm04+06+152 lacking vRAPs (Ø) (B), with mCMV-WT expressing all three vRAPs (K), or with vRAP gene deletion mutants expressing individual vRAPs and combinations of vRAPs as indicated (C to H). Contamination of MEFs with CD11b⁺ cells (e.g., macrophages) was below the detection limit of cytofluorometric analysis. Two-color cytofluorometric analysis was performed after 16 h in the late early phase of viral gene expression. Contour plots represent fluorescence intensity levels for ∼35,000 live cells analyzed with no further gating. Ordinate fluorescence data represent expression of the viral cytoplasmic early-phase protein m164 (gp38/50), and abscissa fluorescence data represent cell surface expression of the MHC class I molecule L^d. Upregulated L^d expression in uninfected cells present within the infected cultures served as an internal standard. The regulating effect of vRAPs is highlighted by a caliper rule symbol. PE, phycoerythrin.

FIG. 2.

vRAP-modulated pattern of IE1 epitope presentation detected with an IE1-specific CTL line. (A) Recognition of infected BALB/c MEFs as stimulator cells in an 18-h IFN-γ-based ELISPOT assay. (B) Recognition of infected and ⁵¹Cr-labeled BALB/c MEFs as target cells in a 4-h cytolysis assay. Effector cells were cells of a CTL line specific for the mCMV IE1 peptide 168-YPHFMPTNL-176 presented by L^d. Epitope recognition sensitivities of the IE1 CTLs in the assays were compared by exogenous loading of MEFs with synthetic IE1 peptide. Ø, no peptide added or no vRAP expressed. The vRAPs expressed in cells infected with the respective mutants (see Table 1) are indicated (n.i., no infection). Bars represent MPNs from linear regression analysis of dose-response curves, and error bars represent the 95% upper confidence limits. In the ELISPOT assays, dose-response curves were established with 300, 200, 100, and 50 CTLs throughout. In the cytolysis assays, log₂-graded numbers of CTLs were seeded, starting with 40,000 cells in the case of peptide titration and with 60,000 cells in the test of the recognition of infected MEFs. Results for key viruses (see the text) are highlighted in black.

FIG. 3.

vRAP-modulated recognition patterns reflect epitope presentation. (A) vRAP-modulated recognition patterns of IE1 epitope-specific, L^d-restricted CTLs in ELISPOT assays performed with BALB/c-derived MEFs as stimulator cells expressing all three H-2^d haplotype MHC class I molecules K^d, D^d, and L^d (top panel) or with BALB/c-H-2^dm2-derived MEFs as stimulator cells expressing only K^d and D^d (bottom panel). (B) vRAP-modulated recognition pattern of m04 epitope-specific, D^d-restricted CTLs in an ELISPOT assay performed with BALB/c-derived MEFs as stimulator cells. For ELISPOT assay details, see the legend to Fig. 2. Ø, no vRAP expressed after infection with mCMV-Δm04+06+152; n.i., no infection.

FIG. 4.

vRAP-modulated recognition patterns of infected BALB/c MEFs for all currently known MHC class I-restricted mCMV epitopes in the H-2^d haplotype, except for IE1-L^d and m04-D^d, which are shown in Fig. 3. For definitions and ELISPOT assay details, see the legend to Fig. 2.

FIG. 5.

vRAPs operate in like manner in different cell types. In ELISPOT assays, infected BALB/c MEFs were used as an example of a stromal cell type (top panel) and infected BALB/c BMDCs as an example of a professional antigen-presenting cell type of hematopoietic lineage (bottom panel). Effectors were cells of a CTL line specific for the M45-derived peptide 507-VGPALGRGL-515 presented by D^d. The sensitivities of epitope detection by M45-D^d-specific CTLs for the two cell types are compared by exogenous loading with synthetic peptide. Ø, no peptide added or no vRAP expressed; n.i., no infection.

FIG. 6.

vRAP-modulated recognition patterns for epitopes presented by MHC class I molecules of the haplotype H-2^b. (A) Results of ELISPOT assays performed with C57BL/6-derived MEFs as stimulator cells. Epitope specificities of the CTLs and the corresponding presenting class I alleles are indicated. (B) Results of an ELISPOT assay performed with M45-D^b epitope-specific CTLs as effector cells and with C57BL/6-derived BMDCs as stimulator cells. For definitions, see the legend to Fig. 2.

FIG. 7.

Adoptive cell transfer approach for testing vRAP function in vivo. (A) Experimental protocol and probe design for ISH. C57BL/6 transfer recipients were immunocompromised by 7.5-Gy γ-irradiation and were infected subcutaneously (s.c.) with the key viruses indicated. CTLs specific for the M45-derived peptide 985-HGIRNASFI-993 presented by D^b were administered in graded numbers intravenously (i.v.). On day (d) 12 after transfer and infection, liver tissue sections were analyzed by ISH for the presence of infected cells, which are primarily hepatocytes. Maps (not drawn to scale) illustrate the positions of vRAP genes (black boxes) and DNA probes (red bars) in the mCMV genome. (B) Combinatorial ISH images of liver tissue sections for the three key viruses and the respective vRAP gene probes verifying the molecular identities of the viruses in the experimental groups with no adoptive transfer (positive control groups; see Ø columns in panel C). Red staining localizes viral DNA accumulated in intranuclear inclusion bodies of infected hepatocytes. Bar marker, 50 μm. (C) Control of virus replication in the liver. The expressed vRAPs are indicated, corresponding (from left to right) to infection with the key viruses mCMV-Δm04+06, mCMV-Δm06, and mCMV-WT, respectively. For quantitation of infected hepatocytes, ISH was performed with the m152 gene-specific probe. The ordinate values represent the numbers of infected hepatocytes present in 50-mm² areas of liver tissue sections. Black dots represent data for individual adoptive transfer recipients, with the median values marked. Ø, no cell transfer.

FIG. 8.

vRAPs regulate the CD8 T-cell-mediated control of virus replication in the lungs. The principle of the in vivo assay of vRAP function is indicated in Fig. 7. The expressed vRAPs are indicated, corresponding (from left to right) to infection with the key viruses mCMV-Δm04+06, mCMV-Δm06, and mCMV-WT, respectively. (A) Adoptive transfer of M45-D^b (985-HGIRNASFI-993)-specific CTLs into 7.5-Gy-irradiated and infected C57BL/6N recipients. (B) Adoptive transfer of M45-D^d (507-VGPALGRGL-515)-specific CTLs into 6.5-Gy-irradiated BALB/cJ recipients. Throughout, virus replication in the lungs was assessed on day 12 after infection and transfer by a virus plaque assay of organ homogenates. The ordinate values represent the amounts of infectious virus per lung, expressed as PFU. The dotted line indicates the detection limit of the virus plaque assay. Throughout, black dots represent data for individual adoptive transfer recipients, with the median values marked. Ø, no cell transfer. In panel B, P values for significance (Wilcoxon-Mann-Whitney test; two-tailed; P < 0.05) are indicated for the minimal effector cell doses required for protection.