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Comparative Study
. 2005 Oct 17;202(8):1109-19.
doi: 10.1084/jem.20050162. Epub 2005 Oct 10.

Endogenous human cytomegalovirus gB is presented efficiently by MHC class II molecules to CD4+ CTL

Nagendra R Hegde  1 , Claire Dunn, David M Lewinsohn, Michael A Jarvis, Jay A Nelson, David C Johnson
Affiliations
Comparative Study

Endogenous human cytomegalovirus gB is presented efficiently by MHC class II molecules to CD4+ CTL

Nagendra R Hegde et al. J Exp Med. .

Abstract

Human cytomegalovirus (HCMV) infects endothelial, epithelial, and glial cells in vivo. These cells can express MHC class II proteins, but are unlikely to play important roles in priming host immunity. Instead, it seems that class II presentation of endogenous HCMV antigens in these cells allows recognition of virus infection. We characterized class II presentation of HCMV glycoprotein B (gB), a membrane protein that accumulates extensively in endosomes during virus assembly. Human CD4+ T cells specific for gB were both highly abundant in blood and cytolytic in vivo. gB-specific CD4+ T cell clones recognized gB that was expressed in glial, endothelial, and epithelial cells, but not exogenous gB that was fed to these cells. Glial cells efficiently presented extremely low levels of endogenous gB--expressed by adenovirus vectors or after HCMV infection--and stimulated CD4+ T cells better than DCs that were incubated with exogenous gB. Presentation of endogenous gB required sorting of gB to endosomal compartments and processing by acidic proteases. Although presentation of cellular proteins that traffic into endosomes is well known, our observations demonstrate for the first time that a viral protein sorted to endosomes is presented exceptionally well, and can promote CD4+ T cell recognition and killing of biologically important host cells.

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Figures

Figure 1.
Figure 1.
Anti-gB CD4+ T cells are cytotoxic. (A) His16 cells were infected with 30 PFUs/cell of Ad viruses expressing HCMV gB (AdtetgB) or HSV gI (AdtetgI) for 24 h, incubated with CD4+ T cells purified from PBMCs, and ELISPOT assays were performed for IFN-γ or granzyme B (GrB). Error bars denote standard deviations, and data points without error bars represent differences that are too small. His16 cells were infected with 10 PFUs/cell of AdtetgB (B) or 100 PFUs/cell of AdTbH9 (C) for 24 h, labeled with Na2 51CrO4, and then incubated with anti-gB CD4+ T cell clones (gB10/G3, gB10/H5, or gB100/2) or with mtb39-specific CD4+ T cell clone, TbH9-9, at various effector/target ratios for 24 h, and the specific release of 51Cr was determined.
Figure 2.
Figure 2.
Presentation of endogenous and exogenous gB to CD4+ T cell clones. (A) Autologous DCs (104) were incubated with 1 μg of soluble gB and 4 × 104 anti-gB CD4+ T cell clone gB3/F11. His16 glial cells were infected with AdtetgB (200 PFUs/cell) for 24 h, then incubated with 4 × 104 clone gB3/F11 or incubated with 1 or 10 μg of soluble gB and simultaneously with 4 × 104 clone gB3/F11. (B) His16 cells were infected with AdtetgB or AdtetgI (30 PFUs/cell) for 24 h before addition of T cells, or incubated with 10 μg/ml of soluble gB or soluble gE/gI and with gB-specific clones gB3/F5, gB10/G3, gB10/H5, or gB100/2. (C) Class II–expressing gastric epithelial cells (2E12) or IFN-γ–stimulated endothelial cells (BB19, brain; DMVEC, dermal vascular; HPV-AEC, aortic) were infected with AdtetgB or AdtetgI for 24 h or incubated with 10 μg of soluble gB or gE/gI and with CD4+ T cell clone gB10/H5. IFN-γ produced by the CD4+ T cells was measured by ELISA after 24 h.
Figure 3.
Figure 3.
Glial cells internalize soluble gB, and present gB peptides and a soluble TB antigen. (A) His16 cells were incubated with 125I-gB at 4°C, washed, warmed to 37°C, and the cell surface gB was removed with citrate buffer before counting cell-associated (internalized) 125I. Background (cells not warmed to 37°C) was subtracted from each value. (B) His16 cells were incubated with pools of peptides (15 mers overlapping by 10 residues) making up the NH2-terminal (gB-N, residues 1–440) or the COOH-terminal (gB-C, residues 430–907) half of gB or all of UL18, for 6 h before incubation with gB10/G3 CD4+ T cells for 24 h. (C) His16 cells were incubated with medium alone (no Ag) or with 1 μg/ml of soluble mtb39, and TbH9-9 (mtb39-specific) T cells, or were infected with 100 PFUs/cell of AdTbH9 or AdtetgB for 24 h before addition of TbH9-9 T cells. IFN-γ was measured in B and C.
Figure 4.
Figure 4.
Efficiency of presentation of exogenous gB by DCs versus endogenous gB by glial cells. (A) Various numbers of His16 cells or autologous DCs were incubated with 1 μg/ml of soluble gB (sol gB) at the time of adding T cells or were infected with 10 PFUs/cell of AdtetgB for 24 h before adding T cells. 4 × 104 gB100/2 anti-gB CD4+ T cells were used in all cases. (B) His16 cells were infected with various doses of AdtetgB for 24 h before incubation with gB10/G3 or gB10/H5 T cell clones for 24 h. In both cases, IFN-γ was measured.
Figure 5.
Figure 5.
Presentation of endogenous gB occurs in endosomes. (A) His16 cells were treated with 50 μM leupeptin or pepstatin, or 10 μM brefeldin A, bafilomycin, or lactacystin for 30 min, infected with AdtetgB (3 PFUs/cell) for 24 h in the presence of the inhibitors, washed, fixed with 0.1% p-formaldehyde, and then incubated for 24 h with gB100/2 T cells. (B) His16 cells were treated with the indicated concentrations of leupeptin, infected with AdtetgB, and used in T cell assays similar to those in (A), using gB10/H5 and gB100/2 T cell clones. Other His16 cells were incubated with 2 μg/ml soluble mtb39 (sol mtb39) and TbH9-9 (mtb39-specific) T cells. IFN-γ was measured in both cases.
Figure 6.
Figure 6.
Endosomal targeting of endogenous gB is required for presentation. (A) WT gB contains a 135-residue cytosolic domain. gBΔCT is truncated three residues after the transmembrane (TM) domain. gB-KKSL contains the ER retention motif KKSL at the COOH terminus. gB-KKSLAL contains two additional residues that reverse the effects of KKSL. (B) His16 cells were infected with Ad vectors expressing WT gB, gB-KKSL, gB-KKSLAL, or gBΔCT (50 PFUs/cell), and cell surface expression of gB was assessed by FACS. (C) His16 cells were infected with Ad viruses (50 PFUs/cell) for 24 h, fixed, permeabilized, and stained for gB (green) and HLA-DM (red) in the case of WT gB and gBΔCT, or protein disulfide isomerase (red) in the case of gB-KKSL. (D) His16 cells were infected with Ad viruses (1 PFU/cell) expressing WT gB, gBΔCT, gB-KKSL, gB-KKSLAL, or HSV gI for 24 h then incubated with anti-gB CD4+ T cell clones. IFN-γ was measured after 24 h.
Figure 7.
Figure 7.
Presentation of gB in HCMV-infected cells. His16 cells were infected with WT HCMV strain TR or TR-BAC lacking the US2 and US3 genes using 0.5 PFU/cell (A) or 0.05 PFU/cell (B) for 72 h and incubated with anti-gB T cells gB10/G3 or gB10/H5 for 24 h, and IFN-γ was measured.
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