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. 2010 Aug;91(Pt 8):2040-2048.
doi: 10.1099/vir.0.020982-0. Epub 2010 Apr 7.

CD8+ T-cell recognition of human cytomegalovirus latency-associated determinant pUL138

Siok-Keen Tey  1   2 , Felicia Goodrum  3 , Rajiv Khanna  2
Affiliations

CD8+ T-cell recognition of human cytomegalovirus latency-associated determinant pUL138

Siok-Keen Tey et al. J Gen Virol. 2010 Aug.

Abstract

Recent studies have shown that long-term persistence of human cytomegalovirus (HCMV) in mononuclear cells of myeloid lineage is dependent on the UL138 open reading frame, which promotes latent infection. Although T-cell recognition of protein antigens from all stages of lytic HCMV infection is well established, it is not clear whether proteins expressed during latent HCMV infection can also be recognized. This study conducted an analysis of T-cell response towards proteins associated with HCMV latency. Ex vivo analysis of T cells from healthy virus carriers revealed a dominant CD8(+) T-cell response to the latency-associated pUL138 protein, which recognized a non-canonical 13 aa epitope in association with HLA-B*3501. These pUL138-specific T cells displayed a range of memory phenotypes that were in general less differentiated than that previously described in T cells specific for HCMV lytic antigens. Antigen-presentation assays revealed that endogenous pUL138 could be presented efficiently by HCMV-infected cells. However, T-cell recognition of pUL138 was dependent on newly synthesized protein, with little presentation from stable, long-lived protein. These data demonstrate that T cells targeting latency-associated protein products exist, although HCMV may limit the presentation of latent proteins, thereby restricting T-cell recognition of latently infected cells.

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Figures

Fig. 1
Fig. 1
CD8+ T-cell recognition of HCMV-encoded latency determinant pUL138. (a) PBMCs from healthy virus carriers were stimulated with Ad5f35.UL138 and expanded for 10 days, and T-cell specificity was assessed in a 6 h intracellular cytokine-secretion assay. The results show the CD8+ T-cell responses to the pUL138 20mer peptide MDDLPLNVGLPIIGVMLVLI in four responders (N01, N02, N06 and N11) and a representative non-responder (N13). (b) Epitope minimization. pUL138-specific T-cell lines expanded from two donors were tested against peptides of different lengths. The minimum epitope was a 13mer non-canonical sequence, LPLNVGLPIIGVM. (c) Stabilization of surface HLA-B35 molecule on T2.B*3501 cells by LPLNVGLPIIGVM peptide. Results from three independent experiments are shown (mean±sem); the P value was calculated using a paired, two-tailed Student’s t-test.
Fig. 2
Fig. 2
Ex vivo characterization of pUL138-specific T cells from healthy virus carriers. (a, b) Fresh PBMCs were stained with anti-CD8, anti-CD27 and anti-CD57 antibody and HLA-B*3501–LPL tetramers. Flow cytometry dot plots gated on CD8+ T cells are shown. (a) Tetramer staining from three representative HLA-B35+ HCMV-seropositive donors and two HLA-B35+ HCMV-seronegative controls. (b) Expression of CD27, CD57, CD45RA and CD45RO in pUL138 and lytic antigen-specific T cells from two representative donors, N02 (i) and N11 (ii). (c) Expression of T-cell memory markers from five HLA-B35+ HCMV-seropositive individuals. T cells were gated by tetramer staining and had the following specificities: pUL138 (n = 5), pp65 (n = 4) and IE1 (n = 2). Results are shown as means±sem; the P value was calculated using an unpaired, two-tailed Student’s t-test. (d) Flow cytometry dot plots from a 6 h intracellular cytokine-secretion assay performed on PMBCs ex vivo from donor N06.
Fig. 3
Fig. 3
Endogenous processing and presentation of pUL138. (a) HLA-B35+ monocytes or primary fibroblasts were infected with Ad5f35.UL138 or HCMV strain Toledo. At 16 h p.i., these cells were used to stimulate pUL138-specific T-cell lines in intracellular cytokine-secretion assays. Results are shown as representative flow cytometry dot plots from one of three independent experiments. (b) Presentation of lytic HCMV antigens by monocytes (HLA-A1+ and -B35+) 16 h after infection with HCMV strain Toledo. Flow cytometry dot plots were gated on HLA-A*0101–VTE (pp50) or HLA-B*3501–IPS (pp65) pentamer-positive T-cell populations. (c) Time course of presentation of pUL138, pp50 and pp65 CD8+ T-cell epitopes by monocytes (HLA-A1+ and -B35+) after infection with HCMV strain Toledo. Results are shown as T-cell responses to HCMV-infected monocytes after subtracting the responses to non-infected monocytes. Results from two independent experiments are shown (mean±sem).
Fig. 4
Fig. 4
Kinetics of pUL138 expression and antigen presentation. (a) Immunoblot of primary lung fibroblasts (MRC5) infected with HCMV strain Toledo. The graph shows the intensity of pUL138 bands measured by densitometry and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. (b) IFN-γ response of the pUL138-specific T-cell line to HCMV-infected primary fibroblasts (HLA-A1+ and -B35+). The response to pp50, an early–late antigen, was used for comparison. The response was assessed simultaneously by co-staining with IFN-γ (FITC), HLA-B*3501–LPL tetramer (APC) and HLA-A*0101–VTE pentamer (PE). ▪, pUL138-specific T-cell response to HCMV-infected fibroblasts; □, pUL138-specific T-cell response to uninfected fibroblasts; ▾, pp50-specific T-cell response to HCMV-infected fibroblasts; ▿, pp50-specific T-cell response to uninfected fibroblasts. Results are shown as means±sd (n = 2). (c) Effect of MHC–peptide stripping (acid) and cycloheximide treatment on pUL138 antigen presentation and T-cell recognition. T-cell response was normalized against peptide-pulsed controls. Results are shown as means±sem from three independent experiments. **P<0.05 compared with untreated controls; the P value was calculated using a paired, two-tailed Student’s t-test.
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