A dominant role for the immunoproteasome in CD8+ T cell responses to murine cytomegalovirus

Abstract

Murine cytomegalovirus (MCMV) is an important animal model of human cytomegalovirus (HCMV), a β-Herpesvirus that infects the majority of the world's population and causes disease in neonates and immunocompromised adults. CD8(+) T cells are a major part of the immune response to MCMV and HCMV. Processing of peptides for presentation to CD8(+) T cells may be critically dependent on the immunoproteasome, expression of which is affected by MCMV. However, the overall importance of the immunoproteasome in the generation of immunodominant peptides from MCMV is not known. We therefore examined the role of the immunoproteasome in stimulation of CD8(+) T cell responses to MCMV - both conventional memory responses and those undergoing long-term expansion or "inflation". We infected LMP7(-/-) and C57BL/6 mice with MCMV or with newly-generated recombinant vaccinia viruses (rVVs) encoding the immunodominant MCMV protein M45 in either full-length or epitope-only minigene form. We analysed CD8(+) T cell responses using intracellular cytokine stain (ICS) and MHC Class I tetramer staining for a panel of MCMV-derived epitopes. We showed a critical role for immunoproteasome in MCMV affecting all epitopes studied. Interestingly we found that memory "inflating" epitopes demonstrate reduced immunoproteasome dependence compared to non-inflating epitopes. M45-specific responses induced by rVVs remain immunoproteasome-dependent. These results help to define a critical restriction point for CD8(+) T cell epitopes in natural cytomegalovirus (CMV) infection and potentially in vaccine strategies against this and other viruses.

Figure 1. CD8⁺ T cell responses to M45 in LMP7^−/− mice infected with MCMV.

A. LMP7^−/− mice and C57BL/6 control mice were injected intravenously (i.v.) with 1×10⁶ plaque forming units (pfu) MCMV or an equivalent volume of phosphate buffered saline (PBS). Seven days p.i. peripheral blood was sampled by tail bleed. Whole blood was stained with fluorochrome-conjugated soluble tetrameric complexes (M45 H-2 D^{b 985}HGIRNASFI⁹⁹³) and anti-CD8 antibody, and red blood cells lysed, before analysis by FACS. B. Lymphocytes were prepared from peripheral blood and spleen of LMP7^−/− and C57BL/6 control mice 7 days p.i. (n = 5) or PBS treated (blood: n = 2; spleen: n = 4) and subjected to tetramer stain and FACS analysis as above. The plots show the frequency of M45 D^b-specific cells among CD8⁺ T cells. SEM was less than 0.1%. C. Splenocytes were prepared from spleens of LMP7^−/− and C57BL/6 control mice (n = 5) 7 days p.i. and incubated in either media containing synthetic peptide HGIRNASFI or media alone, in the presence of Brefeldin A (BFA). After 5 hours cells were stained for CD8 and intracellular IFN-γ and analysed by FACS. The plots show % CD8⁺ T cells producing IFN-γ in response to peptide or media alone. SEM was less than 0.1%. D. Peripheral blood was sampled from LMP7^−/− and C57BL/6 control mice (n = 3) at 7, 40, 60 and 100 days p.i. and subjected to tetramer stains using M45 D^b HGIRNASFI tetramer and FACS analysis as for Figure 1A. The plot shows the frequency of M45 D^b- specific cells among CD8⁺ T cells over a 100 day time course.

Figure 2. CD8⁺ T cell responses to m141 in LMP7^−/− mice infected with MCMV.

A. Lymphocytes were prepared from peripheral blood of LMP7^−/− and C57BL/6 control mice 7 days p.i. (n = 3) or PBS treated (n = 2) and subjected to tetramer stain and FACS analysis as for Figure 1A, using MCMV m141 tetramer (H-2 K^{b 15}VIDASFRL²³). The plot shows the frequency of m141 K^b-specific cells among CD8⁺ T cells. B. Splenocytes were prepared from spleens of LMP7^−/− and C57BL/6 control mice 7 days p.i. (n = 6), pooled, and incubated in the presence of BFA with either media containing peptide VIDAFSRL or media alone for 5 hours. Cells were stained as in Figure 1C and analysed by FACS. The plot shows % CD8⁺ T cells producing IFN-γ in response to peptide or media alone. C. Peripheral blood was sampled from LMP7^−/− and C57BL/6 control mice (n = 3) at 7, 40, 60 and 100 days p.i. and subjected to tetramer stain and FACS analysis as for A. The plot shows the frequency of m141 K^b-specific cells among CD8⁺ T cells over a 100 day time course.

Figure 3. CD8⁺ T cell response to inflating epitope m139 in LMP7^−/− mice infected with MCMV.

A. Lymphocytes were prepared from the peripheral blood of LMP7^−/− and C57BL/6 control mice 7 days p.i. (n = 3) or PBS (n = 2) and subjected to tetramer stain and FACS analysis as for Figure 1A, using MCMV m139 tetramer (H-2 K^{b 419}TWYGFCLL⁴²⁶). This epitope has been shown to elicit a CD8⁺ T cell response that increases over time, or inflates, in C57BL/6 mice. The plot shows the frequency of m139 K^b-specific cells among CD8⁺ T cells over a 100 day time course. B. Splenocytes were prepared from spleens of LMP7^−/− and C57BL/6 control mice 7 days p.i. (n = 3) and incubated in the presence of BFA with either media containing synthetic peptide TWYGFCLL or media alone for 5 hours. Cells were stained for surface CD8 and intracellular IFN-γ as in Figure 1C and analysed by FACS. The plot shows % CD8⁺ T cells producing IFN-γ in response to peptide or media alone.

Figure 4. CD8⁺ T cell responses to inflating epitope M38 in LMP7^−/− mice infected with MCMV.

A. Splenocytes were prepared from spleens of LMP7^−/− and C57BL/6 control mice 7 days p.i.(n = 6), pooled, and incubated in the presence of BFA with either media containing peptide SSPPMFRV (M38 H-2 K^{b 316}SSPPMFRV³²⁵) or media alone for 5 hours. Cells were stained for surface CD8 and intracellular IFN-γ as in Figure 1C and analyzed by FACS. The plots show % CD8⁺ T cells producing IFN-γ in response to peptide or media alone. B. Splenocytes were prepared from spleens of LMP7^−/− and C57BL/6 control mice 100 days p.i. (n = 3) and subjected to IFN-γ intracellular cytokine stain (IFN-γ ICCS) as for A.

Figure 5. Breadth of CD8⁺ T cell responses to MCMV in LMP7^−/− mice.

A. Splenocytes were prepared from spleens of LMP7^−/− and C57BL/6 control mice 7 days p.i. (n = 5), pooled, and exposed to each of nineteen synthetic peptides corresponding to defined CD8 epitopes of MCMV (Table S1), or media alone, in the presence of BFA for 5 hours. Splenocytes were subjected to CD8 surface stain, IFN-γ ICCS and FACS analysis as for Figure 1C. Frequency of IFN-γ cells among CD8⁺ T cells in infected C57BL/6 or LMP7^−/− mice, 7 days p.i. is shown for each peptide. Data is shown with background subtracted for both mouse strains, only for responses that in C57BL/6 mice were above background. Background is defined as three times the sum of the mean and standard error of the mean response to stimulation with media control alone. B. A similar analysis was to that described in Fig 5A was performed for CD8⁺ T cell responses detectable at 100 days p.i. C. Example ICCS staining for C57BL/6 and LMP7^−/− mice (day 100) in response to M45, m139 and M38 peptides.

Figure 6. CD8⁺ T cell responses to M45 in LMP7^−/− mice infected with M45-expressing rVV.

A. Recombinant vaccinia viruses (rVVs) were constructed containing foreign genes derived from the genomic sequence of MCMV Smith Strain (ATCC VR-194). Foreign genes were full length gene encoding the MCMV protein M45 (M45-VV), or a ‘minigene’, encoding the antigenic fragment of M45 corresponding to the CD8 epitope M45 H-2 D^{b 985}HGIRNASFI⁹⁹³ (M45 epitope-VV). Genes were designed for insertion between restriction sites for restriction endonucleases Apa I (5’) and Kpn I (3’). The diagram shows recombinant genes and proteins encoded by the two rVVs used in this study: M45 VV encoding full-length M45; and M45 epitope-VV, encoding the minimal D^b-restricted CD8 epitope of M45 only. B, C. LMP7^−/− or C57BL/6 control mice were injected i.v. with 1×10⁶ pfu M45-VV (B), 1×10⁶ pfu M45 epitope-VV (C) or an equivalent volume of PBS. Blood was sampled 7 days p.i. by tail bleed. Peripheral blood lymphocytes were subjected to tetramer stain with M45 D^b HGIRNSFI tetramer and FACS analysis as for Figure 1A. The frequency of cells specific for M45 H-2 D^{b 985}HGIRNASFI⁹⁹³ among CD8⁺ T cells in the blood of MVacc and MGVacc - infected and mock-infected C57BL/6 and LMP7^−/− mice, 7 days after infection, is shown.

Figure 7. “2-cell” model to explain different effect of LMP7 on “inflating” memory and classical memory CD8⁺ T cell responses to MCMV.

Infected or cross-presenting DCs during acute infection contain immunoproteasomes and constitutive proteasomes and stimulate responses to epitopes produced by either proteasome. In chronic infection other APCs containing only constitutive proteasomes stimulate responses only to those epitopes produced by the constitutive proteasome resulting in selection of more immunoproteasome-independent responses during chronic infection.