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Clinical Trial
. 2013;9(10):e1003635.
doi: 10.1371/journal.ppat.1003635. Epub 2013 Oct 10.

Human cytomegalovirus latency-associated proteins elicit immune-suppressive IL-10 producing CD4⁺ T cells

Gavin M Mason  1 , Sarah Jackson, Georgina Okecha, Emma Poole, J G Patrick Sissons, John Sinclair, Mark R Wills
Affiliations
Clinical Trial

Human cytomegalovirus latency-associated proteins elicit immune-suppressive IL-10 producing CD4⁺ T cells

Gavin M Mason et al. PLoS Pathog. 2013.

Abstract

Human cytomegalovirus (HCMV) is a widely prevalent human herpesvirus, which, after primary infection, persists in the host for life. In healthy individuals, the virus is well controlled by the HCMV-specific T cell response. A key feature of this persistence, in the face of a normally robust host immune response, is the establishment of viral latency. In contrast to lytic infection, which is characterised by extensive viral gene expression and virus production, long-term latency in cells of the myeloid lineage is characterised by highly restricted expression of viral genes, including UL138 and LUNA. Here we report that both UL138 and LUNA-specific T cells were detectable directly ex vivo in healthy HCMV seropositive subjects and that this response is principally CD4⁺ T cell mediated. These UL138-specific CD4⁺ T cells are able to mediate MHC class II restricted cytotoxicity and, importantly, show IFNγ effector function in the context of both lytic and latent infection. Furthermore, in contrast to CDCD4⁺ T cells specific to antigens expressed solely during lytic infection, both the UL138 and LUNA-specific CD4⁺ T cell responses included CD4⁺ T cells that secreted the immunosuppressive cytokine cIL-10. We also show that cIL-10 expressing CD4⁺ T-cells are directed against latently expressed US28 and UL111A. Taken together, our data show that latency-associated gene products of HCMV generate CD4⁺ T cell responses in vivo, which are able to elicit effector function in response to both lytic and latently infected cells. Importantly and in contrast to CD4⁺ T cell populations, which recognise antigens solely expressed during lytic infection, include a subset of cells that secrete the immunosuppressive cytokine cIL-10. This suggests that HCMV skews the T cell responses to latency-associated antigens to one that is overall suppressive in order to sustain latent carriage in vivo.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Mapping of UL138 specific T cell responses to individual 15 amino acid peptides.
PBMC from 17 HCMV seropositive donors were stimulated with 32 individual 15 amino acid overlapping peptides covering UL138 ORF in a 48γ ELISPOT assay. Post incubation IFNγ spot forming units (SFU) were enumerated and the value from an unstimulated control was subtracted before conversion to SFU/106 cells. Data shown is from the 5 donors who made T cell responses to the complete UL138 ORF peptide pool (Table 1).
Figure 2
Figure 2. Mapping of LUNA specific T cell responses to individual 15 amino acid peptides.
PBMC from 17 HCMV seropositive donors were stimulated with 20 individual 15amino acid overlapping peptides covering the LUNA peptide pool in a 48γ ELISPOT assay. Post incubation IFNγ spot forming units (SFU) were enumerated and the value from an unstimulated control was subtracted before conversion to SFU/106 cells. Data shown is from the 6 donors who made T cell responses to the whole LUNA ORF peptide pool (Table 1).
Figure 3
Figure 3. UL138 and LUNA specific CD4+ T cells are stimulated by HCMV lytic infection of monocyte derived dendritic cells.
Dendritic cells were prepared from donor CMV300 and mock or lytically infected with HCMV strain TB40e at MOI 5. After 5 days lytic infection was confirmed by IE expression using immunofluorescence (A) and RT-PCR (B). Autologous mock or TB40e infected dendritic cells were then co-incubated with in vitro expanded antigen specific CD4+ T cells specific to gB, IE, UL138 or LUNA in IFNγ ELISPOT assays in the presence or absence of cognate peptide (C). Post incubation IFNγ spot forming units (SFU/106) were enumerated and the back ground level of IFNγ production for each antigen specificity determined from the mock infected no peptide control (Red dotted line).
Figure 4
Figure 4. UL138 specific CD4+ T cells secrete IFNγ in response to latently infected monocytes.
Monocytes were prepared from donor CMV305 and mock infected or latently infected with TB40e for 10 days at MOI 5. Latent infection was then confirmed by RT-PCR (A). Autologous mock or latently infected monocytes were then co-incubated with in vitro expanded antigen specific CD4+ T cells specific to gB (B) and UL138 (C) in IFNγ ELISPOT assays in the presence or absence of cognate peptide. Post incubation IFNγ spot forming units (SFU/106) were enumerated and the back ground level of IFNγ production for each antigen specificity determined from the mock infected no peptide control (Red dotted line). Error bars are standard error of the mean (n = 5). Statistical analysis were performed using the students t test (* p<0.05;** p<0.01).
Figure 5
Figure 5. CD4+ T cells specific to UL138, but not LUNA can mediate MHC class II restricted cytotoxicity.
Antigen specific CD4+ T cells specific to gB (A), UL138 (B) and LUNA (C) from donor CMV300 were expanded in vitro and incubated with autologous LCL in the presence or absence of cognate peptide at a range of effector to target cell ratios (E∶T) in 6 hour chromium release assays. Percent specific lysis was calculated to determine cytotoxic effector potential of the antigen specific T cells. Values >10% were deemed positive. Error bars are standard error of the mean (n = 6). The LUNA specific T cell line used in (C) was tested for specificity in IFNγ ELISPOT assays (D). Error bars represent standard error of the mean (n = 3). Statistical analysis was performed using the students t test (** p<0.01).
Figure 6
Figure 6. In vitro expanded UL138 and LUNA specific CD4+ T cells secrete IFNγ and IL-10.
CD4+ T cells from donors CMV300 and CMV305 specific to gB and UL138 were expanded in vitro, LUNA specific cells were also expanded from donor CMV300. Antigen specific CD4+ T cells were then incubated with autologous LCL in the presence or absence of peptide and supernatants removed and assayed for 11 cytokines by multi-analyte cytokine assays (A). Supernatants from the same experiment were also used in TGFβ ELISA (B). gB, UL138 and LUNA specific CD4+ T cells were then incubated with autologous irradiated PBMC in the presence and absence of peptide in ELISPOT assays detecting IFNγ (C) and IL-10 (D) including the mitogen PHA as a positive control. Error bars are standard error of the mean (n = 5).
Figure 7
Figure 7. Supernatant from PBMC stimulated with UL138 suppresses CD4+ T cell proliferation.
PBMC were stimulated with either UL138 or gB peptides for 48(control). PBMC were then stimulated or remained unstimulated (Negative) with anti-CD3/CD28 beads for 5 days and where then stained with anti-CD3 and anti-CD4 antibodies, dead cells excluded by the use of 7-amino-actinomycin D. Cells were gated according to (A) and the proliferation of CD4+ T cells was analysed by flow cytometry (B). The same analysis was performed on three individual donors (C). Error bars are standard error of the mean (n = 3). Statistical analysis was performed using the students t test (p<0.01). The same analysis was performed on PBMC from four separate donors and prior to the addition of PBMC supernatants were treated with neutralising anti-IL-10 and/or anti-TGFβ antibodies or isotype control antibodies and proliferation assays performed and analysed as previously (D). Error bars are standard error of the mean (n = 4). Statistical analysis was performed using the students t test (* p<0.05; ** p<0.01).
Figure 8
Figure 8. A subset of UL138 specific CD4+ T cells express phenotypic hallmarks of Treg cells.
CD4+ T cells specific to UL138 (A) or gB (B) from 3 donors were expanded in vitro for 14 days and then stained for stable surface expression of CD25 (C). Numbers in the top right quadrant represent percentage of CD4+ CD25hi cells (A and B). Error bars represent standard error of the mean (n = 3); Statistical analysis was performed using the students t test (C). PBMC were stained directly ex vivo for surface expression of CD25 and intracellular expression of FoxP3 or using an isotype control (D). Quadrant numbers represent percentage of CD4+ cells. In vitro expanded cells from two donors, specific to UL138 and gB (CMV305) or UL138, LUNA and gB (CMV300) were stained for expression of CD4, CD25 and FoxP3 (E). CD4+ CD25hi cells were analysed for expression of FoxP3.
Figure 9
Figure 9. UL138, LUNA, US28 and UL111A specific cells secrete IL-10 and IFNγ directly ex vivo.
PBMC from 13 HCMV seropositive donors were prepared and used in parallel ELISPOT assays detecting IFNγ (black), IL-10 (white), IL-4 (blue) and IL-17 (red). PBMC were stimulated with PHA (A) or peptide pools spanning HCMV open reading frames IE, gB, UL138, LUNA, US28 or UL111A (B). Post incubation spot forming units (SFU) were enumerated for each cytokine response and the proportion of the total T cell response calculated as a percentage. All samples performed in triplicate, data shown is the mean (Error bars not shown).
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References

    1. Staras SA, Dollard SC, Radford KW, Flanders WD, Pass RF, et al. (2006) Seroprevalence of cytomegalovirus infection in the United States, 1988–1994. Clin Infect Dis 43: 1143–1151. - PubMed
    1. Crough T, Khanna R (2009) Immunobiology of human cytomegalovirus: from bench to bedside. Clin Microbiol Rev 22: 76–98 Table of Contents. - PMC - PubMed
    1. Jackson SE, Mason GM, Wills MR (2011) Human cytomegalovirus immunity and immune evasion. Virus Research 157: 151–160. - PubMed
    1. Wills MR, Carmichael AJ, Sissons JG, editors(2006) Adaptive cellular immunity to human cytomegalovirus. 1st ed. Wymondham: Caister Academic press. 25 p.
    1. Einsele H, Roosnek E, Rufer N, Sinzger C, Riegler S, et al. (2002) Infusion of cytomegalovirus (CMV)-specific T cells for the treatment of CMV infection not responding to antiviral chemotherapy. Blood 99: 3916–3922. - PubMed

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