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Transient activation of human cytomegalovirus lytic gene expression during latency allows cytotoxic T cell killing of latently infected cells.

Krishna BA, Lau B, Jackson SE, Wills MR, Sinclair JH, Poole E - Sci Rep (2016)

Bottom Line: Human cytomegalovirus (HCMV) latency in the myeloid lineage is maintained by repressive histone modifications around the major immediate early promoter (MIEP), which results in inhibition of the lytic viral life cycle.We now show that pharmacological inhibition of histone deacetylases (HDACs) relieves this repression of the MIEP and induces transient expression of the viral lytic immediate early (IE) antigens but, importantly, not full virus reactivation.In turn, these latently infected cells now become targets for IE-specific cytotoxic T cells (CTLs) which are present at high frequency in all normal healthy HCMV positive carriers but would normally be unable to target latent (lytic antigen-negative) cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Cambridge, Level 5 Laboratories Block, Addenbrooke's Hospital, Hills Road, Cambridge CB2 0QQ.

ABSTRACT
Human cytomegalovirus (HCMV) latency in the myeloid lineage is maintained by repressive histone modifications around the major immediate early promoter (MIEP), which results in inhibition of the lytic viral life cycle. We now show that pharmacological inhibition of histone deacetylases (HDACs) relieves this repression of the MIEP and induces transient expression of the viral lytic immediate early (IE) antigens but, importantly, not full virus reactivation. In turn, these latently infected cells now become targets for IE-specific cytotoxic T cells (CTLs) which are present at high frequency in all normal healthy HCMV positive carriers but would normally be unable to target latent (lytic antigen-negative) cells. This approach of transiently inducing viral lytic gene expression by HDAC inhibition, in otherwise latently infected cells, offers a window of opportunity to target and purge the latent myeloid cell reservoir by making these normally immunologically undetectable cells visible to pre-existing host immune responses to viral lytic antigens.

No MeSH data available.


Related in: MedlinePlus

Treatment of experimentally latent monocytes with MC1568 or VPA reduces reactivation mediated by IE-specific T cells.Monocytes latently infected with an IE-GFP tagged virus for 5 days were treated with MC1568 and cultured with IE or EBV-specific T cells, following which, monocytes expressing IE-GFP were counted. The graph shows numbers of IE-GFP expressing monocytes relative to IE expression in the absence of T cells (A). Alternatively, 5 day latent monocytes (untreated) were treated with MC1568 or induced to differentiate (differentiated), cultured with IE-specific T cells and analysed by IFN-gamma ELISpot. The graph represents the number of spot forming units (SFUs) above background (B). Additionally, 5 day latent monocytes with (+) or without (−) MC1568 treatment were co-cultured without (no T cells) or with IE-specific T cells or EBV-specific T cells. After this, monocytes were reactivated by differentiation and infectious foci quantified by co-culture on indicator fibroblasts (C). 5 day latent monocytes (latent) monocytes were left untreated (latent) or treated with MC1568 (+MC1568) and co-cultured with IE or EBV-specific T cells, then analysed for CD107a expression. The graph represents fold induction of CD107a expression in IE1-specific T cells after presentation to latently infected cells treated with MC1568 compared to untreated latently infected cells. Values were corrected to CD107a expression following presentation of the same MC1568 treated and untreated latent cells to EBV specific (irrelevant) T cells (D).
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f3: Treatment of experimentally latent monocytes with MC1568 or VPA reduces reactivation mediated by IE-specific T cells.Monocytes latently infected with an IE-GFP tagged virus for 5 days were treated with MC1568 and cultured with IE or EBV-specific T cells, following which, monocytes expressing IE-GFP were counted. The graph shows numbers of IE-GFP expressing monocytes relative to IE expression in the absence of T cells (A). Alternatively, 5 day latent monocytes (untreated) were treated with MC1568 or induced to differentiate (differentiated), cultured with IE-specific T cells and analysed by IFN-gamma ELISpot. The graph represents the number of spot forming units (SFUs) above background (B). Additionally, 5 day latent monocytes with (+) or without (−) MC1568 treatment were co-cultured without (no T cells) or with IE-specific T cells or EBV-specific T cells. After this, monocytes were reactivated by differentiation and infectious foci quantified by co-culture on indicator fibroblasts (C). 5 day latent monocytes (latent) monocytes were left untreated (latent) or treated with MC1568 (+MC1568) and co-cultured with IE or EBV-specific T cells, then analysed for CD107a expression. The graph represents fold induction of CD107a expression in IE1-specific T cells after presentation to latently infected cells treated with MC1568 compared to untreated latently infected cells. Values were corrected to CD107a expression following presentation of the same MC1568 treated and untreated latent cells to EBV specific (irrelevant) T cells (D).

Mentions: Between 0.5–10% of total circulating CD8+ CTLs in healthy HCMV seropositive donors are specific for the viral major IE lytic gene19. However, these IE-specific T cells do not target latently infected (lytic antigen negative) cells; consistent with the fact that latent infection is not cleared despite such high populations of functional IE-specific CTLs. We predicted, however, that treatment of latently infected cells with MC1568 should make them become IE-specific CTL targets. Figure 3A shows that co-culture of MC1568-treated latent monocytes with IE-specific T cell clones, but not irrelevant EBV-specific T cells, resulted in a robust decrease in the number of monocytes positive for IE gene expression. Consistent with the mediation of this effect through T cell effector function, MC1568-treated monocytes also resulted in increased IFN-gamma responses by HCMV-specific T cells to levels comparable to monocytes induced to reactivate by full differentiation (Fig. 3B). Perhaps more importantly, MC1568-treated monocytes, after co-culture with IE-specific CTLs, showed a robust reduction in their ability to reactivate virus after standard differentiation as detected by re-infection of co-cultured indicator fibroblasts (Fig. 3C).


Transient activation of human cytomegalovirus lytic gene expression during latency allows cytotoxic T cell killing of latently infected cells.

Krishna BA, Lau B, Jackson SE, Wills MR, Sinclair JH, Poole E - Sci Rep (2016)

Treatment of experimentally latent monocytes with MC1568 or VPA reduces reactivation mediated by IE-specific T cells.Monocytes latently infected with an IE-GFP tagged virus for 5 days were treated with MC1568 and cultured with IE or EBV-specific T cells, following which, monocytes expressing IE-GFP were counted. The graph shows numbers of IE-GFP expressing monocytes relative to IE expression in the absence of T cells (A). Alternatively, 5 day latent monocytes (untreated) were treated with MC1568 or induced to differentiate (differentiated), cultured with IE-specific T cells and analysed by IFN-gamma ELISpot. The graph represents the number of spot forming units (SFUs) above background (B). Additionally, 5 day latent monocytes with (+) or without (−) MC1568 treatment were co-cultured without (no T cells) or with IE-specific T cells or EBV-specific T cells. After this, monocytes were reactivated by differentiation and infectious foci quantified by co-culture on indicator fibroblasts (C). 5 day latent monocytes (latent) monocytes were left untreated (latent) or treated with MC1568 (+MC1568) and co-cultured with IE or EBV-specific T cells, then analysed for CD107a expression. The graph represents fold induction of CD107a expression in IE1-specific T cells after presentation to latently infected cells treated with MC1568 compared to untreated latently infected cells. Values were corrected to CD107a expression following presentation of the same MC1568 treated and untreated latent cells to EBV specific (irrelevant) T cells (D).
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Related In: Results  -  Collection

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f3: Treatment of experimentally latent monocytes with MC1568 or VPA reduces reactivation mediated by IE-specific T cells.Monocytes latently infected with an IE-GFP tagged virus for 5 days were treated with MC1568 and cultured with IE or EBV-specific T cells, following which, monocytes expressing IE-GFP were counted. The graph shows numbers of IE-GFP expressing monocytes relative to IE expression in the absence of T cells (A). Alternatively, 5 day latent monocytes (untreated) were treated with MC1568 or induced to differentiate (differentiated), cultured with IE-specific T cells and analysed by IFN-gamma ELISpot. The graph represents the number of spot forming units (SFUs) above background (B). Additionally, 5 day latent monocytes with (+) or without (−) MC1568 treatment were co-cultured without (no T cells) or with IE-specific T cells or EBV-specific T cells. After this, monocytes were reactivated by differentiation and infectious foci quantified by co-culture on indicator fibroblasts (C). 5 day latent monocytes (latent) monocytes were left untreated (latent) or treated with MC1568 (+MC1568) and co-cultured with IE or EBV-specific T cells, then analysed for CD107a expression. The graph represents fold induction of CD107a expression in IE1-specific T cells after presentation to latently infected cells treated with MC1568 compared to untreated latently infected cells. Values were corrected to CD107a expression following presentation of the same MC1568 treated and untreated latent cells to EBV specific (irrelevant) T cells (D).
Mentions: Between 0.5–10% of total circulating CD8+ CTLs in healthy HCMV seropositive donors are specific for the viral major IE lytic gene19. However, these IE-specific T cells do not target latently infected (lytic antigen negative) cells; consistent with the fact that latent infection is not cleared despite such high populations of functional IE-specific CTLs. We predicted, however, that treatment of latently infected cells with MC1568 should make them become IE-specific CTL targets. Figure 3A shows that co-culture of MC1568-treated latent monocytes with IE-specific T cell clones, but not irrelevant EBV-specific T cells, resulted in a robust decrease in the number of monocytes positive for IE gene expression. Consistent with the mediation of this effect through T cell effector function, MC1568-treated monocytes also resulted in increased IFN-gamma responses by HCMV-specific T cells to levels comparable to monocytes induced to reactivate by full differentiation (Fig. 3B). Perhaps more importantly, MC1568-treated monocytes, after co-culture with IE-specific CTLs, showed a robust reduction in their ability to reactivate virus after standard differentiation as detected by re-infection of co-cultured indicator fibroblasts (Fig. 3C).

Bottom Line: Human cytomegalovirus (HCMV) latency in the myeloid lineage is maintained by repressive histone modifications around the major immediate early promoter (MIEP), which results in inhibition of the lytic viral life cycle.We now show that pharmacological inhibition of histone deacetylases (HDACs) relieves this repression of the MIEP and induces transient expression of the viral lytic immediate early (IE) antigens but, importantly, not full virus reactivation.In turn, these latently infected cells now become targets for IE-specific cytotoxic T cells (CTLs) which are present at high frequency in all normal healthy HCMV positive carriers but would normally be unable to target latent (lytic antigen-negative) cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Cambridge, Level 5 Laboratories Block, Addenbrooke's Hospital, Hills Road, Cambridge CB2 0QQ.

ABSTRACT
Human cytomegalovirus (HCMV) latency in the myeloid lineage is maintained by repressive histone modifications around the major immediate early promoter (MIEP), which results in inhibition of the lytic viral life cycle. We now show that pharmacological inhibition of histone deacetylases (HDACs) relieves this repression of the MIEP and induces transient expression of the viral lytic immediate early (IE) antigens but, importantly, not full virus reactivation. In turn, these latently infected cells now become targets for IE-specific cytotoxic T cells (CTLs) which are present at high frequency in all normal healthy HCMV positive carriers but would normally be unable to target latent (lytic antigen-negative) cells. This approach of transiently inducing viral lytic gene expression by HDAC inhibition, in otherwise latently infected cells, offers a window of opportunity to target and purge the latent myeloid cell reservoir by making these normally immunologically undetectable cells visible to pre-existing host immune responses to viral lytic antigens.

No MeSH data available.


Related in: MedlinePlus