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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 naturally latent monocytes with MC1568 or VPA reduces reactivation.Monocytes from PBMCs of HCMV seropositive individuals were isolated by plastic adherence. Adherent monocytes were left untreated (no treatment, columns a and b), differentiated to reactivate virus (differentiated, columns c and d), treated with MC1568, (columns e and f) and cultured in the presence (+) or absence (−) of the residual PBMCs (after monocyte depletion). Additionally, wells of MC1568-treated monocytes, after removal of T cells, were also subsequently induced to differentiate (columns g and h). In all cases, any reactivated virus was quantified by fibroblast co-culture and staining for IE positive foci (A). Finally, CD14+ monocytes from PBMCs of two HCMV seropositive individuals were isolated, adhered to plastic and left untreated (none), treated with MC1568 or VPA for 24 h. The PBMCs left after CD14+ isolation were either left untreated (CD14+ depleted PBMC) or further depleted of T cells by CD3+ positive selection (CD14+/CD3+ depleted PBMCs). Subsequently, PBMCs were removed by washing and cells were differentiated and any reactivated virus was quantified by fibroblast co-culture and staining for IE foci (B). Standard deviations are shown and statistical significance was determined using the Student’s t-test.
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f4: Treatment of naturally latent monocytes with MC1568 or VPA reduces reactivation.Monocytes from PBMCs of HCMV seropositive individuals were isolated by plastic adherence. Adherent monocytes were left untreated (no treatment, columns a and b), differentiated to reactivate virus (differentiated, columns c and d), treated with MC1568, (columns e and f) and cultured in the presence (+) or absence (−) of the residual PBMCs (after monocyte depletion). Additionally, wells of MC1568-treated monocytes, after removal of T cells, were also subsequently induced to differentiate (columns g and h). In all cases, any reactivated virus was quantified by fibroblast co-culture and staining for IE positive foci (A). Finally, CD14+ monocytes from PBMCs of two HCMV seropositive individuals were isolated, adhered to plastic and left untreated (none), treated with MC1568 or VPA for 24 h. The PBMCs left after CD14+ isolation were either left untreated (CD14+ depleted PBMC) or further depleted of T cells by CD3+ positive selection (CD14+/CD3+ depleted PBMCs). Subsequently, PBMCs were removed by washing and cells were differentiated and any reactivated virus was quantified by fibroblast co-culture and staining for IE foci (B). Standard deviations are shown and statistical significance was determined using the Student’s t-test.

Mentions: However, a key question is whether drug treatment permits naturally latently infected cells in the peripheral blood of healthy donors to become targetable by blood resident HCMV-specific CTLs. To address this we carried out analyses on total peripheral blood mononuclear cells (PBMC) from two seropositive individuals to allow assessment of the effectiveness of MC1568 in clearing naturally latent HCMV from the peripheral blood. Figure 4A shows that naturally latent cells do not produce infectious virus (column a), as expected (shown by a lack of infectious foci on indicator fibroblasts). Again, as expected, latent cells induced to differentiate, reactivate infectious virus (column c) but this reactivation is ablated in the presence of T cells (column d). This argues that the T cells in the PBMC compartment are able to detect differentiation-dependent virus reactivation. Consistent with the fact that MC1568 treatment does not induce full virus reactivation in latently infected monocytes, HDACi treatment showed no virus production regardless of whether they were cultured with or without autologous T cell-containing PBMCs (columns e and f). However, autologous T cell-containing PBMCs completely ablate the ability of these transiently IE1-expressing latent cells to reactivate endogenous latent virus after their subsequent differentiation (columns g and h). Repeating the analysis using T cell-depleted PBMCs confirmed that the T cell population in these PBMCs were effecting this reduction in latent cells capable of reactivating infectious virus (Fig. 4B).


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 naturally latent monocytes with MC1568 or VPA reduces reactivation.Monocytes from PBMCs of HCMV seropositive individuals were isolated by plastic adherence. Adherent monocytes were left untreated (no treatment, columns a and b), differentiated to reactivate virus (differentiated, columns c and d), treated with MC1568, (columns e and f) and cultured in the presence (+) or absence (−) of the residual PBMCs (after monocyte depletion). Additionally, wells of MC1568-treated monocytes, after removal of T cells, were also subsequently induced to differentiate (columns g and h). In all cases, any reactivated virus was quantified by fibroblast co-culture and staining for IE positive foci (A). Finally, CD14+ monocytes from PBMCs of two HCMV seropositive individuals were isolated, adhered to plastic and left untreated (none), treated with MC1568 or VPA for 24 h. The PBMCs left after CD14+ isolation were either left untreated (CD14+ depleted PBMC) or further depleted of T cells by CD3+ positive selection (CD14+/CD3+ depleted PBMCs). Subsequently, PBMCs were removed by washing and cells were differentiated and any reactivated virus was quantified by fibroblast co-culture and staining for IE foci (B). Standard deviations are shown and statistical significance was determined using the Student’s t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835774&req=5

f4: Treatment of naturally latent monocytes with MC1568 or VPA reduces reactivation.Monocytes from PBMCs of HCMV seropositive individuals were isolated by plastic adherence. Adherent monocytes were left untreated (no treatment, columns a and b), differentiated to reactivate virus (differentiated, columns c and d), treated with MC1568, (columns e and f) and cultured in the presence (+) or absence (−) of the residual PBMCs (after monocyte depletion). Additionally, wells of MC1568-treated monocytes, after removal of T cells, were also subsequently induced to differentiate (columns g and h). In all cases, any reactivated virus was quantified by fibroblast co-culture and staining for IE positive foci (A). Finally, CD14+ monocytes from PBMCs of two HCMV seropositive individuals were isolated, adhered to plastic and left untreated (none), treated with MC1568 or VPA for 24 h. The PBMCs left after CD14+ isolation were either left untreated (CD14+ depleted PBMC) or further depleted of T cells by CD3+ positive selection (CD14+/CD3+ depleted PBMCs). Subsequently, PBMCs were removed by washing and cells were differentiated and any reactivated virus was quantified by fibroblast co-culture and staining for IE foci (B). Standard deviations are shown and statistical significance was determined using the Student’s t-test.
Mentions: However, a key question is whether drug treatment permits naturally latently infected cells in the peripheral blood of healthy donors to become targetable by blood resident HCMV-specific CTLs. To address this we carried out analyses on total peripheral blood mononuclear cells (PBMC) from two seropositive individuals to allow assessment of the effectiveness of MC1568 in clearing naturally latent HCMV from the peripheral blood. Figure 4A shows that naturally latent cells do not produce infectious virus (column a), as expected (shown by a lack of infectious foci on indicator fibroblasts). Again, as expected, latent cells induced to differentiate, reactivate infectious virus (column c) but this reactivation is ablated in the presence of T cells (column d). This argues that the T cells in the PBMC compartment are able to detect differentiation-dependent virus reactivation. Consistent with the fact that MC1568 treatment does not induce full virus reactivation in latently infected monocytes, HDACi treatment showed no virus production regardless of whether they were cultured with or without autologous T cell-containing PBMCs (columns e and f). However, autologous T cell-containing PBMCs completely ablate the ability of these transiently IE1-expressing latent cells to reactivate endogenous latent virus after their subsequent differentiation (columns g and h). Repeating the analysis using T cell-depleted PBMCs confirmed that the T cell population in these PBMCs were effecting this reduction in latent cells capable of reactivating infectious virus (Fig. 4B).

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