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Expansion of murine gammaherpesvirus latently infected B cells requires T follicular help.

Collins CM, Speck SH - PLoS Pathog. (2014)

Bottom Line: X linked lymphoproliferative disease (XLP) is an inherited immunodeficiency resulting from mutations in the gene encoding the slam associated protein (SAP).This failure to proliferate resulted in significantly lower viral loads, and likely accounts for the inability of MHV68 to induce a FIM-like syndrome.This requirement for CD4 T cell help during the germinal center reaction by MHV68 is in contrast with EBV, which is thought to be capable of bypassing this requirement by expressing viral proteins that mimic signals provided by TFH cells.

View Article: PubMed Central - PubMed

Affiliation: Emory Vaccine Center and Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia, United States of America.

ABSTRACT
X linked lymphoproliferative disease (XLP) is an inherited immunodeficiency resulting from mutations in the gene encoding the slam associated protein (SAP). One of the defining characteristics of XLP is extreme susceptibility to infection with Epstein-Barr virus (EBV), a gammaherpesvirus belonging to the genus Lymphocryptovirus, often resulting in fatal infectious mononucleosis (FIM). However, infection of SAP deficient mice with the related Murine gammaherpesvirus 68 (MHV68), a gammaherpesvirus in the genus Rhadinovirus, does not recapitulate XLP. Here we show that MHV68 inefficiently establishes latency in B cells in SAP deficient mice due to insufficient CD4 T cell help during the germinal center response. Although MHV68 infected B cells can be found in SAP-deficient mice, significantly fewer of these cells had a germinal center phenotype compared to SAP-sufficient mice. Furthermore, we show that infected germinal center B cells in SAP-deficient mice fail to proliferate. This failure to proliferate resulted in significantly lower viral loads, and likely accounts for the inability of MHV68 to induce a FIM-like syndrome. Finally, inhibiting differentiation of T follicular helper (TFH) cells in SAP-sufficient C57Bl/6 mice resulted in decreased B cell latency, and the magnitude of the TFH response directly correlated with the level of infection in B cells. This requirement for CD4 T cell help during the germinal center reaction by MHV68 is in contrast with EBV, which is thought to be capable of bypassing this requirement by expressing viral proteins that mimic signals provided by TFH cells. In conclusion, the outcome of MHV68 infection in mice in the setting of loss of SAP function is distinct from that observed in SAP-deficient patients infected with EBV, and may identify a fundamental difference between the strategies employed by the rhadinoviruses and lymphocryptoviruses to expand B cell latency during the early phase of infection.

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Reduced germinal center response in SAP-deficient mice.Mice were infected with either 1,000(low dose) or 4×105 pfu (high dose) of MHV68-H2bYFP, and splenocytes were harvest at day 16–18 post-infection. (A) Representative flow cytometry plots showing defective germinal center B cell formation in SAP-deficient mice. Flow plots were gated on CD3−, B220+ cells, and germinal center B cells were defined as CD95HI, GL7HI. (B) Quantitation of the percentage of total B cells that had a germinal center phenotype. Each symbol represents a single infected mouse. ****, p<0.0001. (C) Representative flow plots showing the percentage of infected B cells that had a GC phenotype. Flow plots were gated on CD3−, B220+, H2bYFP+ cells to identify infected B cells, and cells with a GC phenotype were defined as CD95HI, GL7HI. (D) Quantitation of the percentage of infected B cells that have a GC phenotype. Each symbol represents a single infected mouse. ****, p<0.0001. (E) Percentage of CD3−, B220+, H2bYFP+ cells that express surface IgD after infection with 1,000 pfu of virus and harvested a 16 days post-infection. Each symbol represents a single infected mouse. ***, p = 0.0004. Results in panels B and D were compiled from either 4 (low dose infections) or 2 (high dose infections) independent experiments with 3 to 5 mice per group. Results in panel E were compiled from 2 independent experiments with 4 to 5 mice per group. Each symbol represents a single animal, and the horizontal bar represents the mean.
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ppat-1004106-g002: Reduced germinal center response in SAP-deficient mice.Mice were infected with either 1,000(low dose) or 4×105 pfu (high dose) of MHV68-H2bYFP, and splenocytes were harvest at day 16–18 post-infection. (A) Representative flow cytometry plots showing defective germinal center B cell formation in SAP-deficient mice. Flow plots were gated on CD3−, B220+ cells, and germinal center B cells were defined as CD95HI, GL7HI. (B) Quantitation of the percentage of total B cells that had a germinal center phenotype. Each symbol represents a single infected mouse. ****, p<0.0001. (C) Representative flow plots showing the percentage of infected B cells that had a GC phenotype. Flow plots were gated on CD3−, B220+, H2bYFP+ cells to identify infected B cells, and cells with a GC phenotype were defined as CD95HI, GL7HI. (D) Quantitation of the percentage of infected B cells that have a GC phenotype. Each symbol represents a single infected mouse. ****, p<0.0001. (E) Percentage of CD3−, B220+, H2bYFP+ cells that express surface IgD after infection with 1,000 pfu of virus and harvested a 16 days post-infection. Each symbol represents a single infected mouse. ***, p = 0.0004. Results in panels B and D were compiled from either 4 (low dose infections) or 2 (high dose infections) independent experiments with 3 to 5 mice per group. Results in panel E were compiled from 2 independent experiments with 4 to 5 mice per group. Each symbol represents a single animal, and the horizontal bar represents the mean.

Mentions: We next performed flow cytometric analysis of splenocytes to compare the GC B cell populations in SAP-deficient mice with that seen in wild type mice. As expected, SAP-deficient mice exhibited a severe defect in generating a GC response following MHV68 infection (Fig. 2A,B). Interestingly, analysis of the H2bYFP-positive B cell population revealed that although SAP-deficient mice were severely compromised in their ability to generate GC B cells, a significant fraction of infected B cells exhibited a GC phenotype (Fig. 2C,D). However, the overall percentage of infected B cells that had a GC phenotype in SAP-deficient mice was significantly lower than that seen in wild type mice. Since the inability of SAP-deficient mice to generate a GC response has been shown to be due to inefficient CD4 T cell help [37], this data suggests that MHV68 is at least partially dependent on CD4 T cells for differentiating to a GC phenotype. Consistent with this hypothesis, there was a significantly higher percentage of naïve, IgD+ B cells in the H2bYFP positive B cell fraction in SAP-deficient mice (Fig. 2E).


Expansion of murine gammaherpesvirus latently infected B cells requires T follicular help.

Collins CM, Speck SH - PLoS Pathog. (2014)

Reduced germinal center response in SAP-deficient mice.Mice were infected with either 1,000(low dose) or 4×105 pfu (high dose) of MHV68-H2bYFP, and splenocytes were harvest at day 16–18 post-infection. (A) Representative flow cytometry plots showing defective germinal center B cell formation in SAP-deficient mice. Flow plots were gated on CD3−, B220+ cells, and germinal center B cells were defined as CD95HI, GL7HI. (B) Quantitation of the percentage of total B cells that had a germinal center phenotype. Each symbol represents a single infected mouse. ****, p<0.0001. (C) Representative flow plots showing the percentage of infected B cells that had a GC phenotype. Flow plots were gated on CD3−, B220+, H2bYFP+ cells to identify infected B cells, and cells with a GC phenotype were defined as CD95HI, GL7HI. (D) Quantitation of the percentage of infected B cells that have a GC phenotype. Each symbol represents a single infected mouse. ****, p<0.0001. (E) Percentage of CD3−, B220+, H2bYFP+ cells that express surface IgD after infection with 1,000 pfu of virus and harvested a 16 days post-infection. Each symbol represents a single infected mouse. ***, p = 0.0004. Results in panels B and D were compiled from either 4 (low dose infections) or 2 (high dose infections) independent experiments with 3 to 5 mice per group. Results in panel E were compiled from 2 independent experiments with 4 to 5 mice per group. Each symbol represents a single animal, and the horizontal bar represents the mean.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4006913&req=5

ppat-1004106-g002: Reduced germinal center response in SAP-deficient mice.Mice were infected with either 1,000(low dose) or 4×105 pfu (high dose) of MHV68-H2bYFP, and splenocytes were harvest at day 16–18 post-infection. (A) Representative flow cytometry plots showing defective germinal center B cell formation in SAP-deficient mice. Flow plots were gated on CD3−, B220+ cells, and germinal center B cells were defined as CD95HI, GL7HI. (B) Quantitation of the percentage of total B cells that had a germinal center phenotype. Each symbol represents a single infected mouse. ****, p<0.0001. (C) Representative flow plots showing the percentage of infected B cells that had a GC phenotype. Flow plots were gated on CD3−, B220+, H2bYFP+ cells to identify infected B cells, and cells with a GC phenotype were defined as CD95HI, GL7HI. (D) Quantitation of the percentage of infected B cells that have a GC phenotype. Each symbol represents a single infected mouse. ****, p<0.0001. (E) Percentage of CD3−, B220+, H2bYFP+ cells that express surface IgD after infection with 1,000 pfu of virus and harvested a 16 days post-infection. Each symbol represents a single infected mouse. ***, p = 0.0004. Results in panels B and D were compiled from either 4 (low dose infections) or 2 (high dose infections) independent experiments with 3 to 5 mice per group. Results in panel E were compiled from 2 independent experiments with 4 to 5 mice per group. Each symbol represents a single animal, and the horizontal bar represents the mean.
Mentions: We next performed flow cytometric analysis of splenocytes to compare the GC B cell populations in SAP-deficient mice with that seen in wild type mice. As expected, SAP-deficient mice exhibited a severe defect in generating a GC response following MHV68 infection (Fig. 2A,B). Interestingly, analysis of the H2bYFP-positive B cell population revealed that although SAP-deficient mice were severely compromised in their ability to generate GC B cells, a significant fraction of infected B cells exhibited a GC phenotype (Fig. 2C,D). However, the overall percentage of infected B cells that had a GC phenotype in SAP-deficient mice was significantly lower than that seen in wild type mice. Since the inability of SAP-deficient mice to generate a GC response has been shown to be due to inefficient CD4 T cell help [37], this data suggests that MHV68 is at least partially dependent on CD4 T cells for differentiating to a GC phenotype. Consistent with this hypothesis, there was a significantly higher percentage of naïve, IgD+ B cells in the H2bYFP positive B cell fraction in SAP-deficient mice (Fig. 2E).

Bottom Line: X linked lymphoproliferative disease (XLP) is an inherited immunodeficiency resulting from mutations in the gene encoding the slam associated protein (SAP).This failure to proliferate resulted in significantly lower viral loads, and likely accounts for the inability of MHV68 to induce a FIM-like syndrome.This requirement for CD4 T cell help during the germinal center reaction by MHV68 is in contrast with EBV, which is thought to be capable of bypassing this requirement by expressing viral proteins that mimic signals provided by TFH cells.

View Article: PubMed Central - PubMed

Affiliation: Emory Vaccine Center and Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia, United States of America.

ABSTRACT
X linked lymphoproliferative disease (XLP) is an inherited immunodeficiency resulting from mutations in the gene encoding the slam associated protein (SAP). One of the defining characteristics of XLP is extreme susceptibility to infection with Epstein-Barr virus (EBV), a gammaherpesvirus belonging to the genus Lymphocryptovirus, often resulting in fatal infectious mononucleosis (FIM). However, infection of SAP deficient mice with the related Murine gammaherpesvirus 68 (MHV68), a gammaherpesvirus in the genus Rhadinovirus, does not recapitulate XLP. Here we show that MHV68 inefficiently establishes latency in B cells in SAP deficient mice due to insufficient CD4 T cell help during the germinal center response. Although MHV68 infected B cells can be found in SAP-deficient mice, significantly fewer of these cells had a germinal center phenotype compared to SAP-sufficient mice. Furthermore, we show that infected germinal center B cells in SAP-deficient mice fail to proliferate. This failure to proliferate resulted in significantly lower viral loads, and likely accounts for the inability of MHV68 to induce a FIM-like syndrome. Finally, inhibiting differentiation of T follicular helper (TFH) cells in SAP-sufficient C57Bl/6 mice resulted in decreased B cell latency, and the magnitude of the TFH response directly correlated with the level of infection in B cells. This requirement for CD4 T cell help during the germinal center reaction by MHV68 is in contrast with EBV, which is thought to be capable of bypassing this requirement by expressing viral proteins that mimic signals provided by TFH cells. In conclusion, the outcome of MHV68 infection in mice in the setting of loss of SAP function is distinct from that observed in SAP-deficient patients infected with EBV, and may identify a fundamental difference between the strategies employed by the rhadinoviruses and lymphocryptoviruses to expand B cell latency during the early phase of infection.

Show MeSH
Related in: MedlinePlus