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Murid Gammaherpesvirus Latency-Associated Protein M2 Promotes the Formation of Conjugates between Transformed B Lymphoma Cells and T Helper Cells.

Fontinha D, Lopes FB, Marques S, Simas JP - PLoS ONE (2015)

Bottom Line: Here, we investigate if the MuHV-4 latency associated M2 protein, which assembles multiprotein complexes with B cell signaling proteins, plays a role.Taken together, these findings support that M2 promotes the formation of B-T helper cell conjugates.In an in vivo context this may confer a competitive advantage to the infected B cell in acquisition of T cell help and initiation of a germinal center reaction, hence host colonization.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Medicina Molecular e Instituto de Microbiologia, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal.

ABSTRACT
Establishment of persistent infection in memory B cells by murid herpesvirus-4 (MuHV-4) depends on the proliferation of latently infected germinal center B cells, for which T cell help is essential. Whether the virus is capable of modulating B-T helper cell interaction for its own benefit is still unknown. Here, we investigate if the MuHV-4 latency associated M2 protein, which assembles multiprotein complexes with B cell signaling proteins, plays a role. We observed that M2 led to the upregulation of adhesion and co-stimulatory molecules in transduced B cell lines. In an MHC-II restricted OVA peptide-specific system, M2 polarized to the B-T helper contact zone. Furthermore, it promoted B cell polarization, as demonstrated by the increased proximity of the B cell microtubule organizing center to the interface. Consistent with these data, M2 promoted the formation of B-T helper cell conjugates. In an in vitro competition assay, this translated into a competitive advantage, as T cells preferentially conjugated with M2-expressing B cells. However, expression of M2 alone in B cells was not sufficient to lead to T cell activation, as it only occurred in the presence of specific peptide. Taken together, these findings support that M2 promotes the formation of B-T helper cell conjugates. In an in vivo context this may confer a competitive advantage to the infected B cell in acquisition of T cell help and initiation of a germinal center reaction, hence host colonization.

No MeSH data available.


Related in: MedlinePlus

IFN-γ production in TH cells conjugated with M2-expressing B cells requires specific peptide presentation.A20 B cells stably expressing M2 or M2Y were pulsed overnight, or not, with different concentrations of OVA peptide (OVAp) and incubated with OVAp-specific CD4+ T cells. (A) Extracellular IFN-γ after 20h of incubation. After incubation the supernatant was recovered and analyzed by sandwich ELISA to determine IFN-γ concentration. Supernatants of three independent experiments were tested in duplicate. (B) Upper panel: percentage of T cells producing IFN-γ after 5h of conjugation in the presence of brefeldin A (BFA). Cells were fixed and stained for CD4 with anti-CD4-APC, and for IFN-γ with anti-IFN-γ-PE, and analyzed on a FACS Calibur. Lower panel: IFN-γ mean fluorescence intensity (MFI) of responding T cells. Average of three independent experiments is shown. Statistical significance of the difference between groups was evaluated by a one-tailed unpaired Student’s t test. (C) Representative FACS plots of intracellular IFN-γ production. (D) Representative confocal images of IFN-γ polarization to the contact zone. Prior to incubation B and T cells were labelled with CMFDA (green) and CMAC (blue) live dyes, respectively. Cells were incubated for 2.5h, fixed and stained for IFN-γ (red), and for pTyr (yellow). Images are from one representative experiment out of three and were obtained using a Zeiss LSM 510 META microscope. (E) Quantification of conjugates with IFN-γ polarization per image. Conjugates were evaluated by confocal microscopy based on B-TH cell contact, and IFN-γ polarization (red). 45 to 55 images were acquired per sample from three independent experiments. Only images with a minimum of three T cells were considered for analysis. Statistical significance of the difference between groups was evaluated by a Mann-Whitney U test.
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pone.0142540.g005: IFN-γ production in TH cells conjugated with M2-expressing B cells requires specific peptide presentation.A20 B cells stably expressing M2 or M2Y were pulsed overnight, or not, with different concentrations of OVA peptide (OVAp) and incubated with OVAp-specific CD4+ T cells. (A) Extracellular IFN-γ after 20h of incubation. After incubation the supernatant was recovered and analyzed by sandwich ELISA to determine IFN-γ concentration. Supernatants of three independent experiments were tested in duplicate. (B) Upper panel: percentage of T cells producing IFN-γ after 5h of conjugation in the presence of brefeldin A (BFA). Cells were fixed and stained for CD4 with anti-CD4-APC, and for IFN-γ with anti-IFN-γ-PE, and analyzed on a FACS Calibur. Lower panel: IFN-γ mean fluorescence intensity (MFI) of responding T cells. Average of three independent experiments is shown. Statistical significance of the difference between groups was evaluated by a one-tailed unpaired Student’s t test. (C) Representative FACS plots of intracellular IFN-γ production. (D) Representative confocal images of IFN-γ polarization to the contact zone. Prior to incubation B and T cells were labelled with CMFDA (green) and CMAC (blue) live dyes, respectively. Cells were incubated for 2.5h, fixed and stained for IFN-γ (red), and for pTyr (yellow). Images are from one representative experiment out of three and were obtained using a Zeiss LSM 510 META microscope. (E) Quantification of conjugates with IFN-γ polarization per image. Conjugates were evaluated by confocal microscopy based on B-TH cell contact, and IFN-γ polarization (red). 45 to 55 images were acquired per sample from three independent experiments. Only images with a minimum of three T cells were considered for analysis. Statistical significance of the difference between groups was evaluated by a Mann-Whitney U test.

Mentions: To further investigate if M2 expression in B cells could have an effect in promoting TH cell activation we quantified T cell IFN-γ production and release by ELISA (Fig 5A). After 20h of incubation IFN-γ concentration was measured on culture supernatants by sandwich ELISA. In the absence of peptide we could not detect IFN-γ. For 0.1μM and 1μM of OVAp, M2 expression led to an increase in IFN-γ concentration, compared to the M2Y condition. The IFN-γ production increase observed by ELISA could be due to an increase in production per cell or due to an increase in the number of activated TH cells. To discriminate between these two possibilities, we analyzed IFN-γ production by flow cytometry (Fig 5B and 5C). B and TH cells were incubated in the presence of brefeldin A (BFA) to inhibit protein secretion. As shown in the upper panel of Fig 5B, M2-expressing B cells did not significantly promote IFN-γ production in the absence of peptide. From 0.01μM to 1μM of OVAp concentrations, M2 expression led to an increase in the percentage of TH cells producing IFN-γ, compared to M2Y. Measurement of IFN-γ MFI of responding TH cells showed no significant differences between M2 and M2Y conditions (Fig 5B, lower panel). This can be confirmed in the representative FACS plots (Fig 5C). Therefore, as observed in the calcium measurement experiment, we conclude that the increased IFN-γ production resulted from an increase in the number of activated TH cells, and not from stronger individual responses.


Murid Gammaherpesvirus Latency-Associated Protein M2 Promotes the Formation of Conjugates between Transformed B Lymphoma Cells and T Helper Cells.

Fontinha D, Lopes FB, Marques S, Simas JP - PLoS ONE (2015)

IFN-γ production in TH cells conjugated with M2-expressing B cells requires specific peptide presentation.A20 B cells stably expressing M2 or M2Y were pulsed overnight, or not, with different concentrations of OVA peptide (OVAp) and incubated with OVAp-specific CD4+ T cells. (A) Extracellular IFN-γ after 20h of incubation. After incubation the supernatant was recovered and analyzed by sandwich ELISA to determine IFN-γ concentration. Supernatants of three independent experiments were tested in duplicate. (B) Upper panel: percentage of T cells producing IFN-γ after 5h of conjugation in the presence of brefeldin A (BFA). Cells were fixed and stained for CD4 with anti-CD4-APC, and for IFN-γ with anti-IFN-γ-PE, and analyzed on a FACS Calibur. Lower panel: IFN-γ mean fluorescence intensity (MFI) of responding T cells. Average of three independent experiments is shown. Statistical significance of the difference between groups was evaluated by a one-tailed unpaired Student’s t test. (C) Representative FACS plots of intracellular IFN-γ production. (D) Representative confocal images of IFN-γ polarization to the contact zone. Prior to incubation B and T cells were labelled with CMFDA (green) and CMAC (blue) live dyes, respectively. Cells were incubated for 2.5h, fixed and stained for IFN-γ (red), and for pTyr (yellow). Images are from one representative experiment out of three and were obtained using a Zeiss LSM 510 META microscope. (E) Quantification of conjugates with IFN-γ polarization per image. Conjugates were evaluated by confocal microscopy based on B-TH cell contact, and IFN-γ polarization (red). 45 to 55 images were acquired per sample from three independent experiments. Only images with a minimum of three T cells were considered for analysis. Statistical significance of the difference between groups was evaluated by a Mann-Whitney U test.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4636232&req=5

pone.0142540.g005: IFN-γ production in TH cells conjugated with M2-expressing B cells requires specific peptide presentation.A20 B cells stably expressing M2 or M2Y were pulsed overnight, or not, with different concentrations of OVA peptide (OVAp) and incubated with OVAp-specific CD4+ T cells. (A) Extracellular IFN-γ after 20h of incubation. After incubation the supernatant was recovered and analyzed by sandwich ELISA to determine IFN-γ concentration. Supernatants of three independent experiments were tested in duplicate. (B) Upper panel: percentage of T cells producing IFN-γ after 5h of conjugation in the presence of brefeldin A (BFA). Cells were fixed and stained for CD4 with anti-CD4-APC, and for IFN-γ with anti-IFN-γ-PE, and analyzed on a FACS Calibur. Lower panel: IFN-γ mean fluorescence intensity (MFI) of responding T cells. Average of three independent experiments is shown. Statistical significance of the difference between groups was evaluated by a one-tailed unpaired Student’s t test. (C) Representative FACS plots of intracellular IFN-γ production. (D) Representative confocal images of IFN-γ polarization to the contact zone. Prior to incubation B and T cells were labelled with CMFDA (green) and CMAC (blue) live dyes, respectively. Cells were incubated for 2.5h, fixed and stained for IFN-γ (red), and for pTyr (yellow). Images are from one representative experiment out of three and were obtained using a Zeiss LSM 510 META microscope. (E) Quantification of conjugates with IFN-γ polarization per image. Conjugates were evaluated by confocal microscopy based on B-TH cell contact, and IFN-γ polarization (red). 45 to 55 images were acquired per sample from three independent experiments. Only images with a minimum of three T cells were considered for analysis. Statistical significance of the difference between groups was evaluated by a Mann-Whitney U test.
Mentions: To further investigate if M2 expression in B cells could have an effect in promoting TH cell activation we quantified T cell IFN-γ production and release by ELISA (Fig 5A). After 20h of incubation IFN-γ concentration was measured on culture supernatants by sandwich ELISA. In the absence of peptide we could not detect IFN-γ. For 0.1μM and 1μM of OVAp, M2 expression led to an increase in IFN-γ concentration, compared to the M2Y condition. The IFN-γ production increase observed by ELISA could be due to an increase in production per cell or due to an increase in the number of activated TH cells. To discriminate between these two possibilities, we analyzed IFN-γ production by flow cytometry (Fig 5B and 5C). B and TH cells were incubated in the presence of brefeldin A (BFA) to inhibit protein secretion. As shown in the upper panel of Fig 5B, M2-expressing B cells did not significantly promote IFN-γ production in the absence of peptide. From 0.01μM to 1μM of OVAp concentrations, M2 expression led to an increase in the percentage of TH cells producing IFN-γ, compared to M2Y. Measurement of IFN-γ MFI of responding TH cells showed no significant differences between M2 and M2Y conditions (Fig 5B, lower panel). This can be confirmed in the representative FACS plots (Fig 5C). Therefore, as observed in the calcium measurement experiment, we conclude that the increased IFN-γ production resulted from an increase in the number of activated TH cells, and not from stronger individual responses.

Bottom Line: Here, we investigate if the MuHV-4 latency associated M2 protein, which assembles multiprotein complexes with B cell signaling proteins, plays a role.Taken together, these findings support that M2 promotes the formation of B-T helper cell conjugates.In an in vivo context this may confer a competitive advantage to the infected B cell in acquisition of T cell help and initiation of a germinal center reaction, hence host colonization.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Medicina Molecular e Instituto de Microbiologia, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal.

ABSTRACT
Establishment of persistent infection in memory B cells by murid herpesvirus-4 (MuHV-4) depends on the proliferation of latently infected germinal center B cells, for which T cell help is essential. Whether the virus is capable of modulating B-T helper cell interaction for its own benefit is still unknown. Here, we investigate if the MuHV-4 latency associated M2 protein, which assembles multiprotein complexes with B cell signaling proteins, plays a role. We observed that M2 led to the upregulation of adhesion and co-stimulatory molecules in transduced B cell lines. In an MHC-II restricted OVA peptide-specific system, M2 polarized to the B-T helper contact zone. Furthermore, it promoted B cell polarization, as demonstrated by the increased proximity of the B cell microtubule organizing center to the interface. Consistent with these data, M2 promoted the formation of B-T helper cell conjugates. In an in vitro competition assay, this translated into a competitive advantage, as T cells preferentially conjugated with M2-expressing B cells. However, expression of M2 alone in B cells was not sufficient to lead to T cell activation, as it only occurred in the presence of specific peptide. Taken together, these findings support that M2 promotes the formation of B-T helper cell conjugates. In an in vivo context this may confer a competitive advantage to the infected B cell in acquisition of T cell help and initiation of a germinal center reaction, hence host colonization.

No MeSH data available.


Related in: MedlinePlus