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The HIV-1 envelope protein gp120 is captured and displayed for B cell recognition by SIGN-R1(+) lymph node macrophages.

Park C, Arthos J, Cicala C, Kehrl JH - Elife (2015)

Bottom Line: In contrast, two other antigens, phycoerythrin and hen egg lysozyme, were not captured by these cells.Intravital imaging of mouse LNs revealed persistent, but transient interactions between gp120 bearing interfollicular network cells and both trafficking and LN follicle resident gp120 specific B cells.Our findings reveal a specialized LN antigen delivery system poised to deliver gp120 and likely other pathogen derived glycoproteins to B cells.

View Article: PubMed Central - PubMed

Affiliation: B-cell Molecular Immunology Section, Laboratory of Immunoregulation, National Institutes of Allergy and Infectious Diseases, Bethesda, United States.

ABSTRACT
The HIV-1 envelope protein gp120 is both the target of neutralizing antibodies and a major focus of vaccine efforts; however how it is delivered to B cells to elicit an antibody response is unknown. Here, we show that following local gp120 injection lymph node (LN) SIGN-R1(+) sinus macrophages located in interfollicular pockets and underlying SIGN-R1(+) macrophages form a cellular network that rapidly captures gp120 from the afferent lymph. In contrast, two other antigens, phycoerythrin and hen egg lysozyme, were not captured by these cells. Intravital imaging of mouse LNs revealed persistent, but transient interactions between gp120 bearing interfollicular network cells and both trafficking and LN follicle resident gp120 specific B cells. The gp120 specific, but not the control B cells repetitively extracted gp120 from the network cells. Our findings reveal a specialized LN antigen delivery system poised to deliver gp120 and likely other pathogen derived glycoproteins to B cells.

No MeSH data available.


The injection of gp120 triggers transcription of interferon- γin SIGN-R1+ macrophages as assessed by using ainterferon-γ-eYFP reporter mouse.Flow cytometric analysis of cells prepared from the inguinal LNs ofinterferon-γ-eYFP reporter mice administered gp120 near the tailbase at 0, 3 and 6 hr prior to collection. eYFP vs SIGN-R1 expression incell located in gates a, b and c are shown via a contour plot. WT micewere used as a negative control for eYFP expression in each of thegates.DOI:http://dx.doi.org/10.7554/eLife.06467.005
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fig1s2: The injection of gp120 triggers transcription of interferon- γin SIGN-R1+ macrophages as assessed by using ainterferon-γ-eYFP reporter mouse.Flow cytometric analysis of cells prepared from the inguinal LNs ofinterferon-γ-eYFP reporter mice administered gp120 near the tailbase at 0, 3 and 6 hr prior to collection. eYFP vs SIGN-R1 expression incell located in gates a, b and c are shown via a contour plot. WT micewere used as a negative control for eYFP expression in each of thegates.DOI:http://dx.doi.org/10.7554/eLife.06467.005

Mentions: For these studies we used an early HIV-1 viral isolate subtype A/C gp120, R66M,expressed in 293F cells (Nawaz et al.,2011), and injected 1 μg of fluorescently labeled gp120 near the baseof the mouse tail. Confocal microscopy of thick LN sections prepared 2 hr after gp120injection revealed that labeled gp120 had been captured by LN macrophages thatoverlie and extend into IFCs and that localize at the cortical medullary junction(Figure 1A, top and middle panels). Theasymmetric gp120 signal results from the gp120 accessing the afferent lymphaticsserving the left side of the inguinal LN as it is oriented in the figure. Furtherimmunostaining revealed that gp120 co-localized with SIGN-R1 (Figure 1A, bottom panel), a c-type lectin and functionalortholog of DC-specific ICAM-3-grabbing non-integrin (DC-SIGN), which has beenimplicated in HIV-1 transmission by human DCs (Geijtenbeek et al., 2000; Kang et al.,2003). Intravital TP-LSM revealed the rapid appearance of gp120 in thesubcapsular sinus and identified the same subset of SIGN-R1+macrophages capturing gp120 (Figure 1B).Together the imaging and flow cytometry identified the gp120 binding cells asSIGN-R1+CD169midCD11bmidCD4+/CD11c−F4/80lowsinus macrophages (SIGN-R1+ subcapsular macrophages) andSIGN-R1+/CD169midCD11blowCD4+CD11c−F4/80lowIFM (SIGN-R1+ IFC macrophages) (Figure 1C). These cells are to be distinguished from theSIGN-R1+CD11b+ DCs located in the medullaryregion, previously identified to uptake inactivated influenza virus (Gonzalez et al., 2010). TheSIGN-R1+ DCs also bound gp120 and are likely important for Tcell priming (Figure 1D). Next, weinvestigated the role of SIGN-R1 in gp120 binding. To do this we first checkedwhether gp120 bound in vitro to LNSIGN-R1+CD169midCD11b+ cells andwhether unlabeled gp120 competitively inhibited the binding. We found that gp120bound a phenotypically similar subset of macrophages and that unlabeled gp120 reducedthe binding of the labeled material (Figure1E). When we added a SIGN-R1 blocking antibody with the labeled gp120, thelevel of SIGN-R1 on the LNSIGN-R1+CD169midCD11b+ cellsdeclined as did the fluorescent gp120 binding arguing that SIGN-R1 directlyparticipated in the binding (Figure 1E). Thepercentage of cells that bound gp120 declined by approximately 50% in the presence ofthe SIGN-R1 antibody. To directly visualize these cells in vitro, we sortedGr-1−CD11c−SIGN-R1+CD11b+CD169+cells from mice previously injected with fluorescent gp120. The sorted cells wereimaged (Figure 1—figure supplement1). Because of the rarity of the cells in the LN population the sorted cellswere contaminated with other cell types yet manygp120+SIGN-R1+ cells could be visualized. Wealso cultured the sorted cells with M-CSF. At day 7 the cultured cells were incubatedwith fluorescent gp120 and immunostained for SIGN-R1. The majority of the culturedcells retained SIGN-R1 expression and most of these cells bound gp120 (Figure 1—figure supplement 1). Todetermine whether the uptake of gp120 triggered a biologic response in the IFCmacrophages we injected gp120 locally and checked the intracellularinterferon-γ levels in these cells (Figure1F). Some of the LNSIGN-R1+CD169midCD11b+ cells had anelevated level of intracellular interferon-γ compared to control cells. Weverified these results using an interferon-γ eYFP reporter mouse (Reinhardt et al., 2009). Flow cytometry wasused to assess the percent of eYFP positive cells in the gatedSIGN-R1+ macrophages (Figure1—figure supplement 2), and to examine the induction of eYFPexpression in various other cell populations in the immunized LN (Figure 1—figure supplement 3). To verifythat the eYFP signal arose from the SIGN-R1+ macrophages we sortedtheGr-1−CD11c−SIGN-R1+CD11b+CD169+cells and imaged them. We could readily identifySIGN-R1+eYFP+ cells, while the othercontaminating cells present in the sorted population lacked YFP expression. Finally,we injected non-labeled gp120 near the inguinal lymph of the reporter mouse and 6 hrlater made thick LN sections from the draining LN node and from a distant LN.Confocal microscopy revealed eYFP positiveSIGN-R1+CD169mid cells in the IFC region of thedraining LN, but similar cells were not present in the distant LN (Figure1—figure supplement 3). Togetherthese results identified a subset of mouse subcapsular macrophages that overlie andreside in the IFC, which express SIGN-R1 and rapidly uptake gp120. In addition, ourdata indicates that the local injection of gp120 likely elicits interferon-γproduction by these cells.10.7554/eLife.06467.003Figure 1.SIGN-R1 positive interfollicular channel (IFC) and cortical medullaryjunction macrophages rapidly accumulate lymph borne gp120.


The HIV-1 envelope protein gp120 is captured and displayed for B cell recognition by SIGN-R1(+) lymph node macrophages.

Park C, Arthos J, Cicala C, Kehrl JH - Elife (2015)

The injection of gp120 triggers transcription of interferon- γin SIGN-R1+ macrophages as assessed by using ainterferon-γ-eYFP reporter mouse.Flow cytometric analysis of cells prepared from the inguinal LNs ofinterferon-γ-eYFP reporter mice administered gp120 near the tailbase at 0, 3 and 6 hr prior to collection. eYFP vs SIGN-R1 expression incell located in gates a, b and c are shown via a contour plot. WT micewere used as a negative control for eYFP expression in each of thegates.DOI:http://dx.doi.org/10.7554/eLife.06467.005
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Related In: Results  -  Collection

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fig1s2: The injection of gp120 triggers transcription of interferon- γin SIGN-R1+ macrophages as assessed by using ainterferon-γ-eYFP reporter mouse.Flow cytometric analysis of cells prepared from the inguinal LNs ofinterferon-γ-eYFP reporter mice administered gp120 near the tailbase at 0, 3 and 6 hr prior to collection. eYFP vs SIGN-R1 expression incell located in gates a, b and c are shown via a contour plot. WT micewere used as a negative control for eYFP expression in each of thegates.DOI:http://dx.doi.org/10.7554/eLife.06467.005
Mentions: For these studies we used an early HIV-1 viral isolate subtype A/C gp120, R66M,expressed in 293F cells (Nawaz et al.,2011), and injected 1 μg of fluorescently labeled gp120 near the baseof the mouse tail. Confocal microscopy of thick LN sections prepared 2 hr after gp120injection revealed that labeled gp120 had been captured by LN macrophages thatoverlie and extend into IFCs and that localize at the cortical medullary junction(Figure 1A, top and middle panels). Theasymmetric gp120 signal results from the gp120 accessing the afferent lymphaticsserving the left side of the inguinal LN as it is oriented in the figure. Furtherimmunostaining revealed that gp120 co-localized with SIGN-R1 (Figure 1A, bottom panel), a c-type lectin and functionalortholog of DC-specific ICAM-3-grabbing non-integrin (DC-SIGN), which has beenimplicated in HIV-1 transmission by human DCs (Geijtenbeek et al., 2000; Kang et al.,2003). Intravital TP-LSM revealed the rapid appearance of gp120 in thesubcapsular sinus and identified the same subset of SIGN-R1+macrophages capturing gp120 (Figure 1B).Together the imaging and flow cytometry identified the gp120 binding cells asSIGN-R1+CD169midCD11bmidCD4+/CD11c−F4/80lowsinus macrophages (SIGN-R1+ subcapsular macrophages) andSIGN-R1+/CD169midCD11blowCD4+CD11c−F4/80lowIFM (SIGN-R1+ IFC macrophages) (Figure 1C). These cells are to be distinguished from theSIGN-R1+CD11b+ DCs located in the medullaryregion, previously identified to uptake inactivated influenza virus (Gonzalez et al., 2010). TheSIGN-R1+ DCs also bound gp120 and are likely important for Tcell priming (Figure 1D). Next, weinvestigated the role of SIGN-R1 in gp120 binding. To do this we first checkedwhether gp120 bound in vitro to LNSIGN-R1+CD169midCD11b+ cells andwhether unlabeled gp120 competitively inhibited the binding. We found that gp120bound a phenotypically similar subset of macrophages and that unlabeled gp120 reducedthe binding of the labeled material (Figure1E). When we added a SIGN-R1 blocking antibody with the labeled gp120, thelevel of SIGN-R1 on the LNSIGN-R1+CD169midCD11b+ cellsdeclined as did the fluorescent gp120 binding arguing that SIGN-R1 directlyparticipated in the binding (Figure 1E). Thepercentage of cells that bound gp120 declined by approximately 50% in the presence ofthe SIGN-R1 antibody. To directly visualize these cells in vitro, we sortedGr-1−CD11c−SIGN-R1+CD11b+CD169+cells from mice previously injected with fluorescent gp120. The sorted cells wereimaged (Figure 1—figure supplement1). Because of the rarity of the cells in the LN population the sorted cellswere contaminated with other cell types yet manygp120+SIGN-R1+ cells could be visualized. Wealso cultured the sorted cells with M-CSF. At day 7 the cultured cells were incubatedwith fluorescent gp120 and immunostained for SIGN-R1. The majority of the culturedcells retained SIGN-R1 expression and most of these cells bound gp120 (Figure 1—figure supplement 1). Todetermine whether the uptake of gp120 triggered a biologic response in the IFCmacrophages we injected gp120 locally and checked the intracellularinterferon-γ levels in these cells (Figure1F). Some of the LNSIGN-R1+CD169midCD11b+ cells had anelevated level of intracellular interferon-γ compared to control cells. Weverified these results using an interferon-γ eYFP reporter mouse (Reinhardt et al., 2009). Flow cytometry wasused to assess the percent of eYFP positive cells in the gatedSIGN-R1+ macrophages (Figure1—figure supplement 2), and to examine the induction of eYFPexpression in various other cell populations in the immunized LN (Figure 1—figure supplement 3). To verifythat the eYFP signal arose from the SIGN-R1+ macrophages we sortedtheGr-1−CD11c−SIGN-R1+CD11b+CD169+cells and imaged them. We could readily identifySIGN-R1+eYFP+ cells, while the othercontaminating cells present in the sorted population lacked YFP expression. Finally,we injected non-labeled gp120 near the inguinal lymph of the reporter mouse and 6 hrlater made thick LN sections from the draining LN node and from a distant LN.Confocal microscopy revealed eYFP positiveSIGN-R1+CD169mid cells in the IFC region of thedraining LN, but similar cells were not present in the distant LN (Figure1—figure supplement 3). Togetherthese results identified a subset of mouse subcapsular macrophages that overlie andreside in the IFC, which express SIGN-R1 and rapidly uptake gp120. In addition, ourdata indicates that the local injection of gp120 likely elicits interferon-γproduction by these cells.10.7554/eLife.06467.003Figure 1.SIGN-R1 positive interfollicular channel (IFC) and cortical medullaryjunction macrophages rapidly accumulate lymph borne gp120.

Bottom Line: In contrast, two other antigens, phycoerythrin and hen egg lysozyme, were not captured by these cells.Intravital imaging of mouse LNs revealed persistent, but transient interactions between gp120 bearing interfollicular network cells and both trafficking and LN follicle resident gp120 specific B cells.Our findings reveal a specialized LN antigen delivery system poised to deliver gp120 and likely other pathogen derived glycoproteins to B cells.

View Article: PubMed Central - PubMed

Affiliation: B-cell Molecular Immunology Section, Laboratory of Immunoregulation, National Institutes of Allergy and Infectious Diseases, Bethesda, United States.

ABSTRACT
The HIV-1 envelope protein gp120 is both the target of neutralizing antibodies and a major focus of vaccine efforts; however how it is delivered to B cells to elicit an antibody response is unknown. Here, we show that following local gp120 injection lymph node (LN) SIGN-R1(+) sinus macrophages located in interfollicular pockets and underlying SIGN-R1(+) macrophages form a cellular network that rapidly captures gp120 from the afferent lymph. In contrast, two other antigens, phycoerythrin and hen egg lysozyme, were not captured by these cells. Intravital imaging of mouse LNs revealed persistent, but transient interactions between gp120 bearing interfollicular network cells and both trafficking and LN follicle resident gp120 specific B cells. The gp120 specific, but not the control B cells repetitively extracted gp120 from the network cells. Our findings reveal a specialized LN antigen delivery system poised to deliver gp120 and likely other pathogen derived glycoproteins to B cells.

No MeSH data available.