The HIV-1 envelope protein gp120 is captured and displayed for B cell recognition by SIGN-R1+ lymph node macrophages
- National Institutes of Allergy and Infectious Diseases, United States
Figures
Figure 1 with 3 supplements
SIGN-R1 positive interfollicular channel (IFC) and cortical medullary junction macrophages rapidly accumulate lymph borne gp120.
(A) Confocal microscopy of thick lymph node (LN) sections prepared from mice that had received adoptively transferred B cells (previous day), injected with fluorescently labeled gp120, and …
Figure 1—figure supplement 1
Sorted SIGN-R1+ macrophages capture gp120.
(A) Confocal microscopy image of FACS sorted SIGN-R1+ macrophage 2 hr after sorting. Differential interference contrast (DIC) visualized the cell body and nucleus and was used as a background …
Figure 1—figure supplement 2
The injection of gp120 triggers transcription of interferon- γ in SIGN-R1+ macrophages as assessed by using a interferon-γ-eYFP reporter mouse.
Flow cytometric analysis of cells prepared from the inguinal LNs of interferon-γ-eYFP reporter mice administered gp120 near the tail base at 0, 3 and 6 hr prior to collection. eYFP vs SIGN-R1 …
Figure 1—figure supplement 3
The injection of gp120 triggers transcription of interferon- γ in SIGN-R1+ macrophages.
(A) Flow cytometric analysis of various cellular populations prepared from the inguinal LNs of interferon-γ-eYFP reporter mice administered gp120 near the tail base, 0 (shaded), 3 (shaded, darker) …
Figure 2 with 2 supplements
IFC cell processes bearing gp120 directly contact B cells and a subset of splenic marginal zone cells also bind gp120.
(A) Confocal microscopy of a thick LN section from a mouse previously injected with fluorescent gp120 and immunostained for B220 and CD3. Scale bar is 30 μm. Boxed areas in left image were enlarged …
Figure 2—figure supplement 1
The IFC network macrophages contact CD11c positive cells.
Confocal microscopy of a thick LN section from a mouse previously injected with fluorescent gp120 and immunostained for CD169 and CD11c. The image is centered over an IFC. Scale bar is 30 μm. Boxed …
Figure 2—figure supplement 2
Overlay of gp120 visualizes DC-SIGN+/CD163+ macrophages in a human LN section.
Confocal microscopy of human frozen LN sections overlaid with fluorescent gp120 and immunostained for the indicated markers. (A) Section was stained with CD19 (blue) and CD4 (gray). Scale bar is 400 …
Figure 3
The IFC network of macrophages extends from the subcapsular sinus to the cortical sinus.
(A) Intravital TP-LSM images of the inguinal LN 8 min after injection of fluorescent gp120 and 30 min after CD169. Yellow circles indicate sinus macrophages (upper panel) or network cells in the IFC …
Figure 4
IFC network macrophages do not uptake hen egg lysozyme (HEL) or nitrophenylacetyl (NP)-phycoerythrin (PE).
(A, B) Confocal microscopy of a thick LN section immunostained for CD4, white, following injection of NP-PE, red, fluorescent gp120, green, and fluorescent HEL, blue, near the inguinal LN. Three …
Figure 5 with 2 supplements
IFC network macrophages differentially uptake two different R66M gp120 preparations.
(A) Intravital TP-LSM image of the inguinal lymph following the injection of differentially labeled R66M gp120 expressed in either 293F or CHO-S cells. Scale bars are 100 μm. (B) A z-projection (50 …
Figure 5—figure supplement 1
Deglycosylated gp120 loses its binding specificity to SIGN-R1+ macrophages.
Confocal microscopy of thick LN sections overlaid with fluorescent gp120, which is treated with PNGase F or not, and immunostained for the indicated markers. (A) LN section image (sagittal, tiled) …
Figure 5—figure supplement 2
SIGN-R1+ IFC and cortical medullary junction macrophages rapidly accumulate lymph borne soluble trimeric gp120.
Confocal microscopy of thick LN sections prepared from mice that had been injected with biotinylated soluble trimeric gp120. Biotinylated gp120 was detected by AlexaFluor 488 conjugated …
Figure 6
Recently arrived LN B cells that express the b12 antigen receptor can extract gp120 from IFC network cells.
(A) Flow cytometry to evaluate the binding of labeled R66M gp120 to follicular and marginal zone B cells from either b12 HL or b12 L B cells. Light gray line is background fluorescence. MFI-mean …
Figure 7
LN follicle B cells that express the b12 antigen receptor can extract gp120 from IFC network cells.
(A) Intravital TP-LSM images of b12 HL, red, and WT B, blue, cells in the inguinal LN at 3, 35, and 63 min following injection of fluorescent gp120, green, near the inguinal LN. Blood vessels were …
Figure 8
Individual b12 HL B cell tracks from b12 HL B cells located in the LN follicle near the IFC channel following injection of gp120.
(A) Six tracks, red lines, and displacements, yellow or cyan arrows, of b12 HL B cells were superimposed on 3-D reconstruction image of gp120 loaded IFC network cells, green. 3-D reconstruction …
Videos
Video 1
Intravital two-photon laser scanning microscopy (TP-LSM) images of the interfollicular channel (IFC) network that extends from the subcapsular sinus to the cortical sinus.
Images from two focal planes separated by 30 μm and located over the IFC channel of a mouse inguinal lymph node (LN). The mouse had previously been injected with fluorescent CD169 antibody, red, …
Video 2
Intravital TP-LSM images of the gp120 loaded cellular network in the IFC of the inguinal LN.
An image sequence of a 30 μm z-projection was acquired from a LysM-EGFP mouse, which had previously received by adoptive transfer both B cells, blue, and CD4 T cells, purple. Host endogenous …
Video 3
Intravital TP-LSM images of the dynamic interaction of SIGN-R1+ gp120+ macrophages and a lymphocyte.
An image sequence of a 20 μm z-projection was acquired from the inguinal LN following nearby injection of fluorescent gp120, red, and SIGN-R1, green, antibody (left). Track and displacement (yellow …
Video 4
Intravital TP-LSM images of a newly arriving b12 B cell extracting gp120 from IFC network cells.
An image sequence of a 20 μm z-projection was acquired from the inguinal LN of a mouse, which had fluorescent gp120 (green) delineated IFC network cells. Fluorescently labeled b12 HL B cells (red) …
Video 5
Intravital TP-LSM images of in vivo calcium response of a b12 B cell engaging a gp120 bearing IFC cell.
An image sequence of a 25 μm z-projection was acquired from the inguinal LN of a mouse, which has fluorescent gp120 (green) delineated IFC network cells. b12 HL B cells (blue) were labeled with …
Video 6
Intravital TP-LSM images of resident b12 B cells that extract gp120 from the IFC network cells.
An image sequence of a 20 μm z-projection was acquired from inguinal LN of mouse, which had received by adoptive transfer the previous day b12 HL B cells, red, and wild type B cells, blue. Evans …
