Fig 1.
Visualisation of perifollicular, red pulp and white pulp capillaries centered on three different follicles.
(a,b,c) ROI 1 (a) first section stained for CD34, (b) overlay of all 24 registered sections, (c) first frame of video showing a 3D model with four connections among perifollicular and red pulp capillaries highlighted in different colours. (d,e,f) ROI 2 (d) first section stained for CD34, (e) overlay of all 24 registered sections, (f) first frame of video showing a 3D model with one connection among perifollicular and red pulp capillaries highlighted in red colour. (g,h,i) ROI 3 (g) first section stained for CD34, (h) overlay of all 24 registered sections, (i) first frame of video showing a 3D model with five connections among perifollicular and red pulp capillaries highlighted in different colours. (g,h,i) show two follicles (f in h) and a part of a PALS (asterisk in h) in between. Internal capillaries arise from a central artery in the PALS. Staining of red pulp sinuses has been reduced as documented in suppl. Fig 2 in c,f,i. CD34 is present in capillary endothelia, adventitial fibroblasts of arteries, perifollicular sinus endothelia (weak) and in fibroblasts in trabeculae. All scale bars = 100 μm.
Fig 2.
Visualisation of CD271+ sheaths around red pulp capillaries connected to the perifollicular network of the three follicles.
(a,b,c) ROI 1(a) first section stained for CD34 (brown) and for CD 271 (blue), (b) overlay of all 24 registered sections, (c) first frame of video of the 3D model(d,e,f) ROI 2(d) first section stained for CD34 (brown) and for CD 271 (blue), (e) overlay of all 24 registered sections, (f) first frame of video of the 3D model(g,h,i) ROI 3(g) first section stained for CD34 (brown) and for CD 271 (blue), (h) overlay of all 24 registered sections, (i) first frame of video of the 3D modelCD271 is most strongly expressed in stromal capillary sheath cells. FDCs in follicles, FRCs in a PALS and ubiquitous interstitial fibroblasts are also positive. Staining of red pulp sinuses for CD34 and of ubiquitous fibroblasts for CD271 has been reduced by choosing an appropriate iso-value.(g), (h) and (i) show two follicles (f in h) and a part of a PALS in between. Internal capillaries arise from a central artery in the PALS (asterisk in h).All scale bars = 100 μm.
Fig 3.
Visualisation of capillary networks centered on PALS and follicles.
Distribution of CD34 in an overlay of 24 registered sections of ROI 4 showing a PALS (P) with a branching central artery and two sectioned follicles (f). There is a clear difference in the localisation of the perifollicular capillary network (double arrows) and the perifollicular sinus network (arrows). Internal capillaries of the PALS appear to run in the adventitia of the central artery forming vasa vasorum. Scale bar = 100 μm.
Fig 4.
Localisation of the perifollicular capillary network in relation to stromal cells, B memory cells and plasmablasts.
(a) Double staining for CD34 (brown) and SMA (blue) in a paraffin section. Strongly CD34+ capillary endothelia form a shell around the follicle. Sinus endothelia are weakly stained. A capillary (arrow) in the outer MZ forms a connection to red pulp. Same individual as Figs 1 and 2.(b) Double staining for CD271 (brown) and MAdCAM-1 (blue) in a cryosection. All FDCs are CD271+. MAdCAM-1+ perifollicular MRCs define the MZ and harbour capillaries surrounded by light brown fibroblasts. CD271+ capillary sheaths occur superficially in the MZ. Same individual as Figs 1 and 2.(c) Double staining for CD34 (blue) and CD141 (red) in a paraffin section. CD141 demonstrates venous sinuses in the red pulp and MRCs. Arrow indicates the open end of a perifollicular capillary. Same individual as Figs 1 and 2.(d) Double staining for CD34 (black) and CD27 (brown) in a paraffin section. The CD34+ capillary network is located in a superficial area of the follicle with scattered CD27+ B-cells. Double arrow shows perifollicular capillaries, arrow indicates sinus. Same individual as Figs 1 and 2.(e) Double staining for CD4+ T-cells (brown) and CD271+ FDCs and fibroblasts (blue) in a paraffin section. CD4+ T-cells originating from a PALS (upper part of image) occupy the CD271- area underlying the perifollicular capillary network accompanied by CD271+ fibroblasts. Same individual as Figs 1 and 2.(f) Double staining for Ki-67 (blue) and intracellular IgM (brown) in a paraffin section. A germinal centre is located in the lower right part of the image. IgM+ plasmablasts with blue Ki-67+ nuclei (arrow) are located at the surface of the unstained follicle.Scale bar for all figures = 100 μm.
Fig 5.
Overview of the section stained for CD34 showing localisation of ROI 1–4.
The length of the cut edges is about 1 cm.
Fig 6.
Localisation of a potential barrier towards the open splenic circulation at the follicular surface.
(a) Hemalum-Eosin-stained paraffin section of a typical secondary follicle in an adult human spleen. Arrow indicates a layer of thick fibres and fibroblasts potentially forming a barrier against the open circulation. Same individual as Figs 1 and 2. (b) Double staining for CD3+ T-cells (blue-black) and CD27+ B-cells (brown) in a paraffin section. Arrow indicates fibres and fibroblasts outside the most strongly CD27+ B-cells potentially forming a barrier against the open circulation. Scale bars = 100 μm.
Fig 7.
Schematic drawing visualising stromal cells, capillaries and the location of CD27+ B-lymphocytes at the surface of a secondary follicle in a human spleen.
SMA+MAdCAM-1+ MRCs are depicted in red colour, SMA-MAdCAM-1+ MRCs in violet and CD271+ FDCs in blue. The innermost FDCs (thick blue lines) belong to the germinal centre. Perifollicular capillaries are black and the putative size barrier (formed by the red cells) is light brown. Gray background colour represents the area with CD27+ memory B-cells and blue background shows the follicular mantle zone. Note that the gray area overlaps the outermost blue FDCs and thus the mantle zone. There is no border between the mantle zone and the area of CD27+ B-cells. The follicle-associated T-cell area is not depicted.