Figure S1.
Ig kappa light chain staining identifies plasma cells. (A) Bone
marrow sections from a Blimp-1-GFP reporter mouse were stained for CD41+
megakaryocytes (green) and Ig kappa light chain (red). GFP is visualized in blue. The
white arrow indicates a Blimp-1+/kappa light chain+ plasma cell in contact with a
megakaryocyte. (B) Bone marrow plasma cells express much higher levels of Ig
kappa light chains than other cells in this tissue. Single cell suspensions were stained
for CD138 and intracellular Ig kappa light chains and analyzed by flow cytometry.
A
B
Figure S2.
Individual plasma cells are scattered throughout the bone marrow.
(A) Plasma cells are visualized on a cross section of a murine femur by staining for Ig
kappa light chain (green). Vasculature is labelled by an anti-endothelial antibody
(clone B78, blue). White lines indicate the interface between marrow and bone. (B)
Femur sections stained for Ig kappa (green) and Ova (red) from (I) a non-immunized
mouse (left), (II) a mouse immunized with Ova (middle) and (III) a mouse immunized
with Ova, sample pre-treated with a 10-fold excess of unlabeled Ova (right). White
bars indicate 150 μm. Ova+/Ig kappa+ plasma cells (middle section, arrows) were not
detectable on sections from unimmunized mice (left) or immunized mice when the
staining was blocked with unlabelled Ova (right)
A
B
C
Figure S3. Identification of megakaryocytes and Ova-specific plasma cells (A)
Flow cytometric profile of bone marrow megakaryocytes. Dead cells and debris are
excluded by forward scatter (FSC) and DAPI staining from bone marrow single cell
suspensions (upper right and left). Cells are stained for CD11b, CD41 and CD61 as
indicated (lower panels). (B) Flow cytometric profiles of splenic and bone marrow
Ova-specific plasma cells.
Cell suspensions are fixed and stained for CD138.
Intracellular staining with Ova and anti-Ig-kappa is performed after permeabilization
with saponin. Debris are excluded by FSC (not shown). Representative data from
spleen and bone marrow are shown. (C) Contacts of Ova-specific plasma cells and
megakaryocytes per bone marrow section area as analyzed by confocal microscopy
at day six, nine and 130 after secondary immunization with Ova. Please note that the
reduction of contacts per section area over time results from the drop of Ova-specific
plasma cell numbers from their peak at day 6 after immunization to basal levels later
while the percentage of Ova-specific plasma cells in contact to a megakaryocyte
remains stable over time (Figure 2).
A
B
C
Figure S4.
APRIL and IL-6 staining by megakaryocytes: Controls and
comparison to CD11b+ cells. (A) Isotype controls. Bone marrow sections stained
for CD41 (green) and APRIL, IL-6 or controls (red), as indicated. Of note, the isotype
control for IL-6 (i.e. anti-IL-4) stains some bone marrow cells, but not CD41+
megakaryocytes. (B) Bone marrow sections from APRIL -/- or wt mice stained for
CD41 (green) and APRIL (red) as indicated (upper panels). Bone marrow sections
from IL-6 -/- or wt mice stained for CD41 (green) and IL-6 (red) as indicated (lower
panels). (C) Comparison of IL-6 expression by megakaryocytes with that of CD11b+
cells. While all megakaryocytes express constitutively IL-6, only a few CD11b+ cells
were detected expressing this cytokine.
Figure S5.
Splenic plasma cell counts in c-mpl -/- mice. Wild type and c-mpl -/-
mice were immunized and boosted with NP-KLH. NP-specific IgG1-ASC in spleen
were quantified by ELISPOT at days 5 and 21 after immunization. Means + standard
deviations are shown for groups of three to five mice (statistics: unpaired t-test).
Figure S6.
Kinetics of TPO-induced megakaryopoiesis. Total megakaryocyte
numbers in spleen and bone marrow (left panels). Different megakaryocyte ploidy
stages (PI staining) on day four, eight and ten after initial TPO-injection in bone
marrow (right panels) were analyzed by flow cytometry.
Figure S7.
Effects of TPO-injection on bone marrow cell populations.
The following bone marrow populations were investigated in TPO-treated and control
mice by FACS: population I: Lin-/c-kit+/Sca-1+ primitive hematopoietic cells;
population II: Lin-/CD150+/CD41-/CD48- hematopoietic stem cells; population III:
B220+ total B cells; population IV: CD11b+ monocyte/macrophage lineage cells;
population V: B220+/IgD+ mature B cells; population VI: Gr-1+ granulocyte lineage
cells. Means + standard deviations are shown for groups of five mice (statistics:
unpaired t-test). Representative FACS data are shown (left pannels).
Figure S8.
Functionality of TPO is shown by its ability to stimulate
megakaryocyte differentiation in vitro. Fetal liver cells were isolated and cultured
by a standard method as we described earlier (Shivdasani RA, Schulze H. Culture,
Expansion and Differentiation of Murine Megakaryocytes. Current Protocols in
Immunology. 2005; Suppl. 67 Chapter 22F.6.1-22F.6.13). Picture shows a
representative phase contrast picture of megakaryocytes generated after two days
with TPO stimulation. Routinely, TPO stimulation results in about 4% large polyploidy
CD61+ megakaryocytes (white arrows) already at day two, compared to less than 1%
in unstimulated /mock stimulated cultures. At day 5, TPO stimulation yields up to 10%
of megakaryocytes.