Supplementary Information
Negative Regulation of Lymphocyte Activation and Autoimmunity by
the Molecular Adaptor Cbl-b
Kurt Bachmaier*, Connie Krawczyk*, Ivona Kozieradzki*, Young-Yun Kong*, Takehiko
Sasaki*, Antonio Oliveira-dos-Santos*, Sanjeev Mariathasan**, Dennis Bouchard*,
Andrew Wakeham*, Annick Itie*, Jenny Le*, Pamela S. Ohashi**, Ildiko Sarosi #, Hiroshi
Nishina* Stan Lipkowitz§, and Josef M. Penninger*
*Amgen Institute, Ontario Cancer Institute, Departments of Medical Biophysics and
Immunology, University of Toronto, Toronto, Ontario, Canada M5G 2C1
**Ontario Cancer Institute, Departments of Medical Biophysics and Immunology,
University of Toronto, Toronto, Ontario, Canada
# Department
§
of Pathology, Amgen Inc., Thousand Oaks, California, USA
Genetics Department, Medicine Branch, National Cancer Institute, Bethesda Naval
Hospital, Bethesda, Maryland, USA
Cbl-b
2
Supplementary Information
Facial features, skull preparations, and osteoclasts in cbl-b-/- mice
From 6 months of age onwards, cbl-b-/- mice developed progressive changes in facial
features diagnostic for their genotype. The snouts of older cbl-b-/- mice were shorter
than those of cbl-b+/- or cbl-b+/+ littermates (Supplementary fig. a,b). Since no changes in
connective or muscle tissue were apparent in cbl-b deficient mice, we analysed the
head bone structure. Skull preparations revealed that the head was increased in width
relative to the length of the skull in cbl-b-/- mice when compared to wild type littermates
(Supplementary fig. c,d). Histological analysis revealed that older cbl-b-/- mice (from the
age of 6 months onwards) had significantly increased numbers of osteoclasts compared
to their wild type littermates (Supplementary fig. e,f). Because this phenotype developed
with age, we analysed whether the ability of bone marrow precursors to differentiate into
osteoclasts in vitro was already compromised in young (8wks old) mutant mice.
Compared to wild type controls, bone marrow-derived precursor cells from 8wks old cblb-/- mice displayed enhanced osteoclastogenesis as determined by TRAP solution assay
(not shown). Moreover, mature cbl-b-/- osteoclasts were dramatically larger than their
wild type counterparts (Supplementary fig. g,h). Thus, Cbl-b is a negative regulator of
osteoclastogenesis in vitro and in vivo. However, despite the increased numbers of
osteoclasts and the progressive alterations to the facial skeleton, there were no
significant anomalies in the structures of cbl-b-/- long bones. Since the facial skeleton
and long bones follow different developmental paths, it is conceivable that Cbl-b has a
role in establishing facial bone morphogenesis.
Supplementary Information
Bachmaier et al.
3
Figure Legend
(a,b) Photographs depicting the heads of a 14 month old cbl-b+/+ mouse (a) and its
cbl-b-/- littermate (b). Progressive changes in the facial features, evident only in the
cbl-b-/- mouse, are diagnostic for the cbl-b-/- genotype. Asterisk in the cbl-b-/- mouse
indicates the large submandibulary mass (see also Fig. 2b).
(c,d) Skull preparations of 14 month old cbl-b+/+ and cbl-b-/- littermates. The head is
increased in width relative to the length of the skull in a cbl-b-/- mouse (d) compared to
the skull of its wild type littermate (c). Lines 1 to 5 connect the following points on the
skull: line 1, the points at which the orbital arches reach the snout; line 2, the widest
point of the orbital arches before they turn toward the snout; line 3, the narrowest point
of the nose bone; line 4, the widest point of the orbital arches after they leave the skull;
line 5, the widest points before the orbital arches emerge from the skull. Bars indicate
4.5mm. Cartilage (blue) is stained with alcian blue. Bone is stained with murexide red.
Changes in the facial features develop with age and are not apparent in young mice.
(e,f) Histological cross-sections of the femoral shafts of a 9 month old cbl-b+/+ mouse
and its cbl-b-/- littermate. (e) No osteoclasts are present in the shaft region of the cbl-b+/+
femur. (f) The same region in the cbl-b-/- littermate mouse contains several osteoclasts
(arrows); that is large cells staining positively (red) for TRAP activity. Hematoxylin
counterstain. Magnification x 20.
(g,h) In vitro differentiation of mature osteoclasts from bone marrow precursor cells of a
young (8 week old)cbl-b+/+ mouse and its cbl-b-/- littermate. (g) Osteoclasts of the
expected size and TRAP activity (red staining) in cbl-b+/- mice. (h) Osteoclasts in the
Supplementary Information
Cbl-b
4
culture derived form the cbl-b-/- littermate mouse are significantly larger. Bars indicate
0.5mm. Magnification x 200.
Methods for Supplementary Information
Bone tissue was decalcified using a formic acid solution and embedded in paraffin. The
expression of tartrate-resistant acid phosphatase (TRAP) activity was determined using
a method of enzyme histochemistry that specifically stains osteoclasts red. Alcian blue
was used for cartilage staining and murexide for mineral staining of the bone matrix.
Skull preparations and staining with alcian blue for cartilage and murexide for mineral
staining
of
the
bone
matrix
were
performed
as
described1.
For
in
vitro
osteoclastogenesis, bone marrow cells were cultured overnight IMDM (10% FCS). 1 x
106/ml non-adherent cells were cultured with various concentrations (0.16-500 ng/ml) of
murine OPGL (aa 158 to 316) in the presence of murine CSF-1 (30 ng/ml). Osteoclast
differentiation was evaluated by quantitation of large multinucleated cells staining
positively for TRAP activity and ELISA2.
Reference for Supplementary Information
1) Tarpley, J. E. Adult rodent double skeletal stain. Biotech Histochem;74, 116-8 (1999)
2) Kong, Y. Y., et al. OPGL is a key regulator of osteoclastogenesis, lymphocyte
development and lymph-node organogenesis. Nature 397, 315-323 (1999).
Supplementary Information