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T-bet expressing B cells are required for thedevelopment of
autoimmunity
Kira Rubtsova
1,2
, Anatoly V. Rubtsov
1,2
, John W. Kappler
1,2,3,5
and
Philippa Marrack
1,2,4,5
1
Howard Hughes Medical Institute, Denver, CO,80206.
2
Department of Immunology, National Jewish Health and University
of ColoradoHealth Sciences Center, Denver, CO 80206, USA.
4
Department of Pharmacology, University of Colorado School of
Medicine, Aurora,CO 80045, USA.
5
Department of Medicine, University of Colorado School of Medicine,
Aurora, CO80045, USA.
6
Department of Biochemistry and Molecular Genetics, University of
ColoradoHealth Sciences Center, Aurora, CO 80045, USA.
Autoimmune diseases are common, affecting about 8% of the
population of the USA. Since there is no cure for autoimmunity, it
is extremely important to study the mechanisms that trigger these
diseases.Themajority of autoimmune diseasesaffect predominantly
females more than males, indicating a strong gender bias.
We have recently identified a subset of B cells that accumulates in
aged female mice and can be characterized by the expression of
CD11c and the transcription factor,T- bet. A related subset is found
in autoimmune mice, appearing at the onset of the disease. T-bet+
B cells obtained from autoimmune mice produce high levels of
auto- antibodies upon stimulation. Expression of T-bet in B cells is
necessary andsufficient for the appearance these cells.
These data led us to hypothesize that T-bet expressing B cells are
major precursors of autoantibody producing B cells during the
autoimmune response. Therefore ablationof this subset should lead
to the ablation of or significant delay in the onset of autoimmunity.
In this study we explored the role of T-bet expressing B cells in the
appearance of autoantibodies in SLE1,2,3 mice, a model of lupus.
We have generated mice thatlack
T-bet expression only in B cells by crossing (SLE1,2,3 x T-bet
flox/flox
x CD19
CRE
) animals We followed the appearance of autoantibodies
in these mice, comparing them to (SLE1,2,3 xT-bet
flox/flox
) littermate
controls. Our data indicate that kidney function and survival is
significantly improved in (SLE1,2,3 xT-bet
flox/flox
x CD19
CRE
)mice. At the
same time the titers of autoantibodies were dramatically decreased
in the (SLE1,2,3xT- bet
flox/flox
xCD19
CRE
) mice when compared to
littermate controls. Analysis of the T and B cell compartments
revealed a reduction of germinal cente B cells, pre-plasmablasts,
activated/memory T cells and IFNγ producing T cells. These data
indicate that T-bet expression in B cells plays an important role
during the onset of lupus-like
autoimmunity. Moreover, T-bet expression in B cells was not
required for the development of germinal centers and production
of antibodies in response to vaccination, suggesting that T-bet in B
cells can serve as a novel target for the treatment of autoimmunity
without dramatic impact on the other aspects ofimmune system.
In conclusion our data suggest that T-bet expression in B cells is
required for the generation of autoimmune responses, appearance
of pathogenic autoantibodies and destruction of kidney function.
Therefore conditional deletion of T-bet from B cells leads to the
overall improvement in autoimmune-pronemice.
A high throughput method to characterize monoclonal
antibodies and select native antigen specific IgG hybridoma
cells
Haolin Liu
1
,Janice White
1
,Frances Crawford
1,2
,Gongyi Zhang
1
,Philippa
Marrack
1,2
and John W.Kappler
1,2
1
Department of Biomedical Research, National Jewish Health, Denver,
CO 80206 and
2
Howard Hughes Medical Institute
B cell hybridomas are important source of monoclonal antibodies.
However, the traditional method of using antigen-coated ELISA
plate to detect antibody secretion and using limiting dilution to
select hybridoma cells is time and labor consuming. Moreover,
antigen coated ELISA can’t distinguish antibodies against
the native and denature antigen. To solve these problems, we
developed a high throughput method to characterize mouse IgG
antibodies using Surface Plasmon Resonance (SPR) technology.
This assay rapidly determines their sub-isotypes, whether they bind
native antigen and their approximate affinities for the antigen with
only microliters of hybridoma cell culture supernatant. It could be
used to find out pairs of antibodies that can be used as coating
and detecting antibodies for commercial ELISA kit and blocking
antibodies of virus entry into host. Meanwhile, we found that
mouse hybridomas secreting IgG antibodies also have membrane
form IgG expression. Based on this surface IgG, we isolated rare
γ2a isotype switched variants from a γ2b antibody secreting
hybridoma cell line. We also used fluorescent antigen to single
cell sort native antigen binding hybridoma cells from bulk fusion
mixture thus saving time and labor.