os B cells will not express
appreciable ranges of LFA-1 and that LFA-1 effector sys-
tems are downstream from Ca
flux, it was not surprising
Figure 2. Pharmacological inhibition of anti-IgM
(immunoglobulin M) mediated calcium flux in Ramos B cells. (A,
B) Ramos B cells Alisertib Stattic Entinostat have been incubated with compound at numerous
concentrations prior to stimulation with anti-IgM. IC50
the device compounds are reported in Table 1. The data from
representative experiments are proven as imply �� SD for every
concentration performed in triplicate.
that these inhibitors had no impact on Ca
flux (Fig. 2B and
Additionally, each LFA inhibitors had no effect on
flux in RL cells, even further supporting that LFA-1/ICAM
association happens downstream of Ca
From a routine-profiling standpoint, the FLIPR-based
calcium flux platform yielded robust Z�� statistics according to
DMSO versus CGI-1746 (10 ��M) treated cells. The average
Z�� was 0.75��0.
03, along with the Z�� array was 0.69�C0.79. The
signal�Cbackground (s:b) was 13.4��1.5, the s:b variety was
Development of the Label-Free Platform to
Measure B Cell Activation
As described, RL is really a human Alisertib Stattic Entinostat non-Hodgkin��s lymphoma B
cell line. RL cells express the integrin LFA-1, and associa-
tion with its ligand ICAM-1 mediates B cell adhesion. The
propensity for LFA-1 to associate with ICAM-1 is largely
dependent around the conversion of LFA-1 to an intermediate-
affinity conformation (Fig.
The signaling cascades
elicited on BCR activation contribute towards the conformational
shift needed for LFA-1/ICAM-1 interactions. The princi-ple in the EPIC platform is according to association of LFA-1
expressing RL cells to ICAM-1 coated over the EPIC plate
(Suppl. Fig. 3). We hypothesized that treatment of RL cells
with anti-IgM should shift LFA-1 expressed in RL cells to
an intermediate conformation capable of associating with
ICAM-1 rendered on the EPIC plate.
Treatment of RL cells
with inhibitors on the BCR signaling pathway really should abro-
gate the LFA-1/ICAM-1 association (Suppl. Fig. 3). RL
cells have been seeded onto 384-well EPIC plates precoated with
or without having ICAM-1 and permitted to equilibrate for approxi-
mately 2 h from the EPIC. The equilibration time permitted the
cells to settle, leading to a steady-state baseline.
of anti-IgM elicited a positive shift in response that corre-
sponded to an increased mass in the sensing volume in wells coated with ICAM-1 (Fig. 3A). The peak response
was around 25 min submit anti-IgM application, fol-
lowed by a slow decay (Fig. 3A). The slow decay and
decreased mass inside the sensing volume would suggest
the probable release Alisertib Stattic Entinostat of RL cells through the ICAM-1-coated
surface. From a functional standpoint, this might be con-
sistent with immune cell extravasion and endothelial migra-
tion. Indeed, the erythromyeloblastoid leukemia cell line,
K562, is reported to display dynamic LFA-1/ICAM-1 adhe-
. Not long ago, a number of small-molecule inhibitors of B cell
activation have already been reported. As an example, CGI-1746 is a
small-molecule BTK inhibitor that blocks BCR-dependent B
cell proliferation and minimizes autoantibody levels in colla-
Dasatinib can be a small-molecule kinase
inhibitor that has a broad array of targets that contains SYK and
Each SYK and BTK are downstream effectors of BCR activation (see Fig. 1). Indeed, dasatinib is reported to
inhibit calcium release and PI3K activation in response to
BCR crosslinking in continual lymphocytic leukemia (CLL) cells.
In addition, dasatinib inhibits the release of hista-
mine from human major basophils as well as secretion of pro-
inflammatory cytokines in immune cells.
class of small-molecule inhibitors though is reported to inhibit B cell
signaling, also through a BTK-dependent mechanism; displays
efficacy in the rheumatoid arthritis model; and it is at present in
Small-molecule inhibitors of B cell activation
that target effectors downstream of BTK and Ca
include things like BMS-587101. BMS-587101 is an LFA-1 small-
molecule antagonist that in vitro inhibits LFA-1-mediated
adhesion of T cells to endothelial cells and blocks subsequent
T cell activation.
Importantly, BMS-587101 protects mice
against irritation and bone destruction in the collagen-
induced arthritis study.
Collectively, these information support tar-
geting pathways involved with B cell activation for the prospective
treatment method of autoimmune and inflammatory ailments.
On this examine, we formulated an EPIC-based phenotypic
platform to assess B cell activation, with LFA-1/ICAM-1
adhesion currently being the endpoint readout.
We demonstrate that
the EPIC platform can detect alterations in B cell adhesion to
ICAM-1-coated plates triggered by stimulation of your BCR
and/or CD40R. In contrast, the FLIPR assay was unable to
detect CD40R-mediated improvements in calcium flux under
these experimental problems. Importantly, utilizing a seriesof pharmacological equipment, we can block B cell activation in
both the EPIC and FLIPR platforms.
Whereas the FLIPR assay
is actually amenable to HTS, the EPIC label-free engineering pro-
vides a complementary platform that measures B cell activa-
tion from a holistic perspective. To our knowledge, this is certainly the
to start with report utilizing the EPIC technologies as a phenotypic
screening platform to measure LFA-1/ICAM-1 adhesion as
a readout for B cell activation.
Elements and Procedures
The Ramos B cells and RL B cells have been obtained from your
American Variety Culture Entinostat collections (ATCC, Rockville, MD).
Goat anti-IgM (immunoglobulin M) was obtained from Acris
Antibodies (San Diego, CA) and Southern Biotech
(Birmingham, AL). The rhICAM-1/Fc chimer and the human
CD40/TNFRSF5 antibody had been bought from R & D
Systems (Minneapolis, MN). Recombinant human mega
CD40L was obtained from Enzo Life Sciences (Farmingdale,
NY). The neutralizing anti-CD40L antibody (Anti-hCD40L-
IgA) was obtained from InvivoGen (San Diego, CA). Hank��s
balanced salt solutio