Basic HTML Version
48
ICCS 2012
P32
QUALIFICATION OF A HUMAN BLOOD PSTAT3
ASSAY FOR ANALYSIS OF CLINICAL SAMPLES: AN
INVOLVEMENT OF ITS NUCLEAR TRANSLOCATION IN
CD4+ CELLS
Susan Pleasic-Williams
1
, Ming Zhu
2
, David Wunderlich
1
,
Jaime Masferrer
2
, Fei Hua
2
, John Cheng
1
1
Pfizer, Inc,
2
Pfizer, Inc
The JAK3 signaling pathway transmits information from
IL21 receptors in lymphocytes. Measurements of JAK3
phosphoryation (pSTAT) in these cells may serve as a
pharmacodynamic endpoint for assessing IL21R activation
and blockage. Using the FACSCanto-II and ImageStream
cytometer (ISC), we developed IL21 mediated pSTAT
assays with blood CD4+T cells. Incubation of 10ng/mL of
IL21 for 15min at 37oC in human blood triggered a pSTAT
response measured by phospho-flow. TheFACSCanto assay
was used to test the effect of ATR-107, a human IgG 1l
mAb against IL21R, in a Phase Ia study to supplement the
receptor occupancy assay. In blood samples from healthy
volunteers at day 42 after treatment with a single dose of
ATR107, the pSTAT response in CD4+T cells was inhibited
by >90% in treated subjects (n=4) as compared to placebo
(n=3), consistent with IL21 receptor occupancy results.
Separately, we also investigated a possibility of nuclear
translocation of pSTAT3 using ISC. Analysis of samples by
the ISC revealed a high similarity between the image pairs
of pSTAT-AF647 and nuclear DAPI dye in the cell, indicating
pSTAT translocation upon stimulation. Furthermore, we
demonstrated an acceptable intertube precision (CV= 6.9%)
and sample stability (<30% decrease post 48hr storage
at 4oC) using K2EDTA Vacutainer™. Collectively, we
have demonstrated that IL-21 stimulation results in rapid
translocation of pSTAT3 into the nucleus of CD4+T cells; and
that the pSTAT assay could be used to detect inhibition of
the IL-21 pathway in clinical samples.
P33
MATRIX METALLOPROTEINASES INDUCER (EMMPRIN/
CD147) EXPRESSION ON B CELLS OF HEALTHY
DONORS AND CHRONIC LYMPHOID LEUKEMIA
Cecilia M Rodriguez
1
, Monica B Gilardoni
2
, Dario A Sastre
1
,
Viviana B Heller
1
, Carolina Ramello
2
, Ana C Donadío
2
1
Hospital Nacional de Clinicas,
2
Facultad de Ciencias
Quimicas-UNC
Matrix metalloproteinases (MMPs) play a pivotal role in cell
migration and invasion through extracellular matrix. CD147,
a cell surface glycoprotein, stimulates production of MMPs
on tumor and stromal cells. In this study, we analyzed the
CD147 and MMP9 expression in B lymphocytes of healthy
donors (HD B cell) and neoplastic B lymphocytes (B-CLL) of
patients with Chronic Lymphocytic Leukemia (CLL) and their
regulation through B-CLL-fibroblasts (Fb) contact (coculture)
by Flow Cytometry. CD147 median fluorescence intensity
(MFI) range in HD B cell (21,6 - 63,7) allowed us to separate
CLL in low MFI (19,1 ± 3,2; n=3), middle MFI (42,9 ± 12,5;
n=18) and high MFI (77,7 ± 8,3; n=7). CD147 expression
was significantly higher in high MFI CLL group compared
with moderate (p<0.01), low (p<0.05) and HD B cells
(p<0.01). Higher CD147 expression was associated mainly
with CD19+/CD5+high fraction. The MMP-9 concentration
in culture supernatants after 72 hours of coculture was
heterogeneous, exhibiting higher levels than B-CLL cultured
alone in 3/5 patients. Our results showed an increase in
CD147 expression and MMP-9 levels in B-CLL induced by
Fb contact. These findings could suggest the role of CD147
in the promotion of B-cells movement and migration towards
tissues.
POSTER ABSTRACTS