Multiplexing Phenotype and Function for More Biologically Relevant Insights - 20

More Biologically Relevant Insights | APPLICATION NOTE

in the Jurkat target cells (Figures 5A, B), it increases caspase 3/7 activation in NK-92
effector cells (Figure 5B), while leaving cell membrane permeability in NK-92 cells
unaffected. This effect on NK-92 cells would not have been visible using a traditional
Cr51 release assay, and raises questions on the interpretation of the Jurkat caspase
3/7 data. A second intriguing result comes from U73122. At the highest concentration of U73122, Jurkat target cells show a reverse in the trend of decreased cell
membrane permeability and caspase 3/7 activation. Examination of these two
readouts in NK-92 cells shows that at higher concentrations of U73122, NK-92 cells
also display increased cell membrane permeability and caspase 3/7 activation
(Figure 5E, F), suggesting that U73122 might possess general cytotoxic activity at
these concentrations instead of specific CMC inhibition. This is in contrast to other
inhibitors, such as PP2 and SP600125, which show no increased cell membrane
permeability or caspase 3/7 activation at any concentration in NK-92 cells. Thus, the
ability to measure endpoints in both effector and target cells enabled a possible
explanation of the anomalous U73122 data at high concentrations. Importantly, if
only the highest concentration of U73122 had been examined, its effectiveness as
an inhibitor of CMC would have been underestimated. In the context of a HTS where
primary screening is typically performed using a single concentration of the test
compound, this type of underestimation could lead to early elimination of a potentially potent compound.
Conclusion
The iQue Screener CMC assay is a sensitive, specific, and high-throughput assay that
can provide a rich data set of quantitative information in a cell type-specific manner.
It overcomes the problems of the traditional Cr51 release assay by removing the
need to work with a radiolabeled isotope, reducing hands-on time, and increasing
reproducibility and measurement precision. Like flow-cytometry approaches, the
iQue Screener CMC assay enables discrimination of signal generated by effector cells
versus target cells. However, the iQue Screener CMC assay goes one step further and
improves on flow-cytometry-based approaches through its HTS format. Further, the
potential for additional multiplexing with readouts such as quantification of secreted
cytokines provides even more biological context. By simplifying and miniaturizing
the CMC assay, the iQue Screener enables researchers to get results faster while
conserving precious sample, thus providing an opportunity to accelerate discovery
and development of new immunotherapeutics. n

20

| January, 2019

References
1. Anguille S, Smits EL, Bryant C, et al. Dendritic Cells as Pharmacological Tools for Cancer
Immunotherapy. Pharmacol Rev. 2015;67(4):731-753. doi:10.1124/pr.114.009456.
2. Chester C, Marabelle A, Houot R, Kohrt HE. Dual antibody therapy to harness the innate
anti- tumor immune response to enhance antibody targeting of tumors. Curr Opin Immunol.
2015;33:1-8. doi:10.1016/j.coi.2014.12.010.
3. Zamarin D, Postow MA. Immune checkpoint modulation: rational design of combination
strategies. Pharmacol Ther. 2015;150:23-32. doi:10.1016/j.pharmthera.2015.01.003.
4. Fousek K, Ahmed N. The Evolution of T-cell Therapies for Solid Malignancies. Clin Cancer Res
Off J Am Assoc Cancer Res. 2015;21(15):3384-3392. doi:10.1158/1078-0432.CCR-14-2675.
5. Pennock GK, Chow LQM. The Evolving Role of Immune Checkpoint Inhibitors in Cancer
Treatment. The Oncologist. 2015;20(7):812-822. doi:10.1634/theoncologist.2014-0422.
6. Jedema I, Werff NM van der, Barge RMY, Willemze R, Falkenburg JHF. New CFSE-based assay
to determine susceptibility to lysis by cytotoxic T cells of leukemic precursor cells within a
heterogeneous target cell population. Blood. 2004;103(7):2677-2682. doi:10.1182/ blood-200306-2070.


https://www.intellicyt.com/

Multiplexing Phenotype and Function for More Biologically Relevant Insights

Table of Contents for the Digital Edition of Multiplexing Phenotype and Function for More Biologically Relevant Insights

Contents
Multiplexing Phenotype and Function for More Biologically Relevant Insights - 1
Multiplexing Phenotype and Function for More Biologically Relevant Insights - Contents
Multiplexing Phenotype and Function for More Biologically Relevant Insights - 3
Multiplexing Phenotype and Function for More Biologically Relevant Insights - 4
Multiplexing Phenotype and Function for More Biologically Relevant Insights - 5
Multiplexing Phenotype and Function for More Biologically Relevant Insights - 6
Multiplexing Phenotype and Function for More Biologically Relevant Insights - 7
Multiplexing Phenotype and Function for More Biologically Relevant Insights - 8
Multiplexing Phenotype and Function for More Biologically Relevant Insights - 9
Multiplexing Phenotype and Function for More Biologically Relevant Insights - 10
Multiplexing Phenotype and Function for More Biologically Relevant Insights - 11
Multiplexing Phenotype and Function for More Biologically Relevant Insights - 12
Multiplexing Phenotype and Function for More Biologically Relevant Insights - 13
Multiplexing Phenotype and Function for More Biologically Relevant Insights - 14
Multiplexing Phenotype and Function for More Biologically Relevant Insights - 15
Multiplexing Phenotype and Function for More Biologically Relevant Insights - 16
Multiplexing Phenotype and Function for More Biologically Relevant Insights - 17
Multiplexing Phenotype and Function for More Biologically Relevant Insights - 18
Multiplexing Phenotype and Function for More Biologically Relevant Insights - 19
Multiplexing Phenotype and Function for More Biologically Relevant Insights - 20
Multiplexing Phenotype and Function for More Biologically Relevant Insights - 21
Multiplexing Phenotype and Function for More Biologically Relevant Insights - 22
Multiplexing Phenotype and Function for More Biologically Relevant Insights - 23
Multiplexing Phenotype and Function for More Biologically Relevant Insights - 24
Multiplexing Phenotype and Function for More Biologically Relevant Insights - 25
Multiplexing Phenotype and Function for More Biologically Relevant Insights - 26
Multiplexing Phenotype and Function for More Biologically Relevant Insights - 27
Multiplexing Phenotype and Function for More Biologically Relevant Insights - 28
Multiplexing Phenotype and Function for More Biologically Relevant Insights - 29
Multiplexing Phenotype and Function for More Biologically Relevant Insights - 30
Multiplexing Phenotype and Function for More Biologically Relevant Insights - 31
Multiplexing Phenotype and Function for More Biologically Relevant Insights - 32
Multiplexing Phenotype and Function for More Biologically Relevant Insights - 33
Multiplexing Phenotype and Function for More Biologically Relevant Insights - 34
Multiplexing Phenotype and Function for More Biologically Relevant Insights - 35
Multiplexing Phenotype and Function for More Biologically Relevant Insights - 36
Multiplexing Phenotype and Function for More Biologically Relevant Insights - 37
Multiplexing Phenotype and Function for More Biologically Relevant Insights - 38
https://www.nxtbookmedia.com