Page 27 - Live-cellanalysis handbook

Kinetic Proliferation Assays

Additionally, the IncuCyte system was used to study effects of a bid to block antigen-driven T-cell proliferation and induce T-cell
L-kynurenine (L-kyn) on T-cell proliferation and clustering. L-kyn death, thus allowing cancer cells to escape immune surveillance.
is a metabolite formed from the catabolism of L-tryptophan by Inhibitors of IDO and/or TDO are therefore promising therapeutic
the enzymes indoleamine 2,3-dioxygenase (IDO) and tryptophan targets for the treatment of cancer. Figure 11 shows time-course
2,3-dioxygenase (TDO). Some cancers increase L-kyn production in results of L-kyn inhibition of T-cell proliferation and clustering.

A Proliferation B Cluster formation
Cluster count
% confluence (per well)
100 anti-CD3/IL-2 (10 ng/mL) 1500
37.5 µM
L-kyn 18.75 µM
75 µM L-kyn 18.75 µM
80 37.5 µM
anti-CD3/IL-2 (10 ng/mL)
1000 75 µM

60
150 µM
500
150 µM
40
n=6 replicates
300 µM
20 0
24 48 72 96 120 144 168 24 48 72 96 120
Time (h) Time (h)
C AUC x 10 3 D AUC x 10 4
(48 – 168 h) (24 – 120 h)
9 7

8 6

7 5

6 IC =110 µM 4 IC =170 µM
50
50
(slope=2.9) (slope=2.9)
5 3
4 2

3 1
-5.5 -5.0 -4.5 -4.0 -3.5 -5.5 -5.0 -4.5 -4.0 -3.5
Log [L-kyn] (M) Log [L-kyn] (M)

E IL-2/anti-CD3 F + L-kyn (150 µM)

Figure 11. L-kynurenine inhibits T-cell
proliferation and clustering. Data
demonstrates a clear concentration-related
inhibition of IL-2/anti-CD3 activated T-cell
proliferation (A) and clustering (B) with the
addition of exogenous L-kyn, over time.
Time-course profiles enabled AUC analysis
and generation of concentration-response
curves from which IC50 values for inhibition
of proliferation (C) and clustering (D)
were determined. Images reveal reduction
of clustered morphology with addition
of L-kyunerenine (F) when compared to
activated t-cells (E).

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