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Complex Biology In Vitro Assays: Immuno-OncologyT Cell Proliferation CTG AssayTCR-dependent Stimulation on the Proliferation of Isolated Human PBMCs The immune system has an important role on tumor progression; hence, it is crucial to characterize the possible impact of a drug candidate (antibody or small molecule therapeutic) on the immune system. Through our immune cell activation assays, we can assess the impact of candidate compounds on multiple super antigen-stimulated T cell receptor (TCR) engagement pathways. Proliferation endpoint measurement can be measured using the CellTiter-Glo® assay readout. The CellTiter-Glo® Luminescent Cell Viability Assay is a homogeneous method to determine the number of viable cells in culture based on quantitation of the ATP present, which signals the presence of metabolically active cells. The homogeneous assay procedure involves adding a single reagent directly to cells cultured in serum-supplemented medium. The Immuno-oncology assay can be multiplexed to evaluate individual or multiple cytokines in the supernatant prior to performing the cell viability assay. Identified cytokines can be future studied in vivo across an immunology platform.

Assay Principle Freshly isolated human PBMCs from healthy donors are seeded in the absence or presence of plate-bound anti-CD3 antibody, soluble anti-CD28 antibody, or phytohemagglutinin (PHA) for the indicated time. Isolated human PBMCs are activated using plate-bound anti-CD3 antibody in the absence and presence of standard of care antibodies (SOC). After 2 days and 5 days, the cell culture supernatant is removed for cytokine analysis by MSD assays. The cell proliferation is measured by the addition of the CellTiter-Glo reagent to the cell plate, and luminescence measured on the EnVision™ or EnSpire™ plate reader to assess cell viability.

OverviewPowerful new in vitro assays

provide a translational method

to study biologics or small

molecule modulators of immune

responses. We’ve developed an

optimized panel of T cell assays

available to help clients better

understand the complex biology

of immuno-oncology.

Isolate PBMCs & seed Harvest supernatant

Trigger αCD3/αCD28/PHAor

Trigger αCD3 +/- SOCMeasure viability with CellTiter-Glo®

Day 0 Day 2 or Day 5

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Assay SetupT cell-mediated cytotoxicity protocol has been developed for optimum analysis of toxicity in a culture of labelled MCF7 cells,

Annexin V Reagent, and stimulated PBMCs.

PBMCs

2×105 cells/well

anti-CD3 alone, anti-CD3 + anti-CD28 or PHA

48 hours post trigger

CellTiter-Glo®

Donor

Seeding density

Trigger

Incubation

Readout

Assay PerformanceRepresentative dose response data shown below from one donor measured at 48 hours post stimulation with anti-CD3, anti-CD3

and anti-CD28 antibodies or PHA. Where both anti-CD3 and anti-CD28 stimulation were performed, a dose response to anti-CD3

was tested in the presence of a fixed concentration of anti-CD28 (10 ug/mL).

Figure 2.

Figure 1.

A. B. C.

No stimulation 10 µg/mL PHA-stimulated 10 µg/well anti-CD3 + 10 µg/mL anti-CD28 antibody

T cell proliferation with various stimuation_CTG 48hr

Conc µg/well or mL

antiCD3

antiCD3+CD28

PHA

% M

ax re

spon

se

Complex Biology In Vitro Assays: Immuno-Oncology

PBMCs

T cell Proliferation Assay with Standard of Care Antibodies (SOC)

75,000 cells/well

anti-CD3 alone

IGg1 and IGg4

48 and 120 hours post trigger

CellTiter-Glo®

Donor

Seeding density

Trigger (positive control)

Negative control

Incubation

Readout

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Figure 3.

Summary The homogeneous procedure to measure ATP using the CellTiter-Glo Assay is quicker than other ATP assay methods that

require multiple steps to extract ATP and measure luminescence. CellTiter-Glo data (Fig 1) shows anti-CD3/anti-CD3 +

anti-CD28 to be the stronger inducer of proliferation than phytohemagglutinin.

The cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and programmed death 1 (PD-1) immune checkpoints are

negative regulators of T cell immune function. Inhibition of these targets, resulting in increased activation of the immune

system, has led to new immunotherapies for melanoma, non-small cell lung cancer, and other cancers. Ipilimumab, an

inhibitor of CTLA-4, is approved for the treatment of advanced melanoma. Nivolumab and pembrolizumab, both PD-1

inhibitors, are approved to treat patients with advanced or metastatic melanoma and patients with metastatic, refractory

non-small cell lung cancer. CTLA-4 blockade allows for activation and proliferation of more T cell clones, and reduces

Treg-mediated immunosuppression. PD-1 pathway blockade restores the activity of antitumor T cells that have become

quiescent. The results show that there is an increase in T cell proliferation (Fig 3) after 5 days when treated with ipilimumab,

nivolumab and pembrolizumab. The results show that pembrolizumab enhances T cell proliferation more significantly

(greater than two-fold) when compared to nivolumab and ipilimumab.

The indoleamine 2,3-dioxygenase (IDO) pathway regulates immune responses by suppressing T cell function and enabling

local tumor immune escape. IDO is an enzyme that catalyzes tryptophan to kynurenine. Tryptophan depletion enhances the

function of the suppressive Treg and inhibits the effector T cells. lndoximod is an orally-available tryptophan mimetic with

immuno-activating and anti-neoplastic activities, which inhibits the IDO pathway by counteracting immunosuppressive effects

of kynurenine, activates multiple immune (effector) cells, and prevents activation of regulatory T cells and reprograms Tregs into

helper T cells. Indoximod treatment for 5 days showed an enhancement in T cell proliferation by approximately two-fold. (Fig 3).

Using the CellTiter-Glo proliferation assay methodology, the immune-enhancing effects of both antibodies and small molecules

such as indoximod can be demonstrated.

CellTiter-Glo® 48-hour Time Point

[Antibody]

RLU

CellTiter-Glo® 120-hour Time Point

[Antibody]

askcharlesriver@crl.com • www.criver.com

Need a custom version of this assay? Visit criver.com/ds-vitro-assay

© 2019, Charles River Laboratories International, Inc.askcharlesriver@crl.com • www.criver.com

Assay Reference Code

OTS110-TcellProliferation CTG

Complementary Immuno-Oncology Assays

T Cell Proliferation CTG Assay

T Cell Cytokine Response Assay

T Cell Exhaustion Assay

T Cell-Mediated Chemotaxis Assay

3D Spheroid T Cell Cytotoxicity Assay

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