Human Treg Suppresion Assay

Human Treg Suppresion Assay2020-10-05T13:27:32-07:00
  • Help predict the potential effects of your Treg-targeted molecule with iQ’s T regulatory cell suppression assay bioservice
  • Personalized experimental design ensures that the most relevant data for your molecule are generated
  • All assays are performed with high-quality reagents and strict controls by experienced immunologists
  • Data are provided in appropriate formats, including PowerPoint slides and/or direct data files
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Service Overview

About iQ Biosciences’ Human Treg Suppression Assay Service

Regulatory T cells (Tregs) are currently being targeted as a modality to reshape the disease environment to modulate T cell function. In cases of autoimmunity, therapeutics are being developed to enhance Treg function, while in cancer, molecules are developed to abrogate Treg function. To develop these molecules, Treg suppression assays are commonly used to assess whether a potential therapeutic can inhibit or promote Treg function. In these assays, Tregs are co-cultured with responder cells, such as T cells or PBMCs, and functional readouts of the responder cells are performed at the end of the culture period. Proliferation and cytokine production are often used to assess function.

At iQ Biosciences, we have experience with these human Treg suppression assays and are now offering it as a bioservice to our customers. The Tregs in our assays are generated from conventional CD4 cells isolated from PBMCs using a proprietary process. This is followed by validation of Treg generation through immunophenotyping (Figure 1) and functional analysis (Figure 2). For immunophenotyping, cells are analyzed for the expression of CD4, CD25 (Figure 1), FOXP3 (data not shown), and CD127 (data not shown) while suppressive abilities are tested by co-culturing them with responder cells and assessing them for proliferation (Figure 2).

CD4 and CD25 expression after conversion from conventional CD4 T cells.

Figure 1. CD4 and CD25 expression after conversion from conventional CD4 cells. Conventional CD4 (left) cells were converted into iTregs (right) using a proprietary method. A small aliquot was taken after induction to evaluate expression of CD4 and CD25 by flow cytometric analysis (right).

 

CD4 and CD25 expression after conversion from conventional CD4 T cells.

Figure 2. iTreg suppression in different culture and suppression conditions. CD8 T cells were labeled with CFSE and co-cultured with iTregs at different responder:iTreg ratios and stimulated under high (solid line) or low (dotted line) conditions. After 4 days of culture, cells were harvested and analyzed for CFSE dilution by flow cytometry. Gating was first set on live cells, followed by CFSE positive cells. Percent of proliferation was determined by setting a marker for CFSE-diluted cells as compared to unstimulated control.

In our assays, the responder cells are labeled with CFSE to simplify downstream interrogation of proliferation. The CFSE-labeled responder cells are co-cultured with Tregs at different ratios in the presence of stimuli and the Treg-targeted molecule(s). After the culture period, the cells are harvested and analyzed with a flow cytometer to examine dilution of CFSE in responder cells, which reflects proliferative function (Figure 2). Molecules that promote Treg function will demonstrate less proliferation while those that inhibit Treg function will demonstrate more proliferation relative to untreated control.

In addition, the supernatant from these co-cultures can also be taken for cytokine analysis to add another measure of function. Pro-inflammatory cytokines, such as IL-2, IFN-g, and TNF-a can easily be measured with iQ’s cytokine bead array assay. Similarly, soluble factors produced by Tregs, such as IL-10 and TGF-b, can be measured to determine if the therapeutic molecules modulated Treg activity.

Factors Important for Treg Suppression Activity

iQ has extensive experience with Treg function and can help you design the best experiment based on key factors that can influence suppressive abilities. Some factors that can influence suppression are:

    1. Type of Tregs – There are 2 types of Tregs, iTregs and nTregs, and each has their own unique properties. nTregs are believed to maintain peripheral tolerance to prevent autoimmunity and immune system hyper-responsiveness while iTregs are thought to develop from conventional T cells in a suppressive tumor environment. Depending on your goals, the type of Treg used in the suppression assay may play an important role. For example, if the goal is to promote suppressive abilities in the context of autoimmunity, then nTregs may be the more relevant choice since they are important for peripheral tolerance. In contrast, if Tregs in the tumor environment are targeted to release their suppressive abilities, then iTregs may be the more relevant type.
    2. Responder:Treg ratio – The number of Tregs and responders will vary depending on the microenvironment. In addition, the ratios may vary depending on the cancer type. Therefore, it will be important to understand how a Treg-targeted therapeutic operates under conditions with the different responder:Treg ratios.
    3. Stimulation conditions – A titration of stimulation conditions with proper controls, such as a culture without Tregs, will help establish the point in which Treg modulation can be properly observed. Treg modulation experiments should be performed at or above that stimulation point as the responders need to be activated in order to evaluate Treg suppression. If a titration is not properly performed and a stimulation condition is arbitrarily chosen below that point, the Treg modulator will have no effect as the Tregs do not have activated responders to regulate, and this would read out as false negative for the modulating molecule. Therefore, it is important to perform a titration to properly evaluate if the Treg modulator is affecting Tregs to regulate responders.

At iQ, we have refined our Treg suppression assay protocol with quality-controlled human Tregs, responder cells, and stimulation reagents. In addition, with our extensive knowledge of factors that influence Treg suppression and the different readouts for T cell function, iQ can help design the right experiments for you to determine the potential of your Treg-targeted therapeutic molecule.

The iQ Experience

Tailored Experiments

Your research is important to us. Our scientists will work through each step of the process with you, including assay design, data analysis, and recommendations for future studies.

Streamlined Process

Simplify your workflow. Bypass the middle-man: at iQ Biosciences, you'll get immediate access to our biospecimen inventory, saving you both valuable time and money.

Expertise

We're experts- so you don't have to be. Augmenting years of experience in immunology and working with immune assays, our scientists stay current with the latest publications and technology.

Exceptional Service

We're here to help. We know the challenges you're facing: whether it be through expedited service or our complimentary consulting services, our team is dedicated to helping you reach your goals.

Order Information

Pricing

At iQ Biosciences, we understand that each client is unique. Upon quote request, we will provide complimentary consulting services to determine how to tailor our services to best meet your goals. Based on this preliminary assessment, we will be able to provide a price quote.

Expected Timetable

Fit a timetable appropriate to your research goals. We work with you to determine your expected turn-around on a case-by-case basis and ensure that our services fit your needs. Some experiments may require an expedited service – please inquire for more information.