You can add co stimulatory molecules to drive differentiation and activation of T cell. But I recommend you to add one more control group without the molecules to see the true potential of your allergen, which also minimize the bias of the results.

Article Direct stimulation of cytokines (IL-1 beta, TNF-alpha, IL-6,...

Article Antigen-induced IL-17 response in the peripheral blood monon...

It appears that you can add antigen only, after titirating the dose. You must have control wells that include known stimulants such as LPS. I would certainly isolate proteins from a dust mite that are antigenic, and not apply the whole mite because of LPS (and other) contamination concerns.

A complementary assay that you will likely be expected to accomplish if you wish to publish is a proliferation assay.

Hi,

I think adding co-stimulatory is a good thing as you can produce cytokines ina controlled manner. As we know from Maria and Erin statements you should be very peculiar about the control group too so as you are not supposed to be biased.

You need to be very conscious about the use of house dust mites, since it can have lot of factors associated with it which can lead to numerous concluding remarks while used for activation as whole.

Best,

Hamid

Thanks for the insights... Erin Dobrinen Maria Apriliani Gani Hamid Yousf Dar

But.... may i know, how can allergens stimulate T cells in PBMC? because as far as I know, T cells will only be activated if the system has antigen presenting cells (dendritic cells), whereas in PBMC there is no such cell...

is the stimulation produced against T cells that have been active before due to primary exposure? (just produce secondary responses?)

Do you intend to separate the t-cells from the other PBMCs? There will be an assortment of cells including APCs.

T-cells- cannot react to soluble antigens.

There are a subset of t cells that can react to antigens without antigen (gamma deltas) but they require an increased cytokine signal from other cells to react.

Erin Dobrinen

apparently not, I just want to know how the cytokines from T cells in PBMC can be stimulated without the presence of antigen presenting cells (dendritic cells) ..

Does another APC play a role here? (e.g. macrophages and B lymphocytes)? or memory T cells that play a role?

I'm sorry ... I'm new in immunology and want to examine the effects of allergen stimulation on PBMC (specifically measuring the cytokines released by T cells in PBMC)

APCs are very important to elicit a T cell response- eosinophils in particular are implicated in allergic responses.

Immunology is complex so it's ok to ask questions. I'll help as much as I can. You are starting with a complex experiment as a beginner so you'll want to proceed in stages and do literature research at each step for guidance.

It would be best for you to first review the different cell types in PBMCs and their roles. Allergens are often not available to PBMCs so route of antigen exposure (ingestion, inhalation, mucosal) should be considered. Would the allergen(s) you'd like to study enter the blood? If not you almost have to consider doing in vivo experiments.

You could isolate PBMCs first and experiment on them in culture but an easier method is to expose the animal (or person??) to the allergen and take blood samples before and after. Then isolate the T-cells (there's many methods, but magnetic beads are inexpensive) and measure their cytokines (those are expensive tests but can be obtained) pre- and post exposure. I don't know what equipment you have available so I'll recommend pre-made kits.

Thanks a lot Erin Dobrinen , for all of your kindness

so I will observe the stimulating effect of house dust mites (HDM) on PBMC allergic asthma patients, to monitor the cytokines released by them (specifically Interleukin-4 and Interleukin-10) ...

Of the several possible models (including in vivo animal testing) for this study, so far this PBMC model is the most likely to be done in our lab and there is already a lot of published evidence that PBMC can be stimulated with HDM allergen extract ... so we determined to research this ...

however, we do not yet know what makes PBMC stimulated by HDM (because as far as our understanding in PBMC there are no dendritic cells, even though dendritic cells are very important in T cell activation) I really hope to get an explanation about this, or can get literature that I can use as a basis (so far my main literature is Cellular and Molecular Immunology 9th Ed., by Abbas et al. 2017)

Great explanation of your goals, Ahmad. And a good starting reference. So I looked at this

https://www.stemexpress.com/blogs/peripheral-blood-mononuclear-cells/

and saw that dendritic cells can be present in PBMCs, as well as B cells.

I then looked at pubmed ( https://pubmed.ncbi.nlm.nih.gov/ ) for "PBMCs AND dust" and found relevant articles including this one which may help you:

https://link.springer.com/protocol/10.1007%2F978-1-4939-9591-2_9

I recommend you browse Pubmed and find review articles.

Article Innate Immune Responses in House Dust Mite Allergy

Here's an article you may find interesting! It discusses innate responses to HDM.

Erin Dobrinen thanks a lot for all of your explanation and all of your recommendation.

I'll try my best to learn more about immunology...

I hope that you will always be success and healthy in this global pandemic era.

I think this article will help you a lot. The authors incubated PBMCs with nine common food and environmental allergens or a non-specific mitogen, and measured levels of interleukin (IL)-5, IL-10, IL-13 and interferon-y in acellular supernatants.

Allergen-Specific In Vitro Cytokine Production in Adult Patients with Eosinophilic Esophagitis

Kiyoshi Yamazaki et al, Digestive Diseases and Sciences.