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A Flow Cytometry Approach to describe T Cell Activation Induced Markers and Intracellular Cytokines after SARS-Cov-2 Exposure

Interview with Gianluca Rotta, PhD, BD Biosciences Scientific Affairs Manager–Europe

 

Measuring T-cell activation-induced markers (AIMs) and intracellular cytokines may be crucial to better understand the human immune response to SARS-CoV-2. By optimising a flow cytometry protocol, we can recover T cells while evaluating their cytokine profile1. Following his talk on this topic at CYTO 2022 in Philadelphia, PA, USA, here is an interview with Gianluca Rotta, PhD, Scientific Affairs Manager–Europe at BD Biosciences.

 

1. Why were you invited to speak at CYTO 2022?

 

I presented the results of a research project that I carried out in collaboration with researchers at the University of Genoa’s IRCCS San Martino Polyclinic Hospital. We finetuned a flow cytometry protocol to detect antigen-specific T-cell activation markers while evaluating intracellular cytokines.

 

This flow cytometry approach was used to precisely describe a subset of T cells, which are able to recognise the spike protein and mount a specific immune response against SARS-CoV-2, following vaccination or infection, to potentially protect individuals. This was not a BD-sponsored talk. I was invited and selected based on the science presented in our abstract.

 

2. How is T-cell mediated immunity important to fighting SARS-CoV-2?

 

There are three key reasons why T cells are absolutely crucial in mounting an adaptive immune response to SARS-CoV-2 and pathogens in general:

 

T follicular helper cells: We all know that antibodies are necessary to protect ourselves from pathogens. But the reality is that B cells, which produce antibodies can’t make the decision to produce antibodies by themselves.

 

They need an authorisation, which can only come from a specific subset of T cells. These are called T follicular helper cells. So, a T-cell consensus is required for B cells to produce antibodies.

 

Cytotoxic or CD8+ T cells: Antibodies circulate in bodily fluids several days after infection along with the virus. But once the virus is inside the body’s cells, it remains unaffected by the antibodies that circulate in bodily fluids.

 

At this point, the immune system has a different challenge. It must eliminate the infected cells. And this can be done using a subset of T cells called cytotoxic or CD8+ T cells. They are able to recognise infected cells and eliminate them.

 

Resistance to mutations: And the third reason why T cells are very important is because their ability to recognise a virus is much less sensitive to mutations that are generated by new variants.

 

T cells only recognise very small pieces of the virus, which are less likely to be affected by mutations. As a result, T-cell immunity to SARS-CoV-2 is stable and resistant to all variants of the virus2. This is an extremely positive aspect.

 

3. What are the advantages of detecting AIMs?

 

Historically, the detection of antigen-specific T cells with flow cytometry required extremely specific reagents, called major histocompatibility complex (MHC) multimers or dextramers (initially developed as tetramers). They recognise antigen-specific T cells at a single cell level. Studying antigen-specific T cells can be quite challenging due to the need for such very specific and sensitive reagents.

 

AIM tests represent a paradigm shift because the specificity of T-cell activation in this context is provided by the specificity of the stimulus3. When we put the T cells in contact with a spike protein peptide pool, only the T cells recognising these specific peptides will undergo activation.

 

Then, we simply look at activation markers and cytokine production. The onset of the AIM approach shifted the paradigm from needing specific reagents to requiring a specific stimulus with the use of generic reagents to study T cells.

 

4. Can you describe the partnership between BD and the University of Genoa?

 

It was critical because the researchers at the University of Genoa are experts in T-cell research. They are very familiar with the literature. They also have the skills to culture T cells to obtain high-quality biological samples.

 

When we decided to combine an AIM test with cytokine detection, I immediately thought of them because of their knowledge and our long-term collaboration  (over 10 years).

 

We tested the approach and in the very beginning we found many issues in finding results according to our expectations. We had to modify the protocol with respect to the initial plan to allow for proper activation of T cells along with intracellular cytokine detection. Our cooperation with the University of Genoa was necessary to finetune the protocol and perform troubleshooting.

 

I’m really glad I was able to participate in this project and collaborate with other researchers. The opportunity to build something, which may help improve the quality of life for others, is the best part of my job.

 

Recommendation: Look at the data first without any expectations. Let the data guide you. Sometimes it tells you something different from what you were expecting. I think some key discoveries in science occurred because something unexpected happened.

 

5. What’s the future of this research?

 

This kind of research helps drive vaccine discovery. It’s crucial to describe the complexity of immune response, so it’s widely used by pharmaceutical companies, probably at a higher level of complexity than what we did. It gives us a deep understanding of the immune response to pathogens.

 

It’s important to point out that this approach is for research purposes only. We are far from clinical practice. In the future, I believe this approach will have the potential to influence vaccination strategy. But in current vaccine clinical trials, antibody production is the primary endpoint.

 

There are many researchers who are pushing to change the design of vaccine clinical trials because it’s obvious that T cells play a key role in mounting a specific immune response to protect individuals. Antibody production is just one part of the adaptive immune response. T cells are also crucial.

 

Right now, we know that the protocol is reproducible at the University of Genoa. But we want to check if the same conditions can be reproduced wherever people may have an interest in repeating this data.

 

Our next step is to validate the approach in about 10 laboratories in different countries. We are planning to collect a bigger dataset to have a broader technical validation of this approach. Once this is done, we will be able to proactively promote this approach as part of the BD solution.

 

The research discussed here warrants further study to corroborate the findings and has yet to be certified by peer review.

 

Read preprint of the manuscript “An Optimised Flow Cytometry Protocol for Detecting T-Cell Activation-Induced Markers and Intracellular Cytokines during the Immune Response to SARS-CoV-2”.

References

  1. Altosole T, Rotta G, Bornheimer SJ, Fenoglio D. An optimized flow cytometry protocol for simultaneous detection of T cell activation induced markers and intracellular cytokines: application to SARS-Cov-2 vaccinated individuals. medRxiv. Published online April 22, 2022:2022.04.14.22273819. doi:10.1101/2022.04.14.22273819
  2. Moss P. The T cell immune response against SARS-CoV-2. Nat Immunol. 2022;23(2):186-193. doi:10.1038/s41590-021-01122-w
  3. Reiss S, Baxter AE, Cirelli KM, et al. Comparative analysis of activation induced marker (AIM) assays for sensitive identification of antigen-specific CD4 T cells. PLoS One. 2017;12(10):e0186998. doi:10.1371/journal.pone.0186998

     

     

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