Production and use of antigen tetramers to study antigen-specific B cells

Production and use of antigen tetramers to study antigen-specific B cells
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When I was talking with Dr. Justin Taylor about a “Behind the Paper” for this tetramer protocol, we landed on the concept of stories that go untold. These are the personal journeys and happenstance interactions that exist behind every paper. Those exciting times that an author reveals that the initial observation of the big paper was, actually, panel D in figure 3, and they had absolutely no idea what was going on for three years. I have learned a tremendous amount about myself and the scientist that want to be from the stories that go untold, but they are incredibly hard to find. So, here’s this untold story- and some things I have learned along the way.

Studying antigen-specific B cell responses has been a part of my scientific journey since the very beginning. In Dr. Manel Jordana’s lab, and under the supervision of then-post-doctoral-fellow Dr. Rodrigo Jiménez-Saiz, we used commercially-available monomers of ovalbumin conjugated to FITC or AF647 to detect allergen-specific B cells in allergic mice. My first immunology publication was a very important finding by Manel, Rod, and Dr. Derek K. Chu that IgE production wanes after allergic sensitization and mice naturally lose allergic reactivity to allergens like peanut over time. We found that OVA-specific IgE PCs failed to form long-lived niches, and that continued IgE production required antigen. Since this observation conflicts with what happens in humans, this paper shifted the direction of our research. We started in on follow-up questions that pushed the limits of what the monomer platform could answer; new antigens, smaller populations, and potentially lower affinity responses.

I struck gold the day I met Justin in the poster session of a Keystone Symposium in early 2019. At the time, as a PhD student, I had voraciously read his and Dr. Kathy Pape’s work enriching B cells based on their antigen specificity. I recall thinking that the experiments were far too complex for us; we had never made a bespoke reagent before. The only enrichments we had ever performed were using commercial kits and I had somehow managed to botch even those a few times. In one evening of conversation, Justin had convinced me that we should give it a try. We wouldn’t need to make the tetramer either; he would send us an ovalbumin PE tetramer and a control tetramer, and all the protocols we would need to do the experiment. The craziest thing? Every protocol Justin gave me worked exactly as he said it would on the first try.

I was shocked by the kindness and support. I had been warned, time and time again, by supervisors, committee members, peers, folks at conferences – everyone really – that biomedical research is extremely competitive. Getting scooped was a common occurrence. The ideals for success were defending ideas, holding your cards close to your chest, and keeping any technological development away from competitors so you have the advantage. Here was a PI who was willing to give away the keys to the castle, without resistance or insane conditions? I was perplexed. The ethos of support and collaboration continued from there, I have consulted Justin so frequently over the years, about wide-ranging topics from project ideas to how to start my own lab, that I was certain I would one day receive the “okay, that’s enough, think for yourself” email. It hasn’t happened yet.

What I took away from this happenstance interaction is that the magic really does happen during the poster sessions at conferences. Trainees- if resources allow, attend a field-specific, relatively small meeting. Read the work of the attendees before you go. Really listen to others and engage with their work. Ask them about problems you’re facing- they may have ideas or access to resources that they can share. Be open and kind. Science can be competitive, sure, but increasingly labs are willing to publish findings back-to-back, which can help mitigate the impact of scooping. The friendships and collaborations that you can build from these meetings will stay with you for the rest of your life. 

Another very important aspect of science that I learned is the power of having a close colleague to bounce ideas off. This idea was recently discussed in a perspective by Dr. Itai Yanai and Dr. Martin Lercher. The decision to start building our own tetramers was made together with Allyssa Phelps. We worked to figure out the intricacies of building these reagents and bounced ideas off each other constantly in the process. Allyssa built tetramers for peanut allergens, like Ara h 1 and 2, that are present in this paper. We also had the opportunity to help our colleagues at ALK Abello build a tetramer for the birch protein Bet v 1. Together, we used these reagents to identify a novel phenotype of type 2 polarized memory B cells  (“MBC2”) that we clonally connected to recall IgE responses. We showed that nearly every B cell purified from allergic individuals using the birch tetramer expressed MBC2 markers, and these cells were greatly enriched in Ara h 2 tetramer-binding cells from peanut allergic, but not non-allergic donors. While MBC2s are enriched in IgG1-expressing cells, which are thought to be the reservoir of IgE upon recall, we demonstrated in a recent preprint that sequential switching through IgG1 is not required for IgE memory. This argues that the phenotype, rather than the isotype, of a memory B cell is the crucial determinant of the capacity to regenerate IgE. Allyssa and Dr. Kelly Bruton followed the MBC2 work with a preprint showing that allergic B cell memory retains plasticity, offering hope that therapies may be able to reprogram the memory B cell repertoire away from IgE production. Each of these observations required high-resolution phenotyping of allergen-specific B cells, which was afforded by the tetramer production and enrichment protocol. 

Allyssa and I had envisioned a potential protocol manuscript for generating tetramers of allergens after a few years working with the tools. They were transformative for us, and we thought that others could benefit from them too. Perhaps the real driver of the careful validation that we performed on this platform, which populated most of the figures in the paper, was the extensive and constructive skepticism of a past-mentee and long-time friend Dr. Adam Wade-Vallance. Adam developed a unique eye for good science during his PhD with Dr. Christopher D.C. Allen at UCSF. As we would generate data with this platform, we would excitedly show it to Adam. Unconvinced, he would ask frustratingly good questions like “if IgG PCs downregulate their BCR, can you detect them?” and “that looks like a lot of IgE-expressing cells, how do you know that’s real?” These questions initiated an entire line of experiments to validate the platform (read: prove him wrong). When we would excitedly show him the new data, another set of questions would emerge, which drove more experiments.

Allyssa and I eventually sat down with the intention of putting the protocol idea further into action. Around this time, an excellent scientist named Dr. Diego Pazos-Castro joined the lab on exchange from Dr. Araceli Diaz-Perales’ lab in Spain, and took the platform by the horns, generating multiple tetramers and testing them in a relatively short period of time. With Diego’s experiments in hand, we laid out multiple figures’ worth of comparisons and validation experiments, both in humans and mice, that emerged organically more than intentionally. The manuscript had grown much bigger than an allergen tetramer paper. We also knew from many conversations with Justin that there was a plethora of experiments that he had performed to validate the technique. He had published some of these, but many he told us about in passing and had not found their way into a manuscript. We pitched the idea to try for a manuscript targeted towards a broader audience, Justin agreed to put in his work, and here we are. This partnership only made sense to me; he drove the development of the protocol.

 Well, that’s not really the whole story. Justin had actually been aware of our intention of putting together the protocol for a while; but he had originally encouraged us to go for it alone. He once wrote to me in an email: “I would push back and say that your group has done all the work making these allergen tools, so it is really your story to share.” I couldn’t quite understand that at the time; he did give us the original protocol. Truthfully, I still struggle to understand it today, but reading through Justin’s “Behind the Paper”  post sheds some light on how a great scientist creates incredible impact through sharing, building community, and shining the spotlight on those around them. I hope to follow this lead throughout my own career; we’ve started by building tetramers for others who need them, though most are still too early to talk about.

Antigen tetramers are used in pretty well every project that we have ongoing, today. As I have recruited and trained my own students, these experiments have become the norm so much so that I have to remind them that flow cytometry can be run without tetramer staining and enrichment. It makes me think about the day that Allyssa and I first considered building tetramers and thought “maybe we could do that”.

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