How targeting B cells could lead to improved immunotherapies for lung cancer

Despite their predominance in the tumor microenvironment (TME) and key role in the adaptive immune response, target B cells and tertiary lymphoid structures (TLS) are currently not considered within the development of immunotherapies. In this webinar, Dr. Tullia Bruno describes how her team at the University of Pittsburgh Medical Center is evaluating the role of B cells and TLS in lung cancer to better predict disease progression and identify targets for improving the efficacy of immunotherapies.

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Q: Are there key TME components that influence TLS formation and maturity?

TB: The short answer is that we're still determining all of the key factors that play a role in TLS formation and maturity. I talked about differences in the TME from the standpoint of having virally-induced cancer versus carcinogen-induced cancer, but there's obviously a lot of other components beyond that. We’re also thinking about the non-immune cells because the bottom line is that the stromal cells within the tumor can also lay the foundation for a lot of TLS formation, and this has been shown in normal lymphoid tissues. It’s important to think about not only the key immune cells that are playing a role but also the non-immune cells. We can also think about other factors that might be specific to a tumor from an environment standpoint. These are all things that can be investigated to better understand what cues can determine formation and maturity.

Q: What is the main function of B cells within the TME? If it's antibody production, do these antibodies recognize the tumor?

TB: When we think about B cells, we think about antibody production and so one thing that we're trying to understand in the laboratory is if the B cells that are in the tumor microenvironment are making antibodies, and if they are always targeting or recognizing tumors. There are several ways that we can go about this and there are other groups that are doing this as well.

From this, you can then start to think about what type of isotypes those antibodies are, and this will predict function, such as antibody-dependent cellular cytotoxicity (ADCC). Another function we consider in the tumor microenvironment is antigen presentation, and our group and others have shown that B cells can present antigens. I think they're underappreciated as an antigen-presenting cell. In the tumor microenvironment, they can rapidly pick up proteins and process them quickly in a more efficient way than a dendritic cell, for example. We know if dendritic cells are in contact with the tumor bed they get shut down, whereas for B cells in the TLS, this will not happen. Then there are studies that show that the impact of tumor B cell killing. B cells can directly kill tumor cells, and this is a little less investigated, but I think it's worth thinking about because we always consider the CD8+ T cells as the major killing immune cell, but other cells do and can have effect or function and I think it's understudied.

Q: Do you notice any changes in the TLS heterogeneity over time from the onset of the disease or condition?

TB: This is a little bit difficult to study in people because we are getting a snapshot. One way we are trying to do this in humans is by looking at individuals that have premalignancy or have premalignant lesions. Oftentimes we don't have matched samples or longitudinal samples from those patients, but we can look at different cohorts of individuals that may be at different stages of malignancy or progression, and then we can examine tertiary lymphoid structures. I think the jury's still out on that. We have a little bit of data based on lungs that suggest they do evolve quite a bit from the time somebody is a smoker versus a COPD patient versus a full-blown lung cancer patient, for example, but this is still under investigation. I think a better way to do this is using mouse models. A lot of the mouse models that are out there have B cells, but often those B cells do not organize into tertiary lymphoid structures, so we have also been putting a lot of effort into creating a mouse model that has tertiary lymphoid structures and this is ongoing research. If we do this more, we can then start to get at that question of tracking the disease as the mice are getting full-blown cancer.

Q: What is the implication of increased tox in the context of HPV-positive TLS, especially regarding exhausted CD8+ T cells?

TB: It’s important to remember that those data are coming from TLS that are selected with the NanoString technology and within those TLSs, there are actually very few CD8+ T cells. We are actually going back now because we did vector staining to pair with the NanoString studies we did, and we can now quantify the number of CD8+ T cells that were in the TLS and start to look at that in a more granular way.

We have started to see that sometimes B cells can express tox and that the other cell type that is present is CD4s. I think we have to improve how we correlate the different cell types that are present with the expression that we're seeing by DSP, and this will be something that we do when we fine-tune these studies. I think it's a great point, but we don't really see CD8s within those TLS so it could just be a different cell type that we're underappreciating for tox.

Q: Is tumor vascularization a prerequisite for the maturation of the TLS?

TB: I think having high endothelial venules and vascularization within a TLS is a hallmark of a TLS in general. We don’t know that it is necessarily a marker or a requirement for maturity. This is something that is ongoing that we will continue to think about. The reason I say that is because of how we currently think about TLS in the lab; TLS that are less mature can also have high endothelial venules, so you don't have to see a germinal center to see vascularization. It's possible that it helps, it's just hard to look at that inpatient samples when it's a snapshot, so that's why I think that using a mouse model would be advantageous for some of this, and that's some of what we're moving towards now.

Q: What is the impact of immune checkpoint blockade therapy on TLS?

TB: There were actually three papers that were published last January that showed pretty definitively that when patients have TLS, they do better on immunotherapy. In some of those papers, they even showed this was the case if they have mature TLSs. Now, as far as impact on the actual TLS pre and post, that is still under investigation because those papers really didn't get into a lot of the granularity of all the different hallmarks for TLS. This is something that I think we need to do as a field, to start to understand what's happening to the TLS pre-and post-treatment.

These are sometimes harder experiments to do because you need tissue, and it is often good to have tissue from treatment-naive patients so that they aren’t exposed to any other immunotherapy. Then they're given immunotherapy and you have the post tissue with a new adjuvant setting. This is something we can start to think about, but it is challenging because those tissues are less and less available sometimes and more precious in that regard. The strategy we're actually taking is trying to come up with an algorithm we can use to branch into some of that in a little bit more aggressive way. I think that once we get a good idea of what we're looking for we can then start to dive in and understand how those immunotherapies modulate TLS.

Q: What about regulatory B cells? Are they present in the TME, and if so, what are their roles?

TB: I think this is a great question, and my short answer is I don't know. Regulatory B cells (Bregs) have certainly been defined outside of cancer and there are some studies that have started to describe them within cancer. However, I think that phenotype is a bit tricky. We're not sure exactly what the hallmarks within cancer are for a regulatory B cell. We could try to apply some of what we know about Bregs from other areas of the literature, but we haven't aggressively done this, and part of the reason is, that we don’t know if they're the majority in the tumor types that we study.

I presented studies that had neck and lung cancer, but we also are working in ovarian as well now, and we're not really seeing a huge signature for them in some of the multi-level approaches that we're taking, but that also maybe just because they don't look the same in cancer. The other problem with assaying for regulatory B cells is, for a Treg cell, for example, the best way to tell it is a Treg cell is to show that it is suppressive. I think this is a harder thing to do when you don't know the markers. I think the jury's still out on those, regarding both how many are there and if there is a role, and I think some of the studies that we're doing now will start to reveal if there is something that looks similar, but it's not 100% clear at this point.

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