Understanding What Shapes the Epigenetic Landscape of B Cells
Dr. Nelson received his PhD from North Carolina State University while investigating protein subunit production for vaccine development. He arrived at the NIH in 2005 as a Postdoctoral Fellow, where he studied antibody-mediated neutralization of viral infections. Subsequently, Dr. Nelson worked in Dr. Rafael Casellas' lab investigating factors that regulate the epigenetic landscape of B cells during development and activation, and how these factors play a role in the development of B-cell lymphoma.
- Epigenetic landscape of B cells
- Use of genome editing on immune cells for clinical applications
1. What made you choose scientific research as a career path?
I was actually in a pharmacy program in Boston. During my first year, I had to do some really basic lab work and realized I really liked bench work research, so I switched programs. I received an undergraduate biochemistry degree from the University of Massachusetts at Boston and then got my PhD at North Carolina State University working with Dr. Dennis Brown in the Biochemistry Department. I did lots of virus work, developing protein subunits for potential vaccines. Then I came to the NIH in 2005 as a postdoc in Dr. Ted Pierson's lab. My work focused on understanding viral neutralization and antibodies produced subsequent to infection. It was a natural progression from antibody work to B cell work with Rafael's lab, understanding the epigenetic landscape and what factors modifies the epigenetic landscape that regulates B cell development. But it really was that initial exposure to basic bench work that made me realize I would be interested in doing this for a career.
2. Who is your scientific idol?
There is this gentleman, Jeff Ravetch, who is a tremendous scientist. For me, his work is something I would absolutely love to emulate, but there is only one Jeff! He is an extremely intelligent researcher and does really cool science, but really it is the breadth of his research I love - it's so all encompassing. His seamless transition from one interesting topic to another is fascinating. Overall, he is just a very good scientist.
3. What is your role in the Casellas lab?
I was a biologist, participating in investigating how CTCF impacts the epigenetic landscape of B cells. I was trying to understand several aspects pertaining to the recognition sequence of CTCF and how CTCF binds to target sequence. CTCF in an 11-zinc finger protein so part of what we were trying to do is further determine which component of the protein is important for binding and what would happen if we change the sequence. A lot of it has to do with understanding how this one protein is shaping the genetic landscape of B cells.
1. Can proteins that alter the genetic landscapes of B cells cause malignancies?
Yes. AID (activation-induced cytidine deaminase), for example, is a DNA deaminase that catalyzes a process that results in mutations in the B cell genome that is critical to generate a broad array of antibodies. AID can have off target effects that may promote malignancy and cause lymphomagenesis. Unfortunately, we cannot therapeutically target AID because AID is needed for an effective immune response.
2. In your opinion, what has been the most important advancement in the field in the last five years?
Genome editing. The ability to modify the genome, any genome for any given cell, and then go back and deconstruct to see the importance of a particular sequence of a binding site or of a particular region. From using zinc finger protein to using TALEN (Transcription Activator-Like Effector Nucleases) and more recently the CRISPR/Cas9 system to edit genome, I think it has definitely become the way forward.
3. So you see genome editing being used not just to understand biology, but also as a clinical application in the future?
Absolutely. It has been done already at various levels using different approaches, with different levels of success. Basic research is very important, but the holy grail is to be able to cure patients. I think this field is aiming for more from-bench-to-bedside research. The idea is to get cells from a specific patient, identify the deficiency of that patient compared to a normal healthy person, modify the patient's genome and make sure you can rescue whatever the deficiency is in the patient's cells. Once you do that then clearly the next step is putting those modified cells back into the patient to see if you can help the patient. That is the ultimate.
4. What are the technical challenges currently facing the B cell biology field?
Modifying the genome in such a way that gives you a permanent output is very technically challenging, but the main challenge for a lot of biology research is being able to replicate in vitro some natural events observed in vivo. For example, there may be a particular cancer that you recover from a patient, but then to be able to replicate that in vitro, to get the cells to behave the same way in the lab can be challenging. These technical challenges are especially true in the B cell biology field.
1. Why does your lab use EasySep™?
We often isolate resting B cells using EasySep™. This cell separation platform is very fast, very efficient and the purity is definitely good (always above 95%). Once we tried it, we were very happy and shifted completely to the EasySep™ technology.
2. How has EasySep™ enabled your research?
It enables us to isolate B cells from the spleen really fast with good viability. That is what we need. You don't have to stand in front of a column and wait for it to drip. You don't have to worry and wonder if your purity is OK. You don't have to worry about if you need to put your cells on ice or because it is sitting there at room temperature for 20 minutes while you are waiting for the column to drip. The viability of the cells that you recover tends to be higher just because it is so fast. I think the speed, purity and ease are very good.
On the epigenetic landscape of B cells
B cells are capable of producing a wide array of antibodies with different specificities and isotypes. B cells first acquire this diversity during V(D)J rearrangement in their development. This process is mediated by RAG endonuclease and results in the generation of a specific immunoglobulin, which is used as a B cell receptor. In the periphery, B cells undergo further recombination to generate antibodies of different isotypes. This process is mediated by the deaminase AID1, which is induced by the small GTPase Rab72. The dysregulation of these genetic rearrangements can lead to malignancies. For example, off-target AID-mediated recombination can result in B cell lymphomagenesis3. Interestingly, the Casellas lab discovered that highly-transcribed B cell super-enhancers are the key sites of AID's tumorigenic activity4. Future studies are required to determine how to prevent or target AID's tumorigenic activity without interfering with its normal function in antibody class-switching.
On the use of genome editing on immune cells for clinical applications
The ability to genetically engineer a patient's immune cells and adoptively transfer them back into the patient, holds a tremendous potential for immune therapy against various diseases. With the latest technological advancements in genetics, various genome editing methodologies are now available. For example, the use of site-specific DNA endonucleases such as ZFN, TALEN and Cas9 has allowed for genetic modifications in various cell types5,6, including primary human T cells7. Chimeric antigen receptor (CAR) T cells is a prime example of the use of genetic engineering for cellular therapy. In this system, patient T cells are genetically engineered ex vivo to recognize and eliminate tumor cells, and infused back into the patient for cancer therapy8. Genome editing can also be performed in vivo with the use of lentiviral vectors, and lineage-specific vectors have been developed to allow for cell-type specific genome editing. For example, B cell-lineage lentiviral vectors can be used in vivo to correct B cell disorders9. The use of genetically engineered immune cells for clinical applications is becoming a reality, and clinical trials are ongoing to test the use of CAR T cells for cancer therapy. The results of these trials will set the pace and expectations for the use of genetically engineered immune cells as cellular therapy in other diseases.
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- Park SR. (2012) Activation-induced cytidine deaminase in B cell immunity and cancers. Immune Netw 12(6): 230–239.
- Qian J et al. (2014) B cell super-enhancers and regulatory clusters recruit AID tumorigenic activity. Cell 159(7): 1524–1537.
- Pone EJ et al. (2015) B cell Rab7 mediates induction of activation-induced cytidine deaminase expression and class-switching in T-dependent and T-independent antibody responses. J Immunol 194(7): 3065–3078.
- Liu J et al. (2015) Efficient delivery of nuclease proteins for genome editing in human stem cells and primary cells. Nat Protoc 10(11): 1842–1859.
- Ran FA et al. (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8(11): 2281–2308.
- Schumann K et al. (2015) Generation of knock-in primary human T cells using Cas9 ribonucleoproteins. Proc Natl Acad Sci 112(33): 10437–10442.
- Barrett DM et al. (2014) Chimeric antigen receptor therapy for cancer. Annu Rev Med 65(1): 333–347.
- Sather BD et al. (2011) Development of B-lineage predominnt lentiviral vectors for use in genetic therapies for B cell disorders. Mol Ther 19(3): 515–525.