What is the history of memory BCR sequences that have V and J out of frame from one another? There appears to be some disagreement in the literature on this point. For the purposes of this discussion, let’s call them non-functional receptors. There are two potential sources.
Non-functional BCRs matched with a functional receptor
We have two copies of the BCR locus. Thus, when the first V(D)J rearrangement results in an out of frame receptor, B cells can rearrange the second locus. If this one succeeds in affinity maturation and its B cell is exported from the germinal center, both loci can be sequenced from DNA. There seems to be broad agreement that this is the dominant source of out of frame sequences. However, there’s another option…
BCRs rendered non-functional from somatic hypermutation (SHM) indels
Insertion/deletion (indel) mutations happen in the course of affinity maturation, and if one gets an indel between the V and J templated regions that is of length not divisible by 3, this will lead to V and J going out of frame. This matters for people working to understand the neutral processes underlying affinity maturation, as said in [CD2016]:
some mutations in nonfunctional sequences may be subject to selection pressures if the sequences were rendered nonfunctional during the affinity maturation process
Because of this, [CD2016] develops a very clever but rather complex mouse system in which they are able to sequence lots of non-functional receptors, which are dominated by sequences with V and J in different frames. You can read the paper to hear how it works, but the outcome is that these sequences are enriched in BCRs that were always non-functional, rather than those that became out of frame after indel.
Thinking a little more I’d like to understand better exactly how sequences which were originally in frame but later had disruptive indels would make it into the blood and thus onto the sequencer. The situation seems like it must proceed as follows:
- In frame BCR enters germinal center
- Indel, leading to out of frame sequence
- The indel-containing B cell is exported from the germinal center
- That cell is sampled in the blood as a plasma or memory cell.
How frequently are B cells with faulty BCRs exported from the germinal center? Well, there are two ways out of the germinal center that don’t involve death: export as plasma cells or memory cells.
[VN2012] describe how positive selection is required for plasma cell differentiation:
In addition to cyclic re-entry, positively selected GC cells are exported from the GC as plasmablasts-- the precursors of the plasma cells that will secrete antibody into serum and secretions
For this they cite the work of [PP2006], which shows that there is a strong selective sieve for export as a plasma cell. This is a complex process that isn’t completely understood, but certainly requires BCR signaling or T cell help, both of which require a functioning BCR.
Memory B cells
For memory B cells, the situation seems a little less clear. [VN2012] summarize
… whereas very high-affinity cells have an increased probability of being directed to the plasma cell fate, lower-affinity cells that pass positive selection can be directed into either the memory or recycling GC cell pools…
So although the bar isn’t as high as for plasma cells, they do indicate that cells have to pass positive selection to exit the germinal center. I’d think that a cell without a functional antibody would be unable to pass positive selection.
Another possibility could be that after an indel event, the second BCR locus could rearrange. However, there is no evidence for peripheral RAG recombination. Furthermore, even if the second rearrangement could happen in the GC it’s unlikely to bind well to whatever is being presented on the FDCs.
Other options include that when the GC dissolves some cells with non-functional receptors survive and make their way into the memory pool. Or perhaps indels could arise by themselves during the decades that memory B cells need to keep up a minimum expression level. But…
How are these cells maintained?
For us to sequence these cells in a memory sort, they have to be kept alive by homeostatic mechanisms. Athough there has been some disagreement in the literature, [BE2009] provide evidence that in vivo, memory B cells need BCR stimulation to proliferate even at low levels. On the other hand, [ML2000] show that switching a high affinity receptor for a low affinity receptor doesn’t reduce lifespan, so high affinity engagement of the BCR isn’t essential. Having a low affinity receptor is different than having a non-functional receptor, though.
What else is missing here? Are there experiments that could directly address this question? Are there ways in which we can filter out-of-frame sequences that arise from indels? Although the system of [CD2016] is super cool, the resulting neutrally evolving sequences are limited to a single light chain of a transgenic mouse. If we could confidently treat out-of-frame sequences as coming from faulty rearrangments, or even a good estimate of the frequency of non-functional sequences coming from other sources, we would have many more sequences with which to learn about the neutral somatic hypermutation process.
Thank you to Laura Noges, @bussec, @krdav, and @wsdewitt for discussions. Please add your thoughts below! I’m especially hoping to hear from team @steven.kleinstein.
[BE2009] Benson, M. J., Elgueta, R., Schpero, W., Molloy, M., Zhang, W., Usherwood, E., & Noelle, R. J. (2009). Distinction of the memory B cell response to cognate antigen versus bystander inflammatory signals. The Journal of Experimental Medicine, 206(9), 2013–2025. https://doi.org/10.1084/jem.20090667
[CD2016] Cui, A., Di Niro, R., Vander Heiden, J. A., Briggs, A. W., Adams, K., Gilbert, T., … Kleinstein, S. H. (2016). A Model of Somatic Hypermutation Targeting in Mice Based on High-Throughput Ig Sequencing Data. Journal of Immunology , 197(9), 3566–3574. https://doi.org/10.4049/jimmunol.1502263
[ML2000] Maruyama, M., Lam, K. P., & Rajewsky, K. (2000). Memory B-cell persistence is independent of persisting immunizing antigen. Nature, 407(6804), 636–642. https://doi.org/10.1038/35036600
[PP2006] Phan, T. G., Paus, D., Chan, T. D., Turner, M. L., Nutt, S. L., Basten, A., & Brink, R. (2006). High affinity germinal center B cells are actively selected into the plasma cell compartment. The Journal of Experimental Medicine, 203(11), 2419–2424. https://doi.org/10.1084/jem.20061254
[VN2012] Victora, G. D., & Nussenzweig, M. C. (2012). Germinal centers. Annual Review of Immunology, 30, 429–457. https://doi.org/10.1146/annurev-immunol-020711-075032