In the immune system, B cells produce billions of different antibodies that have the potential to recognize familiar invaders, and to respond to brand new ones. To generate that prodigious diversity, the cells use genetic recombination to shuffle their immunoglobulin genes and deal each B cell a unique hand. But the maneuver, which requires cells to sever and rejoin strands of DNA, comes with a danger. If the breaks are not repaired correctly, they can lead to chromosomal translocations, where aberrant fusions between immunoglobulin genes and other loci can lead to B cell cancers.

Because it is a double-edge sword, recombination is tightly controlled in both time and space. The prevailing view has been that the first step of antibody production, the breakage and rejoining between the variable (V) diversity (D) and joining (J) segments of immunoglobulin genes, was completed in the bone marrow, before the cells emerged into the wider circulation.

Now, research from the lab of Fred Alt, (Senior Investigator and Scientific Director of PCMM/IDI) has overturned that idea by showing that B cells continue V(D)J recombination after they travel out to peripheral lymph nodes. The findings, published in the July 9 issue of Nature, are important for a basic understanding B cell development, and help explain the origins of chromosomal translocations found in B cell cancers.

Mysterious Joinings

The first hint that peripheral B cells might indeed be undergoing V(D)J gene recombination came from Alt's work looking at the origins of B cell tumors in mice. Alt developed a line of mice that lacked the tumor suppressor protein p53 and had a targeted deletion of the enzyme that normally repairs V(D)J breaks. Last year, Alt lab researchers Jing Wang, Monica Gostissa and Catherine Yan reported that the animals developed B cell tumors with unusual translocations. The tumors looked like they came from B cells where early, V(D)J -type recombination was occurring at the same time as a later stage of recombination known as class switching. Because the mice lack DNA repair selectively in peripheral B cells, the only possible explanation was that both types of switching were occurring in peripheral B cells.

To test this controversial contention, Wang and colleagues devised a way catch B cells in the act of V(D)J recombination. By putting back p53 while keeping the block on DNA repair, they found they could prevent B cells from progressing to tumors. When they did this, they started to detect unrepaired DNA breaks in the V(D)J regions of the lambda light chain immunoglobulin gene in normal activated peripheral B cells. Further, the investigators showed that just as in bone marrow cells, the breaks depended on the V(D)J recombinase-activating gene RAG.

"Because DNA repair was shut off only in peripheral B cells, and not in the bone marrow, we knew that the breaks must be happening in the periphery, and that at least for this lambda locus, they were being initiated primarily by V(D)J recombination," Alt explains.

The results settle a long-standing controversy in B cell development, but also raise more questions about the physiological role of the process. "There has been a huge debate about this, and many people had concluded that there is no V(D)J recombination in the periphery. This shows it can occur," Alt says.

The process looks similar to receptor editing, a recombination event that occurs when bone marrow B cells tweak the specificity of their antigen receptors to prevent the production of autoreactive antibodies. But the ultimate purpose of V(D)J recombination in later B cell development remains to be seen. "We don't know yet the relevance of peripheral recombination, whether it is important for normal functioning of the immune system or if it it's an aberrant thing that, when it goes wrong, gives rise to tumors," Alt says. "We have to figure that out." 

From Breaks to Translocations

In peripheral B cells, the researchers found that some translocations, especially between immunoglobulin light and heavy chain genes occurred much more frequently than previously suspected. This is partly because both of the loci are frequently broken in the peripheral B cells, the light chain because of V(D)J recombination and the heavy chain from class switching.

Another reason the translocations were so frequent became apparent when the researchers looked at the 3-dimensional arrangement of chromosomes in the cell nucleus. Using the technique of spinning disc confocal microscopy, Alt and colleagues were able to obtain high-resolution images of gene topology, and found that the light and heavy chain genes sit close together in space. That proximity, along with frequent breaks, seemed to promote the formation of translocations.

The heavy-light chain translocation does not cause cancers, but the researchers found a similar situation for translocations between the proto-oncogene c-myc and the immunoglobulin heavy chain gene, a cause of many B cell lymphomas. In that case, the researchers found that the two loci lie close to each other, but translocation occurs rarely because the myc gene is rarely broken. When the researchers induced targeted breaks in the myc gene, the rate of translocations went way up.

The results identify three critical parameters governing translocations: frequency of breaks, proximity of breaks and the failure of normal DNA repair. "If you have these three things, all of a sudden you can have a very high frequency of translocation, much higher than people would have suspected previously," Alt says.

Alt credits colleague Tomas Kirchhausen, PCMM/IDI Senior Investigator, for suggesting the spinning disc confocal technique. Kirchhausen was using the system to image transport processes in living cells, and helped Alt adapt it to visualize gene proximity. The technique gave the lab a head start on the translocation work, and they plan to continue to collaborate with Kirchhausen to look at localization dynamics in living cells.

"This is just the beginning of studying the 3-dimensional organization of genes in the nucleus," Alt says. "We are out to understand more about how they can be brought together, and what that means."