Gabriel Victora was used to practising alone and at length when he was a professional pianist. Today, as an immunologist, he spends hours alone in a dark, cold room in a laboratory trying to understand how B lymphocytes, the cells that produce antibodies, mature. He still thinks that playing the piano is more difficult than marking cells, but the discipline he inherited from music helped him persist in research led by Brazilian immunologist, Michel Nussenzweig, at the Rockefeller University in the United States, whose results were presented in November in the journal, Cell. The researchers are now beginning to understand an old phenomenon that has been known and little understood by immunologists: affinity maturation – the production and selection of the B lymphocytes that generate more effective antibodies as an infection progresses.
It is believed that this refinement occurred as the B lymphocytes came into contact with antigens, molecules that are recognized by the antibodies. It has now been seen that it is the interaction with other immune system cells that determines which B lymphocytes will become producers of antibodies. “It’s necessary to consider the interaction between these cells when designing vaccines. Looking only at the interaction of the B lymphocyte with the antigen is not necessarily the solution”, says Victora. The work received prominence in Cell and earned a comment by Jason Cyster, from the University of California in São Francisco, one of the world’s leading researchers in the area, in the same edition of the journal.
In an infection the B lymphocytes migrate from the blood to the lymphoid organs, like the tonsils or the lymph nodes in the armpits. There they group together in the so-called germinal center, where there is a high concentration of pieces of infectious agents (antigens) trapped on the surface of other cells of the immune system, the follicular dendritic cells, in addition to the T lymphocytes recruited by these antigens. In these centers the B lymphocytes introduce random changes in the genes that codify the antibodies and generate cells with a genome that is different from those of the other cells in the body.
Most of the mutant cells are less efficient than the original B lymphocyte, but a few become highly effective and are selected to produce antibodies. In this sense, germinal centers are like libraries: they have a large amount of information that can encourage and improve skills or propagate data after a stimulating suggestion. “It’s there that the antibodies evolve in real time and allow a response to pathogens that have an evolving cycle that is faster than ours”, explains Victora. “Without this, we would always lose the evolving race against infections.”
The germinal center is home to happenings that control the path and destination of the maturing cells. To know how B lymphocytes are selected Victora had to understand the dynamics of the two regions of these centers: one with few cell nuclei, the light zone; and the other full of B lymphocytes, the dark zone.
In the light zone B lymphocytes mix with follicular dendritic cells charged with antigens and with the T lymphocytes. The immunologists believe that the B lymphocytes replicate in the dark zone and migrate to the light zone. With the evolution of techniques for obtaining images they began to note a bidirectional movement, with cells from the light zone returning to the dark zone. All that was missing was to find out how they migrate and how this has an influence on selection.
To delineate the two zones Victora developed a way of marking the cells of the germinal centers with micro-atomic accuracy and then monitored their paths in real time in the live animal, before retrieving them for pheotypical characterization and genic expression profile studies. This was only possible with the use of transgenic mice that express a modified version of the green fluorescent protein, GFP, activated by the light of a two-photon laser. As it has a longer wave-length, this laser penetrates intact organs and activates deep regions. It is as if Victora used a torch to illuminate a specific region in the cells.
Using a combination of techniques he activated the B lymphocytes in each zone and measured how long they took to go from one to another. After four hours of photo-activation, half of the B lymphocytes in the dark region migrated to the light region. But after six hours only 15% of the B lymphocytes had gone from the light area to the dark, suggesting that it is in this return that selection of the most suitable for fighting infection happens.
After separating the B lymphocytes from the two regions the researchers assessed their genic expression. In the cells from the dark zone the activation of genes linked to cell division and the occurrence of mutations predominated. In the light zone the lymphocytes had more active genes involved with selection, which depends on the recognition of antigens. They also showed that by facilitating the interaction of the T lymphocytes of the light zone with the Bs, the latter migrate en masse to the dark region where they embark on another cycle of cell division and mutation. Recent work indicates that the presence of a high number of T lymphocytes in the light zone may lead B cells to produce high quantities of antibodies against the organism itself, as happens in autoimmune diseases, like lupus.
In the congress of the Brazilian Society of Immunology, held in November, Nussenzweig pointed out that if a way is found of extending the selection time of B lymphocytes in the germinal centers, perhaps a large diversity of high affinity antibodies, effective for capturing and rendering inactive invading pathogens like HIV, can be generated.
VICTORA, G. D. et al. Germinal center dynamics revealed by multiphoton microscopy with a photoactivatable fluorescent reporter. Cell. v. 143, p. 1-14. 12 nov. 2010.