What changes does the vascular tree of the lymph node undergo during immune responses? According to the research groups of Marc Bajénoff from the Centre d’Immunologie de Marseille-Luminy (CIML, CNRS, INSERM, Aix-Marseille University) and Frederick Klauschen from the Pathology Institute of the Faculty of Medicine in Berlin, it forms new vascular cells that are joined end-to-end, before eliminating both old and new vessels. This discovery, and the technological advance it represents, is presented in the issue of Immunity published on October 18, 2016.
The immune response to aggression is initiated in small peripheral organs less than one centimeter in diameter, the lymph nodes. It is here that the naive lymphocytes first encounter the antigen that will activate them. As a result of this encounter, the lymph node may double in size in less than 24 hours, subsequently returning to its original size once its work is done. Over the last few years, the team of Marc Bajénoff has been working to improve our understanding of the role of the various support cells present in the lymph node in this remodeling process. After partially elucidating the role of the stromal cells, the researchers turned their attention to another key component of this phenomenon: the vascular network of the lymph node.
Lymphocytes must travel from the blood into the tissues of the lymph node, which is no easy task, because the vessel walls form a hermetic barrier separating the blood from the surrounding tissues. In practice, this transendothelial migration involves tiny venules known as high endothelial venules (HEVs). Under normal conditions, these highly differentiated cells express a molecule on their surface to which the lymphocytes can attach, enabling them to roll along the vessel wall, before eventually crossing it and entering the lymph node. The triggering of the immune response causes these cells to “rejuvenate” and generate new vessels, increasing the capacity for lymphocyte recruitment and the supply of oxygen and nutrients to the expanding lymph node.
A rainbow in the lymph node vascular tree!
Until the groundbreaking work reported in Immunity, researchers had no technique at their disposal for observing the vascular tree at the cellular scale and at high resolution. Intravital microscopy cannot be used to observe phenomena taking place over several weeks, such as lymph node remodeling, and the resolution of tomography was too low. The research team overcame these problems by combining single-cell imaging with a macroscopic approach and adapting a multicolored labeling system known as Brainbow, which was initially developed for following the dynamics of neuronal circuits.
"To identify and follow the vascular endothelial cells of the lymph node, we set up a system in which each cell produced a fluorescent marker of a different color (yellow, orange, red, green, violet or blue)" Marc Bajénoff explains. "Thanks to this genetic labeling, each endothelial cell passes its color on to its descendants, making it possible to visualize the origin and fates of the different branches of the vascular tree." The researchers then developed a method for imaging this 3D model at the cellular scale, allowing them to study the complexity of vascular tree organization. This work required the coupling of confocal microscopy with a clarification technique, to obtain sections that were sufficiently thick (200-250 µm, as opposed to 20 µm for traditional imaging), but also transparent.
The result? The researchers found that, during immune reactions, the HEV cells were the principal cells contributing to the segmented growth of the vascular tree, by assembling blocks of cells of the same color, generating a structure resembling a rainbow.
The return to homeostasis: a random process
So, what happens to this vascular network when the lymph node returns to its normal size once the immune response is over? Again, this question had remained unresolved due to the lack of an appropriate model. The researchers overcame this problem by using a mouse model in which cells lacking oxygen could be labeled, and their fate followed. By observing these mice for two months (corresponding to the complete immune reaction), the researchers were able to show that the reorganization of the lymph node involved the entire vascular network, not just the new vessels. The vascular tree had become larger than required to satisfy the needs of the lymph node, and was therefore less well-irrigated, leading to an intense competition between the capillaries for oxygen and nutrients.
"The vascular system probably can’t allow half the cells to randomly die because the capillaries are hermetic systems. It’s therefore more likely that entire segments are sacrificed. Logically, the most hypoxic ones would be preferentially eliminated" Marc Bajénoff concludes.
Clonal proliferation and stochastic pruning orchestrate lymph node vasculature remodeling
Isabelle Mondor, Audrey Jorquera, Cynthia Sene, Sahil Adriouch, Ralf Heinrich Adams, Bin Zhou, Stephan Wienert, Frederick Klauschen and Marc Bajénoff.
http://dx.doi.org/10.1016/j.immuni.2016.09.017
