The lymph node and the social network of immune cells

The immune system's response to aggression is initiated in small peripheral organs less than one centimeter in diameter, the lymph nodes. We see what happens when the ganglion is involved in a location accessible to the touch: it swells and becomes painful. This is a sign that intense activity is occurring, originating in the countless interactions between cells that are preparing the response to aggression.

Thanks to genetics and colored three-dimensional models the team of Marc Bajénoff is trying to understand how the anatomy regulates the movements and functions of immune cells in lymph nodes and their sites of action.

 

Lymph nodes have a complex structure: the density leaves no room for any free space and white blood cells (dendritic cells, B and T cells) crowd along a highly organized network, the stroma. The architecture built by the stromal cells is not inert but is actively involved in the logistics of the immune system by organizing its movements, information exchange and by supplying survival factors.

"We seek to isolate stromal cells to determine their genetic signature, that is to say, the mapping of all genes that are turned on, to better characterize them. As they represent only a tiny fraction of all cells of a ganglion and do not express unique surface markers, this is quite complicated," said Marc Bajénoff. "We also study the dynamics of the stroma during the development of an immune response by observing the behavior of cells in lymph nodes of mice. So we combine a genetic approach with an approach to imaging microscopy in living animals."

Multicolor microscopy to track each immune cell

In this complex cluster of stromal cells, lymphocytes and dendritic antigen-presenting cells, how can we identify who does what?
To resolve this problem, the team draws on the resistance of stromal cells to radiation, the sensitivity of immune cells to the same radiation and plays with colors.

Two-photon excitation microscopy images illustrating the motility of thymocytes (blue) and dendritic cells (red) compared to the network of stromal cells (yellow) within the thymus of a mouse.
Copyright: Marc Bajenoff CIML.


By a simple genetic manipulation, a green fluorescent protein can be expressed in all cells of a mouse. Irradiation of the mouse causes the destruction of the entire immune system, but the stroma remains intact. "We then reconstitute the immune system by grafting the mouse bone marrow from a normal donor mouse. This produces a chimeric mouse, which has green stroma and colorless immune cells," says Marc Bajénoff.
"For example, if one wishes to study the movements of T cells, it is sufficient to isolate a few million of them from a wild type animal, charge them with a fluorescent red dye and re-inject them into the chimeric mouse. Movement of the transferred red cells in the ganglion can be observed by microscopy (endogenous cells are non-fluorescent, thus invisible). We thus discovered that lymphocytes are highly mobile within a ganglion and that they crawl on the surface of stromal cells." Marc Bajénoff also stresses that the role played by architecture in the lymphoid organ function is solvable by the same universal principles in the functioning of the spleen, bone marrow or thymus.

"However, to find out what molecules control the system, the answer will rather be provided by the study of cell genetics"

The team of Marc Bajénoff has many objectives. One of them is to understand how the ganglia are reshaped in response to inflammation: these organs can double in size in 24 hours, which involves extremely fast remodeling logistics. Is this the result of the maturation of precursors that the node contained from the beginning, or have many cells also been recruited to remodel the lymph node?
"To answer the first question, dynamic imaging studies are most relevant," comments Marc Bajénoff. "However, to find out what molecules control the system, that is to say which agents orchestrate adhesion and movement of immune cells on the "roads" formed by the stroma, the answer will rather be provided by the study of cell genetics."

Finally, the team of Marc Bajénoff also plans larger studies of movements of cells once they leave the lymph node. This type of stromal cell, so important in initiating the immune response, is present only in lymphoid organs. However, immune system cells must then migrate to other sites.
"For example, in response to injury or infection, lymphocytes must leave the lymph node to infiltrate the skin, where these special stromal cells are not present to guide their movements and actions," said Marc Bajénoff. "We are therefore seeking the type(s) of cell(s) that may play an equivalent role for lymphocytes outside the lymph nodes. This issue is more complicated to address with mouse chimeras (all skin cells are resistant to radiation, so we can not selectively destroy only some of them), but we are working on different systems to visualize networks of stromal cells from transplanted tumors."