Projects

Cupedo Lab

Biology of human LTi cells 
We characterized human fetal LTi cells as Lineage-negative RORt+CD127+ cells that express lymphotoxin, TNF and RANKL. Fetal LTi cells can differentiate into RORC+ cells that co-express several NK-related molecules (CD56, NKp44), but lack effector functions associated with NK cells such as cytotoxicity and IFN production. Both cells are found in fetal lymphoid organs and intestines but also in tonsils postnatally. Whereas these cells produce IL-17A in the sterile fetal environment, after birth IL-22 becomes the predominant cytokine made by LTi cells and their progeny in the tonsils. This is most likely regulated by the increase in microbial pressure post partum.
Current projects include dissecting lineage relationships between LTi cells and their progeny using gene expression arrays and clonal differentiation cultures; identifying the signals that induce IL-17 or IL-22 production using intestinal organ cultures; and determining functional properties of LTi cells during lymph node development in in-vivo xenograft systems.
The overall goal of this work is to be able to manipulate LTi cells and as a result influence ongoing inflammatory reactions.

Human lymph node stromal cells
Mesenchymal derived stromal cells form the framework of lymphoid organs. In murine studies stromal lymphoid tissue organizer (LTo) cells have been identified that interact with LTi cells and in response to LTR and TNFR ligation start to express adhesion molecules and homeostatic chemokines. Upon colonization with T and B cells, distinct stromal subsets are formed in the respective T cell zone and B cell follicles. We are working towards the identification of human fetal LTo cells, as well as the specific stromal cells in fetal and postnatal human T and B cell areas. This work involves extensive immunophenotyping of developing human lymph nodes by confocal and fluorescence microscopy, as well as flow cytometry, purification and gene expression analysis of the various cells.
A thorough understanding of the cells that define cellular architecture in the developing lymph nodes will be a first step towards identification of similar cells in tertiary lymphoid organs during disease.

Microengineering artificial lymph nodes
In an HFSP-funded collaborative research program with Mark Coles (University of York, UK) and Abraham Stroock (Cornell University, US) we are engineering artificial lymph nodes in which we will study the role of physiological forces on the early and late stages of lymph node development.

During mammalian embryogenesis, mesenchymal cells differentiate into stromal lymph node organizer cells (LNo). In the course of a continuous differentiation program, LNo specialize into stromal cells that specifically accommodating either T or B cell clustering, this process forms the characteristic cellular architecture of secondary lymphoid organs. Gene-targeted mice have provided insight into the hematopoietic cells interacting with stromal cells during their differentiation, and several of the molecules essential for this process have been elucidated. However, in vivo studies are intrinsically limited in their ability to allow forthe simultaneous and tightly controlled manipulation of multiple cells and signals. In this project, we will develop artificial lymph node (ALN) environments in vitro that will allow us to study the complex cellular and molecular networks underlying LN organogenesis with unprecedented control. Using the ALN environments, we will unravel 1) Minimal cellular and molecular requirements for LN development; 2) Organization of stromal cells into distinct T and B cell specific areas and 3) A role for non-paracrine forces including mechanical stresses, oxygen tension, cell movement and extracellular matrix remodeling in development of LNs. To achieve this, 3D remoldable collagen scaffolds will be microfabricated that capture the anatomy of the developing LN microenvironment. The scaffolds will be seeded with appropriate human or mouse cells and connected to externally controlled fluidics to allow for direct modulation of the cellular (LTi, stroma, lymphocytes) and chemical (signaling molecules, inhibitors) microenvironments within the scaffold at any given time. Additionally, this approach will allow for direct modulation of physiological forces (oxygen, shear stress and wall tension). Seeded cells will be allowed to differentiate and organize while these parameters are varied. Stromal cells, LTi cells, lymphocytes and the extracellular matrix will be monitored in situ by real time 4D multicolor imaging and cells will be harvested from the scaffolds such that gene expression signatures between different conditions can be measured and compared and correlated with imaging results. From this data we will develop both physiological and genomic maps of LN formation and generate fully functional ALNs, providing a unique platform to study LN development and function in real time and allow visualization and modulation of biological and pathological processes.

Ectopic germinal center formation by non-Hodgkin lymphomas
Follicular lymphomas arise as an indolent malignancy from germinal center B cells in the lymph node. Similar to their non-malignant counterparts, follicular lymphoma cells need stromal derived signals in order to survive and proliferate. Upon diagnosis, up to 70% of patients present with bone marrow involvement. Within the bone marrow, follicular lymphoma cells arrange in follicular structures and induce ectopic germinal centers. This involves the modulation of the bone marrow microenvironment to resemble the stromal composition of the lymph node follicle. In this way, follicular lymphoma cells ensure their survival and proliferation at ectopic locations like the bone marrow.
We are interested in dissecting the signaling pathways underlying the formation of lymph node-like stromal cells in the bone marrow. On the one hand this will serve as a model system to study early events in B cell follicle formation, while on the other hand such signals are potential therapeutic targets aimed at disrupting ectopic follicles. To achieve this use a xenotransplant model in which primary human follicular lymphoma cells are transferred into immunodeficient mice. In addition, 3D-culture systems based on primary human stromal cells are being developed that will allow for the generation of B cell follicles in vitro.
The ultimate goal of this work is to gain the ability to specifically disrupt malignant B cell follicles and in this way influence survival and proliferation of the lymphoma cells.