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IMMUNOBIOLOGY OF LYMPHOID STROMAL CELLS

Periodic Reporting for period 3 - STROMA (IMMUNOBIOLOGY OF LYMPHOID STROMAL CELLS)

Reporting period: 2018-10-01 to 2020-03-31

In 1984, Nossal wrote ‘‘A readership consisting of primarily anatomists has every right to question the
favorite sport of research workers in cell immunology. This is to take a lymphoid tissue and totally destroy
its beautiful and elaborately designed architecture to obtain simple cell suspension of lymphocytes, which are
then asked to do more or less all the jobs of the original anatomic masterpiece’’. Growing evidence that
lymph node (LN) stromal cells control the motility, activation and survival of lymphocytes has reinforced
this view. These architectural cells assemble in 3D networks that regulate LN homeostasis and control its
ability to remodel during inflammation. Understanding stromal cell biology is thus mandatory to our full
comprehension of the immune system but this ambitious objective is technically challenging. As the
complexity of the LN cannot be modelled in culture, knowledge gained from in vitro experiments is limited
and will not address many relevant questions related to the biology of LN stromal cells, in particular (i) the
elucidation of their origin and the precursor/product relationships that link them, (ii) the determination of
their behavior in inflamed LNs and (iii) their subsequent fate in LNs that have returned to homeostasis. To
this aim, we have developed several original, cutting-edge multicolour fluorescent reporter mouse models and
computational modelling approaches to map the fate of single stromal cells and their progeny in situ. Using
this innovative approach, my group is investigating the spatiotemporal behaviour and molecular cues
that orchestrate the development and dynamics of the major LN stromal cell populations in vivo, at
steady state and under inflammatory conditions, at the single cell level.
Clonal Proliferation and Stochastic Pruning Orchestrate Lymph Node Vasculature Remodeling.
Lymph node (LN) expansion during an immune response relies on the transient remodeling of its vasculature. Although the mechanisms driving LN endothelial cell division are beginning to be understood, a comprehensive view of LN endothelial cell dynamics at the single-cell level is lacking. Here, we used multicolored fluorescent fate-mapping models to track the behavior of blood endothelial cells during LN expansion upon inflammation and subsequent return to homeostasis. We found that expansion of the LN vasculature relied on the sequential assembly of endothelial cell proliferative units. This segmented growth was sustained by the clonal proliferation of high endothelial venule (HEV) cells, which act as local progenitors to create capillaries and HEV neo-vessels at the periphery of the LN. Return to homeostasis was accompanied by the stochastic death of pre-existing and neo-synthesized LN endothelial cells. Thus, our fate-mapping studies unravel-at a single-cell level-the complex dynamics of vascular-tree remodeling during LN expansion and contraction.

T Cell Zone Resident Macrophages Silently Dispose of Apoptotic Cells in the Lymph Node.
In lymph nodes (LNs), dendritic cells (DCs) are thought to dispose of apoptotic cells, a function pertaining to macrophages in other tissues. We found that a population of CX3CR1+ MERTK+ cells located in the T cell zone of LNs, previously identified as DCs, are efferocytic macrophages. Lineage-tracing experiments and shield chimeras indicated that these T zone macrophages (TZM) are long-lived macrophages seeded in utero and slowly replaced by blood monocytes after birth. Imaging the LNs of mice in which TZM and DCs express different fluorescent proteins revealed that TZM-and not DCs-act as the only professional scavengers, clearing apoptotic cells in the LN T cell zone in a CX3CR1-dependent manner. Furthermore, similar to other macrophages, TZM appear inefficient in priming CD4 T cells. Thus, efferocytosis and T cell activation in the LN are uncoupled processes designated to macrophages and DCs, respectively, with implications to the maintenance of immune homeostasis.

Hemogenic endothelial fate mapping reveals dual developmental origin of mast cells.
Hematopoiesis occurs in distinct waves. ‘Definitive’ hematopoietic stem cells (HSC) with the potential for all blood lineages emerge in the aorta-gonado-mesonephros, while ‘primitive’ progenitors, whose potential is thought to be limited to erythrocytes, megakaryocytes and macrophages (MΦ), arise earlier in the yolk sac (YS). Here, we questioned whether other YS lineages exist that have not been identified, partially owing to limitations of current lineage tracing models. We established the use of Cdh5-CreERT2 for hematopoietic fate mapping, which revealed the YS origin of mast cells (MC). YS derived MC are replaced by definitive MC, which maintain themselves independently from the bone marrow in the adult. Replacement occurs with tissue specific kinetics. MC in the embryonic skin, but not other organs, remain largely YS derived prenatally and are phenotypically and transcriptomically distinct from definite adult MC. We conclude that within myeloid lineages, dual hematopoietic origin is shared between MΦ and MC.
Our original approach combining advanced imaging techniques and innovative lineage tracing models will allow us to extend our understanding of stromal cell biology.
We expect to unravel:
- new lymphoid stromal cell subsets
- new biological functions (including immune and non immune)
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