Abstract
Mesenteric lymph nodes (mLNs) are sentinel sites of enteral immunosurveillance and immune homeostasis. Immune cells from the gastrointestinal tract (GIT) are constantly recruited to the mLNs in steady-state and under inflammatory conditions resulting in the induction of tolerance and immune cells activation, respectively. Surgical dissection and transplantation of lymph nodes (LN) is a technique that has supported seminal work to study LN function and is useful to investigate resident stromal and endothelial cell biology and their cellular interactions in experimental disease models. Here, we provide a detailed protocol of syngeneic mLN transplantation and report assays to analyze effective mLN engraftment in congenic recipients. Transplanted mLNs allow to study T cell activation and proliferation in preclinical mouse models. Donor mLNs proved viable and functional after surgical transplantation and regenerated blood and lymphatic vessels. Immune cells from the host completely colonized the transplanted mLNs within 7-8 weeks after the surgical intervention. After allogeneic hematopoietic cell transplantation (allo-HCT), adoptively transferred allogeneic CD4+ T cells from FVB/N (H-2q) mice homed to the transplanted mLNs in C57BL/6 (H-2b) recipients during the initiation phase of acute graft-versus-host disease (aGvHD). These CD4+ T cells retained full proliferative capacity and upregulated effector and gut homing molecules comparable to those in mLNs from unmanipulated wild-type recipients. Wild type mLNs transplanted into MHCII deficient syngeneic hosts sufficed to activate alloreactive T cells upon allogeneic hematopoietic cell transplantation, even in the absence of MHCII+ CD11c+ myeloid cells. These data support that orthotopically transplanted mLNs maintain physiological functions after transplantation. The technique of LN transplantation can be applied to study migratory and resident cell compartment interactions in mLNs as well as immune reactions from and to the gut under inflammatory and non-inflammatory conditions.
Highlights
Lymph nodes (LNs), spleen and Peyer’s patches (PPs) are secondary lymphoid organs that serve as sites for immune cell interaction and activation
We determined the viability of mLNs after surgical transplantation, in this set of experiments mLNs from B6.L2G85.CD45.1 expressing firefly luciferase were transplanted into congenic B6 albino mice
Positioned between the layers of the mesentery, mLNs locate in the center of the gastrointestinal tract (GIT) and their dysfunction [44]
Summary
Lymph nodes (LNs), spleen and Peyer’s patches (PPs) are secondary lymphoid organs that serve as sites for immune cell interaction and activation. Lymphocytes primed in mLNs are imprinted for gut tropism and the ligands specific for their homing receptors a4b7 integrin and CCR9 are found in the gastrointestinal tract (GIT) [5, 7, 20, 21] These allow for efficient homing of immune effector cells to the lamina propria of the intestinal tract via the blood stream to protect from intestinal infections but are relevant in pathological inflammatory conditions such as inflammatory bowel disease [17, 22,23,24,25,26,27] and intestinal acute graft-versus-host disease (aGvHD) [28,29,30,31,32,33,34,35,36]. - Enzyme mix for small intestine digestion: 10 ml solution
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