On-chip human lymph node stromal network for evaluating dendritic cell and T-cell trafficking

Introduction:: Lymph nodes are small organs found throughout our bodies that regulate the trafficking of adaptive immune cells, including T-cells. One region of the lymph node known as the paracortex (i.e. the T-cell zone) is made up of a network of mesenchymal stromal cells called fibroblastic reticular cells (FRCs). FRCs deposit an extracellular matrix conduit network and secrete chemokines that recruit T-cells and dendritic cells (DCs) to the paracortex. The FRC and conduit network also serve as a cellular and molecular network which DCs and T-cells adhere to and migrate along. To date, our mechanistic understanding of the paracortex and FRCs has mostly relied on in vivo mouse models, as there are limited in vitro human models of lymph nodes. In the present work, we engineered a multichannel microfluidic system with a 3D stromal network made of human FRCs in a collagen-fibrin hydrogel. The purpose of this study was to evaluate how our engineered lymph node stromal network can model the human paracortex, as well as regulate human DC and T-cell migration.

Materials and Methods:: To engineer our 3D human lymph node stromal network, we utilized fluorescence activated cell sorting to enrich for CD45-CD31-PDPN+ FRCs from commercially available human lymphatic fibroblasts (ScienCellTM). The sorted FRCs were embedded in a collagen I-fibrin composite hydrogel in two different custom designed, multi-channel polydimethylsiloxane devices. The FRCs were then cultured for four days to form a 3D, interconnected cell network. After four days, primary human immune cells isolated or cultured from peripheral blood mononuclear cells were embedded into a neighboring channel in the same collagen-I fibrin hydrogel. The migration of the immune cells into the engineered stromal network was evaluated over three days and compared to the migration of the immune cells toward a blank hydrogel.

Results, Conclusions, and Discussions:: In the collagen I-fibrin hydrogel, the FRCs self-assembled into a 3D, interconnected network and secreted the extracellular matrix proteins entactin, collagen IV, and fibronectin. Furthermore, the FRC network expressed an array of immune cell trafficking chemokines, including CXCL8, IL-6, and CCL2. Although the engineered FRC network did not secrete characteristic CCR7-ligand chemokines (i.e. CCL19 and CCL21), human primary TNF-α matured monocyte-derived dendritic cells, pan T-cells, CD45RA+ T-cells, and CD45RA- T-cells migrated toward the lymph node stromal network to a greater extent than toward a blank hydrogel. The migration of these cells toward the FRC network was likely due to the combined action of multiple chemokines secreted by the FRCs. A fraction of these immune were also found to be in physical contact with the FRC network. Furthermore, the FRCs co-recruited dendritic cells and pan T-cells into the same region of the lymph node stromal network (Fig. 1).

In conclusion, this study has yielded an 3D lymphatic stromal network that can evaluate the ability of TNF-α matured monocyte-derived dendritic cells and primary T-cells to migrate toward an engineered human lymph node paracortex. This engineered lymph node stromal network may help evaluate how human dendritic cells and T-cells migrate into the lymph node paracortex via CCR7-independent mechanisms. Future work will explore how to engineer a human lymph node stromal network that secretes characteristic CCR7-ligand chemokines that are found to be secreted in vivo by lymph nodes.

Acknowledgements (Optional): : This work was supported in part by B.J.K.’s appointment to the Research Participation Program at CBER administered by the Oak Ridge Institute for Science and Education through the U.S. Department of Education and U.S. Food and Drug Administration. This work is also supported by B.J.K.’s appointment to the Interagency Oncology Task Force Fellowship through the National Cancer Institute and the U.S. Food and Drug Administration.

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