Aim 2: Perfusion of the lymph node with lymphatic and blood endothelial cells
  1. Perfusion of hLN-on-chip with lymphatic and blood endothelial cells by coculture of human dermal lymphatic endothelial cells or umbilical vein endothelial cells (HUVEC) will be achieved by moving to a more complex chip design (Fig 6 below) where the LN stroma can be perfused by a microengineered channel on either side.
    The channels are separated only by a slight constriction (Fig. 6B), thus enabling cell-cell contact between the channels. To mimic in vivo like vasculature, we will use the side channels to culture lymphatic or vascular endothelium either side of the exposed surface of the gel. We have shown that endothelial cells in such channels form an in-vivo like monolayer with barrier function and active transport. Further studies from other laboratories have identified the role of hydrostatic pressure on the growth of lymphatic and vascular vessels. Thus, we will optimize the hydrostatic pressure on the lymphocyte containing gel to perfuse it with microvessels. With the help of collaborators (Esak Lee, postdoctoral researcher in Chris Chen’s lab), we can also induce lymph and vascular neoangiogenesis using cytokine gradients within the lymph node stroma. The major caveat here is donor mismatch which can be addressed by a) HLA typing or b) obtaining matched patient tumor (as source of tumor cells and immune cells for in Aim 3 and endothelial cells) and PBMC. If need be, we may limit our experiments to contact independent interactions where the endothelium is separated from the lymphocytes by a porous membrane (0.4-1uM pores to prevent cellular traffic). However, we will start with endothelial and PBMC samples from different donors as previously data have successfully demonstrated HUVEC and PBMC culture from separate donors (13).
  2. Impact of 3D perfusion with endothelial cells on immune stimulation and checkpoint blockade will be assessed by experiments discussed above in bullet e. Now, we will be able to test how access to lymph node via vessels impacts organization of T and B cell zones, TCR polarization and therapeutic treatment of the lymph node with OKT3, CTLA-4 blockade. Further in new collaboration with pharmaceutical companies we could assess novel targets for antibody mediated cancer immunotherapy such as inhibitory receptors LAG-3, TIM-3 (14) and stimulatory receptors like 4-1BB (15). At this stage, we could also study lymphocyte egress from the hLN-on-chip in response to the perturbations described above; we can collect the effluent to quantify and determine the phenotype of emigrant lymphocytes and visualize the process in situ by microscopy.