FIGURE 6.

GFP + cell homing in serial transplants reveals preference for organ of origin that is lost in liver-derived cells that have been educated in the spleen. (A) Experimental strategy displayed as a cartoon depicting serial liver and spleen transplantation to quantify GFP cell homing bias. Liver-derived GFP⁺ cells from four individual animals were adoptively transferred into four separate non-GFP hosts (round one transfer). Seven days post transfer GFP⁺ cells were collected from livers and spleens in parallel (i.e., four liver-liver and four liver-spleen) These parallel GFP⁺ cell preparations were then adoptively transferred into one naïve host recipient per cell preparation (round 2 transfer). After another 7 days, both liver and spleens were isolated and profiled as paired samples (i.e., one liver and one spleen per host) by flow cytometry. (B) Spleen-derived GFP⁺ cells were tested as described for (A) using spleens from four GFP + animals as original donor tissue. (i.e., four spleens −>4 spleen-spleen + 4 spleen-liver transfers). (C) Flow cytometry cell counting of terminal organs containing GFP⁺ cells from serially transplanted organs. The ratio between terminal livers and spleens was quantified per recipient with a value of 1 indicating an equal preference of GFP⁺ cells for liver or spleen from round 2 adoptive transfers. Values higher than 1 indicate a preference for homing to host liver and values lower than 1 indicating a preference for homing to host spleen tissue. Only liver-derived GFP⁺ cells isolated from host spleen (condition 2, liver-spleen, indicated with blue arrow) show education that allows equal migration to liver/spleen from round two adoptive transfers. Bar graph showing the ratio of GFP cells in the liver and spleen from serial transplant experiments. Error bars represent mean ± SEM of four biological samples. (D,E) Intravenously transplanted GFP⁺ cells from the liver and spleen display differential leukocyte trafficking to host lymphoid organs with a preference for the tissue of origin. Spleen-derived GFP⁺ cells also show robust homing to thymic nodes. Representative ex vivo images of host lymphoid organs four days after transplantation of 1–5 × 10⁵ liver or spleen leukocytes. N = 6 host recipients in each transplant group.