The Role of Lymph Node Transfer and Lymphangiogenesis in Vascularized Composite Allotransplantation Catherine Tsai1,2, Mai Abd El Hafez1, Radu Olariu2, Anastasia Milusev1, Ioana Lese2, Mihai Constantinescu2, Robert Rieben1, Esther Vögelin2, Adriano Taddeo1,2 1 Department for BioMedical Research, University of Bern, Switzerland, 2 Department of Plastic and Hand Surgery, Inselpital, University Hospital, University of Bern, Switzerland Objectives Methods Results Two surgical groups Vascularized composite allotransplantation (VCA) is an A Effect of lymph node transfer and lymphatic targeting on VCA increasingly performed procedure defined by the D+ D- survival. transfer of multiple tissues as a functional unit. Inguinal LN Inguinal LN N=8 N=8 (A) Graft survival represented with Advanced reconstructive microsurgical techniques are Donor with LN in place Donor with lymphadenectomy Kaplan-Meier survival curves. Recipient with LN in place Recipient with LN in place Rejection was graded used in patients who have suffered severe tissue loss, macroscopically from 0 to 3 (see methods), rats were sacrificed at including limb and hand amputations. The nature of Popliteal LN Popliteal LN these grafts poses several immunological challenges Grade 3 rejection. No significant B difference in survival between the requiring lifelong immunosuppression to prevent graft Experimental timeline two groups was found. (B) Rejection-free period (i.e., time rejection. The lymphatic system is involved in Daily lymphography with VisionSense near-infrared scanner after indocyanine green (ICG) injection before showing first signs of modulating inflammation, autoimmune disease, and rejection, reaching grade 1) was organ immune-rejection processes. The lymph node shorter in the D- group, POD 0: POD 2 POD 7: represented with Kaplan-Meier (LN) is the first site of contact between donor and Hindlimb Daily lymphography Blood collection for Endpoint at Grade 3 survival curves. * p<0.05 calculated transplantation FACS (cell sorting) rejection: by Mantel-Cox test. recipient immune cells and plays an important role in Lymphatic Sample collection: blood, Effect of lymph node transfer and lymphatic tracts spleen, thymus, transplant skin, ICG v targeting on peripheral blood chimerism and inducing peripheral tolerance after transplantation. contralateral skin (control), Characterization of the transplant LN, contralateral circulating Treg number, POD7. (control) LN, bone marrow, lymphangiogenic process. However the role of lymphatic reconstitution after VCA Quantitative summaries for the absolute number Ventral view for FACS (cell sorting) has not previously been studied. The objective of this When lymphangiogenesis was (expressed as cell/µL of blood) of donor leukocytes study is to assess the impact of regional donor LN and observed, it occurred earlier in the D+ (CD45+RT1Ac+ cells), donor monocytes Rejection grade scoring Lymphography group compared to the LN- group (CD45+RT1Ac+CD4+CD3-), and of the frequency of Crossover of ICG (mean 3.5 days vs 5 days, + + + + + lymphangiogenesis on graft survival after VCA in a rat Treg (CD45 CD3 CD4 CD25 FoxP3 ) among the Th respectively). Lymphangiogenesis model of limb transplantation. Suture line cells and the absolute number of donor Treg occurred significantly earlier than grade (CD45+CD3+CD4+CD25+FoxP3+RT1Ac+). There were 3 rejection for the D+ group (p=0.002, significantly more monocytes, total and donor Tregs in but not significantly earlier than grade 1 the D+ group compared to the D- group. *p<0.05 Methods ICG dye rejection. * p<0.05 calculated by calculated by unpaired parametric student t-test. Mantel-Cox test. Grade 0 Grade 1 Grade 2 Grade 3 Ventral view transplant limb No rejection Edema, erythema Epidermolysis, Desquamation, 16 orthotopic Brown-Norway to Lewis rat hind-limb transplantations exudation necrosis, Lymphatic reconstitution= crossover mummification of ICG across the suture line Brown-Norway Lewis Conclusion Flow Cytometry Cell Sorting (FACS) gating strategy Viable cells Single Single Leukocytes Donor LN transfer within a VCA promotes faster lymphangiogenesis as well as increased regulatory cells cells Height SSC-A SSC-H T cells and monocyte chimerism in the recipient. Furthermore, lymphography can be a potential Viability Viability method to detect rejection before it macroscopoically occurs. These results underline the potential of A total of 16 Brown-Norway to Lewis rats hind-limb transplantations FSC-A Width SSC-A FSC-A CD45 specifically targeting the LN component of a VCA to influence graft rejection. were performed; 8 received grafts with popliteal and inguinal LN Helper T cells Regulatory T cells (Group LN+) and 8 received grafts depleted of all LN (Group LN-). Donor regulatory CD4 FoxP3 CD4 T cells This project was funded by the US Department of Defense (DoD) Contact: Catherine Tsai, [email protected] CD3 CD25 RT1Ac RT1Ac: Marker for Brown-Norway (donor) cells.