*Purpose: Blocking lymphangiogenesis is thought to diminish immune responses to donor tissues. However, lung transplantation (Tx), which involves airway and vascular but not lymphatic anastomoses, is plagued by primary graft dysfunction and acute and chronic allograft rejection, leaving unanswered the importance of lymphoangiogenesis in graft outcomes. We have undertaken a reductionist approach to investigate this issue, by undertaking lymphatic disruption in healed murine lung isografts in which lymphatic reconstitution has already occurred.

*Methods: Using fully MHC disparate BALB/c and C57BL/6 mice, left lungs from triply transgenic donors (mice with a floxed stop codon associated with the diphtheria toxin receptor and VEGFR3, plus tamoxifen-regulated CreERT2) were engrafted into WT recipients treated with conjunction with CD154 mAb/DST plus 28 days of rapamycin (2 mg/kg/d via Alzet pumps). In addition, RAG-/- lung allograft recipients were adoptively transferred with conventional T cells (1×10⁶) alone or plus Foxp3+ Treg cells (2×10⁶).

*Results: Mice treated with costimulation blockade plus RPM showed long-term allograft survival (>150 days). Likewise, Treg cell therapy induced long-term allograft survival (>100 days) in contrast to mice receiving conventional T cells alone (rejection by 14 d). Installation of fluorescent microparticles at 3 weeks post-Tx and subsequent detection in draining pulmonary lymph nodes demonstrated restoration of lymphatic drainage within lung Tx. After 3 weeks, we deleted intrapulmonary lymphatics by administration of tamoxifen and then use of diphtheria toxin. We confirmed lymphatic deletion by lack of fluorescent bead uptake by draining lymphatics, and by staining for lung lymphatic endothelial cells that were identified in control non-diphtheria toxin treated mice lungs as Lyve-1 and Prox1 positive cells. Analysis at 30 days of mice with depletion of intrapulmonary lymphatics showed dense bronchovascular mononuclear cell infiltrates (A3, B1R) despite costimulation blockade and RPM.

*Conclusions: These studies show that pulmonary lymphatics are essential to costimulation blockade induced allograft survival. Ongoing studies are directed towards assessing whether Tregs can still induce allograft survival in the absence of pulmonary lymphatics. Our work highlights how lymphatics are more than simply drain tubes and suggest that they play key roles in leukocyte recirculation, immune surveillance and allograft acceptance.

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