*Purpose: Vascular Composite Tissue Allotransplantation (VCA) has become a clinical reality. High frequencies of acute rejections remain an unsolved problem. Here, we delineated components of VCA for their contribution to graft immunogenicity, antigen presentation and early immune responses.

*Methods: Early immune responses following skin and orthotopic hind limb (HL) Tx in allogeneic mouse models dissected the role of antigen presenting cells (APC) with a focus on classical dendritic cells (DC). Using standard depletion protocols, grafts of zDC-DTR (diphteria toxin receptor) transgenic and C57BL/6 wild type (WT) mice, pretreated with DT or clodronate liposomes (CL) for APC/DC depletion, respectively (CL), were transplanted into DBA2 recipients. Grafts from untreated WT mice served as controls. Immune cell phenotyping was performed by flow cytometry in blood, spleen and lymph nodes (LN) of recipients. Intragraft gene expression of relevant cytokines was analyzed by qPCR, serum cytokine levels by Luminex assays. Rejections were assessed serially (POD 1,3,5) based on the BANFF criteria (hair loss, erythema, edema, desquamation, mummification, grading 0-4).

*Results: Both, WT hindlimb and skin grafts showed increased signs of acute rejection by POD 5 (grade 3) compared to DC-depleted HL groups (grade 2). DC counts (CD11c⁺, CD11c⁺MHCII⁺/CD40⁺) were markedly increased in recipients of skin WT grafts compared to HL WT grafts (blood, spleen, LN, POD6, p<0.0001). Depletion of donor DC or APC significantly reduced DC counts in skin graft recipients (p<0.05 vs. WT), but had no impact on DC numbers following HL Tx. Recipients of HL grafts showed significantly higher cDC1 subset counts (CD11b^–B220^–CD11c⁺CD8⁺) compared to skin Tx (blood, spleen, LN, p<0.05); cDC2 subset counts (CD11b⁺CD11c^highMHCII^high) were significantly increased in recipients of skin grafts (p<0.01). Numbers of CD4⁺IL17A⁺ T cells were augmented significantly following skin Tx (p<0.0001 vs. HL) while IL-6, TNF-α, IL-17, and TNF-β protein levels were elevated in WT recipients of HL grafts (p<0.05 vs. skin Tx).

*Conclusions: Early immune responses including DC activation differed significantly between skin and hindlimb transplant recipients. Focusing on DC and Th17 immune responses may provide specific therapeutic targets in VCA transplantation.

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