OBJECTIVES/SPECIFIC AIMS: Objective: To summarize the diagnosis and management of two acute rejection (AR) episodes in the first penis transplant patient in the U.S. Background: Vascularized composite allotransplantation (VCA) has been utilized for state-of-the-art reconstruction of devastating craniofacial defects, limb loss, and recently, severe genitourinary defects. To date, more than 200 VCA’s have been performed, of which four successful penis transplants have been achieved worldwide (Two in the U.S.). However, despite the technical success of VCAs in general, acute rejection episodes remain a significant postoperative management problem, with 80-85% experiencing at least one episode in the first-year post-transplantation. The incorporation of skin in VCAs, which is highly immunogenic, allows early visible recognition of rejection but requires prompt management to prevent allograft failure as well as the progression of chronic rejection, which has been associated with the frequency of acute rejection episodes in preclinical models. We present the first report of acute rejection in a penile allograft. AR episodes in VCA typically manifest with erythema of the allograft skin and/or maculopapular lesions that can be either patchy, focal or diffuse. Histopathologic assessment is essential for diagnosis and management. The Banff classification of histopathologic criteria for degree of AR is most commonly utilized to direct clinical management. METHODS/STUDY POPULATION: We reviewed the clinical course of the first American patient who underwent penis transplantation at Massachusetts General Hospital in 2016. Postoperatively, routine clinical and chemical assessment (immunosuppression levels, routine blood work) was performed, with increased frequency during AR episodes. Skin punch biopsies were obtained during (suspected) AR episodes, analyzed and graded according to the Banff 2007 classification of rejection of skin-containing composite allografts. Histopathologic tissue assessment included CD3, C4d, CD4/8, CD20 FOXP3 and cellular infiltration (hyper keratinization, lymphocytic infiltrate, dermal erosion, macrophage, eosinophilia, T-cell infiltration) and epidermal or perivascular fibrosis. RESULTS/ANTICIPATED RESULTS: The patient is a 65-year-old male with history of penile carcinoma requiring subtotal penectomy in 2012. He is currently 30-months post penile transplantation (as of 11/15/2018). First Rejection Episode: At 28 days post-transplantation, the patient noted induration, swelling and erythema of the allograft, which was diagnosed as AR clinically (Image 1A). Biopsy showed a Banff Grade III AR, with focal keratinocyte apoptosis with lymphocytic infiltration in epidermis and arteriolar endothelialitis with perivascular inflammation. Initially this episode was treated for 2 days with 2 pulse doses of methylprednisolone (500mg/d IV) with clinical improvement. However, recurrent allograft erythema was observed on postoperative day 32 and an acute rejection grade III according the Banff classification was confirmed by a second biopsy that demonstrated epidermal perivascular lymphocytic infiltrates, spongiosis and dyskeratosis, deep dermis focal lymphocytic infiltrates and focal infiltrates in arterioles as well as endothelialitis in venules. Donor specific antibodies and C4d were negative. CD3+ T cells were present in the epidermis and perivascular space. This was treated with anti-thymocyte globulin (thymoglobulin) course for 4 days (1.5mg/kg/day IV) and 3 more pulse doses of methylprednisolone (500mg/d IV.), followed by a prednisone (250mg/d) taper to baseline. This resulted in complete resolution of AR. Second Rejection Episode: At 10.8 months post VCA the patient presented with penile erythema and scrotal swelling suggestive of AR and received three doses of methylprednisolone (day 1: 500mg/d IV, day 2: 1000mg/d IV and day 3: 500mg/d IV respectively) followed by increased baseline prednisone (10mg PO daily; increased dose compared to previous AR episode). A skin biopsy confirmed Banff Grade III AR. Compared to the previous biopsy, this biopsy demonstrated an increased density of lymphocytic inflammation of the dermis with endarteritis. Prominent involvement of epidermis and adnexal structures corresponding to acute T-cell mediated rejection was also observed (Figure 1). Donor specific antibodies and C4d were again negative. Three doses of ATG (1.5mg/kg/day IV) were administered. In addition, tacrolimus was increased and local tacrolimus (1% ointment) treatment was begun. Clinical signs of rejection improved and repeat biopsy showed dramatic histopathological improvement. Current maintenance immunosuppressive regimen consists of tacrolimus, sirolimus, prednisone, mycophenolic mofetil acid (MMF), rapamycin, and tacrolimus ointment, with no new clinical or histopathological signs of rejection (Image 1B). DISCUSSION/SIGNIFICANCE OF IMPACT: We report the first described case of acute T-cell mediated rejection in penile transplantation. These rejection episodes demonstrated that, even on stringent immunosuppressive regimens, severe acute rejection episodes in VCA may still occur. Edema and acute induration preceded the development of erythema in our cases, representing a harbinger for the more severe grade of rejection that eventually developed. Our experience was consistent with other VCAs in that donor specific antibodies did not develop, despite a severe Banff Grade. Consistent use of topical calcineurin inhibitor based immunosuppression on the allograft skin may be helpful in warding off future episodes, as our patient has been rejection free now for 18 months. To date, no histologic signs of chronic rejection were present on 2-year protocol surveillance biopsy. We have added rapamycin to the current drug regimen, with concurrent reduction of tacrolimus dosing for renal protection, which has been demonstrated in cardiac transplantation to deter the intimal hyperplasia/vasculopathy associated with chronic rejection.