Donor and recipient pigs used in this study were genotyped for their swine lymphocyte antigen (SLA) class I and II haplotypes by running low-resolution PCR screening assays on ear punch biopsies (sows) and testicular tissues (boars). Genomic DNA was isolated using commercial kits following the manufacturer’s instructions (QIAamp DNA Micro Kit, Qiagen, Hilden, Germany; E.Z.N.A. Tissue DNA Kit, Omega Bio-tek, Inc., Norcross, GA, USA). SLA class I (SLA-1, SLA-2, and SLA-3) and SLA class II (DRB1, DQB1 and DQA) low-resolution haplotypes were identified by a sequence-specific primed PCR-based typing assay. The criteria and nomenclature used for SLA class I and SLA class II haplotyping were based on those proposed by the international SLA ISAG/IUIS-VIC nomenclature Committee in the IPD-MHC database of suids (www.ebi.ac.uk/ipd/mhc/group/SLA) (43, 44).

TGMS-TAC was prepared as described previously (12). Briefly TGMS (AK Scientific, Union City CA, USA) and TAC (R&S Pharmchem Co., Shanghai, China) were dissolved in dimethyl sulfoxide (DMSO; Merck, Darmstadt, Germany). After complete dissolution, preheated MilliQ water (ThermoFisher Scientific, Waltham, MA, USA) was added to obtain the final formulation with a TAC concentration of 7 mg/mL and 20% v/v DMSO. TGMS TAC was subsequently transferred into Fisherbrand Luer-Lock sterile syringes (ThermoFisher). The formulation was tested for the presence of endotoxins using a Pierce Chromogenic Endotoxin Quant Kit (ThermoFisher). TGMS-TAC was prepared using endotoxin-free water and then tested according to the manufacturer’s instructions using 405 nm absorbance measured in a microplate reader (Tecan, Männedorf, Switzerland). TGMS-TAC enzyme-responsive drug release was measured as previously described. Briefly, 200 mL of TGMS-TAC hydrogel (TAC7 mg/mL) were diluted 1:4 in PBS. The dialysis bag (Float-A-Lyzer G2, Spectropore, molecular weight cutoff 8–10 kDa) was placed into a Falcon tube filled with PBS and pre-heated to 37°C. The tubes were kept at 37°C under horizontal shaking (50 rpm) for the whole duration of the release study. At different time points, aliquots were withdrawn for further analysis and the complete release medium replaced with fresh PBS. As an enzymatic challenge, on day 28 a volume of 100 mL of lipase type II solution (258 mg/mL) from porcine pancreas (SIGMA, Ref: SLCD5418, activity 388 units/mg) was added to these diluted samples and, at different time points, the TAC concentration was analyzed by HPLC.

All experimental procedures were approved by the Veterinary Office of the Canton of Bern (approval number: BE48/19), and animals were treated according to the Animal Welfare Act and Ordinance of the Swiss Animal Welfare Legislation during the entire experiment. Experimental protocols were refined according to 3R principles. Males and females were included in both donor and recipient groups. Animal well-being was regularly assessed by both scientists and board-certified veterinarians using a pre-designed score sheet for health and behavior appraisal to minimize any potential suffering and a clear guideline of early termination criteria.

MHC-mismatched Swiss landrace pigs, aged 11–14 weeks, underwent a modified osteomyocutaneous flap allotransplant as a representative model of VCA (45). Briefly, a graft weighing approximately 800 grams, comprised of vascularized skin, muscles, knee joint, distal femur, and proximal tibia, was placed in the flank of a recipient pig, where the skin island of about 12x8 cm was exteriorized for graft monitoring purposes ( Figure 1A ). Before transplantation, the graft was weighed to determine the necessary dose of immunosuppressive medication. As previously described (46), a port-a-cath (Braun, Melsungen, Germany) central venous catheter was positioned during the surgical procedure in the external jugular vein with the port placed subcutaneously in the posterior neck region, in order to facilitate postoperative venous blood sampling without the need for sedating the animal for manipulation. Subsequently, pigs were allocated into three groups: group 1 (untreated), where no immunosuppressive therapy was given; group 2 (TGMS-TAC), where TGMS-TAC was given at a dose of 140 mg per kg of graft weight only at postoperative day (POD) 0 immediately after the procedure; and group 3 (TGMS-TAC (R)), where TGMS-TAC was injected at the same dose as in group 2 at PODs 0, 30 and 60. Evenly distributed 0.5 mL depots of TGMS-TAC were injected subcutaneously into the skin of the graft. All animals were followed up until either grade III rejection or POD 90 was reached. A 30-day dosing interval, as well as TGMS-TAC dose per kg of graft weight, was estimated based on the findings of tacrolimus tissue concentration after intra-graft TGMS-TAC therapy associated with graft rejection changes from prior published rat and porcine hind limb studies (13, 14). After transplantation, tissue and blood samples were collected at defined time points ( Figure 1B ).

Macroscopic changes characteristics of graft rejection were assessed in the exteriorized skin of the VCA graft by two blinded investigators and were graded as previously described as 0 = no difference between graft skin and native skin, I = mild erythema, II = moderate erythema with the beginning of scaling and scabbing, III = severe erythema and scabbing with areas of epidermolysis, IV = full-thickness graft epidermolysis with areas of necrosis (47).

For histopathological analyses, skin and muscle samples collected from the graft at endpoint were fixed in 4% paraformaldehyde (PFA), sectioned, and stained with Hematoxylin and Eosin (H&E). Images were acquired using a Pannoramic 250 Flash III microscope (3D Histech, Budapest, Hungary) with the 10x objective. Skin rejection was assessed and classified using the Banff 2007 scoring for skin-containing composite tissue allografts (48). Muscle samples were analyzed for the presence of necrosis and/or atrophy and inflammatory infiltrates. A four-tiered system was used depicting the severity of changes from none (0), minimal (1) to moderate (2), and finally to extensive (3).

TAC levels were measured systemically (from blood) at baseline, POD1, 3, 7, and then weekly until endpoint. TAC levels in tissue were measured in skin biopsies collected from both the graft and contralateral skin at baseline, POD7, and then every 2 weeks until endpoint.

For systemic TAC levels, peripheral blood was collected from the vascular access port in EDTA-coated tubes (Sarstedt, Nümbrecht, Germany) and immediately processed. TAC concentrations in blood were assessed using the MS1100 Kit (ClinMass Complete Kit for Immunosuppressants in Whole Blood, RECIPE Chemicals + Instruments GmbH, Munich, Germany) and quantified by Ultra-High Performance Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS).

Skin biopsies, 5 mm size, from the graft and contralateral side were excised, weighed, snap-frozen, and stored at -80°C until further use. Care was taken to ensure that each skin sample was taken from a different region of the skin island over time. Local concentrations of TAC in the graft and contralateral side were measured from skin biopsies. Samples were processed and analyzed by LC-MS/MS as previously described (13, 14), where a modification in the calibration curve range was set to 30ng/mL, and the capillary and the cone voltages were set at 3.2 kV and 40 V, respectively.

To measure off-target toxicity, markers of liver and kidney function were analyzed from whole blood collected at baseline and then weekly until endpoint. Analyzed markers included cholesterol, triglyceride, creatinine, alanine aminotransferase, and aspartate aminotransferase. Paraffin-embedded kidney and liver samples retrieved from the animals at endpoint were sectioned and H&E stained for histopathological assessment of TAC-related changes by a blinded pathologist and compared to naive, age-matched landrace pigs. H&E slides were scanned using a Pannoramic 250 Flash III microscope (3D Histech, Budapest, Hungary) with the 10x objective.

A complete blood count was performed using EDTA-blood and an ADVIA 2120i analyzer (Siemens Healthcare AG, Zurich, Switzerland) running multispecies software (version 6.3.2-MS).

Peripheral blood from the vascular access port was collected in EDTA tubes and promptly centrifuged for plasma storage and peripheral blood mononuclear cells (PBMCs) collection. PBMCs were isolated using a sterile density gradient medium (Ficoll-Paque PLUS, MERCK, REF: 17144002) and stored at -150°C until use.

Circulating T cells were quantified from the previously isolated PBMCs through flow cytometry. Briefly, after washing with FACS buffer (PBS + 2% FBS) cells were incubated for 30 minutes with LIVE/DEAD Fixable Yellow Dead Cell Stain Kit 405nm. (ThermoFisher, Ref: L34959). Next, non-specific binding was blocked using FcR Blocking Reagent (MACS Ref 130–059-901). Subsequently, cells were incubated for 30 minutes with the following labeled antibodies: CD3-FITC (Clone PPT3. Bio-Rad, Ref: MCA5951F), CD3-RPE (Clone PPT3. Bio-Rad, Ref: MCA5951PE), CD4-PE Cy5 (Clone 74–12-4, ThermoFisher, Ref: MA5–28734), CD8b – FITC (Clone PPT23. Bio-Rad, Ref: MCA5954F). For intranuclear staining, cells were fixed, permeabilized, and washed using a FoxP3/Transcription Factor Staining Buffer set (eBioscience, Ref: 00–5523-00) and incubated 30 min with an anti-FoxP3 eFluor 450 antibody (Clone FJK-16s. ThermoFisher, Ref: 48–5773-82). Cells were acquired using a CytoFLEX S cytometer equipped with CytExpert Software (Beckman Coulter Life Science, California, United States). Data were analyzed using Flow-Jo software (Tri-Star, Ashland, United States).

Plasma levels of C3a were measured by ELISA using a porcine C3a ELISA kit (MyBioSource, Ref MBS2509360) according to the manufacturer’s instructions. Absorbance values were measured at 450 nm using a Varioskan LUX multimode microplate reader (ThermoFisher).

Cytokine levels were measured from EDTA plasma and skin samples collected at different time points. For tissue protein extraction, skin samples were weighed and manually homogenized in RIPA lysis and extraction Buffer (ThermoFisher Scientific, Ref: 89900) supplemented with protease inhibitors (Halt, ThermoFisher, Ref: 1861280). Samples were further lysed using a TissueLyser II (Qiagen), 2 cycles of 30 seconds at 20 Hz. After centrifugation, the supernatant was collected and stored at -20°C until used. A Luminex multiplex assay (MILLIPLEX Porcine Cytokine/Chemokine Magnetic Bead Panel. MERCK, Ref: PCYTMG-23K-13PX) was performed on tissue lysates and plasma collected at baseline and endpoint for the following markers: IFN-γ, IL-1α, IL-1β, IL-1ra, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, and TNF-α.

Skin and muscle samples collected at endpoint were embedded in TissueTec - O.C.T., (Sakura Finetek, Alphen aan den Rijn, The Netherlands) and cryostat sectioned at 5 µm. Tissue slides were stained with DAPI (4’,6-diamidino-2-phenylindole. Invitrogen, Ref: D1306) and the following antibodies: CD31 (Clone 377537. R&D system, Ref: MAB33871), CD3 (Clone PPT3. Biotech, Ref: 4510–13), C3b/c - FITC (Agilent Dako – Ref: F0201), Myeloperoxidase (Clone 2C7. Abcam, Ref: ab25989), H4Cit (Millipore, Ref: 07–596). The following secondary antibodies were used: goat anti-rat Alexa Fluor 680 (Invitrogen, Ref: A21096), donkey anti-rabbit Alexa Fluor 488 (Invitrogen, Ref: A32790), donkey anti-mouse Alexa Fluor 568 (Invitrogen, Ref: A10037). Slides were imaged using confocal microscopy (20x objective, Zeiss LSM 980, Zen Blue software), and processed using ImageJ software (https://imagej.nih.gov/ij/). For quantification of infiltrating T cells in tissue, the percentage of CD3+ cells was calculated as [number of CD3+ cells/total number of cells per field] x 100, from 5 representative immunofluorescence images/sample/pig.

Neutrophils in allografts were assessed by western-blot analysis. Graft skin protein extractions were performed as described above and quantified using the Pierce Coomassie (Bradford) Protein Assay Kit (ThermoFisher, Ref: 23200). Equal amounts of protein (50 µg) were loaded and electrophoretically resolved using Bolt 4–12%, Bis-Tris gels (Invitrogen, Ref: NW04120BOX). Proteins were then transferred to PVDF membranes (Invitrogen, Ref: IB24001). Membranes were blocked using Intercept Blocking buffer (LI-COR, Ref: 927–70001), and then incubated with a primary antibody against MPO (Clone 2C7, Abcam, Ref: ab25989) followed by a goat anti-mouse, IRDye 800cW secondary antibody (LI-COR, Ref: 926–32210). Blots were imaged using the Odyssey Imaging System 9120 (LI-COR Biosciences, Nebraska, United States). An antibody against beta-actin (LI-COR, Ref: 926–42212) was used to assess sample loading. Bands were quantified using ImageJ software (https://imagej.nih.gov/ij/) by dividing the mean intensity value of the band of interest by the intensity of the loading control. The amount of MPO in allograft skin samples was calculated as a fold-change compared to the amount found in healthy naive tissue.

DNA-MPO complexes were measured by ELISA (49, 50) in EDTA-plasma and in graft skin lysate obtained as previously described. In brief, Nunc MaxiSorp plates were coated with 5 mg/mL of anti-myeloperoxidase (MPO) antibody (Clone 4A4. Bio-Rad, Ref: 0400–0002). Prior to sample addition, wells were blocked with 5% BSA for 2 hours. To increase assay sensitivity, sample NETs were digested using 2 U/mL of DNAse I (Sigma, DN25) for 15 minutes, after which 2.5 mM of EDTA was added to stop the digestion. Samples were incubated with a peroxidase-conjugated anti-DNA antibody from an ELISA kit for cell death detection (Roche, Ref: 11544675001) for 2 hours. After washing, TMB substrate (3,3’,5,5’-tetramethylbenzidine. ThermoFisher, Ref: N301) was added to each well and incubated for a maximum of 20 minutes. The reaction was stopped using 0.5M sulfuric acid. Complexes were quantified as optical density values at 405 nm using a Varioskan LUX multimode microplate reader. DNA-MPO levels were calculated as fold-change with respect to levels measured in pooled plasma or skin lysates obtained from 8 naive healthy pigs.

Porcine and human neutrophils were both isolated from EDTA-blood from healthy individuals by density centrifugation with Histopaque-1119 (Merck, Ref: 11191) and density-gradient centrifugation with Percoll (Merck, Ref: P4937). The purity of isolated neutrophils was confirmed using the Sysmex KX21N Hematology Analyzer (Sysmex Corporation. Hyogo, Japan). To assess the effect of TAC on NET formation, neutrophils were cultured in RPMI 1640 medium (Gibco, Ref: 72400047) and stimulated with 5 µM ionomycin (ThermoFisher, Ref: I24222) in the presence of different concentrations (0 - 100 ng/mL) of TAC (R&S Pharmchem, Ref: 104987–11-3). To identify NETs, cellular DNA was stained with DAPI and then visualized using confocal microscopy (Zeiss LSM 980). NETs were defined morphologically as neutrophils with extruded DNA content in a web-like structure and quantified as percentages using [number of NETs + neutrophils/total neutrophils per field] x 100, from 5 representative images/sample.

Inter-group t-tests and analyses of variance (ANOVA) were performed using Prism software (GraphPad, La Jolla, CA, United States). The following symbols represent statistical significance based on p values determined by the specific group tests described in each figure legend: *p<0.05; **p<0.01; ***p<0.001; and ****p<0.0001.

To evaluate the effect of TGMS-TAC on VCA in a clinically relevant setting, a series of heterotopic limb allotransplantations were performed in Swiss landrace pigs ( Figure 1A ). Donor and recipient pigs were selected based on their Swine Leukocyte Antigen (SLA) typing to achieve a constant mismatch of 8–10 out of 12 alleles within and between groups ( Supplementary Figure S1 ). TGMS-TAC is an enzyme-responsive hydrogel, where TAC release is boosted by the presence of proteolytic enzymes such as matrix metalloproteinases (MMPs) and lipase, which are upregulated in tissue during inflammation ( Supplementary Figure S2A ). We tested the inflammation-based delivery profile of TGMS-TAC and, as shown in Supplementary Figure S2B , found a large cumulative increase in TAC release upon the addition of lipase. Next, to assess the in-vivo stability of the hydrogel after intra-graft injection, a syngeneic transplant (n = 1, MHC-mismatch 0/12), where no immunological challenge is expected, was performed. After transplantation, the recipient animal received a single dose of TGMS-TAC and was then followed until either postoperative day (POD) 90 or grade III rejection. As expected, the syngeneic transplanted animal reached 90 days of rejection-free graft survival. Ultrasound evaluation of the graft 30 days after injection of the hydrogel confirmed the presence of multiple intra-graft depots of TGMS-TAC ( Supplementary Figure S2C ), indicating that without an inflammatory environment, the hydrogel is stable, and TAC is not significantly released. In addition, no TAC levels were observed in circulation (data not shown).

Having established the delivery system for TGMS-TAC, the multiple MHC-mismatched transplanted animals were either untreated (group 1), received a single dose of TGMS-TAC at POD 0 (group 2), or received multiple doses of TGMS-TAC (R) at 30-day intervals starting from POD 0 (group 3; Figure 1B ). Recipient pigs were then followed until macroscopic grade III graft rejection or POD 90. As shown in Figure 2A , a single dose of TGMS-TAC (group 2, n=4) was sufficient to increase the median survival time (MST) to 45 days, compared to the untreated group (group 1, n = 4), which had an MST of 7 days. Monitoring of signs of rejection is depicted in Figures 2B, C .

Although treatment prolonged graft survival, Banff histopathological evaluation of skin graft samples collected at endpoint confirmed end-stage graft rejection for all pigs in both groups ( Figures 2B–D ). Graft muscle tissue isolated from pigs injected with TGMS-TAC was less damaged ( Figure 2D , panel 2), showing muscle damage scores ranging from 0–2, compared to scores of 3 from muscles obtained from the untreated group. These muscle grafts from the untreated control group showed extensive damage, including necrosis and subtotal atrophy with moderate inflammation, whereas muscle samples from the TGMS-TAC treated groups only exhibited lipomatosis with mild to moderate perivascular infiltration of inflammatory cells.

Interestingly, all pigs treated with multiple TGMS-TAC injections (30-day intervals, TGMS-TAC (R) group, n = 4) reached the experimental endpoint of POD 90 ( Figure 2A ). However, while graft survival was prolonged, the grafts still showed macroscopic signs of rejection at the POD 90 endpoint ( Figures 2B, C ). Three grafts had Banff-skin scores of 2 showing moderate perivascular inflammation and mild epidermolysis, and one graft had extensive necrosis characteristic of Banff grade 4. As seen in Figure 2D , histologically, muscle-tissue samples of the TGMS-TAC (R) group had a similar profile: three of the grafts showed minimal perivascular-accentuated lymphohistiocytic infiltration, corresponding to a muscle rejection score of 2, and one exhibited extensive acute inflammatory infiltrate and necrosis with a muscle damage score of 3.

Our hypothesis was that local TGMS-TAC administration would both allow a higher intra-graft concentration of TAC and avoid off-target toxicity due to its low systemic levels. We, therefore, assessed the distribution profile of TAC in both tissue and whole blood using Ultra-High Performance Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS). As illustrated in Figure 3A , the levels of tacrolimus in the skin of TGMS-TAC treated animals were notably elevated, ranging from 4- to 12-fold higher in the injected grafts compared to the levels detected in the contralateral skin. This substantial difference strongly suggests a localized release of tacrolimus at the site of injection, underscoring the effectiveness of TGMS-TAC to specifically deliver TAC to the desired area. Skin tissue levels of TAC measured over time revealed that the release peaked around POD 7 after the injection, corresponding to the time where rejection occurs in the untreated animals, further suggesting the inflammatory-based release profile. After POD 7, TAC levels gradually decreased, becoming undetectable by POD 28 (4 weeks after the initial injection). Pigs of the TGMS-TAC (R) group that received multiple injections of TGMS-TAC showed a similar profile ( Figure 3A ). A peak in TAC levels was observed 1 - 2 weeks after injection (POD 7, 37, 67) and then was non-detectable after 4 weeks (POD28 and POD56). These findings highlight that multiple TGMS-TAC injections are necessary to maintain appropriate drug levels in the VCA grafts.

We further evaluated the blood levels of TAC. As shown in Figure 3B , TAC blood levels corresponded to the profile found in graft skin tissue. Similar to what other DDS described (14, 21, 23), an initial burst release was observed within 7 days after the TGMS-TAC injection, which then progressively declined, reaching subtherapeutic levels 2 weeks after injection (<10 ng/mL). Unlike in the skin, blood TAC levels were higher than therapeutic target concentrations (>20ng/mL) from POD 1 to POD 7. This burst release of TAC was not observed with the subsequent TGMS-TAC administrations at POD 30 and POD 60 in the TGMS-TAC (R) group, where blood TAC levels were always below 20 ng/ml ( Figure 3B ). This finding suggests that the first systemic TAC peak was probably caused by the excess inflammation induced by the surgical procedure.

To determine whether the transiently high concentration of TAC observed in the blood (>20 ng/mL) might cause off-target toxicity, plasma levels of ALAT, ASAT, triglyceride, cholesterol, and creatinine were measured ( Figures 4A-E ). While no effect on liver biomarkers was observed ( Figures 4A-D ), pigs of the TGMS-TAC (R) group, treated with multiple TGMS-TAC injections, showed minor elevations in creatinine ( Figure 4E ). However, these higher levels did not correspond to kidney damage. As shown in Figure 4F , histological evaluation of kidney sections revealed that glomeruli and tubules in cortical and medullary areas appeared normal compared to kidney biopsies from healthy naive pigs. Furthermore, no infiltration of inflammatory cells or signs of tubular damage were observed. As shown in Figure 4F , histological liver sections also confirmed the absence of hepatic injury. Lobular hepatic parenchyma and portal tracts were of regular appearance, and no signs of hepatocellular degeneration or damage to the biliary epithelium or sinusoidal dilation were detected.

To better understand the role of intragraft tacrolimus administration in VCA, we evaluated T cells, neutrophils, complement, and inflammatory cytokines at endpoint both systemically in plasma samples, and locally in skin from grafts of treated and untreated animals. In VCA, skin is considered the most immunogenic component (51, 52), and was therefore chosen to analyze graft survival and/or rejection. Complement is known to participate in the immunological rejection of transplanted organs. Once activated, complement proteolytic fragments can directly mediate tissue injury and can also be important stimuli for tissue-resident immune cells (e.g., neutrophils and B cells) to produce chemokines and other inflammatory mediators that result in graft rejection (53). Since different activation pathways of the complement cascade merge at the complement protein C3, its C3a and C3b/c proteolytic fragments, were measured respectively in plasma and tissue. Surprisingly, no increase in C3a was observed in plasma samples at endpoint compared to baseline ( Figure 5A ). An average of 350 ng/mL of C3a was found in plasma samples from all groups. In contrast, C3b/c deposition was observed in all skin samples from rejected grafts ( Figure 5B ). Skin samples of all TAC-treated animals showed more prominent C3b/c staining when compared to grafts from untreated animals. It is known that tacrolimus activates complement during SOT (54), therefore, the increase of C3b/c deposition in rejected skin tissues of tacrolimus-treated animals is expected.

Next, as T cells are one of the major players in graft rejection, subsets of circulating T-cells (i.e., T-helper, T-cytotoxic, and T-reg cells) were measured using flow cytometry. The gating strategies used for identifying the different T-cell subsets are shown in Supplementary Figures S3A, B . Interestingly, the frequency of the different T-cell subsets did not change at rejection compared to baseline. As shown in Figures 6A–C , TAC treatment did not alter the different T-cell subtypes. Similar amounts of T-helper ( Figure 6A ), T-cytotoxic ( Figure 6B ) and T-reg cells ( Figure 6C ) were found in all TAC-treated and untreated pigs. Among the subtypes, T-helper cells were found to be most abundant, with an average frequency of approximately 40%, followed by 15% T-cytotoxic cells and 2–4% T-reg cells in both TAC treated and untreated animals ( Supplementary Figures S3C-E ).

Since no difference in T-cell subsets was observed systemically, we explored whether local intra-graft TAC treatment would alter T-cell infiltration. A significant increase in T-cell infiltration (denoted as CD3+ cells) was seen in skin samples at rejection ( Figure 6D ). Interestingly, local treatment with TAC led to a 50% reduction of the amount of infiltrating T cells compared to untreated pigs. However, a 10% to 15% level of T-cell infiltration was still observed ( Figure 6E ) in the single- as well as multiple TGMS-TAC-treated pigs. This suggests that although intra-graft TGMS-TAC dampens local T-cell infiltration, it is not sufficient to completely prevent rejection by itself.

Next, we measured the plasma concentrations of various T-cell-derived cytokines (e.g., IFN-γ, IL-1β, IL-4, and TNF-α). Again, no differences in cytokine concentrations were identified at endpoint vs. baseline ( Supplementary Figure S4 ), nor after treatment with TAC. Only the anti-inflammatory cytokine lL-1ra was found to increase in TGMS-TAC (R) treated pigs.

Taken together, these data indicate that although tacrolimus treatment delays rejection by dampening local T-cell infiltration, this is not sufficient to completely avoid rejection, suggesting that other cells might be involved.

As neutrophils are also known to be involved in SOT rejection (36), we investigated their role in our VCA animal model. Similar to T cells, neutrophil levels in plasma did not increase at rejection ( Figure 7A ), nor did TGMS-TAC treatment influence neutrophil blood counts.

Since both complement activation and T-cell infiltration changes were mainly observed locally in rejected grafts, we analyzed neutrophil infiltration in rejected skin tissues. As shown in Figures 7B, C , neutrophils, identified as protein levels of MPO by western blot, were found in rejected skin grafts from all untreated animals. As observed in Figure 7C , MPO levels were significantly reduced in rejected graft skin lysates from animals treated with TAC.

During inflammation, neutrophils can release extracellular traps, a meshwork of decondensed DNA associated with neutrophilic proteins like MPO and citrullinated histones (37, 38). We, therefore, assessed the presence of NETs in skin of rejected grafts. NETs were visualized in all rejected grafts as DNA structures co-localizing with MPO and citrullinated Histone 4 (H4Cit). As shown in Figures 7D–G , NETs were less abundant in skin grafts after local treatment with TAC ( Figures 7F, G ) compared to grafts from untreated animals ( Figure 7E ). Next, NETs were quantified as DNA-MPO complexes by an in-house developed ELISA. ( Figure 7H ). A 40% to 60% decrease in NETs was detected in rejected skin tissue isolated from TAC-treated grafts compared to the untreated group. This indicates that TAC not only inhibited T-cell infiltration but also influenced neutrophil responses during rejection. NETs were also measured in plasma samples; however, in agreement with our previous findings that the events leading to graft rejection occur mainly locally, no NETs were detected ( Figure 7I ).

As TAC appears to influence neutrophil responses during rejection, we also analyzed the effect of TAC on NET formation (NETosis) in-vitro. Porcine peripheral neutrophils were activated with ionomycin alone (a calcium salt known to induce NETosis) or after pre-incubation with different concentrations of TAC ( Figures 8A, B ). Preincubation of neutrophils with either 25, 50, or 100 ng/mL of TAC resulted in 62%, 77%, and 84% decreases in NETosis, respectively, when compared to ionomycin-stimulated neutrophils. This inhibitory effect of TAC on NET formation was further confirmed using human peripheral neutrophils ( Supplementary Figures S5A, B ). Taken together, these findings suggest that the presence of NETs may play an important role in VCA graft rejection and that their formation may be modulated by local immunosuppression.