Skin Allograft Rejection Research Articles

Electrostimulation suppresses allograft rejection via promoting lymphatic regulatory T cell migration mediated by lymphotoxin - lymphotoxin receptor β signaling

Conventional immunosuppressants that suppress allograft rejection cause various side effects. Although regulatory T cells (Tregs) are essential for allograft survival, the limited efficacy of Treg therapy demands improvement. Thus, it is imperative to seek new approaches to enhancing Treg suppression. Low-intensity electrostimulation (ES) has been shown to exert antiinflammatory effects without causing major adverse reactions. However, it remains unknown whether and how ES regulates alloimmunity. Here, we found that regional ES delayed murine skin allograft rejection and promoted long-term allograft survival induced by an mTOR inhibitor, rapamycin. ES also extended islet allograft survival. Mechanistically, ES enhanced the expression of lymphotoxin α (LTα) on Tregs after transplantation. Blockade of lymphotoxin β receptor–mediated nonclassical NFκB signaling suppressed lymphatic Treg migration and largely reversed the effects of ES on allograft survival. Moreover, ES failed to extend allograft survival when recipients lacked LTα/lymph nodes or if transferred Tregs lacked LTα. Therefore, ES promoted the lymphatic migration of CD4+Foxp3+ Tregs by upregulating their surface expression of LTα. Finally, ES augmented expression of LTα on murine or human Tregs, but not conventional T cells, while promoting their calcium influx in vitro. This ES-mediated upregulation of LTα relied on calcium influx. Thus, our findings have unveiled novel mechanisms underlying ES-mediated immunoregulation.

Siglec-E is a novel co-inhibitory receptor in the modulation of innate activation after transplantation

Abstract Early after transplantation, inflammation and tissue injury are associated with significant activation of the innate immune cells, such as DCs. This activation drives adaptive immune responses, triggering acute rejection while hindering the development of transplant tolerance. Current immunosuppressive drugs primarily target T cells, while innate immunity is not efficiently inhibited in transplantation. The sialic acid–binding immunoglobulin-like lectin E (Siglec-E, or SigE) is an innate receptor that binds to ligands carrying sialic acids and inhibits TLRs-triggered inflammatory responses. Here, we demonstrate the role of SigE in experimental solid organ transplantation mouse models. Upon heart transplantation in mice, SigE is upregulated in graft-infiltrating APCs, including DCs. In humans, Siglec-7 and -9, the human homologs of SigE, are also upregulated in human heart biopsies following transplantation. Genetic ablation of recipient SigE leads to accelerated rejection of heart, kidney, and skin allografts. Mechanistically, SigE-deficient DCs are more susceptible to activation by DAMPS with an enhanced NF-kB activation and production of TNF-a. Moreover, DCs lacking SigE have a higher ability to induce allo-T cell responses in vitro and in vivo. In contrast, overexpression of SigE on DCs decreases their activation by DAMPs and reduces their T cell allostimulatory capacity. Thus, Siglec-E is a major inhibitory receptor in transplantation by controlling APC activation.

RORγt inverse agonist TF-S14 inhibits Th17 cytokines and prolongs skin allograft survival in sensitized mice

Chronic antibody mediated rejection (AMR) is the major cause of solid organ graft rejection. Th17 contributes to AMR through the secretion of IL17A, IL21 and IL22. These cytokines promote neutrophilic infiltration, B cell proliferation and donor specific antibodies (DSAs) production. In the current study we investigated the role of Th17 in transplant sensitization. Additionally, we investigated the therapeutic potential of novel inverse agonists of the retinoic acid receptor-related orphan receptor gamma t (RORγt) in the treatment of skin allograft rejection in sensitized mice. Our results show that RORγt inverse agonists reduce cytokine production in human Th17 cells in vitro. In mice, we demonstrate that the RORγt inverse agonist TF-S14 reduces Th17 signature cytokines in vitro and in vivo and leads to blocking neutrophilic infiltration to skin allografts, inhibition of the B-cell differentiation, and the reduction of de novo IgG3 DSAs production. Finally, we show that TF-S14 prolongs the survival of a total mismatch grafts in sensitized mice. In conclusion, RORγt inverse agonists offer a therapeutic intervention through a novel mechanism to treat rejection in highly sensitized patients.

Coordinated elimination of bacterial taxa optimally attenuates alloimmunity and prolongs allograft survival

This study aimed to dissect the relationship between specific gut commensal bacterial subgroups, their functional metabolic pathways, and their impact on skin allograft outcome and alloimmunity. We previously showed that oral broad-spectrum antibiotic (Abx) pretreatment in mice delayed skin, heart, and lung allograft rejection and dampened alloimmune responses. Here, rationally designed Abx combinations targeting major bacterial groups were used to elucidate their individual contribution to modulating alloimmune responses. Abx cocktails targeting intestinal gram-negative, gram-positive, or anaerobic/gram-positive bacteria by oral gavage, all delayed skin allograft rejection, and reduced alloreactive T cell priming to different extents. Notably, the most pronounced extension of skin allograft survival and attenuation of alloimmunity were achieved when all gut bacterial groups were simultaneously targeted. These results suggest a model in which the strength of the alloimmune response is additively tuned up by gut microbial diversity. Shotgun metagenomic sequencing enabled strain-level resolution and identified a shared commensal, Parabacteroides distasonis, as the most enriched following all Abx treatments. Oral administration of P.distasonis to mice harboring a diverse microbiota significantly prolonged skin allograft survival, identifying a probiotic with therapeutic benefit in transplantation.

The adaptation model of immunity: allograft rejection versus allograft adaptation

Abstract Rejection of allografts and acceptance of grafts between identical twins appear to be associated with allogeneity or nonself entity of allografts as suggested by the self-nonself (SNS) model of immunity. Nevertheless, such guilt by association does not necessarily follow a cause-effect direction. The adaptation model proposes that graft rejection or tolerance is determined by the expression of the tissue-specific adaptation receptors (AdRs) which are linked to anti-apoptotic pathways for tolerating the recipient’s alloreactive immune response induced by surgical procedure during organ transplantation. A better engraftment of less matched living-donor organs compared with matched cadaver-donor organs is because of a severely altered AdRs in the latter. To this end, tissue adaptation to an anti-graft immune response suggests that AdRs can be restored in allografts if treated properly. Re-transplantation studies demonstrated that second fresh skin allograft rejection while the first adapted allograft from the same donor remaining viable in the presence of all-reactive T cells. Also, constitutive expression of a bidirectional AdR, B7-H1 linked to Bcl-xL anti-apoptotic pathway, in the cornea and retina protects them from rejection while block-ade of B7-H1 accelerates allograft rejection. Also, severe GVHD following autologous stem cell transplantation could be due to chemotherapy-induced alterations in the AdRs in target tissues. This is supported by demonstrating that autologous T RMcells that are involved in local tissue homeostasis but not alloreactive T EMcells are involved in GVHD. Interestingly, restoration of the putative AdR, B7-H1, expression by parenchymal tissues could prevent or ameliorate GVHD in humans. This work was supported by the Office of the Assistant Secretary of Defense for Health Affairs through the Breast Cancer Research Program under Award No. W81XWH2210793. Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the U.S. Department of Defense. This work was also supported by multi-investigator pilot funding from the VCU Massey Cancer Centre, supported in part with funding from NIH/NCI Cancer Centre support grants P30 CA016059.

Donor Batf3 inhibits murine lung allograft rejection and airway fibrosis

Chronic lung allograft dysfunction (CLAD) limits survival after lung transplantation. Noxious stimuli entering the airways foster CLAD development. Classical dendritic cells (cDCs) link innate and adaptive immunity and exhibit regional and functional specialization in the lung. The transcription factor basic leucine zipper ATF-like 3 (BATF3) is absolutely required for the development of type 1 cDCs (cDC1s), which reside in the airway epithelium and have variable responses depending on the context. We studied the role of BATF3 in a mouse minor alloantigen-mismatched orthotopic lung transplant model of CLAD with and without airway inflammation triggered by repeated administration of intratracheal lipopolysaccharide (LPS). We found that cDC1s accumulated in allografts compared with isografts and that donor cDC1s were gradually replaced by recipient cDC1s. LPS administration increased the number of cDC1s and enhanced their state of activation. We found that Batf3-/- recipient mice experienced reduced acute rejection in response to LPS; in contrast, Batf3-/- donor grafts underwent enhanced lung and skin allograft rejection and drove augmented recipient cluster of differentiation 8+ T-cell expansion in the absence of LPS. Our findings suggest that donor and recipient cDC1s have differing and context-dependent roles and may represent a therapeutic target in lung transplantation.

Natural Compound 2,2′,4′-Trihydroxychalcone Suppresses T Helper 17 Cell Differentiation and Disease Progression by Inhibiting Retinoid-Related Orphan Receptor Gamma T

Retinoid-related orphan receptor γt (RORγt), a vital transcription factor for the differentiation of the pro-inflammatory Th17 cells, is essential to the inflammatory response and pathological process mediated by Th17 cells. Pharmacological inhibition of the nuclear receptor RORγt provides novel immunomodulators for treating Th17-driven autoimmune diseases and organ transplant rejection. Here, we identified 2,2',4'-trihydroxychalcone (TDC), a natural chalcone derivant, binds directly to the ligand binding domain (LBD) of RORγt and inhibited its transcriptional activation activity. Using three mice models of Th17-related diseases, it was found that the administration of TDC effectively alleviated the disease development of experimental autoimmune encephalomyelitis (EAE), experimental colitis, and skin allograft rejection. Collectively, these results demonstrated TDC targeting RORγt to suppress Th17 cell polarization, as well as its activity, thus, indicating the potential of this compound in treating of Th17-related autoimmune disorders and organ transplant rejection disorders.

Research Highlights

Research Highlights

A novel formulation of cyclosporine A/phosphatidylserine-containing liposome using remote loading method: Potential product for immunosuppressive effects

In this study, two PEGylated and non-PEGylated liposomal formulations of Cyclosporine A (CsA) were prepared by remote loading procedure to improve the tissue distribution profile of CsA and the therapeutic efficacy thereof. DOPE/DOPS/Chol/α-Tocopherol/mPEG2000 (35/40/19.5/0.5/5) and DOPE/DOPS/Chol/α-Tocopherol (40/40/19.5/0.5) were examined in terms of their physicochemical stability, immunosuppressive efficiency, and pharmacokinetic profiles. Besides, the delayed-type hypersensitivity reaction (DTH) was studied from sheep red blood cells (SRBC) injection to male Wistar rats and skin allograft rejection transplanted from male C57BL into BALB/c mouse. Both liposomes had a size of about 162.2 ± 75 nm with a polydispersity index of <0.14, exhibited a high drug encapsulation efficacy (49.9–61.3%), and showed a long-term (6 months) shelf-life stability. Moreover, the liposomes enhanced the delivery of CsA and the improved immunosuppressive efficiency in vitro compared to non-liposomal CsA. In the animal studies, the liposomes also enhanced the drug half-life in the serum of the rat and improved the tissue distribution of CsA compared to non-liposomal CsA. Besides, the liposomes minimized the DTH and delayed the skin allograft rejection up to 17 days compared to CsA. To sum up, the liposomal formulations of CsA improved both the delivery profile and therapeutic efficacy of CsA. However, further studies need to be done to translate this formulation into the clinics.

IL-34 deficiency impairs FOXP3+ Treg function in a model of autoimmune colitis and decreases immune tolerance homeostasis.

BackgroundImmune homeostasis requires fully functional Tregs with a stable phenotype to control autoimmunity. Although IL‐34 is a cytokine first described as mainly involved in monocyte cell survival and differentiation, we recently described its expression by CD8+ Tregs in a rat model of transplantation tolerance and by activated FOXP3+ CD4+ and CD8+ Tregs in human healthy individuals. However, its role in autoimmunity and potential in human diseases remains to be determined.MethodsWe generated Il34 −/− rats and using both Il34 −/− rats and mice, we investigated their phenotype under inflammatory conditions. Using Il34 −/− rats, we further analyzed the impact of the absence of expression of IL‐34 for CD4+ Tregs suppressive function. We investigated the potential of IL‐34 in human disease to prevent xenogeneic GVHD and human skin allograft rejection in immune humanized immunodeficient NSG mice. Finally, taking advantage of a biocollection, we investigated the correlation between presence of IL‐34 in the serum and kidney transplant rejection.ResultsHere we report that the absence of expression of IL‐34 in Il34 −/− rats and mice leads to an unstable immune phenotype, with production of multiple auto‐antibodies, exacerbated under inflammatory conditions with increased susceptibility to DSS‐ and TNBS‐colitis in Il34 −/− animals. Moreover, we revealed the striking inability of Il34 −/− CD4+ Tregs to protect Il2rg−/− rats from a wasting disease induced by transfer of pathogenic cells, in contrast to Il34 +/+ CD4+ Tregs. We also showed that IL‐34 treatment delayed EAE in mice as well as GVHD and human skin allograft rejection in immune humanized immunodeficient NSG mice. Finally, we show that presence of IL‐34 in the serum is associated with a longer rejection‐free period in kidney transplanted patients.ConclusionAltogether, our data emphasize on the crucial necessity of IL‐34 for immune homeostasis and for CD4+ Tregs suppressive function. Our data also shows the therapeutic potential of IL‐34 in human transplantation and auto‐immunity.Highlights –Absence of expression of IL‐34 in Il34−/− rats and mice leads to an unstable immune phenotype, with a production of multiple auto‐antibodies and exacerbated immune pathology under inflammatory conditions.– Il34−/− CD4+ Tregs are unable to protect Il2rg−/− rats from colitis induced by transfer of pathogenic cells.–IL‐34 treatment delayed EAE in mice, as well as acute GVHD and human skin allograft rejection in immune‐humanized immunodeficient NSG mice.

Vγ2+ γδ T Cells in the Presence of Anti-CD40L Control Surgical Inflammation and Promote Skin Allograft Survival

γδ T cells represent a small fraction of total T cells in the body and do not use classical polymorphic major histocompatibility complex‒loaded peptides for mounting an immune response. The importance of the effector and regulatory function of γδ T cells in infections, autoimmunity, and tumor models are well characterized. In this study, we investigated the mechanistic role of γδ T cells in costimulatory blockade‒induced transplantation tolerance. We used donor-specific transfusion and anti-CD40L treatment in C57BL/6 mice to induce tolerance to BALB/c skin allografts. We show that depletion of γδ T cells, specifically Vγ2+ γδ T cells, led to the acute rejection of skin allografts despite tolerogen treatment. Tolerogen treatment promoted CD39+Vγ2+ γδ T cells and suppressed IFN-γ‒producing Vγ2+ γδ T cells in the spleen and allografts. Vγ2+ γδ T cells isolated from tolerized mice suppress T helper type 1 cell differentiation. Adoptive transfer of these regulatory Vγ2+ γδ T cells prolonged the survival of allografts in an untreated recipient and Tcrδ‒/‒ mice. Together, our data show that the Vγ2+ subset promotes costimulatory blockade‒induced survival of skin allografts and that tolerogenic Vγ2+ T cells can be used as an adoptive cellular therapy to promote the survival of allografts.

Adeno-Associated Viral Vector–Mediated Interleukin 10 Gene Transfer in Sprague-Dawley Rat Skin Allograft

Viral vector-mediated interleukin 10 gene transfer is one of the most promising methods for the prevention of graft rejection. Adeno-associated virus (AAV) is a nonpathogenic helper-dependent parvovirus. This is one of the most promising vehicles for gene delivery. Our study aimed to analyze the immune-suppressive potential of recombinant adeno-associated virus-mediated rat IL-10 in rat skin allograft. Dorsal skin from Sprague-Dawley rats was grafted to Fischer 344 rats. In vivo AAV-viral IL-10 (vIL-10) gene transfer was done in the experimental group by direct subcutaneous injection. Observation of graft appearance, cytologic and immunohistochemical testing, and confocal immunofluorescence were performed at postoperative days 5 and 10. The viral IL-10 gene-treated group showed improved graft survival and reduced acute rejection response compared to the control group. IL-10 expression on skin was identified by immunohistochemistry and confocal microscopy. AAV IL-10 gene transfer could be an effective immunosuppressive method for preventing skin allograft rejection.

Genetically targeting the BATF family transcription factors BATF and BATF3 in the mouse abrogates effector T cell activities and enables long-term heart allograft survival

T cells must be activated and become effectors first before executing allograft rejection, a process that is regulated by diverse signals and transcription factors. In this study, we studied the basic leucine zipper ATF-like transcription factor (BATF) family members in regulating T cell activities in a heart transplant model and found that mice deficient for both BATF and BATF3 (Batf-/- Batf3-/- mice) spontaneously accept the heart allografts long-term without tolerizing therapies. Similarly, adoptive transfer of wild type T cells into Rag1-/- hosts induced prompt rejection of heart and skin allografts, whereas the Batf-/- Batf3-/- T cells failed to do so. Analyses of graft-infiltrating cells showed that Batf-/- Batf3-/- T cells infiltrate the graft but fail to acquire an effector phenotype (CD44high KLRG1+ ). Co-transfer experiments in a T cell receptor transgenic TEa model revealed that the Batf-/- Batf3-/- T cells fail to expand in vivo, retain a quiescent phenotype (CD62L+ CD127+ ), and unable to produce effector cytokines to alloantigen stimulation, which contrasted sharply to that of wild type T cells. Together, our data demonstrate that the BATF and BATF3 are critical regulators of T effector functions, thus making them attractive targets for therapeutic interventions in transplant settings.

Skin and heart allograft rejection solely by long-lived alloreactive TRM cells in skin of severe combined immunodeficient mice.

Whether induced tissue-resident memory T (TRM) cells in nonlymphoid organs alone can mediate allograft rejection is unknown. By grafting alloskin or heart into severe combined immunodeficient or Rag2KO mice in which a piece of induced CD4+ and/or CD8+ TRM cell–containing MHC-matched or syngeneic skin was transplanted in advance, we addressed this issue. The induced CD4+ TRM cells in the skin alone acutely rejected alloskin or heart grafts. RNA-seq analysis showed that induced CD4+ TRM cells in skin favorably differentiated into TH17-like polarization during the secondary immune response. Inhibition of the key TH17 signaling molecule RORγt attenuated TRM cell–mediated graft rejection. Thus, we offer a unique mouse model to specifically study TRM cell–mediated allograft rejection without the involvement of lymphocytes in lymphoid organs and tissues. Our study provides strong evidence supporting the hypothesis that long-lived alloreactive TRM cells resident in other organs/tissues substantially contribute to organ allograft rejection.

Immunological discrepancy in aged mice facilitates skin allograft survival.

More and more aged people are undergoing organ transplantation. Understanding aging effects on immunity will be helpful for post-transplantation care and adjustment of immunosuppressants for aged recipients. A mouse model, using C3H mice as donors and aged/young C57BL/10J mice as recipients, was employed to study aging effects on immunity. The results showed that frequency of myeloid-derived suppressor cells (MDSC) and level of TGF-β was higher in aged mice than in young mice (4.4 ± 1.4% versus 1.6 ± 1.1%, p = 0.026 for MDSC; 21.04 ± 3.91 ng/ml versus 15.26 ± 5.01 ng/ml, p = 0.026 for TGF-β). In vivo, skin allograft survived longer on the aged than on young mice (19.7 ± 5.2 days versus 11.9 ± 4.1 days, p = 0.005). When entinostat was applied to block MDSC, the survival of skin allografts on aged mice was shorten to 13.5 ± 4.7 days which was not different from the survival on young mice (p = 0.359). In conclusion, allogeneic immunity was different in aged from young mice in high frequency of MDSC and high serum level of TGF-β. Blocking the function of MDSC reversed the low immunity in aged mice and caused skin allograft rejection similar to young recipients.

Viral Vector Mediated Interleukin-10 Gene Transfer in Skin Allograft

BackgroundExpression of genes with immunoregulatory capacity can potentially decrease rejection of allograft. According to recent studies, viral interleukin (IL)-10 can reduce immune response during allotransplantation and is one of the most promising methods for the prevention of rejection. Our study aimed to analyze the immunosuppressive potential of recombinant adenovirus-mediated rat IL-10 in rat skin allograft. MethodsWe performed skin graft surgery 1 hour after infecting the donated skin with adenovirus-mediated rat IL-10. On day 7 postoperatively, the skin allografts were harvested, and acute rejection was graded histologically. ResultsViral IL-10 gene transfer into rat skin allografts improved graft survival and reduced acute rejections. ConclusionThe results of our study suggest that the therapeutic potential of graft viral IL-10 gene transfer is an effective immunosuppressive method for preventing skin allograft rejection.

IFN-γ Control of an Effector/Target Combination for Skin Allograft Rejection: Macrophage/Skin Components in Normal Mice or T Cell/Endothelial Cells in IFN-γ-Deficient Mice.

Organ, skin, or cell allografts are acutely rejected from normal mice, whereas vascularized organ allografts, but not allografted Meth A cells, are rejected from interferon-γ (IFN-γ)-deficient mice. Here we explored effector/target combinations for i.p. allografted Meth A (cytotoxic T lymphocyte [CTL]-resistant) or RLmale1 (CTL-susceptible) cells into or for BALB/c skin (skin components: CTL resistant) onto normal or IFN-γ-deficient C57BL/6 mice. After allografting, normal mice showed more infiltration but only a little thrombosis/hemorrhage. Monocyte/macrophage MHC receptor (MMR)+ macrophages (on days 5-10) and T cell receptor (TCR)+ CTLs (on days 7-9) were cytotoxic against Meth A cells or skin components and RLmale1 cells, respectively, and the allografts were rejected. After allografting into IFN-γ-deficient mice, MMR- macrophages and highly activated TCR+ CTLs were induced, and the mice died of hemorrhagic ascites with Meth A cells and more acutely rejected RLmale1 cells. The CTLs on days 4-6 were inactive toward skin components at an in vivo effector/target ratio but injured endothelial cells to cause severe thrombosis/hemorrhage and more acute rejection of skin allografts. These results indicate that IFN-γ-dependent MMR expression was essential for macrophage-mediated cytolysis of allogeneic skin components and that IFN-γ-deficient mice more acutely rejected skin allograft by causing CTL-induced injury to endothelial cells.

The Effect of in vivo Macrophage Depletion on Skin Allograft Rejection in Wild-type and CD8 Knockout Mice

The Effect of in vivo Macrophage Depletion on Skin Allograft Rejection in Wild-type and CD8 Knockout Mice

Mechanisms of Graft-versus-Host Disease Prevention by Post-transplantation Cyclophosphamide: An Evolving Understanding.

Post-transplantation cyclophosphamide (PTCy) has been highly successful at preventing severe acute and chronic graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT). The clinical application of this approach was based on extensive studies in major histocompatibility complex (MHC)-matched murine skin allografting models, in which cyclophosphamide was believed to act via three main mechanisms: (1) selective elimination of alloreactive T cells; (2) intrathymic clonal deletion of alloreactive T-cell precursors; and (3) induction of suppressor T cells. In these models, cyclophosphamide was only effective in very specific contexts, requiring particular cell dose, cell source, PTCy dose, and recipient age. Achievement of transient mixed chimerism also was required. Furthermore, these studies showed differences in the impact of cyclophosphamide on transplanted cells (tumor) versus tissue (skin grafts), including the ability of cyclophosphamide to prevent rejection of the former but not the latter after MHC-mismatched transplants. Yet, clinically PTCy has demonstrated efficacy in MHC-matched or partially-MHC-mismatched HCT across a wide array of patients and HCT platforms. Importantly, clinically significant acute GVHD occurs frequently after PTCy, inconsistent with alloreactive T-cell elimination, whereas PTCy is most active against severe acute GVHD and chronic GVHD. These differences between murine skin allografting and clinical HCT suggest that the above-mentioned mechanisms may not be responsible for GVHD prevention by PTCy. Indeed, recent work by our group in murine HCT has shown that PTCy does not eliminate alloreactive T cells nor is the thymus necessary for PTCy's efficacy. Instead, other mechanisms appear to be playing important roles, including: (1) reduction of alloreactive CD4+ effector T-cell proliferation; (2) induced functional impairment of surviving alloreactive CD4+ and CD8+ effector T cells; and (3) preferential recovery of CD4+ regulatory T cells. Herein, we review the history of cyclophosphamide's use in preventing murine skin allograft rejection and our evolving new understanding of the mechanisms underlying its efficacy in preventing GVHD after HCT. Efforts are ongoing to more fully refine and elaborate this proposed new working model. The completion of this effort will provide critical insight relevant for the rational design of novel approaches to improve outcomes for PTCy-treated patients and for the induction of tolerance in other clinical contexts.

Mast Cell Degranulation Exacerbates Skin Rejection by Enhancing Neutrophil Recruitment.

Recent evidences indicate an important role of tissue inflammatory responses by innate immune cells in allograft acceptance and survival. Here we investigated the role of mast cells (MC) in an acute male to female skin allograft rejection model using red MC and basophil (RMB) mice enabling conditional MC depletion. Kinetic analysis showed that MCs markedly accelerate skin rejection. They induced an early inflammatory response through degranulation and boosted local synthesis of KC, MIP-2, and TNF. This enhanced early neutrophil infiltration compared to a female-female graft-associated repair response. The uncontrolled neutrophil influx accelerated rejection as antibody-mediated depletion of neutrophils delayed skin rejection. Administration of cromolyn, a MC stabilizer and to a lesser extent ketotifen, a histamine type I receptor antagonist, and absence of MCPT4 chymase also delayed graft rejection. Together our data indicate that mediators contained in secretory granules of MC promote an inflammatory response with enhanced neutrophil infiltration that accelerate graft rejection.