Prolonged Skin Graft Survival Research Articles

Adoptive Cell Therapy in Mice Sensitized to a Grass Pollen Allergen.

The proportion of patients with type I allergy in the world population has been increasing and with it the number of people suffering from allergic symptoms. Recently we showed that prophylactic cell therapy employing allergen-expressing bone marrow (BM) cells or splenic B cells induced allergen-specific tolerance in naïve mice. Here we investigated if cell therapy can modulate an established secondary allergen-specific immune response in pre-immunized mice. We sensitized mice against the grass pollen allergen Phl p 5 and an unrelated control allergen, Bet v 1, from birch pollen before the transfer of Phl p 5-expressing BM cells. Mice were conditioned with several combinations of low-dose irradiation, costimulation blockade, rapamycin and T cell-depleting anti-thymocyte globulin (ATG). Levels of allergen-specific IgE and IgG1 in serum after cell transfer were measured via ELISA and alterations in cellular responses were measured via an in vitro proliferation assay and transplantation of Phl p 5+ skin grafts. None of the tested treatment protocols impacted Phl p 5-specific antibody levels. Transient low-level chimerism of Phl p 5+ leukocytes as well as a markedly prolonged skin graft survival were observed in mice conditioned with high numbers of Phl p 5+ BMC or no sensitization events between the day of cell therapy and skin grafting. The data presented herein demonstrate that a pre-existing secondary allergen-specific immune response poses a substantial hurdle opposing tolerization through cell therapy and underscore the importance of prophylactic approaches for the prevention of IgE-mediated allergy.

Blockade of Common Gamma Chain Cytokine Signaling with REGN7257, an Interleukin 2 Receptor Gamma (IL2RG) Monoclonal Antibody, in Combination with Costimulatory Blockers Delayed Skin Graft Rejection in Mice

There is a current medical need to identify innovative maintenance therapies to prevent allograft rejection in solid organ transplantation. During transplant rejection, recipient immune cells identify a grafted organ as foreign and attack it: CD4 + T cells orchestrate transplant rejection by recruiting a range of effector cells responsible for the damage of rejection including CD8 + T cells, NK cells, B cells and macrophages. These pathogenic immune cell responses can be driven by cytokines of the common gamma chain (γ c) cytokine family (IL2, IL4, IL7, IL9, IL15, and IL21). γ c cytokines signal through their corresponding receptors, expressed primarily on immune cells, that share a common coreceptor, interleukin 2 receptor subunit gamma (IL2RG) that is required for signaling. To understand the role of γ c cytokines in driving transplant rejection, we generated REGN7257, a fully human IL2RG monoclonal antibody that inhibits γ c cytokine-induced signaling, and we tested its ability to suppress allogeneic immune cell responses in a mouse model of skin graft rejection when delivered as a monotherapy and in combination with mycophenolate mofetil (MMF) or T cell costimulatory pathway blockers. In this model, recipient B6/129 (mixed background) Il2rg hu/hu mice were transplanted with tail skin from BALB/cJ donor mice. Graft survival was analyzed using a log rank Mantel-Cox test to detect statistically significant differences across groups (*=p<0.033; **= p<0.002; ***=p<0.0002; ****=p<0.0001). In a mouse model of skin graft rejection, γ c cytokine signaling blockade with REGN7257 given as maintenance immunosuppression effectively improved overall graft survival (Median Survival Time [MST] IL2RG=13) compared to mice that received a control antibody (MST CTRL=9 [****vs IL2RG Ab]), and with similar efficacy to that observed with the calcineurin inhibitor tacrolimus (MST TAC=12.5 [**vs control, n.s. vs IL2RG Ab]). In treatment combination experiments, while monotherapy treatment with the antiproliferative agent MMF was not as efficient as REGN7257 at delaying skin graft rejection (MST MMF=10 [*vs control] and MST IL2RG=12 [*vs MMF]), simultaneous blockade of cell proliferation and γ c cytokine signaling conferred an advantage over monotherapies (MST MMF+IL2RG=15.5 [****vs control, **vs IL2RG Ab]). Similarly, induction therapy consisting of γ c cytokine signaling blockade followed by maintenance immunosuppression using the CTLA4-Ig fusion protein abatacept, significantly improved overall skin graft survival (MST IL2RG+CTLA4=19.5 [****vs control]) compared to REGN7257 induction only (MST IL2RG=11 [****vs control and combo]) or abatacept maintenance only (MST CTLA4=10 [*vs control, ****vs combo]). Finally, maintenance immunosuppression combining blocking of γc cytokine signaling together with blocking of both CD28 and CD40L costimulatory signals prolonged skin graft survival better than any combination therapies of only 2 pathway blockers (MST TRIPLE=26 [**vs control] and MST DOUBLE=16-21 [n.s.-** vs triple]). Overall, our study showed that γ c cytokine blockade could potentially be an alternative to calcineurin inhibitor-based regimens to prevent rejection by specifically targeting immune cells. Our data also highlighted the inherent complexity of effector mechanisms and pathways involved in allograft rejection and showed that prolonged allograft survival requires simultaneous blockade of multiple T cell activating signals, including inhibition of cytokine signaling and costimulatory signals. These data provide evidence of γ c cytokines as key drivers of allogeneic immune cell responses during acute transplant rejection, offering a potentially novel strategy for the management of transplant patients.

Naringenin Impedes the Differentiation of Mouse Hematopoietic Stem Cells Derived from Bone Marrow into Mature Dendritic Cells, thereby Prolonging Allograft Survival.

The use of immature dendritic cells (imDCs) to induce donor-specific immunotolerance following in vivo stimulation is limited by their low rate of induction and their tendency to undergo maturation. We derived imDCs from bone marrow hematopoietic stem cells (HSCs-imDCs). We then tested the ability of naringenin (Nar) to impede the maturation of HSCs-imDCs for inducing transplantation immune tolerance. HSCs derived from bone marrow were collected and induced to differentiate into imDCs by treating with Nar (Nar-HSCs-imDCs). Flow cytometry was used to evaluate DC surface markers, apoptosis, and endocytic ability. The ability of DCs to influence the in vitro proliferation of T cells and of regulatory T cells (Tregs) was analyzed by mixed lymphocyte reaction assays. Enzyme-linked immunoassays were used to quantify cytokine levels in supernatants from co-cultured DCs and Tregs, as well as in the serum of experimental animals. The level of immunotolerance induced by Nar-HSCs-imDCs was evaluated by skin grafting in recipient Balb/c mice, while the Kaplan-Meier method was used to statistically evaluate graft survival. Compared with HSC-imDCs, Nar-HSCs-imDCs showed higher expression of cluster of differentiation 11c (CD11c), but lower expression levels of CD80, CD86, and major histocompatibility complex class II. Nar-HSCs-imDCs also showed stronger inhibition of T cells and higher Treg cell proliferation. Interleukin 2 (IL-2) and interferon gamma levels were downregulated in Nar-HSCs-imDCs, whereas IL-4, IL-10, and transforming growth factor beta levels were upregulated. The rate of apoptosis and endocytic capacity of Nar-HSCs-DCs increased significantly after treatment with lipopolysaccharide. HSCs-imDCs or Nar-HSCs-imDCs were injected into Balb/c mice via the tail vein 7 days before skin grafting. Significantly reduced donor-specific CD4+ T cells and induced proliferation of CD4+CD25+FoxP3+ Treg cells were observed in the spleen of mice from the Nar-HSCs-imDCs group, especially at a dose of 106 Nar-HSCs-imDCs. The latter group also showed significantly prolonged survival of skin grafts. Nar-HSCs-imDCs markedly improved the acceptance of organ allografts, offering a potentially new strategy for inducing immune tolerance in transplantation.

Effects of periodontal pathogen-induced intestinal dysbiosis on transplant immunity in an allogenic skin graft model

Periodontal disease can induce dysbiosis, a compositional and functional alteration in the microbiota. Dysbiosis induced by periodontal disease is known to cause systemic inflammation and may affect transplant immunity. Here, we examined the effects of periodontal disease-related intestinal dysbiosis on transplant immunity using a mouse model of allogenic skin graft in which the mice were orally administered the periodontal pathogen Porphyromonas gingivalis (Pg). For 6 weeks, the Pg group orally received Pg while the control group orally received phosphate-buffered saline solution. After that, both groups received allogenic skin grafts. 16 s rRNA analysis of feces revealed that oral administration of Pg significantly increased three short chain fatty acids (SCFAs) producing genera. SCFA (acetate and propionate) levels were significantly higher in the Pg group (p = 0.040 and p = 0.005). The ratio of regulatory T cells, which are positively correlated with SCFAs, to total CD4+ T cells in the peripheral blood and spleen was significantly greater (p = 0.002 and p < 0.001) in the Pg group by flowcytometry. Finally, oral administration of Pg significantly prolonged skin graft survival (p < 0.001) and reduced pathological inflammation in transplanted skin grafts. In conclusion, periodontal pathogen-induced intestinal dysbiosis may affect transplant immunity through increased levels of SCFAs and regulatory T cells. (198 words).

Wasabi Component 6-(Methylsulfinyl)hexly Isothiocyanate and Derivatives Improve the Survival of Skin Allografts.

We tested the effect of 6-(Methylsulfinyl)hexyl Isothiocyanate (6-MITC) and derivatives (I7447 and I7557) on the differentiation and maturation of human myeloid dendritic cells (DCs) in vitro, and skin transplantation in vivo. Triggering of CD14+ myeloid monocyte development toward myeloid DCs with and without 6-MITC and derivatives to examine the morphology, viability, surface marker expression, and cytokine production. Stimulatory activity on allogeneic naive T cells was measured by proliferation and interferon-γ production. The skin allograft survival area model was used to translate the 6-MITC and derivatives’ antirejection effect. All of the compounds had no significant effects on DC viability and reduced the formation of dendrites at concentrations higher than 10 μM. At this concentration, 6-MITC and I7557, but not I7447, inhibited the expression of CD1a and CD83. Both 6-MITC and I7557 exhibited T-cells and interferon-γ augmentation at lower concentrations and suppression at higher concentration. The 6-MITC and I7557 prolonged skin graft survival. Both the 6-MITC and I7557 treatment resulted in the accumulation of regulatory T cells in recipient rat spleens. No toxicity was evident in 6-MITC and I7557 treatment. The 6-MITC and I7557 induced human DC differentiation toward a tolerogenic phenotype and prolonged rat skin allograft survival. These compounds may be effective as immunosuppressants against transplant rejection.

The Protective Effect of the Soluble Egg Antigen of Schistosoma japonicum in A Mouse Skin Transplantation Model.

BackgroundOrgan transplantation is currently an effective method for treating organ failure. Long-term use of immunosuppressive drugs has huge side effects, which severely restricts the long-term survival of patients. Schistosoma can affect the host’s immune system by synthesizing, secreting, or excreting a variety of immunomodulatory molecules, but its role in transplantation was not well defined. In order to explore whether Schistosoma-related products can suppress rejection and induce long-term survival of the transplant, we used soluble egg antigen (SEA) of Schistosoma japonicum in mouse skin transplantation models.Materials and methodsEach mouse was intraperitoneally injected with 100 μg of SEA three times a week for four consecutive weeks before allogenic skin transplant. Skin transplants were performed on day 0 to observe graft survival. Pathological examination of skin grafts was conducted 7 days post transplantation. The skin grafts were subjected to mRNA sequencing. Bioinformatics analysis was conducted and the expression of hub genes was verified by qPCR. Flow cytometry analysis was performed to evaluate the immune status and validate the results from bioinformatic analysis.ResultsThe mean survival time (MST) of mouse skin grafts in the SEA-treated group was 11.67 ± 0.69 days, while that of the control group was 8.00 ± 0.36 days. Pathological analysis showed that Sj SEA treatment led to reduced inflammatory infiltration within skin grafts 7 days after allogenic skin transplantation. Bioinformatics analysis identified 86 DEGs between the Sj SEA treatment group and the control group, including 39 upregulated genes and 47 downregulated genes. Further analysis revealed that Sj SEA mediated regulation on cellular response to interferon-γ, activation of IL-17 signaling and chemokine signaling pathways, as well as cytokine–cytokine receptor interaction. Flow cytometry analysis showed that SEA treatment led to higher percentages of CD4+IL-4+ T cells and CD4+Foxp3+ T cells and decreased CD4+IFN-γ+ T cells in skin transplantation.Conclusion Sj SEA treatment suppressed rejection and prolonged skin graft survival by regulating immune responses. Sj SEA treatment might be a potential new therapeutic strategy to facilitate anti-rejection therapy and even to induce tolerance.

Strategies to Induce Blood Vessel Ingrowth into Skin Grafts and Tissue-Engineered Substitutes.

Skin is a multilayer organ consisting of several tissues and appendages residing in a complex niche. Adequate and physiologically regulated vascularization is an absolute requirement for skin homeostasis, regeneration, and wound healing. The lack of vascular networks and ischemia results in delayed wound closure. In addition, vascularization is critical for the prolonged function and survival of skin grafts and tissue-engineered skin substitutes. This study highlights the clinical challenges associated with the limited vascularization in the cutaneous wounds. Then, we highlight the novel approaches for the development of vascular networks in the skin autografts, allografts, and artificial substitutes. Also, the future directions to overcome the existing vascularization complications in skin grafting and synthetic skin substitutes are presented. Statement of Significance Delayed closure of large dermal wounds, such as burn injuries, results from the lack of vascular networks and ischemia. The amount of blood supply in the skin graft is the primary factor determining the quality of the transplanted grafts. The current skin grafts and their fabrication methods lack the appropriate features that contribute to the vascularization and integration of the wound bed and graft and adherence to the skin layers. Therefore, the new generation of skin grafts should consider advanced technologies to induce vascularization and overcome current challenges.

Impact of Graft-Resident Leucocytes on Treg Mediated Skin Graft Survival.

The importance and exact role of graft-resident leucocytes (also referred to as passenger leucocytes) in transplantation is controversial as these cells have been reported to either initiate or retard graft rejection. T cell activation to allografts is mediated via recognition of intact or processed donor MHC molecules on antigen-presenting cells (APC) as well as through interaction with donor-derived extracellular vesicles. Reduction of graft-resident leucocytes before transplantation is a well-known approach for prolonging organ survival without interfering with the recipient’s immune system. As previously shown by our group, injecting mice with IL-2/anti-IL-2 complexes (IL-2cplx) to augment expansion of CD4 T regulatory cells (Tregs) induces tolerance towards islet allografts, and also to skin allografts when IL-2cplx treatment is supplemented with rapamycin and a short-term treatment of anti-IL-6. In this study, we investigated the mechanisms by which graft-resident leucocytes impact graft survival by studying the combined effects of IL-2cplx-mediated Treg expansion and passenger leucocyte depletion. For the latter, effective depletion of APC and T cells within the graft was induced by prior total body irradiation (TBI) of the graft donor. Surprisingly, substantial depletion of donor-derived leucocytes by TBI did not prolong graft survival in naïve mice, although it did result in augmented recipient leucocyte graft infiltration, presumably through irradiation-induced nonspecific inflammation. Notably, treatment with the IL-2cplx protocol prevented early inflammation of irradiated grafts, which correlated with an influx of Tregs into the grafts. This finding suggested there might be a synergistic effect of Treg expansion and graft-resident leucocyte depletion. In support of this idea, significant prolongation of skin graft survival was achieved if we combined graft-resident leucocyte depletion with the IL-2cplx protocol; this finding correlated along with a progressive shift in the composition of T cells subsets in the grafts towards a more tolerogenic environment. Donor-specific humoral responses remained unchanged, indicating minor importance of graft-resident leucocytes in anti-donor antibody development. These results demonstrate the importance of donor-derived leucocytes as well as Tregs in allograft survival, which might give rise to new clinical approaches.

Inhibition of NLRC5 regulates cytokine expression in CD4+ T helper lymphocytes and prolongs murine islet and skin allograft survival

The purpose of this study was to study the effect of inhibiting NLRC5 expression and function on CD4 + T cells, and islet and skin transplantation in mice. A murine skin graft model and islet cell transplantation model were established, and the expression of NLRC5 was compared in rejection and immune tolerance groups. Mice spleen-derived CD4 + T cells were cultured, purified, and enriched in vitro, and transfected with the shRNA lentiviral vector NLRC5-RNAi-GFP. Changes in cytokine secretion were detected to understand changes in immunological function. Murine islet and skin transplantation models were injected with CD4 + T cells transfected with the lentivirus, and the survival time of the grafts and the levels of IFN-γ and IL-10 were compared between groups. The expression of NLRC5 mRNA in islet and skin grafts was significantly increased. In vitro experiments showed that the expression of IL-4 and IL-10 was up-regulated in CD4 + T cells, and T cells differentiation turned to Th2 after inhibition of NLRC5. In vivo experiments showed that inhibition of NLRC5 prolonged islet and skin graft survival. Pathological examination showed that the rejection of transplanted skin and islets in the NLRC5-RNAi group was mild, and there was a correlation between high expression of NLRC5 and rejection of mouse islet and skin grafts. In summary, inhibition of NLRC5 can prolong islet and skin graft survival induce transplant immune tolerance through induction of the secretion of Th2 cytokines by CD4 + T cells.

CTLA-4-IG PROLONGS GRAFT SURVIVAL SPECIFICALLY IN YOUNG BUT NOT IN OLD RECIPIENTS

Introduction: The proportion of older transplant recipients has increased dramatically. Age-specific effects of established immunosuppressants are poorly understood. Here, we assessed the impact of CTLA4-Ig, a fusion protein blocking costimulatory signaling between APCs and T cells through CD28. We hypothesized that the immunosuppressive capacity of CTLA4-Ig will depend on an age-specific expression of CD28 on alloreactive T-cells. Methods: Heart and skin grafts from young DBA2/J mice were transplanted into either young or old (2-3 and 18 mths, resp.) allogeneic C57BL/6 recipients treated with CTLA4-Ig or PBS; T-cell frequencies and subsets in addition to cytokine expression were assessed by flow cytometry and ELISA; expression of CD28 was assessed on young and old T-cell subsets. To delineate the age-specific immunosuppressive potential of CTL4-Ig, T-cells were transfected with a lentiviral vector inducing CD28 expression; transfected T-cells were subsequently transferred into young RAG2-/- mice receiving a fully mismatched skin graft. Results: Treatment with CTLA4-Ig prolonged skin graft survival only in young (p<0.01) but not in old recipients (p=0.33). Age-specific immunosuppressive effects of CTLA4-Ig were also confirmed in a SOT model: cardiac allografts in young mice treated with CTLA4-Ig survived indefinitely. In contrast, 80% of old recipients treated with CTLA4-Ig had lost their graft by 100 days. CTLA4-Ig reduced CD4+ central-memory and effector-memory T cells and diminished systemic IFN-γ levels only in young (p<0.05), but not old recipients (p=0.8). These differences correlated with a reduced expression of CD28 on antigen-experienced CD4+ T cells in old mice (p<0.001). Interestingly, Tregs displayed an increased expression of CD28 with age making them more prone to be targeted by CTLA4-Ig. Strikingly, adoptive transfer of old CD4+ T cells that were transfected with a lentiviral vector augmenting the expression of CD28 accelerated the rejection of allogeneic skin grafts in young RAG2-/- recipient mice (p<0.01 vs. controls animals that received adoptively transferred, non-transfected old T cells) demonstrating the cardinal role of CD28 expression on alloreactive CD4+ T cells. Conclusion: Immunosuppressive effects of CTLA4-Ig showed distinct age-dependent effects in both skin and cardiac transplantation linked to a reduced CD28 expression of alloreactive old T-cells in parallel to an augmented CD28 expression on old T-regs. Transfection experiments confirmed the critical role of CD28 in mounting robust alloimmune responses. These findings carry relevant clinical consequences.

B lymphocytes contribute to indirect pathway T cell sensitization via acquisition of extracellular vesicles.

B cells have been implicated in transplant rejection via antibody-mediated mechanisms and more recently by presenting donor antigens to T cells. We have shown in patients with chronic antibody-mediated rejection that B cells control the indirect T cell alloresponses. To understand more about the role of B cells as antigen-presenting cells for CD4+ T cell with indirect allospecificity, B cells were depleted in C57BL/6 mice, using an anti-CD20 antibody, prior to receiving MHC class I-mismatched (Kd ) skin. The absence of B cells at the time of transplantation prolonged skin graft survival. To study the mechanisms behind this observation, T cells with indirect allospecificity were transferred in mice receiving a Kd skin transplant. T cell proliferation was markedly inhibited in the absence of recipient B cells, suggesting that B cells contribute to indirect pathway sensitization. Furthermore, we have shown that a possible way in which B cells present alloantigens is via acquisition of MHC-peptide complexes. Finally, we demonstrate that the addition of B cell depletion to the transfer of regulatory T cells (Tregs) with indirect alloresponse further prolonged skin graft survival. This study supports an important role for B cells in indirect T cell priming and further emphasizes the advantage of combination therapies in prolonging transplant survival.

Cellular Therapy with Donor Mesenchymal Stem Cells Combined with Co-Stimulation Blockade Prolongs Solid Organ Allograft Survival.

<h3>Introduction</h3> Co-stimulation blockade (CoB) has emerged as a promising immunosuppression strategy and potential alternative to calcineurin inhibitors in solid organ transplantation. Despite recent advancements, CoB based regimens are hampered by increased rates of acute cellular rejection. Bone marrow derived mesenchymal stem cells (BM-MSCs) offer a clinically relevant cellular therapy to suppress T cell alloreactivity and prolong allograft survival. <h3>Objectives</h3> Here we studied the effects of combined donor BM-MSCs and CoB on allograft survival in both murine and non-human primate (NHP) models of transplantation. <h3>Methods</h3> BM-MSCs were cultured and serially passaged in an alpha Minimum Essential Medium based media and subsequently characterized by flow cytometry. A mouse model of MHC mismatched skin transplantation (Balb/C C57BL/6) was used to assess the effect of donor splenocytes vs. BM-MSCs on allograft survival. Next, we investigated whether donor BM-MSCs could prolong renal allograft survival in a fully MHC mismatched NHP model. Mixed lymphocyte reactions (MLRs) were also performed to assess the impact of NHP BM-MSCs on T cell responses <i>in vitro</i>. <h3>Results</h3> Balb/C BM-MSCs administered intravenously on days 1,3, and 5 post-transplant (10⁵ cells per infusion) significantly prolonged skin graft survival compared to identical administration of Balb/C splenocytes in the setting of CoB treatment (CTLA-4Ig + anti-CD154) (MST 40 days vs. 21 days, p = 0.04*). In NHP, BM-MSCs were characterized by expression of CD3-/CD11b-/CD16-/CD20-/CD34-/HLA-DR-/CD44+/CD73+/CD90+/CD105+. Repeated infusions of donor BM-MSCs (2 × 10⁶ cells/kg per infusion) combined with CoB (anti-CD28 + anti-CD154) (n=4) prolonged renal allograft survival compared to CoB alone (n = 3) (MST 228.5 days vs. 41 days, p = 0.09). <i>In vitro</i>, NHP BM-MSCs suppressed allo-stimulated T cell proliferation and cytokine effector function in a contact independent fashion. <h3>Conclusion</h3> Donor BM-MSCs prolong allograft survival in both murine and NHP models of transplantation when combined with CoB treatment. These data suggest BM-MSCs are a promising cellular therapy to suppress co-stimulation resistant T cell alloreactivity and prevent cellular rejection in clinical solid organ transplantation.

Netrin-1 prolongs skin graft survival by inducing the transformation of mesenchymal stem cells from pro-rejection to immune-tolerant phenotype.

Mesenchymal stem cells (MSCs) induce allograft immune tolerance, but low efficacy severely limits their wide application. In this work, Netrin-1 was used to maintain MSC function in an IR environment to study its role in the immune tolerance induction of the allograft. The experiments were divided into three groups: the control group, the IR group and the Netrin-1 group (Netrin-1 was added to MSC medium and then cultured for 48 h). After digestion, MSCs were mixed with TLR4 and TLR3 antibodies (BD), incubated for 20 min, and washed with Phosphate-Buffered Saline (PBS) three times. The mean fluorescence intensity (MFI) of TLR4 and TLR3 was detected by flow cytometry. Isolated lymphocytes were divided into four groups: the control group (no treatment), the MSC group (lymphocytes were co-cultured with MSCs in the control group), the rejection group (lymphocytes were co-cultured with MSCs in the IR group), and the Netrin-1 group (MSCs in the IR group) was stimulated by Netrin-1 for 48h. Our study found that compared with control mice, toll-like receptor (TLR3) expression in bone marrow MSCs decreased as the expression of TLR4 increased, the secretion of transforming growth factor-β (TGF-β) and interleukin-10 (IL-10) was reduced, while the secretion of IL-6 significantly increased in immune rejection (IR) mice. MSCs in IR mice promoted T-cell proliferation and reduced the ratio of Treg cells. Netrin-1 inhibited the pro-rejection effect of these MSCs, further inhibited T-cell proliferation and facilitated an increase in the ratio of Treg cells. The animal experiment results showed that MSC transplantation in the rejection group would shorten the mean survival time of the skin graft and induce the infiltration of lymphocytes. Netrin-1 prolonged the mean survival time of the skin graft by enhancing MSC function. The immunohistochemistry results showed that, compared with the rejection group, the T cell number in the skin graft significantly decreased in the Netrin-1 group. MSC can be divided into immune-tolerant and pro-rejection types in organ transplantation and Netrin-1 can induce the transformation of MSC from the pro-rejection to immune-tolerant type and markedly prolong the skin graft survival time.

Effects of resveratrol on Th17 cell-related immune responses under tacrolimus-based immunosuppression

BackgroundWe previously reported that tacrolimus (Tac) does not decrease T helper 17 cells (Th17) response in kidney transplantation. In this study, we evaluated whether Resveratrol (Resv) has immunosuppressive effects by decreasing Th17 responses in Tac-based immunosuppression.MethodsWe investigated the effects of Resv under Tac-treatment conditions, on CD4+ T cell differentiation to Th17 cells in peripheral blood mononuclear cells (PBMCs), and proliferation of CD4+ T cells co-cultured with human renal proximal tubular epithelial cells (HRPTEpiCs). The effects of Resv on Th17 cells were tested in the murine skin transplant model.ResultsIn PBMCs, Tac did not but combination of Tac and Resv further suppressed Th17 immune response. In the co-culture study, combination of Resv to Tac significantly decreased HRPTEpiC-induced T cell proliferation compared to Tac alone. Resv treatment in the Jurkat cell induced the expression of AMP-activated protein kinase and suppressed the expression of mammalian target of rapamycin (mTOR), suggesting blocking Th17 pathway by Resv. In the murine skin transplant model, combination of Resv to Tac significantly prolonged skin graft survival accompanied by the suppression of Th17 cells, compared to either the Tac-alone or control groups.ConclusionThe results of our study suggest that Resv provides additional immunosuppressive effects to Tac by suppressing effector CD4+ T cells, especially Th17 cells, in the transplantation setting.

Prolongation of skin graft survival in mice by an azaphenothiazine derivative

Azaphenothiazines are predominantly immunosuppressive compounds. We evaluated the efficacy of an azaphenothiazine derivative, 6-chloroethylureidoethyldiquino[3,2-b;2′,3′-e][1,4]thiazine (DQT) in prolongation of survival of skin allografts between BALB/c and C57Bl/6 mice. The mice were treated intraperitoneally (i.p.) with 100 μg of DQT on alternate days, on days 1–13 of the experiment (7 doses). The effect of DQT on a two-way mixed lymphocyte reaction (MLR) in the human model, as well as its effect on production of TNF α and IL-10 in a whole blood cell culture, stimulated by lipopolysaccharide (LPS), were evaluated. In addition, DQT effects were investigated regarding the proportion of T cell subsets in human peripheral blood lymphocytes (PBMC) by flow cytometry. Lastly, the effect of DQT on expression of signaling molecules involved in pro apoptotic pathways was determined by RT PCR. The results showed that DQT significantly extended skin graft survival. The compound also strongly suppressed two-way MLR in the human model at a concentration range of 2.5–5.0 μM. In addition, DQT inhibited LPS-inducible TNF α, but not IL-10 production. The compound preferentially caused a loss of the CD3-CD8+CD11b + PBMC cell subset, and transformed CD3+CD8+high into CD3+CD8+low cells. Lastly, we demonstrated significant increases in expression of caspases (in particular caspase 8) and of p53 in a culture of Jurkat T cells. We conclude that the immunosuppressive actions of the compound in allograft rejection may be predominantly associated with induction of cell apoptosis and inhibition of TNF α production. The apoptosis could be predominantly selective for the CD3-CD8+CD11b + cell phenotype.

Heme oxygenase-2 suppresses acute inflammation and improves the survival of skin allografts

The heme oxygenase (HO) system is an important regulatory arm of the intrinsic cytoprotective and anti-inflammatory system. HO-2 plays an important role in regulating inflammation following injury. The aim of this study was to evaluate the effect of HO-2 overexpression on inflammatory responses. A skin transplantation model, involving the application of skin grafts from wild-type or HO-2 overexpressing mice to BALB/c mice, was used for investigation. HO-2 overexpression suppressed the production of pro-inflammatory cytokines (IL-1β, TNF-α, and IL-6) in macrophages compared to that in macrophages obtained from control mice. HO-2 overexpression also significantly prolonged the survival of allografted skin. Our findings suggest that HO-2 attenuates inflammatory responses and effectively prolongs skin graft survival.

Costimulatory Blockade with CTLA4-Ig Abrogates Prolonged Graft Survival in Old Recipients

Introduction Although they represent a rapidly growing population, elderly organ transplant recipients are underrepresented in clinical trials. Age-specific aspects of established immunosuppressants are therefore poorly understood. Methods Here, we assessed the impact of immunosuppressive treatment with CTLA4-Ig, a fusion protein blocking costimulatory signaling between APCs and T cells through CD28, on alloimmune responses in old and young recipients (2-3 months vs. 16 months) in a fully MHC-mismatched murine transplantation models. Results While treatment with CTLA4-Ig prolonged skin graft survival in young recipients, the same treatment was unable to prolong graft survival in old recipients. Conversely, cardiac allografts in young mice treated with CTLA4-Ig survived indefinitely, while 80% of old recipients treated with CTLA4-Ig had lost their graft after 100 days (log-rank test, p<0.001; n=5/group; Fig. 1).{{AbstractFigure.1}} CTLA4-Ig reduced the in-vivo proliferation of CD4+ and CD8+ T cells (as assessed by BrdU incorporation) uniformly in both young and old recipients; in contrast, CTLA4-Ig reduced CD4+ central-memory and effector-memory T cells only in young but not old recipients. Moreover, systemic frequencies of CD4+IFN-γ+ T cells and systemic levels of IFN-γ cytokine production were reduced in young recipients, but remained unchanged in old. CTLA4-Ig caused significant perturbation in the Treg compartments with reduced frequencies and compromised proliferation of CD4+CD25+Foxp3+ cells. These differences correlated with a significant reduction in expression of CD28 on T cells in old mice, while levels of CTLA4 remained stable.Conclusion Immunosuppressive effects of costimulatory blockade with CTLA4-Ig showed distinct age-dependent effects in both skin and cardiac transplantation. Reduced expression of CD28 with aging may represent an escape mechanism for old alloreactive T cells, with unique clinical consequences for immunosuppression in the growing population of elderly transplant recipients.

Gut microbes contribute to variation in solid organ transplant outcomes in mice

BackgroundSolid organ transplant recipients show heterogeneity in the occurrence and timing of acute rejection episodes. Understanding the factors responsible for such variability in patient outcomes may lead to improved diagnostic and therapeutic approaches. Rejection kinetics of transplanted organs mainly depends on the extent of genetic disparities between donor and recipient, but a role for environmental factors is emerging. We have recently shown that major alterations of the microbiota following broad-spectrum antibiotics, or use of germ-free animals, promoted longer skin graft survival in mice. Here, we tested whether spontaneous differences in microbial colonization between genetically similar individuals can contribute to variability in graft rejection kinetics.ResultsWe compared rejection kinetics of minor mismatched skin grafts in C57BL/6 mice from Jackson Laboratory (Jax) and Taconic Farms (Tac), genetically similar animals colonized by different commensal microbes. Female Tac mice rejected skin grafts from vendor-matched males more quickly than Jax mice. We observed prolonged graft survival in Tac mice when they were exposed to Jax mice microbiome through co-housing or fecal microbiota transplantation (FMT) by gastric gavage. In contrast, exposure to Tac mice did not change graft rejection kinetics in Jax mice, suggesting a dominant suppressive effect of Jax microbiota. High-throughput sequencing of 16S rRNA gene amplicons from Jax and Tac mice fecal samples confirmed a convergence of microbiota composition after cohousing or fecal transfer. Our analysis of amplicon data associated members of a single bacterial genus, Alistipes, with prolonged graft survival. Consistent with this finding, members of the genus Alistipes were absent in a separate Tac cohort, in which fecal transfer from Jax mice failed to prolong graft survival.ConclusionsThese results demonstrate that differences in resident microbiome in healthy individuals may translate into distinct kinetics of graft rejection, and contribute to interpersonal variability in graft outcomes. The association between Alistipes and prolonged skin graft survival in mice suggests that members of this genus might affect host physiology, including at sites distal to the gastrointestinal tract. Overall, these findings allude to a potential therapeutic role for specific gut microbes to promote graft survival through the administration of probiotics, or FMT.

Importance of B cells to development of regulatory T cells and prolongation of tissue allograft survival in recipients receiving autologous bone marrow transplantation.

We previously showed that congenic bone marrow transplantation (BMTx) post myeloablation augmented tissue allograft survival in association with increased regulatory T (Treg) cells of both host and bone marrow donor origin. Regulatory B (Breg) cells can also modulate T-cell immunity and B cells may be implicated in the development of Treg cells. Accordingly, we explored the effect of B-cell depletion invivo on augmented graft survival post BMTx. C57BL/6 mice received BALB/c skin allografts followed 7days later by myeloablation using cyclophosphamide and busulphan. Mice then received T-cell-depleted bone marrow from CD45.1 congenic donors, and ongoing immunosuppression with rapamycin (to day 28 after BMTx). Control mice received cyclophosphamide and busulphan followed by rapamycin, but not congenic bone marrow. At different times post BMTx, mice received B-cell-depleting antibody treatment, and the effect on both skin graft survival, and induction of Treg cells was assessed. BMTx resulted in significantly prolonged skin graft survival versus control mice, in association with attenuated donor-specific alloreactivity relative to controls, increased splenic Treg cells and significantly diminished anti-donor IgG. In mice receiving infusion of B-depleting antibodies for 12days from day 15 post BMTx, both graft survival and Treg cell activity were diminished, particularly for functional Treg cells of donor origin. Adoptive transfer of Breg cells from mice harvested at 15days post BMTx prolonged survival in naive transplanted mice and increased Treg cell levels. Thus, autologous BMTx augmentation of graft survival is dependent in part upon a population of Breg cells that can modulate the function of donor-derived Treg cells.

Rapamycin Prolongs Graft Survival and Induces CD4+IFN-γ+IL-10+ Regulatory Type 1 Cells in Old Recipient Mice.

Although the elderly represents a rapidly growing population among transplant recipients, age-specific aspects have not been considered sufficiently in clinical trials. Moreover, age-specific effects of immunosuppressive therapies remain poorly understood. Here, we assessed the impact of rapamycin on alloimmune responses in old recipients using a fully major histocompatibility complex-mismatched murine transplantation model. Old untreated recipients displayed a prolonged skin graft survival compared to their young counterparts, an observation that confirmed data of our previous experiments. Rapamycin led to a significant prolongation of graft survival in both young and old recipients. However, graft survival was age-dependent and extended in old versus young recipients (19 days vs 12 days, P = 0.004). This age-specific effect was not linked to changes in frequencies or subset composition of either cluster of differentiation (CD)8 or CD4 T cells. Moreover, antiproliferative effects of rapamycin on CD8 and CD4 T cells as assessed by in vivo bromdesoxyuridine incorporation were comparable and age-independent. In contrast, the systemic production of IL-10 was markedly elevated in old recipients treated with rapamycin. In parallel to this shift in cytokine balance, IFN-γ/IL-10 double-positive regulatory type 1 cells emerged during T helper type 1 differentiation of old T helper cells in presence of rapamycin. Similarly, CD4IFN-γIL-10 cells expanded among Foxp3-negative cells after in vivo treatment of old recipients with rapamycin. Our results highlight novel aspects of age-dependent immunosuppressive effects of rapamycin, with relevance for age-specific immunosuppressive regimens.