Skin Allograft Survival Research Articles

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.

Novel approach for enhancing skin allograft survival by bioadhesive nanoparticles loaded with rapamycin

Skin graft rejection is a significant challenge in skin allografts for skin defects, particularly in extensive burn injury patients when autografts are insufficient. Enhancing the survival duration of allogeneic skin grafts can improve the success rate of subsequent autologous skin grafting, thereby promoting the therapeutic efficacy for wound healing. Rapamycin (Rapa), a potent immunosuppressant with favorable efficacy in organ transplantation, is limited by its systemic administration-associated toxicity and side effects. Therefore, addressing the short survival time of allogeneic skin grafts and minimizing the toxicity related to systemic application of immunosuppressive agents is an urgent requirement. Here, we present a topical formulation based on bioadhesive poly (lactic acid)-hyperbranched polyglycerol nanoparticles (BNPs) with surface-modified encapsulation of Rapamycin (Rapa/BNPs), applied for local immunosuppression in a murine model of allogeneic skin grafts. Our Rapa/BNPs significantly prolong nanoparticle retention, reduce infiltration of T lymphocytes and macrophages, decrease the level of pro-inflammatory cytokines and ultimately extend skin allograft survival with little systemic toxicity compared to free Rapa or Rapamycin-loaded non-bioadhesive nanoparticles (Rapa/NNPs) administration. In conclusion, Rapa/BNPs effectively deliver local immunosuppression and demonstrate potential for enhancing skin allograft survival while minimizing localized inflammation, thus potentially increasing patient survival rates for various types of skin defects.

Tregs integrate native and CAR-mediated costimulatory signals for control of allograft rejection.

Tregs expressing chimeric antigen receptors (CAR-Tregs) are a promising tool to promote transplant tolerance. The relationship between CAR structure and Treg function was studied in xenogeneic, immunodeficient mice, revealing advantages of CD28-encoding CARs. However, these models could underrepresent interactions between CAR-Tregs, antigen-presenting cells (APCs), and donor-specific Abs. We generated Tregs expressing HLA-A2-specific CARs with different costimulatory domains and compared their function in vitro and in vivo using an immunocompetent model of transplantation. In vitro, the CD28-encoding CAR had superior antigen-specific suppression, proliferation, and cytokine production. In contrast, in vivo, Tregs expressing CARs encoding CD28, ICOS, programmed cell death 1, and GITR, but not 4-1BB or OX40, all extended skin allograft survival. To reconcile in vitro and in vivo data, we analyzed effects of a CAR encoding CD3ζ but no costimulatory domain. These data revealed that exogenous costimulation from APCs can compensate for the lack of a CAR-encoded CD28 domain. Thus, Tregs expressing a CAR with or without CD28 are functionally equivalent in vivo, mediating similar extension of skin allograft survival and controlling the generation of anti-HLA-A2 alloantibodies. This study reveals a dimension of CAR-Treg biology and has important implications for the design of CARs for clinical use in Tregs.

Inhibition of DAI refrains dendritic cells from maturation and prolongs murine islet and skin allograft survival.

Central to allograft rejection is the T cell-mediated adaptive immune response initiated by activated dendritic cells (DCs). Previous studies have shown that the DNA-dependent activator of IFN regulatory factors (DAI) is involved in the maturation and activation of DCs. Therefore, we hypothesized that inhibition of DAI could prevent DCs from maturation and prolong murine allograft survival. Donor mouse bone marrow-derived dendritic cells (BMDCs) were transduced with the recombinant adenovirus vector (AdV-DAI-RNAi-GFP) to inhibit DAI expression (DC-DAI-RNAi), and the immune cell phenotype and function of DC-DAI-RNAi upon lipopolysaccharide (LPS) stimulation were evaluated. Then DC-DAI-RNAi was injected into recipient mice before islet transplantation and skin transplantation. The survival times of islet and skin allograft were recorded and the proportions of T cell subsets in spleen and secretion levels of cytokines in serum were measured. We identified that DC-DAI-RNAi inhibited the expression of main co-stimulatory molecules and MHC-II, exhibited strong phagocytic ability, and secreted high levels of immunosuppressive cytokines and low levels of immunostimulating cytokines. Recipient mice treated with DC-DAI-RNAi had longer islet and skin allograft survival times. In the murine islet transplantation model, we observed an increase in Treg cells proportion, a reduction in Th1 and Th17 cells proportions in spleen, and similar trends in their secreted cytokines in serum in the DC-DAI-RNAi group. Inhibition of DAI by adenovirus transduction inhibits the maturation and activation of DCs, affects the differentiation of T cell subsets as well as their secreted cytokines, and prolongs allograft survival.

Reduced Satb1 expression predisposes CD4+ T conventional cells to Treg suppression and promotes transplant survival

Limiting CD4+ T cell responses is important to prevent solid organ transplant rejection. In a mouse model of costimulation blockade-dependent cardiac allograft tolerance, we previously reported that alloreactive CD4+ conventional T cells (Tconvs) develop dysfunction, losing proliferative capacity. In parallel, induction of transplantation tolerance is dependent on the presence of regulatory T cells (Tregs). Whether susceptibility of CD4+ Tconvs to Treg suppression is modulated during tolerance induction is unknown. We found that alloreactive Tconvs from transplant tolerant mice had augmented sensitivity to Treg suppression when compared with memory T cells from rejector mice and expressed a transcriptional profile distinct from these memory T cells, including down-regulated expression of the transcription factor Special AT-rich sequence-binding protein 1 (Satb1). Mechanistically, Satb1 deficiency in CD4+ T cells limited their expression of CD25 and IL-2, and addition of Tregs, which express higher levels of CD25 than Satb1-deficient Tconvs and successfully competed for IL-2, resulted in greater suppression of Satb1-deficient than wild-type Tconvs invitro. In vivo, Satb1-deficient Tconvs were more susceptible to Treg suppression, resulting in significantly prolonged skin allograft survival. Overall, our study reveals that transplantation tolerance is associated with Tconvs' susceptibility to Treg suppression, via modulated expression of Tconv-intrinsic Satb1. Targeting Satb1 in the context of Treg-sparing immunosuppressive therapies might be exploited to improve transplant outcomes.

231.5: Anti-CD40L Antibody Promotes the Differentiation of Vy2+ Regulatory Gamma-delta T Cells and Prolongs the Survival of Skin Allografts

231.5: Anti-CD40L Antibody Promotes the Differentiation of Vy2+ Regulatory Gamma-delta T Cells and Prolongs the Survival of Skin Allografts

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.

Therapeutic effect of multiple functional minicircle vector encoding anti-CD25/IL-10/CXCR3 in allograft rejection model.

Background/AimsWe previously proposed minicircle vector technology as the potential platform for the development and production of new biologics. In this study, we have designed a novel target molecule for the treatment of allograft rejection and evaluated its feasibility as the therapeutic agent in this disease using the minicircle vector system.MethodsWe engineered vectors to carry cassette sequences for anti-CD25, interleukin-10 (IL-10), and C-X-C motif chemokine receptor 3 (CXCR3) fusion protein, and then isolated minicircle vectors from the parent vectors. We verified the substantial production of anti-CD25/IL-10/CXCR3 fusion protein from minicircles and their duration in HEK293T cells and mice models. We also evaluated whether minicircle-derived anti-CD25/IL-10/CXCR3 has therapeutic effects in a skin allograft in mice model.ResultsWe confirmed the production of anti-CD25/IL-10/CXCR3 from minicircle by its significant availability in cells transfected with the minicircle and in its conditioned media. After a single injection of minicircle by hydrodynamic injection via mouse tail vein, luminescence or red fluorescence was maintained until 40 days in the liver tissue, suggesting the production of anti-CD25/IL-10/CXCR3 protein from minicircles via protein synthesis machinery in the liver. Mice treated with the minicircle encoding anti-CD25/IL-10/CXCR3 showed prolonged skin allograft survival times accompanied by improved immunologic regulation e.g., reduction of the lymphocyte population of Th1, Th2, and Th17 and an induction of regulatory T cells.ConclusionsThese findings implied that self-generated anti-CD25/IL-10/CXCR3 protein drug by minicircle technology is functionally active and relevant for reducing allograft rejection. The minicircle vector system may be useful for developing new biological drugs, avoiding manufacturing or practical problems.

Ablation of BATF Alleviates Transplant Rejection via Abrogating the Effector Differentiation and Memory Responses of CD8+ T Cells.

Allogeneic CD8+ T cells are prominently involved in allograft rejection, but how their effector differentiation and function are regulated at a transcriptional level is not fully understood. Herein, we identified the basic leucine zipper ATF-like transcription factor (BATF) as a key transcription factor that drives the effector program of allogeneic CD8+ T cells. We found that BATF is highly expressed in graft-infiltrating CD8+ T cells, and its ablation in CD8+ T cells significantly prolonged skin allograft survival in a fully MHC-mismatched transplantation model. To investigate how BATF dictates allogeneic CD8+ T cell response, BATF–/– and wild-type (WT) CD8+ T cells were mixed in a 1:1 ratio and adoptively transferred into B6.Rag1 –/– mice 1 day prior to skin transplantation. Compared with WT CD8+ T cells at the peak of rejection response, BATF–/– CD8+ T cells displayed a dysfunctional phenotype, evident by their failure to differentiate into CD127–KLRG1+ terminal effectors, impaired proliferative capacity and production of pro-inflammatory cytokines/cytotoxic molecules, and diminished capacity to infiltrate allografts. In association with the failure of effector differentiation, BATF–/– CD8+ T cells largely retained TCF1 expression and expressed significantly low levels of T-bet, TOX, and Ki67. At the memory phase, BATF-deficient CD8+ T cells displayed impaired effector differentiation upon allogeneic antigen re-stimulation. Therefore, BATF is a critical transcriptional determinant that governs the terminal differentiation and memory responses of allogeneic CD8+ T cells in the transplantation setting. Targeting BATF in CD8+ T cells may be an attractive therapeutic approach to promote transplant acceptance.

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.

Intermittent Exposure of Hypercapnia Suppresses Allograft Rejection via Induction of Treg Differentiation and Inhibition of Neutrophil Accumulation.

Background: In the management of major burn wounds, allogeneic skin transplantation is a critical procedure to improve wound repair. Our previous works found that intermittent exposure to carbon dioxide leads to permissive hypercapnia (HCA) and prolongs skin allograft survival. However, the modulatory effects of HCA exposure on the immune system are not well understood. Objectives: Our purpose was to investigate how intermittent exposure to HCA can effectively reduce the immune reaction to allogeneic skin graft rejection. Methods: A fully major histocompatibility complex-incompatible skin transplant from BALB/c to C57BL/6 mice model was utilized. Immune cells from splenic and draining lymph nodes were analyzed by flow cytometry. Serum proinflammatory cytokines were analyzed by ELISA. Results: Serum levels of IFN-γ, IL-2, IL-6, and TNF-α were significantly decreased in the HCA group. Additionally, the percentage of CD8+ cells in draining lymph nodes was significantly lower in HCA than in the control group. Moreover, the generation rate of FoxP3+ regulatory T cells (Tregs) from spleen naïve CD4+ T cells was increased by intermittent exposure to carbon dioxide. The infiltrated neutrophils were also eliminated by HCA. Taken together, we concluded that intermittent hypercapnia exposure could effectively suppress skin rejection by stimulating Treg cell generation and suppressing immune reactions.

The Inability of Ex Vivo Expanded Mesenchymal Stem/Stromal Cells to Survive in Newborn Mice and to Induce Transplantation Tolerance.

An encounter of the developing immune system with an antigen results in the induction of immunological areactivity to this antigen. In the case of transplantation antigens, the application of allogeneic hematopoietic cells induces a state of neonatal transplantation tolerance. This tolerance depends on the establishment of cellular chimerism, when allogeneic cells survive in the neonatally treated recipient. Since mesenchymal stem/stromal cells (MSCs) have been shown to have low immunogenicity and often survive in allogeneic recipients, we attempted to use these cells for induction of transplantation tolerance. Newborn (less than 24h old) C57BL/6 mice were injected intraperitoneally with 5 × 106 adipose tissue-derived MSCs isolated from allogeneic donors and the fate and survival of these cells were monitored. The impact of MSC application on the proportion of cell populations of the immune system and immunological reactivity was assessed. In addition, the survival of skin allografts in neonatally treated recipients was tested. We found that in vitro expanded MSCs did not survive in neonatal recipients, and the living MSCs were not detected few days after their application. Furthermore, there were no significant changes in the proportion of individual immune cell populations including CD4+ cell lineages, but we detected an apparent shift to the production of Th1 cytokines IL-2 and IFN-γ in neonatally treated mice. However, skin allografts in the MSC-treated recipients were promptly rejected. These results therefore show that in vitro expanded MSCs do not survive in neonatal recipients, but induce a cytokine imbalance without induction of transplantation tolerance.

The impact of CD160 deficiency on alloreactive CD8 T cell responses and allograft rejection

CD160 is a member of the immunoglobulin superfamily with a pattern of expression mainly restricted to cytotoxic cells. To assess the functional relevance of the HVEM/CD160 signaling pathway in allogeneic cytotoxic responses, exon 2 of the CD160 gene was targeted by CRISPR/Cas9 to generate CD160 deficient mice. Next, we evaluated the impact of CD160 deficiency in the course of an alloreactive response. To that aim, parental donor WT (wild-type) or CD160 KO (knock-out) T cells were adoptively transferred into non-irradiated semiallogeneic F1 recipients, in which donor alloreactive CD160 KO CD4 T cells and CD8 T cells clonally expanded less vigorously than in WT T cell counterparts. This differential proliferative response rate at the early phase of T cell expansion influenced the course of CD8 T cell differentiation and the composition of the effector T cell pool that led to a significant decreased of the memory precursor effector cells (MPECs) / short-lived effector cells (SLECs) ratio in CD160 KO CD8 T cells compared to WT CD8 T cells. Despite these differences in T cell proliferation and differentiation, allogeneic MHC class I mismatched (bm1) skin allograft survival in CD160 KO recipients was comparable to that of WT recipients. However, the administration of CTLA-4.Ig showed an enhanced survival trend of bm1 skin allografts in CD160 KO with respect to WT recipients. Finally, CD160 deficient NK cells were as proficient as CD160 WT NK cells in rejecting allogeneic cellular allografts or MHC class I deficient tumor cells. CD160 may represent a CD28 alternative costimulatory molecule for the modulation of allogeneic CD8 T cell responses either in combination with costimulation blockade or by direct targeting of alloreactive CD8 T cells that upregulate CD160 expression in response to alloantigen stimulation.

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.

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.

Cyclic Helix B Peptide Prolongs Skin Allograft Survival via Inhibition of B Cell Immune Responses in a Murine Model.

Antibody-mediated rejection (AMR) represents a major cause of allograft dysfunction and results in allograft failure in solid organ transplantation. Cyclic helix B peptide (CHBP) is a novel erythropoietin-derived peptide that ameliorated renal allograft rejection in a renal transplantation model. However, its effect on AMR remains unknown. This study aimed to investigate the effect of CHBP on AMR using a secondary allogeneic skin transplantation model, which was created by transplanting skin from BALB/c mice to C57BL/6 mice with or without CHBP treatment. A secondary syngeneic skin transplantation model, involving transplantation from C57BL/6 mice to C57BL/6 mice, was also created to act as a control. Skin graft rejection, CD19+ B cell infiltration in the skin allograft, the percentages of splenic plasma cells, germinal center (GC) B cells, and Tfh cells, the serum levels of donor specific antibodies (DSAs), and NF-κB signaling in splenocytes were analyzed. Skin allograft survival was significantly prolonged in the CHBP group compared to the allogeneic group. CHBP treatment also significantly reduced the CD19+ B cell infiltration in the skin allograft, decreased the percentages of splenic plasma cells, GC B cells, and Tfh cells, and ameliorated the increase in the serum DSA level. At a molecular level, CHBP downregulated P100, RelB, and P52 in splenocytes. CHBP prolonged skin allograft survival by inhibiting AMR, which may be mediated by inhibition of NF-κB signaling to suppress B cell immune responses, thereby decreasing the DSA level.

CHBP induces stronger immunosuppressive CD127+ M-MDSC via erythropoietin receptor

Erythropoietin (EPO) is not only an erythropoiesis hormone but also an immune-regulatory cytokine. The receptors of EPO (EPOR)2 and tissue-protective receptor (TPR), mediate EPO’s immune regulation. Our group firstly reported a non-erythropoietic peptide derivant of EPO, cyclic helix B peptide (CHBP), which could inhibit macrophages inflammation and dendritic cells (DCs) maturation. As a kind of innate immune regulatory cell, myeloid-derived suppressor cells (MDSCs) share a common myeloid progenitor with macrophages and DCs. In this study, we investigated the effects on MDSCs differentiation and immunosuppressive function via CHBP induction. CHBP promoted MDSCs differentiate toward M-MDSCs with enhanced immunosuppressive capability. Infusion of CHBP-induced M-MDSCs significantly prolonged murine skin allograft survival compared to its counterpart without CHBP stimulation. In addition, we found CHBP increased the proportion of CD11b+Ly6G−Ly6Chigh CD127+ M-MDSCs, which exerted a stronger immunosuppressive function compared to CD11b+Ly6G−Ly6Chigh CD127− M-MDSCs. In CHBP induced M-MDSCs, we found that EPOR downstream signal proteins Jak2 and STAT3 were upregulated, which had a strong relationship with MDSC function. In addition, CHBP upregulated GATA-binding protein 3 (GATA-3) protein translation level, which was an upstream signal of CD127 and regulator of STAT3. These effects of CHBP could be reversed if Epor was deficient. Our novel findings identified a new subset of M-MDSCs with better immunosuppressive capability, which was induced by the EPOR-mediated Jak2/GATA3/STAT3 pathway. These results are beneficial for CHBP clinical translation and MDSC cell therapy in the future.

Impact of Injection Frequency of Adipose-Derived Stem Cells on Allogeneic Skin Graft Survival Outcomes in Mice

Previous studies indicated that mesenchymal stem cells (MSCs) exhibit immunomodulatory properties in composite tissue allotransplantation. However, due to the high immunogenicity of skin, although the single administration of MSCs improves survival of the skin allotransplant, immune rejection is still inevitable. The aim of our study was to evaluate whether multiple administrations of MSCs would improve immune tolerance in the allogeneic skin graft, compared to that with a single administration in a mouse model. After full-thickness skin allotransplantation on the backs of the mice, the recipient mice were infused with phosphate-buffered saline and isogenic 1.5 × 105/mL adipose-derived stem cells (ADSCs). ADSCs were transplanted into different mice according to the different injection frequencies such as single, once a week, and twice a week. Skin sections were taken on days 7 and 21 post-transplantation in all groups for gene expression and histological studies. ADSCs increased skin allograft survival compared to that in control mice (P < 0.05). Interleukin-6 and tumor necrosis factor-alpha messenger RNA levels were decreased, and the abundance of lymphocytes, based on immunohistochemistry, was also decreased in ADSC-infused mice (P < 0.05). However, among the different ADSC injection frequency groups, multiple ADSC infusion did not improve the survival rate and decreased proinflammatory cytokines and lymphocytes, compared to those with the single administration of ADSCs (P > 0.05). Conversely, the results with single administration were slightly better than those with multiple administrations. Our study demonstrated that ADSCs have the potential for immunomodulation in vivo. However, the results with multiple ADSC administration were not as good as those with single administration, which indicates the complexity of ADSCs in vivo and implying the need for adequate preclinical experimentation.

IL-33 drives the production of mouse regulatory T cells with enhanced in vivo suppressive activity in skin transplantation.

Regulatory T cells (Tregs) are crucial mediators of immune homeostasis with the ability to modulate allogeneic response and control transplant rejection. Although Treg-based cell therapies have shown immense promise, methods to optimize current strategies are critical for successful implementation within the clinic. IL-33 is a cytokine with pleiotropic properties and effects on Treg function and development. In this study, we explored the unique properties of Treg populations activated through the IL-33/ST2 pathway, aiming to exploit their tolerogenic properties for cell therapy. We show that treatment with exogenous IL-33 results in a generalized downregulation of genes critical to T cell biology together with an upregulation of Treg-associated genes. Tregs that develop in response to IL-33 upregulate critical Treg-associated markers, yet without developing enhanced in vitro suppressive capacity. Conversely, these Tregs display potent regulatory activity in vivo, promoting long-term skin allograft survival in a stringent transplantation model. Detailed transcriptomic and immunophenotypic analyses of IL-33–expanded Tregs reveal an enhancement in graft-homing chemokine receptors, which may be partly responsible for their superior in vivo activity that is not reflected in vitro. IL-33 treatment is therefore an attractive adjunctive strategy for patients receiving Treg cell therapeutics.