Allogeneic Transplantation Model Research Articles

CTLA4-Ig alleviates the allogeneic immune responses against insulin-producing cells in a murine model of cell transplantation.

The adoptive transfer of insulin-producing cells (IPCs) is one of the promising treatments for insulin-dependent diabetes mellitus. While the use of allogeneic cell resources is inevitable in the case of a series of patients, alloimmune responses are a major barrier ahead of the successful implementation of allogeneic therapeutic cells. This study is aimed at evaluating the potential of CTLA4-Ig, as an approved immunomodulatory biologic, in protecting the IPCs against allogeneic immune responses. The C57BL/6 and BALB/c mice were used to establish a murine model of allogeneic cell transplantation. The mouse bone-marrow-derived mesenchymal stem cells were in vitro differentiated into IPCs, and the in vitro as well as the in vivo immune responses against IPCs were evaluated in the presence and absence of CTLA4-Ig. The allogeneic IPCs induced the in vitro activation of CD4+ T-cells, IFN-γ release, and the proliferation of lymphocytes, which all were controlled by CTLA4-Ig. Upon in vivo transfer of IPC into an allogeneic host, the splenic CD4+ and CD8+ T-cells exhibited a significant activation, and there was a significant donor-specific antibody response. Either of the mentioned cellular and humoral responses were modulated by a CTLA4-Ig regimen. This regimen also reduced the infiltration of CD3+ T-cells into the IPC injection site along with the improved overall survival of diabetic mice. CTLA4-Ig could be a complementary therapy for improving the efficacy of allogeneic IPC therapy through modulating the cellular and humoral responses that can lead to prolonged durability of IPCs within an allogeneic host.

PKN1 is a novel therapeutic target to prevent graft-vs-host disease after allogeneic hematopoietic cell transplantation

Abstract We were the first to demonstrate that targeting JAK1/2 using baricitinib (BARI) and ruxolitinib (RUX) prevents and treats GVHD while enhancing multi-lineage hematopoietic reconstitution and graft-vs-leukemia (GvL) effect in mouse models of allogeneic hematopoietic cell transplantation (allo-HCT). However, unlike BARI, RUX fails to completely prevent or treat GvHD. To elucidate the mechanism underlying the superiority of BARI to RUX, we performed both mouse and human kinome analyses. We found that BARI, but not RUX, is a potent inhibitor of both murine and human PKN1, suggesting that the superiority of BARI to RUX in preventing and treating GvHD may result from the off-target effects of BARI on PKN1 in addition to its known inhibitory effects on JAK1/2. While the function of PKN1 in GvHD remains unexplored, PKN1 has been suggested as a protein kinase in signaling pathways involving S1PR1/ PPP1R14A, LFA-1/STK4 and LFA-1/RPH3A for immune cell adhesion/trafficking and the balance of regulatory T cells and effector T cells. To define the role of PKN1 in GvHD, we examined if OTS167, a PKN1 inhibitor, alters the downstream signaling of these candidate pathways. We found that OTS167 significantly reduces the phosphorylation of PPP1R14A and STK4 in murine T cells in a dose-dependent manner. Of note, RPH3A or pRPH3A is not detectable in T cells. In addition, we found that in vivo administration of OTS167 reduces GvHD in a mouse model of allo-HCT (p<0.05). Furthermore, we found that allo-HCT recipient mice transplanted with S1PR1 KO T cells demonstrate significantly improved overall survival (p<0.05). These data suggest that PKN1 and S1PR1/PPP1R14A (and also LFA-1/STK4 which is currently under investigation) are critical components in inducing GvHD. This research was supported by the Alvin J. Siteman Cancer Center through The Foundation for Barnes-Jewish Hospital.

Monomethyl fumarate prevents alloimmune rejection in mouse heart transplantation by inducing tolerogenic dendritic cells.

Dendritic cells (DCs) are important targets for eliciting allograft rejection after transplantation. Previous studies have demonstrated that metabolic reprogramming of DCs can transform their immune functions and induce their differentiation into tolerogenic DCs. In this study, we aim to investigate the protective effects and mechanisms of monomethyl fumarate (MMF), a bioactive metabolite of fumaric acid esters, in a mouse model of allogeneic heart transplantation. Bone marrow-derived DCs are harvested and treated with MMF to determine the impact of MMF on the phenotype and immunosuppressive function of DCs by flow cytometry and T-cell proliferation assays. RNA sequencing and Seahorse analyses are performed for mature DCs and MMF-treated DCs (MMF-DCs) to investigate the underlying mechanism. Our results show that MMF prolongs the survival time of heart grafts and inhibits the activation of DCs in vivo. MMF-DCs exhibit a tolerogenic phenotype and function in vitro. RNA sequencing and Seahorse analyses reveal that MMF activates the Nrf2 pathway and mediates metabolic reprogramming. Additionally, MMF-DC infusion prolongs cardiac allograft survival, induces regulatory T cells, and inhibits T-cell activation. MMF prevents allograft rejection in mouse heart transplantation by inducing tolerogenic DCs.

TNX-1500, a crystallizable fragment–modified anti-CD154 antibody, prolongs nonhuman primate cardiac allograft survival

Blockade of the CD40/CD154 T cell costimulation pathway is a promising approach to supplement or replace current clinical immunosuppression in solid organ transplantation. We evaluated the tolerability and activity of a novel humanized anti-CD154 monoclonal antibody, TNX-1500 (TNX), in a nonhuman primate heterotopic cardiac allogeneic (allo) transplant model. TNX-1500 contains a rupluzimab fragment antigen-binding region and an immunoglobin G4 crystallizable fragment region engineered to reduce binding to the crystallizable fragment gamma receptor IIa and associated risks of thrombosis. Recipients were treated for 6 months with standard-dose TNX (sTNX) monotherapy, low-dose TNX monotherapy (loTNX), or loTNX with mycophenolate mofetil (MMF) (loTNX + MMF). Results were compared with historical data using chimeric humanized 5c8 monotherapy dosed as for loTNX but discontinued at 3 months. Median survival time was similar for humanized 5c8 and both loTNX groups, but significantly longer with sTNX (>265 days) than with loTNX (99 days) or loTNX + MMF (88 days) (P < 0.05 for both comparisons against sTNX). Standard-dose TNX prevented antidonor alloantibody elaboration, inhibited chronic rejection, and was associated with a significantly reduced effector T cells/regulatory T cells ratio relative to loTNX with MMF. No thrombotic complications were observed. This study demonstrated that TNX was well tolerated, prolongs allograft survival, and prevents alloantibody production and cardiac allograft vasculopathy in a stringent preclinical nonhuman primate heart allotransplant model.

Effect of Recombinant Human Thrombopoietin (rhTPO) on Hematopoietic Reconstitution in Allogeneic Hematopoietic Stem Cell Transplantation Model

To explore the effect of recombinant human thrombopoietin (rhTPO) on hematopoietic reconstruction in allogeneic hematopoietic stem cell transplantation (allo-HSCT) model. The C57BL/6 mice were employed as the donors, and BALB/c mice as recipients. The bone marrow mononuclear cells of the donor mice were extracted and pretreated, which then were injected with 5×106 per mouse through the tail vein of the recipient to establish an allo-HSCT model. The implantation of hematopoietic stem cells in the recipient mice was detected by flow cytometry on the 28th day after transplantation. Next, the successfully modeled recipient mice were randomly divided into experimental group and control group. The rhTPO was injected into mice in the experimental group on the first day after transplantation, while the saline was injected into mice in the control group. Both groups were injected for 14 consecutive days. The peripheral blood and bone marrow hematopoiesis of the two groups were observed on day 1, 3, 7, 14, and 21 after transplantation. The expression rate of H-2Kb in the bone marrow of recipient mice was 43.85% (>20%) on the 28th day after transplantation, which indicated that the recipient mice were successfully chimerized. Meanwhile, counts of PLTs on the day 3, 7, 14, and 21 after transplantation in the experimental group were higher than those in the control group with statistical significances (P<0.05). In addition, hematopoietic function of bone marrow was suppressed in both groups on day 1, 3 and 7 after transplantation, but hematopoietic bone marrow hyperplasia was better in the experimental group than in the control group. On day 14 and 21 after transplantation, the hematopoietic function of bone marrow in the two groups was recovered, and the experimental group showed more obvious than the control group. rhTPO can effectively stimulate the production of PLTs and facilitate the recovery of white blood cells and hemoglobin after allo-HSCT, and promote hematopoietic recovery and reconstitution of bone marrow.

Single-Cell RNA Sequencing Maps Immune Cell Heterogeneity in Mice with Allogeneic Cardiac Transplantation

Objective: Immune cells play important roles in mediating allograft rejection and tolerance after cardiac transplantation. However, immune cell heterogeneity at the single-cell level, and how immune cell states shape transplantation immunity, remain incompletely characterized. Methods: We performed single-cell RNA sequencing (scRNA-seq) on immune cells in LNs from a mouse syngeneic and allogeneic cardiac transplantation model. Nine T cell clusters were identified through unsupervised analysis. Pathway enrichment analysis was used to explore the functional differences among cell subpopulations and to characterize the metabolic heterogeneity of T cells. Results: We comprehensively determined the transcriptional landscape of immune cells, particularly T cells, and their metabolic transcriptomes in LNs during mouse cardiac transplantation. On the basis of molecular and functional properties, we also identified T cell types associated with transplantation-associated immune processes, including cytotoxic CD8+ T cells, activated conventional CD4+ T cells, and dysfunctional Tregs. We further elucidated the contribution of JunB to the induction of Th17 cell differentiation and restriction of Treg development, and identified that HIF-1a participates in T cell metabolism and function. Conclusions: We present the first systematic single-cell analysis of transcriptional variation within the T cell population, providing new insights for the development of novel therapeutic targets for allograft rejection.

Amplified Natural Killer Cell Activity and Attenuated Regulatory T-cell Function Are Determinants for Corneal Alloimmunity in Very Young Mice.

Corneal transplantation outcomes are generally less favorable in young children compared with adults. The purpose of this study was to determine the immunological mechanisms underlying this difference. A murine model of allogeneic corneal transplantation was used in the study, and graft survival was determined by evaluating opacity scores for 8 wk. Syngeneic transplantation in the very young host served as a surgical control. The frequencies of total and activated natural killer (NK) cells in cornea posttransplantation were kinetically evaluated using flow cytometry. The regulatory T cell (Treg) frequency and function in naive animals were assessed by flow cytometry and in vitro suppression assays, respectively. Finally, graft survival and immune responses were determined in NK cell-depleted, or adult naive Treg-transferred, young hosts. Corneal allograft survival in the very young recipients was significantly lower than in adult hosts. The frequencies of total NK cells and their interferon gamma-expressing subset in the cornea were significantly higher in the very young mice posttransplantation. In ungrafted mice, frequencies of Treg in draining lymph nodes as well as their capabilities to suppress NK-cell secretion of interferon gamma were lower in the very young compared with adults. In NK cell-depleted or adult Treg--transferred very young recipients, the allograft survival was significantly improved along with the suppressed NK-cell response. Our data demonstrate that amplified activity of NK cells, together with lower suppressive function of Treg, contributes to early rejection of corneal allografts in very young graft recipients.

TCF-1 regulates NKG2D expression on CD8 T cells during anti-tumor responses

Cancer immunotherapy relies on improving T cell effector functions against malignancies, but despite the identification of several key transcription factors (TFs), the biological functions of these TFs are not entirely understood. We developed and utilized a novel, clinically relevant murine model to dissect the functional properties of crucial T cell transcription factors during anti-tumor responses. Our data showed that the loss of TCF-1 in CD8 T cells also leads to loss of key stimulatory molecules such as CD28. Our data showed that TCF-1 suppresses surface NKG2D expression on naïve and activated CD8 T cells via key transcriptional factors Eomes and T-bet. Using both in vitro and in vivo models, we uncovered how TCF-1 regulates critical molecules responsible for peripheral CD8 T cell effector functions. Finally, our unique genetic and molecular approaches suggested that TCF-1 also differentially regulates essential kinases. These kinases, including LCK, LAT, ITK, PLC-γ1, P65, ERKI/II, and JAK/STATs, are required for peripheral CD8 T cell persistent function during alloimmunity. Overall, our molecular and bioinformatics data demonstrate the mechanism by which TCF-1 modulated several critical aspects of T cell function during CD8 T cell response to cancer.Graphical abstractSummary Figure: TCF-1 is required for persistent function of CD8 T cells but dispensable for anti-tumor response. Here, we have utilized a novel mouse model that lacks TCF-1 specifically on CD8 T cells for an allogeneic transplant model. We uncovered a molecular mechanism of how TCF-1 regulates key signaling pathways at both transcriptomic and protein levels. These key molecules included LCK, LAT, ITK, PLC-γ1, p65, ERK I/II, and JAK/STAT signaling. Next, we showed that the lack of TCF-1 impacted phenotype, proinflammatory cytokine production, chemokine expression, and T cell activation. We provided clinical evidence for how these changes impact GVHD target organs (skin, small intestine, and liver). Finally, we provided evidence that TCF-1 regulates NKG2D expression on mouse naïve and activated CD8 T cells. We have shown that CD8 T cells from TCF-1 cKO mice mediate cytolytic functions via NKG2D.

Prevention of alloimmune rejection using XBP1-deleted bone marrow-derived dendritic cells in heart transplantation.

Genetically modified dendritic cells (DCs) modulate the alloimmunity of T lymphocytes by regulating antigen presentation. We generated mice with specific deletion of the X-box-binding protein 1 (XBP1) allele in bone marrow cells and cultured bone marrow-derived DCs (Xbp1-/- BMDCs) from these animals. We then tested the phenotype of Xbp1-/- BMDCs, evaluated their capability to activate allogeneic T cells and investigated their mechanistic actions. We developed a mouse model of allogeneic heart transplantation in which recipients received PBS, Xbp1-/- BMDCs, a suboptimal dose of cyclosporine A (CsA), or Xbp1-/- BMDCs combined with a suboptimal dose of CsA to evaluate the effects of Xbp1-/- BMDC transfusion on alloimmunity and on the survival of heart allografts. The deletion of XBP1 in BMDCs exploited the IRE1-dependent decay of TAPBP mRNA to reduce the expression of MHC-I on the cell surface, altered the capability of BMDCs to activate CD8+ T cells, and ultimately suppressed CD8+ T-cell-mediated allogeneic rejection. The adoptive transfer of Xbp1-/- BMDCs inhibited CD8+ T-cell-mediated rejection. In addition, XBP1-deficient BMDCs were weak stimulators of allogeneic CD4+ T cells despite expressing high levels of MHC-II and costimulatory molecules on their cell surface. Moreover, the adoptive transfer of Xbp1-/- BMDCs inhibited the production of circulating donor-specific IgG. The combination of Xbp1-/- BMDCs and CsA treatment significantly prolonged the survival of allografts compared to CsA alone. The deletion of XBP1 induces immunosuppressive BMDCs, and treatment with these immunosuppressive BMDCs prevents alloimmune rejection and improves the outcomes of heart transplantation. This finding provides a promising therapeutic target in combating transplant rejection and expands knowledge of inducing therapeutic DCs.

MiR-155 Derived from Bone Marrow Mesenchymal Stem Cell-Secreted Exosomes Reduces Kidney Rejection in Rat Allogeneic Transplantation Model via SDF-1/CXCR4

Aberrantly expressed miR-155 is associated with renal rejection after allogeneic transplantation. This study mainly explored the mechanism of miR-155 derived from bone marrow mesenchymal stem cell-secreted exosomes (BMSC-exo) in renal rejection after allogeneic transplantation. Thirty Fischer rats and 40 Lewis rats were used as donors and recipients, respectively. The Lewis rats were randomized into 4 groups (10 rats per group): Control group, miR-155 group, positive control group and CXCR4 agonist group. The following indicators were monitored in BMSC-exo: miR-155 expression, serum creatinine level, renal histopathological changes, CADI score, number of cells that were positive for TGF-β, Smad3 and α-SMA, as well as the protein levels of Smad3, TGF-β, CXCR4 and SDF-1. miR-155 expression in BMSC-exo was significantly higher than that in HKb-20 cells. On the 7th day after surgery, the serum creatinine levels of rats in the miR-155 group and positive control group reduced significantly, while decreasing slowly in the control group and CXCR4 agonist group. The CADI scores of rats in the miR-155 group and positive control group were significantly higher than those in the control group and CXCR4 agonist group (P &lt; 0.05). No significant difference was found either between the miR-155 group and positive control group, or between the control group and CXCR4 agonist group (P &gt; 0.05). Rats in the control group and CXCR4 agonist group had more cells that were positive for TGF-β, Smad3 and α-SMA, while those in the miR-155 group and positive control group showed less. The Smad3, TGF-β, CXCR4 and SDF-1 proteins were weakly expressed in the miR-155 group and positive control group, but strongly expressed in the control group and CXCR4 agonist group. No significant difference in the protein levels was found either between the miR-155 group and positive control group, or between the control group and CXCR4 agonist group (P &gt; 0.05). miR-155 derived from BMSC-exo is protective against allogeneic kidney transplantation. Specifically, BMSC-exo-derived miR-155 blocked the activity of SDF-1/CXCR4 and TGF-β/Smad3 pathways, thereby downregulating the expression of α-SMA. As a result, it ameliorated renal fibrosis and alleviated renal dysfunction, ultimately leading to the prevention and reduction of renal rejection following allograft transplantation.

Transcriptionally Distinct B Cells Infiltrate Allografts After Kidney Transplantation.

Following allogeneic kidney transplantation, a substantial proportion of graft loss is attributed to the formation of donor-specific antibodies and antibody-mediated rejection. B cells infiltrate kidney grafts during antibody-mediated rejection; however, the origins, repertoires, and functions of these intrarenal B cells remain elusive. Here, we use murine allogeneic kidney transplant models to study the origins, transcriptional programming and B cell receptor repertoire of intragraft B cells, and in vitro stimulation assays to evaluate the ability of intragraft B cells to promote CD4+ T cell expansion. B cells infiltrate kidney grafts in settings of allogeneic, but not syngeneic, transplantation. Intragraft B cells have characteristics of activation but are transcriptionally distinct from germinal center B cells and resemble innate-like B cells. B cell receptor sequencing demonstrates that the majority of intragraft B cells do not originate from lymph node germinal center B cells and are largely germline. Class-switched intragraft B cells are rare but can be donor-specific and produce IgG capable of binding to the kidney allograft. Lastly, intrarenal B cells are capable of stimulating naive T cells but have an altered ability to promote T follicular helper cell expansion. Together, these data demonstrate that intrarenal B cells during transplant rejection are transcriptionally distinct from lymph node B cells.

Research Highlights

Research Highlights

Target induction of heme oxygenase-1 attenuates anti-HLA I antibody induced transplant vasculopathy in an allogeneic aortic transplantation model in mice

Abstract Introduction After solid organ transplantation every second patient develops transplant vasculopathy (TV), which still remains the major obstacle for long-term graft survival. The main risk factor for developing TV are de novo synthesized donor-specific antibodies binding molecules of the MHC complex (HLA complex in humans, respectively) on the surface of endothelial cells (EC). EC become activated, vascular smooth muscle cells proliferate, and monocytes are recruited, contributing the development of the neointima a hallmark of TV. We have demonstrated that the anti-HLA I antibody (w6/32) significantly increases transmigration of monocytes across an endothelial monolayer in vitro. These interactions were dependent on modulating the anti-inflammatory enzyme heme oxygenase (HO)-1. Purpose The aim of the project is to investigate which adhesion molecule might mediate the antibody-induced transmigration of monocytes sensitive to HO-1 modulation and whether this modulation has beneficial effects in an allogenic aortic transplantation model in mice. Methods Before w6/32 stimulation, HO-1 was modulated by pretreating Human Coronary Artery Endothelial Cells (HCAEC) either with CoPPIX as a HO-1 activator or with statins to test for a therapeutic approach. Expression of a variety of established adhesion receptors in HCAEC was screened after HO-1 modulation and w6/32 stimulation. Antibody-induced transmigration and adhesion of THP-1 were quantified in the presence of CD62E-blocking antibodies. Thoracic aorta was transplanted into the abdominal aorta between fully MHC mismatched mice and applying the corresponding anti-MHC I antibody (SF1-1.1) induced TV. Mice were treated with CoPPIX, statins or CORM to evaluate the effects of HO-1 modulation or its metabolite CO on TV development. Statistical evaluation was performed with t-test or ANOVA. Results HO-1 modulation in HCAEC diminished w6/32-induced THP-1 transmigration. CD62E-expression in HCAECs was enhanced after w6/32 stimulation on mRNA and protein level, but preincubation with CoPPIX or statins reduced these effects. Incubation of HCAEC with a CD62E-blocking antibody after w6/32 stimulation significantly diminished the rate of transmigration normalized to isotype-stimulated HCAEC (isotype 100%, w6/32 + IgG 226% vs. w6/32 + CD62E-block 138%, p&amp;lt;0.05.). Treatment of mice with CoPPIX resulted in a significant reduction of the SF1-1.1-induced number of transmigrated monocytes (SF1-1.1: 10 vs. CoPPIX: 5.4; p&amp;lt;0.05). Application of statins (SF1-1.1: 10 vs. statin 6.6; p&amp;lt;0.05) or CORM (CORM: 6.1 vs. iCORM: 11.3, p&amp;lt;0.05) significantly reduces monocyte number as well. In all treatment groups, the SF1-1.1-induced neointima formation was ameliorated. Conclusion w6/32-induced monocyte transmigration is CD62E-dependent. SF1-1.1-induced monocytic infiltrate and intimal hyperplasia in a murine aortic transplantation model is also CD62E dependent and amenable to HO-1 modulation. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): This sudy was funded by the German Research Foundation to Jan Larmann

Blocking CCL8-CCR8-Mediated Early Allograft Inflammation Improves Kidney Transplant Function.

In kidney transplantation, early allograft inflammation impairs long-term allograft function. However, precise mediators of early kidney allograft inflammation are unclear, making it challenging to design therapeutic interventions. We used an allogeneic murine kidney transplant model in which CD45.2 BALB/c kidneys were transplanted to CD45.1 C57BL/6 recipients. Donor kidney resident macrophages within the allograft expanded rapidly in the first 3 days. During this period, they were also induced to express a high level of Ccl8, which, in turn, promoted recipient monocyte graft infiltration, their differentiation to resident macrophages, and subsequent expression of Ccl8. Enhanced graft infiltration of recipient CCR8+ T cells followed, including CD4, CD8, and γδ T cells. Consequently, blocking CCL8-CCR8 or depleting donor kidney resident macrophages significantly inhibits early allograft immune cell infiltration and promotes superior short-term allograft function. Targeting the CCL8-CCR8 axis is a promising measure to reduce early kidney allograft inflammation.

Current use of T-cell depleting polyclonal antibodies in liver transplant recipients: Results of a nationwide French survey

Shikonin (SHK) has multifaceted physiological functions, including antitumor, anti-inflammatory, and antimicrobial effects. Recently, SHK has been shown to affect immune responses; however, its detailed immune modulatory function in vivo remains unclear. In this study, we demonstrated that SHK not only inhibited T cell proliferation in vitro, but also intensively inhibited thymopoiesis and eliminated CD4/CD8 double-positive thymic progenitor cells in vivo. Treatment of mice with SHK resulted in immune profile alterations, which promoted myelosis in the bone marrow and increased inhibitory immune cells in central immune organs. A decrease in T cells and B cells was observed in the spleen. Using a murine allogenic skin transplantation model, we revealed that short-term treatment of recipients with SHK significantly inhibited skin graft rejection, in which the levels of myeloid-derived suppressor cells (MDSC) were markedly increased. Taken together, our study suggests that SHK can efficiently eliminate proliferating T cells and inhibit thymopoiesis while promoting the generation of MDSC, indicating its potential role in alleviating immune responses in allogeneic organ/cell transplantation.

Shikonin impairs T lymphocyte proliferation and thymopoiesis while it may increase myeloid-derived suppressor cells to alleviate immune responses

Shikonin (SHK) has multifaceted physiological functions, including antitumor, anti-inflammatory, and antimicrobial effects. Recently, SHK has been shown to affect immune responses; however, its detailed immune modulatory function in vivo remains unclear. In this study, we demonstrated that SHK not only inhibited T cell proliferation in vitro, but also intensively inhibited thymopoiesis and eliminated CD4/CD8 double-positive thymic progenitor cells in vivo. Treatment of mice with SHK resulted in immune profile alterations, which promoted myelosis in the bone marrow and increased inhibitory immune cells in central immune organs. A decrease in T cells and B cells was observed in the spleen. Using a murine allogenic skin transplantation model, we revealed that short-term treatment of recipients with SHK significantly inhibited skin graft rejection, in which the levels of myeloid-derived suppressor cells (MDSC) were markedly increased. Taken together, our study suggests that SHK can efficiently eliminate proliferating T cells and inhibit thymopoiesis while promoting the generation of MDSC, indicating its potential role in alleviating immune responses in allogeneic organ/cell transplantation.

Lactobacillus rhamnosus HN001 Ameliorates BEZ235-Induced Intestinal Dysbiosis and Prolongs Cardiac Transplant Survival.

ABSTRACTCardiac allograft rejection remains a major factor limiting long-term engraftment after transplantation. A novel phosphoinositide 3-kinase (PI3K)/mTOR dual inhibitor, BEZ235, prolonged cardiac allograft survival by effectively suppressing activation of the PI3K/serine/threonine kinase (AKT)/mTOR pathway. However, long-term usage of pharmacological immunosuppressant drugs can cause intestinal microbiota dysbiosis. We established mouse models of allogeneic heterotopic heart transplantation with different treatments. Fecal samples were collected and subjected to 16S rRNA sequencing and targeted fecal metabolomic analysis. Graft samples were taken for immune cell detection by flow cytometry. Inflammatory cytokines in serum were quantified by enzyme-linked immunosorbent assay (ELISA). Compared to single-target approaches (IC-87114 and rapamycin), BEZ235 more efficiently prolongs cardiac transplant survival. Interestingly, BEZ235 reduces the diversity and abundance of the intestinal microbiota community. We demonstrated that Lactobacillus rhamnosus HN001 rescues the intestinal microbiota imbalance induced by BEZ235.IMPORTANCE Our data confirmed that the combination of BEZ235 and Lactobacillus rhamnosus HN001 significantly prolongs cardiac transplant survival. A main metabolic product of Lactobacillus rhamnosus HN001, propionic acid (PA), enriches regulatory T (Treg) cells and serves as a potent immunomodulatory supplement to BEZ235. Our study provides a novel and efficient therapeutic strategy for transplant recipients.

MiR-505-5p alleviates acute rejection of liver transplantation by inhibiting Myd88 and inducing M2 polarizationof Kupffer cells.

The occurrence of acute rejection after liver transplantation seriously impairs the prognosis of patients. miRNA is involved in many physiological and pathological processes of the body, but the mechanism of miRNA action in liver transplantation is not completely clear. In this study, we discuss the role of miR-505-5p in acute rejection after liver transplantation and its putative regulating mechanism. We construct an allogeneic rat liver transplantation model, observe the morphological and pathological changes in liver tissue, detect the expression levels of Myd88, miR-505-5p, IL-10 and TNF-α, and confirm that Myd88 is one of the direct targets of miR-505. The effects of miR-505-5p on the Myd88/TRAF6/NF-κB and MAPK pathways are detected both in vitro and in vivo, and the standard markers of Kupffer cell M1/M2 polarization are also detected. The results of qRT-PCR experiments show that miR-505-5p has a downward trend in rats with acute rejection. Western blot analysis reveals that over-expression of miR-505-5p induces the reduction of NF-κB and MAPK pathways both in vitro and in vivo. The role of miR-505-5p in alleviating acute rejection after transplantation may be accomplished by inducing M2-type polarization of Kupffer cells. In conclusion, we find that miR-505-5p alleviates acute rejection of liver transplantation by inducing M2 polarization of macrophages via the Myd88/TRAF6 axis, which suggests a potential strategy based on miRNAs in the follow-up treatment of liver transplantation.

A Notch/IL-21 signaling axis primes bone marrow T cell progenitor expansion.

Long-term impairment in T cell–mediated adaptive immunity is a major clinical obstacle following treatment of blood disorders with hematopoietic stem cell transplantation. Although T cell development in the thymus has been extensively characterized, there are significant gaps in our understanding of prethymic processes that influence early T cell potential. We have uncovered a Notch/IL-21 signaling axis in bone marrow common lymphoid progenitor (CLP) cells. IL-21 receptor expression was driven by Notch activation in CLPs, and in vivo treatment with IL-21 induced Notch-dependent CLP proliferation. Taking advantage of this potentially novel signaling axis, we generated T cell progenitors ex vivo, which improved repopulation of the thymus and peripheral lymphoid organs of mice in an allogeneic transplant model. Importantly, Notch and IL-21 activation were equally effective in the priming and expansion of human cord blood cells toward the T cell fate, confirming the translational potential of the combined treatment.

Pre-transplant infusion of donor leukocytes treated with extracorporeal photochemotherapy induces immune hypo-responsiveness and long-term allograft survival in murine models

Recipients of solid organ transplantation (SOT) rely on life-long immunosuppression (IS), which is associated with significant side effects. Extracorporeal photochemotherapy (ECP) is a safe, existing cellular therapy used to treat transplant rejection by modulating the recipient’s own blood cells. We sought to induce donor-specific hypo-responsiveness of SOT recipients by infusing ECP-treated donor leukocytes prior to transplant. To this end, we utilized major histocompatibility complex mismatched rodent models of allogeneic cardiac, liver, and kidney transplantation to test this novel strategy. Leukocytes isolated from donor-matched spleens for ECP treatment (ECP-DL) were infused into transplant recipients seven days prior to SOT. Pre-transplant infusion of ECP-DL without additional IS was associated with prolonged graft survival in all models. This innovative approach promoted the production of tolerogenic dendritic cells and regulatory T-cells with subsequent inhibition of T-cell priming and differentiation, along with a significant reduction of donor-specific T-cells in the spleen and grafts of treated animals. This new application of donor-type ECP-treated leukocytes provides insight into the mechanisms behind ECP-induced immunoregulation and holds significant promise in the prevention of graft rejection and reduction in need of global immune suppressive therapy in patients following SOT.