Control Of Allograft Rejection Research Articles

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.

The Role of Regulatory Myeloid Cell Therapy in Renal Allograft Rejection.

Kidney transplantation is a primary therapy for end-stage renal disease (ESRD) all the time. But it does not mean that we have fully unraveling the mystery of kidney transplantation and confer every patient favorable prognosis. Immune rejection has always been a stumbling block when we try to increase the success rate of kidney transplantation and improve long-term outcomes. Even if the immune rejection is effectively controlled in acute phase, there is a high possibility that the immune response mediated by chronically activated antibodies will trigger chronic rejection and ultimately lead to graft failure. At present, immunosuppressive agent prepared chemically is mainly used to prevent acute or chronic rejection, but it failed to increase the long-term survival rate of allografts or reduce the incidence of chronic rejection after acute rejection, and is accompanied by many adverse reactions. Therefore, many studies have begun to use immune cells to regulate the immune response in order to control allograft rejection. This article will focus on the latest study and prospects of more popular regulatory myeloid cells in the direction of renal transplantation immunotherapy and introduce their respective progress from experimental research to clinical research.

A DEEP LEARNING-BASED CAD SYSTEM FOR RENAL ALLOGRAFT ASSESSMENT: DIFFUSION, BOLD, AND CLINICAL BIOMARKERS.

Recently, studies for non-invasive renal transplant evaluation have been explored to control allograft rejection. In this paper, a computer-aided diagnostic system has been developed to accommodate with an early-stage renal transplant status assessment, called RT-CAD. Our model of this system integrated multiple sources for a more accurate diagnosis: two image-based sources and two clinical-based sources. The image-based sources included apparent diffusion coefficients (ADCs) and the amount of deoxygenated hemoglobin (R2*). More specifically, these ADCs were extracted from 47 diffusion weighted magnetic resonance imaging (DW-MRI) scans at 11 different b-values (b0, b50, b100, …, b1000 s/mm2), while the R2* values were extracted from 30 blood oxygen level-dependent MRI (BOLD-MRI) scans at 5 different echo times (2ms, 7ms, 12ms, 17ms, and 22ms). The clinical sources included serum creatinine (SCr) and creatinine clearance (CrCl). First, the kidney was segmented through the RT-CAD system using a geometric deformable model called a level-set method. Second, both ADCs and R2* were estimated for common patients (N = 30) and then were integrated with the corresponding SCr and CrCl. Last, these integrated biomarkers were considered the discriminatory features to be used as trainers and testers for future deep learning-based classifiers such as stacked auto-encoders (SAEs). We used a k-fold cross-validation criteria to evaluate the RT-CAD system diagnostic performance, which achieved the following scores: 93.3%, 90.0%, and 95.0% in terms of accuracy, sensitivity, and specificity in differentiating between acute renal rejection (AR) and non-rejection (NR). The reliability and completeness of the RT-CAD system was further accepted by the area under the curve score of 0.92. The conclusions ensured that the presented RT-CAD system has a high reliability to diagnose the status of the renal transplant in a non-invasive way.

A multimodal computer‐aided diagnostic system for precise identification of renal allograft rejection: Preliminary results

Early assessment of renal allograft function post-transplantation is crucial to minimize and control allograft rejection. Biopsy - the gold standard - is used only as a last resort due to its invasiveness, high cost, adverse events (e.g., bleeding, infection, etc.), and the time for reporting. To overcome these limitations, a renal computer-assisted diagnostic (Renal-CAD) system was developed to assess kidney transplant function. The developed Renal-CAD system integrates data collected from two image-based sources and two clinical-based sources to assess renal transplant function. The imaging sources were the apparent diffusion coefficients (ADCs) extracted from 47 diffusion-weighted magnetic resonance imaging (DW-MRI) scans at 11 different b-values (b0, b50, b100, ..., b1000 s/mm ), and the transverse relaxation rate (R2*) extracted from 30 blood oxygen level-dependent MRI (BOLD-MRI) scans at 5 different echo times (TEs=2, 7, 12, 17, and 22ms). Serum creatinine (SCr) and creatinine clearance (CrCl) were the clinical sources for kidney function evaluation. The Renal-CAD system initially performed kidney segmentation using the level-set method, followed by estimation of the ADCs from DW-MRIs and the R2* from BOLD-MRIs. ADCs and R2* estimates from 30 subjects that have both types of scans were integrated with their associated SCr and CrCl. The integrated biomarkers were then used as our discriminatory features to train and test a deep learning-based classifier, namely stacked autoencoders (SAEs) to differentiate non-rejection (NR) from acute rejection (AR) renal transplants. Using a leave-one-subject-out cross-validation approach along with SAEs, the Renal-CAD system demonstrated 93.3% accuracy, 90.0% sensitivity, and 95.0% specificity in differentiating AR from NR. Robustness of the Renal-CAD system was also confirmed by the area under the curve value of 0.92. Using a stratified tenfold cross-validation approach, the Renal-CAD system demonstrated its reproducibility and robustness by a diagnostic accuracy of 86.7%, sensitivity of 80.0%, specificity of 90.0%, and AUC of 0.88. The obtained results demonstrate the feasibility and efficacy of accurate, noninvasive identification of AR at an early stage using the Renal-CAD system.

HLA-G dimer targets Granzyme B pathway to prolong human renal allograft survival.

Human leukocyte antigen G (HLA-G), a nonclassic HLA class Ib molecule involved in the maintenance of maternal tolerance to semiallogeneic fetal tissues during pregnancy, has emerged as a potential therapeutic target to control allograft rejection. We demonstrate here that the level of soluble HLA-G dimer was higher in a group of 90 patients with a functioning renal allograft compared with 40 patients who rejected (RJ) their transplants. The HLA-G dimer level was not affected by demographic status. One of the potential mechanisms in tissue-organ allograft rejection involves the induction of granzymes and perforin, which are the main effector molecules expressed by CD8+ cytotoxic T lymphocytes and function to destroy allogeneic transplants. Using genomics and molecular and cellular analyses of cells from T-cell-mediated RJ and nonrejected kidney transplant patients, cells from leukocyte Ig-like receptor B1 (LILRB1) transgenic mice, humanized mice, and genetically engineered HLA-G dimer, we demonstrated a novel mechanism by which HLA-G dimer inhibits activation and cytotoxic capabilities of human CD8+ T cells. This mechanism implicated the down-regulation of Granzyme B expression and the essential involvement of LILRB1. Thus, HLA-G dimer has the potential to be a specific and effective therapy for prevention of allograft rejection and prolongation of graft survival.-Ajith, A., Portik-Dobos, V., Nguyen-Lefebvre, A. T., Callaway, C., Horuzsko, D. D., Kapoor, R., Zayas, C., Maenaka, K., Mulloy, L. L., Horuzsko, A. HLA-G dimer targets Granzyme B pathway to prolong human renal allograft survival.

Myeloid-Derived Suppressor Cells as a Regulator of Immunity in Organ Transplantation.

Regulation of allo-immune responses is proposed as a topic for investigation in the current field of organ transplantation. As a regulator, regulatory T cells (Tregs) have received attention due to their ability to control allograft rejection. Concurrently, however, the independent action of Tregs is not enough to achieve tolerance status in many situations. Meanwhile, as a multi-functional regulator, myeloid-derived suppressor cells (MDSCs) can suppress effector T cells as well as induce Tregs or regulatory B cells (Bregs) in certain circumstances. Furthermore, the importance of a crosstalk between MDSCs and natural killer T cells to induce tolerance has been reported. Thus, orchestration between MDSCs, myeloid regulators, T/Bregs and other lymphoid/myeloid regulators can shed light on achieving allogeneic tolerance. Here, we review the current knowledge in terms of immunological regulatory function displayed by MDSCs in the context of organ transplantation. Ideal control of MDSCs would lead to a reduction of allograft rejection and subsequent long-term allograft acceptance.

Immunosuppressive effect of the gut microbiome altered by high-dose tacrolimus in mice.

The alterations induced in gut microbiota by tacrolimus may affect immune function and organ transplantation. Mice were treated with high-dose tacrolimus for 14days. The fecal microbiota were analyzed by pyrosequencing the 16S rRNA genes, and the effect on metabolism was predicted using the sequence data. The subgroups of T cells in the serum, gut-associated lymphoid tissue, and draining lymph nodes were determined by flow cytometry. Tacrolimus treatment significantly altered the relative abundance of Allobaculum, Bacteroides, and Lactobacillus and CD4+ CD25hi FoxP3+ regulatory T cells in the colonic mucosa and the circulation. These were significantly increased after either tacrolimus treatment or treatment by fecal microbiota transfer from tacrolimus-treated donors. Further, treatment with low-dose tacrolimus plus fecal microbiota transfer from high-dose tacrolimus-altered mice increased skin allograft survival rate in a skin transplantation model. Thus, high-dose tacrolimus alters the compositions and taxa of the gut microbiota. Administration of these conditioned gut microbiota plus low-dose tacrolimus resulted in regulation of colonic and systemic immune responses and an increased allograft survival rate. This study demonstrated a new strategy for controlling allograft rejection by combining an immunosuppressive agent with gut microbiome transplantation.

The combination of mitomycin-induced blood cells with a temporary treatment of ciclosporin A prolongs allograft survival in vascularized composite allotransplantation.

Vascularized composite allotransplantation (VCA) is a rapidly expanding field of transplantation and provides a potential treatment for complex tissue defects. Peripheral blood mononuclear cells (PBMCs) shortly incubated with the antibiotic and chemotherapeutic agent mitomycin C (MMC) can suppress allogeneic T cell response and control allograft rejection in various organ transplantation models. MMC-incubated PBMCs (MICs) are currently being tested in a phase I clinical trial in kidney transplant patients. Previous studies with MICs in a complex VCA model showed the immunomodulatory potential of these cells. The aim of this study is to optimize and evaluate the use of MICs in combination with a standard immunosuppressive drug in VCA. Fully mismatched rats were used as hind limb donors [Lewis (RT11)] and recipients [Brown-Norway (RT1n)]. Sixty allogeneic hind limb transplantations were performed in six groups. Group A received donor-derived MICs combined with a temporary ciclosporin A (CsA) treatment. Group B received MICs in combination with a temporarily administered reduced dose of CsA. Group C served as a control and received a standard CsA dose temporarily without an additional administration of MICs, whereas Group D was solely medicated with a reduced CsA dose. Group E received no immunosuppressive therapy, neither CsA nor MICs. Group F was given a continuous standard immunosuppressive regimen consisting of CsA and prednisolone. The endpoint of the study was the onset of allograft rejection which was assessed clinically and histologically. In group A and B, the rejection-free interval of the allograft was significantly prolonged to an average of 23.1±1.7 and 24.7±1.8days compared to the corresponding control groups (p<0.01). Rejection in groups C, D, and E was noted after 14.3±1.1, 7.8±0.7, and 6.9±0.6days. No rejection occurred in control group F during the follow-up period of 100days. No adverse events have been noted. The findings of this study show that the combination of MICs with a temporary CsA treatment significantly prolongs the rejection-free interval in a complex VCA model. The combination of MICs with CsA showed no adverse events such as graft-versus-host disease. MICs, which are generated by a simple and reliable in vitro technique, represent a potential therapeutic tool for prolonging allograft survival through immunomodulation.

Fc-Silent Anti-CD154 Domain Antibody Effectively Prevents Nonhuman Primate Renal Allograft Rejection.

The advent of costimulation blockade provides the prospect for targeted therapy with improved graft survival in transplant patients. Perhaps the most effective costimulation blockade in experimental models is the use of reagents to block the CD40/CD154 pathway. Unfortunately, successful clinical translation of anti-CD154 therapy has not been achieved. In an attempt to develop an agent that is as effective as previous CD154 blocking antibodies but lacks the risk of thromboembolism, we evaluated the efficacy and safety of a novel anti-human CD154 domain antibody (dAb, BMS-986004). The anti-CD154 dAb effectively blocked CD40-CD154 interactions but lacked crystallizable fragment (Fc) binding activity and resultant platelet activation. In a nonhuman primate kidney transplant model, anti-CD154 dAb was safe and efficacious, significantly prolonging allograft survival without evidence of thromboembolism (Median survival time 103 days). The combination of anti-CD154 dAb and conventional immunosuppression synergized to effectively control allograft rejection (Median survival time 397 days). Furthermore, anti-CD154 dAb treatment increased the frequency of CD4+ CD25+ Foxp3+ regulatory T cells. This study demonstrates that the use of a novel anti-CD154 dAb that lacks Fc binding activity is safe without evidence of thromboembolism and is equally as potent as previous anti-CD154 agents at prolonging renal allograft survival in a nonhuman primate preclinical model.

CD4+CD25+ T Regulatory Cells in Transplantation Tolerance: 25 Years On.

In the 1970s, the capacity of T cells to inhibit immunity and those from transplant tolerant hosts to transfer alloantigen-specific suppression to lymphopenic recipients was described. CD4 T suppressor cells that ex vivo reverted to effector cells were described in the 1980s. Their antigen-specific suppressor function could be preserved by stimulation by specific donor alloantigen and cytokines from activated lymphocytes. This led to the finding that alloantigen-specific T suppressor cells express IL-2 receptor (CD25) and that IL-2 in part promotes their survival.Whether these alloantigen-specific CD4CD25FOXP3 regulatory T (Treg) cells are progeny of thymic-derived CD4CD25FOXP3 Treg (tTreg) cells or are induced from peripheral effector CD4CD25FOXP3T cells (iTreg) cells is still debated.In vitro studies of antigen specific Treg has been difficult as they die in the absence of cytokines produced by immune activated cells. The antigen-specific CD4CD25 T cells that control rejection in tolerant hosts differ from the naive tTreg. Thymic-derived Treg cells are not antigen-specific, and very high ratios are required to suppress rejection. Thymic-derived Treg cells can be expanded ex vivo and are currently being tested in trials to control allograft rejection and graft versus host disease.Thymic-derived Treg cells with specific receptors for alloantigen are activated by either IL-2 or IL-4 but rapidly become dependent on other cytokines, respectively, IFN-γ or IL-12 if activated by IL-2, or IL-5 if activated by IL-4. The Th1 and Th2 pathways for activation of tTreg produce more potent antigen-specific Treg that only suppress specific donor rejection. After 25 years, much remains unknown about antigen-specific CD4CD25FOXP3 Treg-mediating transplant tolerance.

Combining autologous dendritic cell therapy with CD3 antibodies promotes regulatory T cells and permanent islet allograft acceptance.

Cell therapy and the use of mAbs that interfere with T cell effector functions constitute promising approaches for the control of allograft rejection. In the current study, we investigated a novel approach combining administration of autologous tolerogenic dendritic cells with short-term treatment with CD3-specific Abs. Permanent acceptance of pancreatic islet allografts was achieved in mice treated with the combination therapy the day before transplantation but not in recipients treated with either therapy alone. The combination treatment induced a marked decrease in T cells infiltrating the allografts and a sustained reduction of antidonor responses. Importantly, CD4(+)Foxp3(+) regulatory T cells appeared to play a crucial role in the long-term graft acceptance. Their frequency increased significantly in the spleen, draining lymph nodes, and transplanted islets and remained elevated over the long term; they exhibited increased donor-specific suppressive functions; and their removal at the time of transplantation abrogated the therapeutic effect of the combined therapy. These results support the therapeutic potential of protocols combining autologous dendritic cells and low-dose CD3 Abs, both currently in clinical development, and that act in synergy to control allogeneic immune responses and favor graft survival in a full-mismatch situation.

Human fibrocytic myeloid-derived suppressor cells express IDO and promote tolerance via Treg-cell expansion.

By restraining T-cell activation and promoting Treg-cell expansion, myeloid-derived suppressor cells (MDSCs) and tolerogenic DCs can control self-reactive and antigraft effector T cells in autoimmunity and transplantation. Their therapeutic use and characterization, however, is limited by their scarce availability in the peripheral blood of tumor-free donors. In the present study, we describe and characterize a novel population of human myeloid suppressor cells, named fibrocytic MDSC, which are differentiated from umbilical cord blood precursors by 4-day culture with FDA-approved cytokines (recombinant human-GM-CSF and recombinant human-G-CSF). This MDSC subset, characterized by the expression of MDSC-, DC-, and fibrocyte-associated markers, promotes Treg-cell expansion and induces normoglycemia in a xenogeneic mouse model of Type 1 diabetes. In order to exert their protolerogenic function, fibrocytic MDSCs require direct contact with activated T cells, which leads to the production and secretion of IDO. This new myeloid subset may have an important role in the in vitro and in vivo production of Treg cells for the treatment of autoimmune diseases, and in either the prevention or control of allograft rejection.

B Cell Dependent Control of Allograft Tolerance Through Expansion of Thymic CD4+ Foxp3+ T Cells.

Introduction: B cells are known to function as producers of antibodies and act as antigen presenting cells, whereas emerging data indicates a role for B cells in transplant tolerance. And although B cells have been reported in the thymus their thymic role is unclear. We have shown that BAFF over-expression can directly control allograft rejection via a peripheral expansion of Tregs. This expansion was shown to be B cell dependent. Results: In new experiments BAFF did not enhance peripheral Treg survival, proliferation or conversion but promoted a ˜3-fold increase in thymic Helios+Treg numbers. Interestingly BAFF also enhanced thymic B cells. Thymic Treg expansion was lost in BAFF-Tg/μMT-/-BM chimeras and B cell null μMT-/-mice exhibited decreased numbers of thymic Tregs. Allograft survival and expansion of thymic Tregs directly correlated to the amount of thymic B cells present resulting in ˜80% islet allograft acceptance. CD19+B cells accumulated in the thymic medulla region close to medullary thymic epithelial cells suggesting a role in thymic Treg selection. Further to this thymic B cells were observed in close proximity to Foxp3+ T cells. In the absence of B cell surface MHC class II, thymic Treg expansion did not occur, indicating the possibility of direct B-Treg interactions in the thymus. Also, thymic Treg expansion did not occur in BAFF-Tg/IgHEL BCRBM chimeras demonstrating a requirement for antigen-specificity. Conclusion: We identify a new role for thymic B cells and present a model whereby intra-thymic B cells provide a source of cognate signals to modulate thymic Treg production. These data demostrate a direct relationship between thymic B cells, thymic Tregs and allograft tolerance.

Attenuation of Donor-Reactive T Cells Allows Effective Control of Allograft Rejection Using Regulatory T Cell Therapy

Regulatory T cells (Tregs) are essential for the establishment and maintenance of immune tolerance, suggesting a potential therapeutic role for Tregs in transplantation. However, Treg administration alone is insufficient in inducing long-term allograft survival in normal hosts, likely due to the high frequency of alloreactive T cells. We hypothesized that a targeted reduction of alloreactive T effector cells would allow a therapeutic window for Treg efficacy. Here we show that preconditioning recipient mice with donor-specific transfusion followed by cyclophosphamide treatment deleted 70–80% donor-reactive T cells, but failed to prolong islet allograft survival. However, infusion of either 5 × 106 Tregs with direct donor reactivity or 25 × 106 polyclonal Tregs led to indefinite survival of BALB/c islets in more than 70% of preconditioned C57BL/6 recipients. Notably, protection of C3H islets in autoimmune nonobese diabetic mice required islet autoantigen-specific Tregs together with polyclonal Tregs. Treg therapy led to significant reduction of CD8+ T cells and concomitant increase in endogenous Tregs among graft-infiltrating cells early after transplantation. Together, these results demonstrate that reduction of the donor-reactive T cells will be an important component of Treg-based therapies in transplantation.

Control Of Allograft Rejection With Mitomycin C Incubated Blood Cells

Immunosuppressive treatment in allogeneic transplantation is an effective way to control allograft rejection. But unfortunately it can cause serious side effects, such as infections or malignancies. Cell therapeutic approaches for induction of specific tolerance are on the rise and may have fewer side effects. We observed that monocytes or peripheral blood mononuclear cells (PBMC) of healthy persons after culture with dendritic cell-inducing cytokines and differentiation blockade with the chemotherapeutic drug mitomycin C (MMC) show the ability to suppress allogeneic T cell responses. Interestingly these cells resemble morphologically as well as phenotypically myeloid-derived suppressor cells (MDSC) which occur in cancer patients impairing the anti-tumor immunity. FACS- and gene expression analyses of these MMC incubated blood cells revealed downregulation of the immunostimulatory cell surface molecules CD80, CD83, CD86 and HLA-DR, and upregulated transcription of immunosuppressive and pro-apoptotic genes such as iNOS, arginase-1, IL-10, COX2, TGF-β, placental growth factor, immunoglobulin-like transcript 3, adrenomedullin, SMAD5, LRDD and of the transcription factor C/EBPβ. For use in preclinical transplantation models in vitro culture of MMC treated donor-derived blood cells with dendritic-cell inducing cytokines was not applicable and was therefore omitted. The tolerance inducing properties of donor-derived MMC incubated blood cells were investigated in two different animal models of solid organ transplantation. When injected into rats, donor-derived PBMC incubated with MMC strongly prolonged heart allograft survival. Suppression of rejection was donor-specific. Recipients showed an increased number of CD4+CD25+FoxP3+ regulatory T-cells and tolerance could be transferred to syngeneic recipients with blood or spleen cells. Donor-derived MMC incubated blood cells also prolonged kidney allograft survival in pigs. Furthermore, in a first clinical use a 6-year-old girl with relapse of acute lymphoblastic leukemia and imminent rejection of a third haploidentical stem cell transplant received donor-derived CD3/CD19-depleted peripheral blood stem cells, which have been treated in vitro with MMC, in order to prohibit graft rejection. We describe a simple method for in vitro generation of tolerance inducing donor-derived blood cells for potential use in clinical organ transplantation. Disclosures: No relevant conflicts of interest to declare.

In vitro-expanded CD4+CD25highFoxp3+ regulatory T cells controls corneal allograft rejection

Natural CD4(+)CD25(+) regulatory cells (nTregs) have been implicated in maintaining peripheral immune tolerance. This study aims to test whether immunotherapy using in vitro-expanded Treg (iTregs) could suppress allograft rejection in corneal transplantation model. Natural CD4(+)CD25(+) T cells were freshly purified from naïve mice and expanded in vitro by culturing with anti-CD3/CD28-coated Dynabeads, interleukin (IL)-2 and transforming growth factor (TGF-β1). Suppression ability of iTregs was assayed by co-culturing with CD4(+)CD25(-) T cells (Teff) in vitro and by targeting corneal allograft rejection in vivo. Tracking of iTreg after adoptive transfer in vivo were examined by CFSE labeling. Natural Treg cells were expanded by culturing with anti-CD3/CD28-coated Dynabeads in the presence of IL-2 and TGF-β1. Compared with nTregs, iTregs had similar expression of CD62L, and PD- L1, lower expression of CD69, higher levels of PD-1, CD25, and Foxp3. iTreg cells exerted stronger suppression function than natural Treg cells when cocultured with CD4(+)CD25(-) T cells in vitro and prevented fully MHC-mismatched corneal allograft rejection. Survival of iTreg cells could suppress alloimmune reaction and most prone to migrate to graft draining LNs and spleens. Moreover, maintaining CD25 expression on iTregs was indicative for preservation of allosuppression. Therapeutic use of in vitro-expanded CD4(+)CD25(+) T cells may be a effective and safe tool for controlling allograft rejection and may help induce allograft tolerance.

Regulation of human dendritic cells by a novel specific nuclear factor–κB inhibitor, dehydroxymethylepoxyquinomicin

Regulation of antigen-presenting cells (APC) is crucial in controlling allograft rejection. Dendritic cells (DC) are the most potent APC and must mature to present antigens to T-cell receptors. During DC maturation, nuclear factor–κB (NF-κB) is a key transcriptional factor. We synthesized dehydroxymethylepoxyquinomicin (DHMEQ), which specifically inhibits the final step of nuclear translocation of activated NF-κB proteins and examined its immunoregulatory effects on human monocyte–derived DC (Mo-DC). Regulatory Mo-DC were generated by pretreatment with DHMEQ before LPS stimulation, which were termed dl-DC. DHMEQ pretreatment (5 μg/ml) completely inhibited nuclear translocation of activated NF-κB. DHMEQ significantly inhibited DC production of proinflammatory cytokines (IL-6, TNF-α, and IL-12 p70) in a dose-dependent manner. IL-12 was most potently inhibited. However, IL-10 production by dl-DC was only moderately affected by DHMEQ. Although CD40 and the expression of HLA-DR (HLA-DR) expression on dl-DC was downregulated, CD80 and CD86 expression was moderately upregulated. Induction of T helper 1 cell responses was efficiently impaired by dl-DC. This confirmed that DHMEQ-treated Mo-DC exhibited immunoregulatory effects. These findings suggest that DHMEQ has potential as an immunosuppressive drug for human immune cells.

IFN‐γ Triggered STAT1‐PKB/AKT Signalling Pathway Influences the Function of Alloantigen Reactive Regulatory T Cells

CD4+CD25+Foxp3+ regulatory T cells (Tregs) play a key role in the induction and maintenance of peripheral tolerance. Rapid and transient production of IFN-γ by Tregs from mice tolerized to alloantigen in vivo has been shown to be critical for their regulatory function. This IFN-γ has the potential to affect the function of cells present in the same local microenvironment as the Tregs, including the Tregs themselves. Here we investigated the mechanism by which IFN-γ produced by Tregs triggered signaling pathways in alloantigen reactive Tregs themselves thereby influencing their function in vivo. We show that IFN-γ production and STAT1 activation was increased, while STAT1-dependent PKB/AKT activation was downregulated in alloantigen reactive Tregs. Further, the activation of STAT1 was blocked in IFN-γ receptor deficient as well as IFN-γ–deficient Tregs, suggesting that IFN-γ produced by the alloantigen reactive Tregs might act in an autocrine manner to induce STAT1 activation. Importantly, STAT1-deficient Tregs failed to control allograft rejection in vivo. Overall, these findings suggest that the IFN-γ–induced STAT1-PKB/AKT signaling pathway plays a key role in upregulating the ability of alloantigen reactive Tregs to control graft rejection in vivo.

English

Treatment of disorders through immune manipulation of the host has come a long way in the last decades and has broadened its applications from infectious diseases to control of allograft rejection, induction of tolerance for the treatment of autoimmunity and break of tolerance to induce cancer rejection. Immunotherapy, biological response modifier therapy or biotherapy uses the immune system to fight disease like cancer. The potentials of immunotherapy are many. The objective of this review article is to increase awareness of contemporary immunologic therapies and recent advances in immunotherapy. Keywords - Immunotherapy, Adjuvant, Interleukin, Monoclonal antibody, Interferon, Autoimmunity Immunotherapy Introduction Immunotherapy uses the immune system to fight disorders and immune based therapies are to harness the sensitivity, specificity and self- regulation of the immune system to eradicate any and all tumor cells (1). Biological response modifiers (BRMs) change the route the body's defenses interact with infected cells and produced in a laboratory and given to patients to enhance immune system. BRMs include nonspecific immunomodulating agents, interferons, interleukins, colony-stimulating factors, monoclonal antibodies, cytokine therapy, and vaccines. Monoclonal antibodies are agents, produced in the laboratories that bind to infected cells. Examples of monoclonal antibody therapy include trastuzumab for breast cancer and rituximab for lymphoma. Chronic disorders including malignant, infectious and autoimmune conditions are difficult to treat and often require the use of multiple agents, alone or in combination. The latter approach is already being attempted in some cancer patients. Herceptin, which targets cell proliferation mediated by HER2 and bevacizumab, which targets tumor angiogenesis mediated by VEGF, are being used in combination in breast cancer patients (2). Research has been undertaken to provide an explanation on how a relatively limited number of antibodies can protect against a vast and almost infinite number of invading antigens (3). Proposed potential explanations include the notion that one antibody may be capable of binding more than one antigen through the selection of specific binding sites on pre-existing antibody conformations, and in this manner antibodies with multiple specificities can be achieved. It has been suggested that multispecificity may evolve and play a role in the efficient antibody repertoire for immune protection with one antibody performing more than one task (4). Autoimmune beneficial from the use of two-in-one antibodies and autoimmune disorders, particularly rheumatoid arthritis and systemic lupus erythematosus, are being treated with single biologic agents directed at specific pathogenic pathways, but one may envision the use of two-in-one antibodies directed at multiple targets in the not too distant future (5). Cytokine therapy is used with advanced melanoma and with adjuvant therapy and reaches all parts of the body to kill infected cells and prevent tumors from growing. Vaccine therapy involves use of vaccines produced in Escherichia coli , yeast or insect cells. Plant- based vaccines are also advantageous in terms of their scalability, no cold chain required, stability, safety, cost-effectiveness and needle- free administration (6). Peptide immunotherapy using dominant T-cell epitopes derived from allergens represents another approach to developing safer treatment, because regions involved in allergenicity i.e., responsible for reactivity with IgE, can be completely removed while retaining immunogenicity (7). Antigen- specific immunotherapy strategies include administration of: proteins/peptides with or without adjuvant; dendritic cells (DCs; transfected with DNA- or RNA-encoding tumor antigens, or loaded with peptides, whole proteins or tumor lysates); recombinant viruses encoding tumor antigens; and autologous or allogeneic tumor cells (8). Future of immune therapy for cancer holds promise with novel combined approaches that simultaneously target cancer-initiating stem cells, restore APC immune-stimulating activity, expand tumor-reactive T cells and down regulate suppressor pathways to generate effective therapy.(9) Immune therapy for lung cancer has high potential. Identification of the cell type capable of sustaining neoplastic growth and directing immune therapy to cells that possess

Effect of in vitroexpanded CD4+CD25+Foxp3+ regulatory T cell therapy combined with lymphodepletion in murine skin allotransplantation

A promising approach for preventing allograft rejection involves shifting the balance between cytopathic and regulatory T cells to dominance of the latter cell type. Nonspecific lymphodepletion was conducted by administration of depleting anti-CD4 and anti-CD8 antibodies to reduce effector T cells and adoptive transfer of ex vivo-expanded host Treg cells by stimulation with donor dendritic cells to augment the Treg cell compartment. Evaluation of an MHC-mismatched skin allograft model revealed that combined therapy with these two protocols consistently induced modest prolongation of allograft survival, although all skin grafts were eventually rejected. The administration of IL-2/anti-IL-2 complexes significantly improved the efficacy of combination therapy via promoting the expansion of adoptively transferred Treg cells as well as endogenous recipient Treg cells. We conclude that Treg cell therapy combined with lymphodepletion is of practical benefit for the control of allograft rejection, but requires supplementary measures to promote immune tolerance.