Tolerogenic Antigen Research Articles

Serum Albumin fused Flt3L expands a tolDC compartment and amplifies tolerogenic antigen immunotherapy for the prevention of pathogenic anti-drug antibody responses

Abstract From antibodies to enzyme replacement therapies, biologics and protein drugs have become a major part of the medical market and revolutionized medicine. Repeated administration of many of these drugs, however, leads to humoral responses and potentially drug failure via neutralization, clearance, or immune related infusion reactions. Once pathogenic anti-drug antibodies are formed, patients must take broad immunosuppressive drugs, undergo continuous B cell depletion, or discontinue treatment entirely. Currently, no drugs are approved by the FDA to prevent the induction of anti-drug antibodies. Herein, we characterize the effect of our engineered cytokine, Flt3L-SA, in vivoand demonstrate how our molecule expands the dendritic cells while enhancing a tolerogenic, TGFβ expressing subpopulation. We then leverage these dendritic cells to attenuate the antibody response to the highly immunogenic drug Rasburicase and ultimately reduce antibodies by 100-fold when administered as part of a tolerogenic regime with low dose Rasburicase. We also show how this reduction in antibodies results in fewer and less severe infusion reactions upon final challenge, evidencing both a reduction in the antibody response as well as pathology. Future work will aim to determine the exact mechanism by which this tolerance is achieved as well as demonstrate efficacy in the context of a wide range of extremely immunogenic therapies, including enzyme replacement therapeutics for lysosomal storage disorders and AAV therapies. Supported via the Chicago Immunoengineering Innovation Center and the Alper Family Foundation

Type 1 regulatory T cell-mediated tolerance in health and disease.

Type 1 regulatory T (Tr1) cells, in addition to other regulatory cells, contribute to immunological tolerance to prevent autoimmunity and excessive inflammation. Tr1 cells arise in the periphery upon antigen stimulation in the presence of tolerogenic antigen presenting cells and secrete large amounts of the immunosuppressive cytokine IL-10. The protective role of Tr1 cells in autoimmune diseases and inflammatory bowel disease has been well established, and this led to the exploration of this population as a potential cell therapy. On the other hand, the role of Tr1 cells in infectious disease is not well characterized, thus raising concern that these tolerogenic cells may cause general immune suppression which would prevent pathogen clearance. In this review, we summarize current literature surrounding Tr1-mediated tolerance and its role in health and disease settings including autoimmunity, inflammatory bowel disease, and infectious diseases.

The Use of a Staggered Herringbone Micromixer for the Preparation of Rigid Liposomal Formulations Allows Efficient Encapsulation of Antigen and Adjuvant

Anionic liposomal formulations have previously shown to have intrinsic tolerogenic capacity and these properties have been related to the rigidity of the particles. The combination of highly rigid anionic liposomes to deliver tolerogenic adjuvants and antigen peptides has potential applications for the treatment of autoimmune and inflammatory diseases. However, the preparation of these highly rigid anionic liposomes using traditional methods such as lipid film hydration presents problems in terms of scalability and loading efficiency of some costly tolerogenic adjuvants like 1-α,25-dihydroxyvitaminD3. Here we propose the use of an off-the-shelf staggered herringbone micromixer for the preparation of these formulations and performed a systematic study on the effect of temperature and flow conditions on the size and polydispersity index of the formulations. Furthermore, we show that the system allows for the encapsulation of a wide variety of peptides and significantly higher loading efficiency of 1-α,25-dihydroxyvitaminD3 compared to the traditional lipid film hydration method, without compromising their non-inflammatory interaction with dendritic cells. Therefore, the microfluidics method presented here is a valuable tool for the preparation of highly rigid tolerogenic liposomes in a fast, size-tuneable and scalable manner.

Antigen-specific tolerization in human autoimmunity: Inhibition of interferon-beta1a anti-drug antibodies in multiple sclerosis: A case report

Background: Antigen-specific tolerance in auto-immune diseases is the goal for effective treatment with minimal side-effects. Whilst this is achievable in animal models, notably via intravenous delivery of the model-specific autoantigen following transient CD4 T cell depletion, specific multiple sclerosis autoantigens remain unproven. However, anti-drug antibodies to human therapeutic proteins represent a model human autoimmune condition, which may be used to examine immune-tolerance induction. Some people with MS (PwMS) on interferon-beta1a (IFNβ1a) develop neutralizing antibodies to IFNβ1a that do not disappear in repeated tests over years.Methods: One PwMS was recruited, as part of a planned phase IIa trial (n = 15), who had developed neutralizing antibodies to subcutaneous IFNβ1a. Mitoxantrone (12 mg/m2) was administered as a lymphocyte depleting agent followed by four days of (88 μg/day + three 132 μg/day) intravenous IFNβ1a. Subcutaneous IFNβ1a three times a week was maintained during follow-up. IFNβ1a neutralizing antibody responses in serum were measured during treatment and three-monthly for 12 months.Findings: One participant was recruited and, within 6 months of tolerization, the neutralizing antibodies were undetectable. The tolerization treatment was well tolerated. However, the study was terminated after the first enrolment, on ethical grounds, as treatment alternatives became available and the potential risks of mitoxantrone use increased.Interpretation: The data suggest that it may be possible to induce antigen-specific tolerance by providing tolerogenic antigen following transient immune depletion. Further studies are warranted.Funding: The study was supported by an unrestricted research grant from Merck-Serono.

Strategies to Use Nanoparticles to Generate CD4 and CD8 Regulatory T Cells for the Treatment of SLE and Other Autoimmune Diseases.

Autoimmune diseases are disorders of immune regulation where the mechanisms responsible for self-tolerance break down and pathologic T cells overcome the protective effects of T regulatory cells (Tregs) that normally control them. The result can be the initiation of chronic inflammatory diseases. Systemic lupus erythematosus (SLE) and other autoimmune diseases are generally treated with pharmacologic or biological agents that have broad suppressive effects. These agents can halt disease progression, yet rarely cure while carrying serious adverse side effects. Recently, nanoparticles have been engineered to correct homeostatic regulatory defects and regenerate therapeutic antigen-specific Tregs. Some approaches have used nanoparticles targeted to antigen presenting cells to switch their support from pathogenic T cells to protective Tregs. Others have used nanoparticles targeted directly to T cells for the induction and expansion of CD4+ and CD8+ Tregs. Some of these T cell targeted nanoparticles have been formulated to act as tolerogenic artificial antigen presenting cells. This article discusses the properties of these various nanoparticle formulations and the strategies to use them in the treatment of autoimmune diseases. The restoration and maintenance of Treg predominance over effector cells should promote long-term autoimmune disease remission and ultimately prevent them in susceptible individuals.

CD1d deficiency limits tolerogenic properties of peritoneal macrophages

Invariant natural killer T (iNKT) cells are involved in various autoimmune diseases. Although iNKT cells are arthritogenic, transforming growth factor beta (TGFβ)-treated tolerogenic peritoneal macrophages (Tol-pMφ) from wild-type (WT) mice are more tolerogenic than those from CD1d knock-out iNKT cell-deficient mice in a collagen-induced arthritis (CIA) model. The underlying mechanism by which pMφ can act as tolerogenic antigen presenting cells (APCs) is currently unclear. To determine cellular mechanisms underlying CD1d-dependent tolerogenicity of pMφ, in vitro and in vivo characteristics of pMφ were investigated. Unlike dendritic cells or splenic Mφ, pMφ from CD1d+/− mice showed lower expression levels of costimulatory molecule CD86 and produced lower amounts of inflammatory cytokines upon lipopolysaccharide (LPS) stimulation compared to pMφ from CD1d-deficient mice. In a CIA model of CD1d-deficient mice, adoptively transferred pMφ from WT mice reduced the severity of arthritis. However, pMφ from CD1d-deficient mice were unable to reduce the severity of arthritis. Hence, the tolerogenicity of pMφ is a cell-intrinsic property that is probably confer-red by iNKT cells during pMφ development rather than by interactions of pMφ with iNKT cells during antigen presentation to cognate T cells.

Immunomodulatory Dual-Sized Microparticle System Conditions Human Antigen Presenting Cells Into a Tolerogenic Phenotype In Vitro and Inhibits Type 1 Diabetes-Specific Autoreactive T Cell Responses.

Current monotherapeutic agents fail to restore tolerance to self-antigens in autoimmune individuals without systemic immunosuppression. We hypothesized that a combinatorial drug formulation delivered by a poly-lactic-co-glycolic acid (PLGA) dual-sized microparticle (dMP) system would facilitate tunable drug delivery to elicit immune tolerance. Specifically, we utilized 30 µm MPs to provide local sustained release of granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor β1 (TGF-β1) along with 1 µm MPs to facilitate phagocytic uptake of encapsulated antigen and 1α,25(OH)2 Vitamin D3 (VD3) followed by tolerogenic antigen presentation. We previously demonstrated the dMP system ameliorated type 1 diabetes (T1D) and experimental autoimmune encephalomyelitis (EAE) in murine models. Here, we investigated the system’s capacity to impact human cell activity in vitro to advance clinical translation. dMP treatment directly reduced T cell proliferation and inflammatory cytokine production. dMP delivery to monocytes and monocyte-derived dendritic cells (DCs) increased their expression of surface and intracellular anti-inflammatory mediators. In co-culture, dMP-treated DCs (dMP-DCs) reduced allogeneic T cell receptor (TCR) signaling and proliferation, while increasing PD-1 expression, IL-10 production, and regulatory T cell (Treg) frequency. To model antigen-specific activation and downstream function, we co-cultured TCR-engineered autoreactive T cell “avatars,” with dMP-DCs or control DCs followed by β-cell line (ßlox5) target cells. For G6PC2-specific CD8+ avatars (clone 32), dMP-DC exposure reduced Granzyme B and dampened cytotoxicity. GAD65-reactive CD4+ avatars (clone 4.13) exhibited an anergic/exhausted phenotype with dMP-DC presence. Collectively, these data suggest this dMP formulation conditions human antigen presenting cells toward a tolerogenic phenotype, inducing regulatory and suppressive T cell responses.

Transfer of antigen-encoding bone marrow under immune-preserving conditions deletes mature antigen-specific B cells in recipients and inhibits antigen-specific antibody production

Background aimsPathological activation and collaboration of T and B cells underlies pathogenic autoantibody responses. Existing treatments for autoimmune disease cause non-specific immunosuppression, and induction of antigen-specific tolerance remains an elusive goal. Many immunotherapies aim to manipulate the T-cell component of T–B interplay, but few directly target B cells. One possible means to specifically target B cells is the transfer of gene-engineered BM that, once engrafted, gives rise to widespread specific and tolerogenic antigen expression within the hematopoietic system. MethodsGene-engineered bone marrow encoding ubiquitous ovalbumin expression was transferred after low-dose (300-cGy) immune-preserving irradiation. B-cell responsiveness was monitored by analyzing ovalbumin-specific antibody production after immunization with ovalbumin/complete Freund's adjuvant. Ovalbumin-specific B cells and their response to immunization were analyzed using multi-tetramer staining. When antigen-encoding bone marrow was transferred under immune-preserving conditions, cognate antigen-specific B cells were purged from the recipient's preexisting B-cell repertoire and the repertoire that arose after bone marrow transfer. ResultsOVA-specific B-cell deletion was apparent within the established host B-cell repertoire as well as that developing after gene-engineered bone marrow transfer. OVA-specific antibody production was substantially inhibited by transfer of OVA-encoding BM and activation of OVA-specific B cells, germinal center formation and subsequent OVA-specific plasmablast differentiation were all inhibited. Low levels of gene-engineered bone marrow chimerism were sufficient to limit antigen-specific antibody production. ResultsThese data show that antigen-specific B cells within an established B-cell repertoire are susceptible to de novo tolerance induction, and this can be achieved by transfer of gene-engineered bone marrow. This adds further dimensions to the utility of antigen-encoding bone marrow transfer as an immunotherapeutic tool.

Biomimetic tolerogenic artificial antigen presenting cells for regulatory T cell induction

Regulatory T cell (Treg)-based therapeutics are receiving increased attention for their potential to treat autoimmune disease and prevent transplant rejection. Adoptively transferred Tregs have shown promise in early clinical trials, but cell-based therapies are expensive and complex to implement, and “off-the-shelf” alternatives are needed. Here, we investigate the potential of artificial antigen presenting cells (aAPCs) fabricated from a blend of negatively charged biodegradable polymer (poly(lactic-co-glycolic acid), PLGA) and cationic biodegradable polymer (poly(beta-amino ester), PBAE) with incorporation of extracellular protein signals 1 and 2 and a soluble released signal 3 to convert naïve T cells to induced Foxp3+ Treg-like suppressor cells (iTregs) both in vitro and in vivo in a biomimetic manner. The addition of PBAE to the aAPC core increased the conjugation efficiency of signal proteins to the particle surface and resulted in enhanced ability to bind to naïve T cells and induce iTregs with potent suppressive function. Furthermore, PLGA/PBAE tolerogenic aAPCs (TolAPCs) supported the loading and sustained release of signal 3 cytokine TGF-β. A single dose of TolAPCs administered intravenously to C57BL/6 J mice resulted in an increased percentage of Foxp3+ cells in the lymph nodes. Thus, PLGA/PBAE TolAPCs show potential as an “off-the-shelf” biomimetic material for tolerance induction. Statement of SignificanceRegulatory T cells (Tregs) are promising for basic research and translational medicine as they can induce tolerance and have the potential to treat autoimmune diseases such as type 1 diabetes and multiple sclerosis. As cell-based therapies are expensive and difficult to manufacture and implement, non-cellular methods of engineering endogenous Tregs are needed. The research reported here describes a new type of biomimetic particle, tolerogenic artificial antigen presenting cells (TolAPCs) fabricated from a blend of negatively charged biodegradable polymer, poly(lactic-co-glycolic acid), and positively charged biodegradable polymer, poly(beta-amino ester), along with key biomolecular signals: extracellularly presented protein signals 1 and 2 and a soluble released signal 3. These TolAPCs bind to naïve T cells and induce Foxp3+ Treg-like suppressor cells with potent suppressive function. In both in vitro and in vivo studies, it is shown that this non-cellular approach is useful to induce tolerance.

Endocrine Disruptor Bisphenol A (BPA) Triggers Systemic Para-Inflammation and is Sufficient to Induce Airway Allergic Sensitization in Mice.

Allergic airway diseases are accompanied by increased permeability and an inflammatory state of epithelial barriers, which are thought to be susceptible to allergen sensitization. Although exogenous drivers (proteases, allergens) of epithelial barrier disruption and sensitization are well studied, endogenous contributors (diet, xenobiotics, hormones, and metabolism) to allergic sensitization are much less understood. Xenoestrogens are synthetic or natural chemical compounds that have the ability to mimic estrogen and are ubiquitous in the food and water supply of developed countries. By interfering with the estrogen produced by the endocrine system, these compounds have the systemic potential to disrupt the homeostasis of multiple tissues. Our study examined the potential of prototypical xenoestrogen bisphenol A (BPA) to disrupt epithelial homeostasis in vitro and promote allergic responses in vivo. We found that BPA exposure in epithelial cultures in vitro significantly inhibited epithelial cell proliferation and wound healing, as well as promoted the expression of the innate alarmin cytokine TSLP in a time-and dose-dependent manner. In vivo, the exposure to BPA through water supply or inhalation induced a systemic para-inflammatory response by promoting the expression of innate inflammatory mediators in the skin, gut, and airway. In a murine tolerogenic antigen challenge model, chronic systemic exposure to BPA was sufficient to induce airway sensitization to innocuous chicken egg ovalbumin in the complete absence of adjuvants. Mechanistic studies are needed to test conclusively whether endocrine disruptors may play an upstream role in allergic sensitization via their ability to promote a para-inflammatory state.

IL-35+ regulatory immune cells in diabetic nephropathy

Abstract Diabetes causes an elevation of the blood glucose level and a long-term hyperglycemia contributes to kidney damage, i.e. diabetic nephropathy (DN). DN exhibits signs of inflammation and infiltration of mononuclear cells in kidney. Regulatory T cells (Tregs), regulatory B cells (Bregs), tolerogenic antigen presenting cells (tolAPCs) maintain the homestasis of the immune system, specifically by producing anti-inflammatory cytokine IL-35. Despite intensive research the role of Tregs, Bregs and tol APCs and IL-35 is not yet clear. Herein, we investigated the role of these immune cells in mice and patients. The circulating concentration of IL-35 was lower in T1D patients with DN and without DN than healthy controls. Proportions of IL35+Treg, IL-35+Bregs, IL-35+tolAPCs were lower but IL-17+Tregs were higher in patients with DN compared to healthy controls, suggesting a phenotypic shift of Tregs. The proportions of IL35+Treg, IL-35+Bregs, IL-35+tolAPCs were lower in kidneys of diabetic mice. Furthermore, IFN-gamma+ Tregs were higher in kidneys of diabetic mice, indicating a phenotypic shift of Treg cells. After the administration of IL-35 in diabetic mice the mice became normoglycemic and had a lowered albumin creatinine ratio compared to control mice. The IL-IL35+Treg, IL-35+Bregs, IL-35+tolAPCs were increased in IL-35 treated mice. IFN-gamma+Tregs were decreased and the number of Foxp3+ cells was decreased, and mononuclear cells infiltration was also decreased in kidneys of IL-35 treated mice. Our data suggest that IL-35+ regulatory immune cells are impaired in DN and systemic administration of IL-35 prevents the development of DN in mouse models of DN.

Kinetics of immune cell responses in the multiple low dose streptozotocin mouse model of type 1 diabetes

Abstract In type 1 diabetes (T1D), the insulin producing β cells are damaged by immune attacks. It has been hypothesized that the very first response in T1D onset comes from innate immune cells, which further activates the adaptive immune cells to attack the β cells. The kinetics of the infiltration of different immune cells in the multiple low dose streptozotocin (MLDSTZ) induced T1D mouse model is still unclear. Therefore, we used flow cytometry to investigate the proportions of innate immune cells such as neutrophils, dendritic cells (DCs), plasmacytoid DCs (pDCs) and macrophages, and adaptive immune cells (T and B lymphocytes) in thymi, pancreatic draining lymph nodes (PDLNs) and spleens of MLDSTZ mice on days 3, 7, 10 and 21 after the first injection of MLDSTZ. The proportions of DCs and pDCs were increased on day 3 while the proportions of B-1a lymphocytes and neutrophils were increased on day 10 in MLDSTZ treated mice, illustrating that the initial immune response is induced by DCs and pDCs. The proportions of T helper 1, Helios+ T cells and cytotoxic T cells were increased from day 7, suggesting that the innate immune cells precede the adaptive immune cell response in MLDSTZ mice. We also found the proportions of interleukin-35 (IL-35) expressing tolerogenic antigen presenting cells (tolAPCs) were decreased in the spleens of MLDSTZ mice than in control mice on day 21. Furthermore, we found the proportions of IL-35+ tolAPCs were decreased in the peripheral blood mononuclear cells from T1D patients than from healthy controls. Altogether, our data demonstrate a possible sequence of events regarding the involvement of DCs, pDCs, B-1a lymphocytes, neutrophils, and Helios+ T-cells at the early stage of T1D development.

Differentially Tolerized Mouse Antigen Presenting Cells Share a Common miRNA Signature Including Enhanced mmu-miR-223-3p Expression Which Is Sufficient to Imprint a Protolerogenic State.

Dendritic cells (DCs) are pivotal for the induction and maintenance of antigen-specific tolerance and immunity. miRNAs mediate post-transcriptional gene regulation and control in part the differentiation and stimulation-induced immunogenic function of DCs. However, the relevance of miRNAs for the induction and maintenance of a tolerogenic state of DCs has scarcely been highlighted yet. We differentiated mouse bone marrow cells to conventional/myeloid DCs or to tolerogenic antigen presenting cells (APCs) by using a glucocorticoid (dexamethasone) or interleukin-10, and assessed the miRNA expression patterns of unstimulated and LPS-stimulated cell populations by array analysis and QPCR. Differentially tolerized mouse APCs convergingly down-regulated a set of miRNA species at either state of activation as compared with the corresponding control DC population (mmu-miR-9-5p, mmu-miR-9-3p, mmu-miR-155-5p). These miRNAs were also upregulated in control DCs in response to stimulation. In contrast, miRNAs that were convergingly upregulated in both tolerized APC groups at stimulated state (mmu-miR-223-3p, mmu-miR-1224-5p) were downregulated in control DCs in response to stimulation. Overexpression of mmu-miR-223-3p in DCs was sufficient to prevent stimulation-associated acquisition of potent T cell stimulatory capacity. Overexpression of mmu-miR-223-3p in a DC line resulted in attenuated expression of known (Cflar, Rasa1, Ras) mRNA targets of this miRNA species shown to affect pathways that control DC activation. Taken together, we identified sets of miRNAs convergingly regulated in differentially tolerized APCs, which may contribute to imprint stimulation-resistant tolerogenic function as demonstrated for mmu-miR-223-3p. Knowledge of miRNAs with protolerogenic function enables immunotherapeutic approaches aimed to modulate immune responses by regulating miRNA expression.

The Ubiquitin Ligase Adaptor NDFIP1 Selectively Enforces a CD8+ T Cell Tolerance Checkpoint to High-Dose Antigen

Escape from peripheral tolerance checkpoints that control cytotoxic CD8+ Tcells is important for cancer immunotherapy and autoimmunity, but pathways enforcing these checkpoints are mostly uncharted. We reveal that the HECT-type ubiquitin ligase activator, NDFIP1, enforces a cell-intrinsic CD8+ Tcell checkpoint that desensitizes TCR signaling during invivo exposure to high antigen levels. Ndfip1-deficient OT-I CD8+ Tcells responding to high exogenous tolerogenic antigen doses that normally induce anergy aberrantly expanded and differentiated into effector cells that could precipitate autoimmune diabetes in RIP-OVAhi mice. In contrast, NDFIP1 was dispensable for peripheral deletion to low-dose exogenous or pancreatic islet-derived antigen and had little impact upon effector responses to Listeria or acute LCMV infection. These data provide evidence that NDFIP1 mediates a CD8+ Tcell tolerance checkpoint, with a different mechanism to CD4+ Tcells, and indicates that CD8+ Tcell deletion and anergy are molecularly separable checkpoints.

40. Transgene Product-Specific Treg Are Initially Induced in the Celiac Lymph Node During Tolerance Induction By Hepatic AAV Gene Transfer

Previous work by us and several other laboratories has shown that liver-directed gene transfer can induce transgene product-specific immune tolerance. Induction of CD4+CD25+FoxP3+ regulatory T cells (Treg) is a critical component of the tolerance mechanism. Gaps in knowledge exist about the identity of the antigen-presenting cells (APCs) and the origin of peripherally induced Treg. To characterize the interaction of adeno-associated virus (AAV) antigen expression in the liver with immune cells, we used AAV8-EF1a vectors, which have a high tropism for murine liver, expressing either a secreted ovalbumin (AAV8-ova) or cytoplasmic ovalbumin (AAV8-Cytoova). In Treg-deficient mice transgenic for an ova-specific CD4+ T cell receptor (DO11.10-tg Rag-2-/- BALB/c), Treg were induced with either vector. For secreted ova, multiple organs (liver, spleen, lymph nodes [LN]) showed a similar level and time course of Treg induction, with greater than 1% of the CD4+ T cells being Treg starting at 4 weeks. Substantial T cell activation using the very early activation marker CD69 was also observed in these tissues starting at 3 weeks after AAV8-ova. However, Treg induction was observed in the celiac LN (one of the liver-draining LN) as early as 3 weeks after AAV8-Cyto-ova gene transfer, followed by induction of Treg in other tissues by 4 weeks. Interestingly, T cell activation was notably higher in liver and celiac LN after AAV8-Cyto-ova with over 70% activated CD4+ T cells after 4 weeks. In addition, T cell activation was observed earlier in all tissues after AAV8-Cyto-ova when compared to AAV8-ova. Literature data document a role for tolerogenic antigen presentation in the liver itself via sinusoidal endothelial cells and possibly hepatocytes. Our data suggest an important role of conventional mechanisms of adaptive immunity involving draining LNs that survey tissue antigens leading to early T cell activation and Treg induction of hepatic expressed non-systemic antigens. To start defining the critical APCs, BALBc mice were given AAV8-ova vector two weeks prior to adoptive transfer of CellTrace Violet-labeled CD4+ T cells from DO11.10 donor mice and their proliferation was measured using flow cytometry after 5 days. Half of these mice had their Kupffer cells and M1 macrophages inactivated with Gadolinium chloride for two days prior to DO11.10 adoptive transfer. DO11.10 cells proliferated in an antigen-specific manner, and proliferation was blocked by Gadolinium chloride, supporting a role for professional APCs in presentation of hepatic transgene product. Our data suggest and reinforce the importance of Kupffer cells as key APCs for hepatic antigens and further characterize the impact of the route of antigen expression on T cell activation and Treg induction.

Apolipoprotein B-100 Peptide p210 Inhibits Proliferation of Na?ve T Effector Cells and Promotes Induction of Tolerogenic Antigen Presenting Cells and Regulatory T Cells in Vitro

Objectives: Modulation of immune responses against LDL antigens through therapeutic vaccines represents a possible new approach for prevention of cardiovascular disease. The mode of action of these vaccines remains to be fully characterized but the protective effect of immunization with the apolipoprotein B-100 (apoB-100) derived peptide p210 has in several studies been associated with activation of regulatory T cells. The present study used an in vitro model to study the effect of p210 on immune cells. Methods and results: CD11c+ antigen presenting cells, CD25-CD4+ naive T effector cells and CD25+CD4+ T regulatory cells were isolated from mouse spleens using antibody-coated magnetic beads. Pre-incubation of antigen presenting cells with p210 conjugated to cationized bovine serum albumin (p210-cBSA) down-regulated the expression of CD86 and MHC class II molecules, inhibited proliferation of pre-activated naive T effector cells and stimulated conversion of these cells into regulatory T cells. These effects were shown to partly be mediated through a suppression of the release of IL-12 from antigen presenting cells. Conclusions: The present findings demonstrate that p210-cBSA inhibits proliferation of naive T effector cells and promotes their conversion into regulatory T cells and this is suggested to be associated with a reduced activation status of antigen presenting cells. Taken together these findings suggest that immunization with p210-based vaccines have the capability of inducing tolerogenic APCs that in turn generate regulatory T cells suppressing T effector cell functions.

The surface carbohydrates of the Echinococcus granulosus larva interact selectively with the rodent Kupffer cell receptor

The larvae of the cestodes belonging to the genus Echinococcus dwell primarily in mammalian liver. They are protected by the laminated layer (LL), an acellular mucin-based structure. The glycans decorating these mucins constitute the overwhelming majority of molecules exposed by these larvae to their hosts. However, their decoding by host innate immunity has not been studied. Out of 36 mammalian innate receptors with carbohydrate-binding domains, expressed as Fc fusions, only the mouse Kupffer cell receptor (KCR; CLEC4F) bound significantly to the Echinococcus granulosus LL mucins. The receptor also bound the Echinococcus multilocularis LL. Out of several synthetic glycans representing Echinococcus LL structures, the KCR bound strongly in particular to those ending in Galα1-4Galβ1-3 or Galα1-4Galβ1-4GlcNAc, both characteristic LL carbohydrate motifs. LL carbohydrates may be optimized to interact with the KCR, expressed only in liver macrophages, cells known to contribute to the tolerogenic antigen presentation that is characteristic of this organ.

Quantification of Blood Dendritic Cells in Colorectal Cancer Patients During the Course of Disease

Colorectal cancer is a malignancy with poor prognosis that might be associated with defective immune function. The aim of the present study was to investigate circulating dendritic cells in colorectal cancer patients, in order to contribute to elucidate tumor-escape mechanisms and to point out a possible correlation with the clinical condition of the disease. Therefore, we enumerated ex vivo myeloid and plasmacytoid dendritic cells, through multicolor flow cytometry, in 26 colorectal patients and 33 healthy controls. Furthermore we performed several analyses at determined time points in order to define the immunological trend of cancer patients after surgery and other conventional treatments. At the pre-operative time point the absolute number of plasmacytoid dendritic cells in cancer patients was significantly reduced in comparison to controls, this result being mainly referred to stage III-IV patients. The number of myeloid dendritic cells did not show any significant difference compared to healthy controls; interestingly the expression of the tolerogenic antigen CD85k was significantly higher on cancer patients' myeloid dendritic cells than controls'. At the following samplings, circulating dendritic cell absolute number did not show any difference compared to controls. Conclusively the impairment of the number of circulating dendritic cells may represent one of the tumor escape mechanisms occurring in colorectal cancer. These alterations seem to be correlated to cancer progression. Our work sheds light on one of dendritic cell-based tumor immune escape mechanisms. This knowledge may be useful to the development of more effective immunotherapeutic strategies.

CD134/CD137 dual costimulation‐elicited IFN‐γ maximizes effector T‐cell function but limits Treg expansion

T cell tolerance to tumor antigens represents a major hurdle in generating tumor immunity. Combined administration of agonistic monoclonal antibodies to the costimulatory receptors CD134 plus CD137 can program T cells responding to tolerogenic antigen to undergo expansion and effector T cell differentiation, and also elicits tumor immunity. Nevertheless, CD134 and CD137 agonists can also engage inhibitory immune components. To understand how immune stimulatory versus inhibitory components are regulated during CD134 plus CD137 dual costimulation, the current study utilized a model where dual costimulation programs T cells encountering a highly tolerogenic self-antigen to undergo effector differentiation. IFN-γ was found to play a pivotal role in maximizing the function of effector T cells while simultaneously limiting the expansion of CD4+CD25+Foxp3+ Tregs. In antigen-responding effector T cells, IFN-γ operates via a direct cell-intrinsic mechanism to cooperate with IL-2 to program maximal expression of granzyme B. Simultaneously, IFN-γ limits expression of the IL-2 receptor alpha chain (CD25) and IL-2 signaling through a mechanism that does not involve T-bet-mediated repression of IL-2. IFN-γ also limited CD25 and Foxp3 expression on bystanding CD4+Foxp3+ Tregs, and limited the potential of these Tregs to expand. These effects could not be explained by the ability of IFN-γ to limit IL-2 availability. Taken together, during dual costimulation IFN-γ interacts with IL-2 through distinct mechanisms to program maximal expression of effector molecules in antigen-responding T cells while simultaneously limiting Treg expansion.

Transgenic rice accumulating modified cedar pollen allergen Cry j 2 derivatives

In order to create a safe tolerogenic antigen with reduced IgE reactivity, we developed transgenic rice that accumulates in the seed endosperm a sufficient amount of Cry j 2, the cedar pollen allergen, in a restructured form of tail-to-head, providing a feasible mucosal allergy vaccine against cedar pollinosis.