State Of Nonresponsiveness Research Articles

Abstract 5130: Immune profiling of malignant effusion from metastatic gastric cancer patients as a predictive biomarker for immune checkpoint inhibitors

Abstract Many types of cancers are able to circumvent immune surveillance by the host's immune system. It has been well established that many tumor infiltrating T cells are in a state of non-responsiveness due to the immune suppressive tumor microenvironment. The main mechanisms of T cells that confer suppressive anti-tumor immune response in tumor microenvironment could be regulatory T cells (Treg) and exhausted T cells. In advanced stage cancer patients, detailed evaluation of Treg cells in tumor microenvironment is hampered by limitation for enough tumor tissue, because surgical resection for sample is not standard treatment in these patients. In order to overcome this limitation of research, we performed the studies using lymphocytes and tumor cells isolated from malignant effusion including pleural effusion or ascites from advanced gastric cancer patients. We collected 25 malignant effusion specimens from advanced gastric cancer patients, including 20 paired peripheral blood samples. Lymphocytes from malignant effusions and peripheral bloods were analyzed for subtype of Memory, Activated T cell and Treg cells, and expressions of inhibitory molecule using by flow cytometry. To evaluate the expression of PD-L1, we used the tumor cells of malignant effusions. Firstly, the percentage of CD8+ T cells in malignant effusion seems to increase compared to those in peripheral blood, but the percentage of CD4+ T in malignant effusion were significantly lower than those in peripheral blood. Interestingly, the percentage of CD45RO+ (Memory) on CD4+ and CD8+ T cells was significantly higher in malignant effusion than peripheral blood. And Early activated molecule (CD69 and CD25) on CD4+ and CD8+ T cells was significantly higher in malignant effusion than peripheral blood. The percentage of PD-1 expression on CD8+ T cells was significantly higher in malignant effusion than peripheral blood (p = 0.017). And the percentage and MFI of Treg cells (Foxp3+/CD4 T cell) tend to increase in malignant effusion was comparable with those of peripheral blood but did not show the statistical significance. Collectively, we found that T cells derived from malignant effusion demonstrated the exhausted phenotype showing highly expression of PD-1, compared to those from peripheral blood. Finally, the PD-L1 expression on tumor cells from malignant effusions was 0.04∼12.78%. The upregulation of PD-1 on Treg cells and PD-L1 on tumor cell of malignant effusions could be a potential predictive marker and therapeutic target for immune checkpoint blockade. Citation Format: Woo Sun Kwon, So Jung Lim, Won Suk Lee, Eunji Jo, Hyun-jeong Kim, Kyung Hee Lee, Hyun Cheol Chung, Sun Young Rha. Immune profiling of malignant effusion from metastatic gastric cancer patients as a predictive biomarker for immune checkpoint inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5130.

What makes children outgrow food allergy?

What makes children outgrow food allergy?

Genetic Basis of Altered Central Tolerance and Autoimmune Diseases: A Lesson from AIRE Mutations

The thymus is a specialized organ that provides an inductive environment for the development of T cells from multipotent hematopoietic progenitors. Self–nonself discrimination plays a key role in inducing a productive immunity and in preventing autoimmune reactions. Tolerance represents a state of immunologic nonresponsiveness in the presence of a particular antigen. The immune system becomes tolerant to self-antigens through the two main processes, central and peripheral tolerance. Central tolerance takes place within the thymus and represents the mechanism by which T cells binding with high avidity self-antigens, which are potentially autoreactive, are eliminated through so-called negative selection. This process is mostly mediated by medullary thymic epithelia cells (mTECs) and medullary dendritic cells (DCs). A remarkable event in the process is the expression of tissue-specific antigens (TSA) by mTECs driven by the transcription factor autoimmune regulator (AIRE). Mutations in this gene result in autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED), a rare autosomal recessive disease (OMIM 240300). Thus far, this syndrome is the paradigm of a genetically determined failure of central tolerance and autoimmunty. Patients with APECED have a variable pattern of autoimmune reactions, involving different endocrine and nonendocrine organs. However, although APECED is a monogenic disorder, it is characterized by a wide variability of the clinical expression, thus implying a further role for disease-modifying genes and environmental factors in the pathogenesis. Studies on this polyreactive autoimmune syndrome contributed enormously to unraveling several issues of the molecular basis of autoimmunity. This review focuses on the developmental, functional, and molecular events governing central tolerance and on the clinical implication of its failure.

HIV-Mediated Phosphatidylinositol 3-Kinase/Serine–Threonine Kinase Activation in APCs Leads to Programmed Death-1 Ligand Upregulation and Suppression of HIV-Specific CD8 T Cells

Recent evidence demonstrates that HIV-1 infection leads to the attenuation of cellular immune responses, which has been correlated with the increased expression of programmed death (PD)-1 on virus-specific CD8(+) T cells. PD-1 is induced upon T cell activation, and its prolonged expression facilitates CD8(+) T cell inhibitory signals when bound to its B7 family ligands, PD-ligand (L)1/2, which are expressed on APCs. Importantly, early reports demonstrated that blockade of the PD-1/PD-L interaction by Abs may help to counter the development of immune exhaustion driven by HIV viral persistence. To better understand the regulation of the PD-1 pathway during HIV infection, we examined the ability of the virus to induce PD-L expression on macrophages and dendritic cells. We found a direct relationship between the infection of APCs and the expression of PD-L1 in which virus-mediated upregulation induced a state of nonresponsiveness in uninfected HIV-specific T cells. Furthermore, this exhaustion phenotype was revitalized by the blockade of PD-L1, after which T cells regained their capacity for proliferation and the secretion of proinflammatory cytokines IFN-γ, IL-2, and IL-12 upon restimulation. In addition, we identify a critical role for the PI3K/serine-threonine kinase signaling pathway in PD-L1 upregulation of APCs by HIV, because inhibition of these intracellular signal transducer enzymes significantly reduced PD-L1 induction by infection. These data identify a novel mechanism by which HIV exploits the immunosuppressive PD-1 pathway and suggest a new role for virus-infected cells in the local corruption of immune responses required for viral suppression.

CCL20 and β-Defensin-2 Induce Arrest of Human Th17 Cells on Inflamed Endothelium In Vitro under Flow Conditions

CCR6 is a chemokine receptor that is expressed at the cell surface of Th17 cells, an IL-17- and IL-22-secreting population of CD4(+) T cells with antipathogenic, as well as inflammatory, properties. In the current study, we have determined the involvement of CCR6 in human Th17 lymphocyte migration toward inflamed tissue by analyzing the capacity of its ligands to induce arrest of these cells onto inflamed endothelium in vitro under flow conditions. We show that polarized, in situ-differentiated, skin-derived Th17 clones activated via the TCR-CD3 complex produce CCL20 in addition to IL-17 and IL-22. The latter cytokines induce, in a synergic fashion, the production of human β-defensin (hBD)-2, but neither hBD-1 nor hBD-3, by epidermal keratinocytes. Both CCL20 and hBD-2 are capable of inducing the arrest of Th17 cells, but not Th1 or Th2 cells, on HUVEC in an CD54-dependent manner that is CCR6 specific and independent from the expression of CXCR4, reported to be an alternative receptor for hBD-2. In addition, Ag-specific activation induces a transient loss of CCR6 expression, both at the transcriptional and protein level, which occurs with slow kinetics and is not due to endogenous CCL20-mediated internalization of CCR6. Together, these results indicate that Ag-specific activation will initially contribute to CCR6-mediated Th17 cell trafficking toward and sequestration in inflamed tissue, but that it eventually results in a transitory state of nonresponsiveness to further stimulation of these cells with CCR6 ligands, thus permitting their subsequent migration out of the inflamed site.

Nicotinic acid adenine dinucleotide phosphate-mediated calcium signalling in effector T cells regulates autoimmunity of the central nervous system

Nicotinic acid adenine dinucleotide phosphate represents a newly identified second messenger in T cells involved in antigen receptor-mediated calcium signalling. Its function in vivo is, however, unknown due to the lack of biocompatible inhibitors. Using a recently developed inhibitor, we explored the role of nicotinic acid adenine dinucleotide phosphate in autoreactive effector T cells during experimental autoimmune encephalomyelitis, the animal model for multiple sclerosis. We provide in vitro and in vivo evidence that calcium signalling controlled by nicotinic acid adenine dinucleotide phosphate is relevant for the pathogenic potential of autoimmune effector T cells. Live two photon imaging and molecular analyses revealed that nicotinic acid adenine dinucleotide phosphate signalling regulates T cell motility and re-activation upon arrival in the nervous tissues. Treatment with the nicotinic acid adenine dinucleotide phosphate inhibitor significantly reduced both the number of stable arrests of effector T cells and their invasive capacity. The levels of pro-inflammatory cytokines interferon-gamma and interleukin-17 were strongly diminished. Consecutively, the clinical symptoms of experimental autoimmune encephalomyelitis were ameliorated. In vitro, antigen-triggered T cell proliferation and cytokine production were evenly suppressed. These inhibitory effects were reversible: after wash-out of the nicotinic acid adenine dinucleotide phosphate antagonist, the effector T cells fully regained their functions. The nicotinic acid derivative BZ194 induced this transient state of non-responsiveness specifically in post-activated effector T cells. Naïve and long-lived memory T cells, which express lower levels of the putative nicotinic acid adenine dinucleotide phosphate receptor, type 1 ryanodine receptor, were not targeted. T cell priming and recall responses in vivo were not reduced. These data indicate that the nicotinic acid adenine dinucleotide phosphate/calcium signalling pathway is essential for the recruitment and the activation of autoaggressive effector T cells within their target organ. Interference with this signalling pathway suppresses the formation of autoimmune inflammatory lesions and thus might qualify as a novel strategy for the treatment of T cell mediated autoimmune diseases.

Mucosal immunology, eosinophilic esophagitis, and other intestinal inflammatory diseases

The gastrointestinal mucosa constitutes the largest host-environment interface of the body. It uses both innate and adaptive immune mechanisms to provide protection from the diverse onslaught of foods, microbes, and other ingested products. The innate immune system is genetically encoded and evolutionarily ancient, possesses no memory, and lacks diversity. In contrast, the adaptive immune system is quite diverse, develops memory, and undergoes expansion after stimulation. The gastrointestinal mucosa is charged with the difficult task of mounting protective responses against invading microorganisms while simultaneously maintaining an overall state of nonresponsiveness or tolerance to innocuous substances, such as commensal bacteria and food antigens. Perturbation or malfunction of these complex protective mechanisms results in diseases, such as inflammatory bowel diseases, celiac disease, or eosinophilic gastrointestinal diseases.

Monkeypox virus evades antiviral CD4 + and CD8 + T cell responses by suppressing cognate T cell activation

Monkeypox virus (MPV) is a virulent human pathogen that has gained increased attention because of its potential use as a bioterrorism agent and inadvertent introduction into North America in 2003. The US outbreak also provided an important opportunity to study MPV-specific T cell immunity. Although MPV-specific CD4(+) and CD8(+) T cells could recognize vaccinia virus (VV)-infected monocytes and produce inflammatory cytokines such as IFNgamma and TNFalpha, they were largely incapable of responding to autologous MPV-infected cells. Further analysis revealed that, unlike cowpox virus (CPV), MPV did not interfere with MHC expression or intracellular transport of MHC molecules. Instead, MPV-infected cells were capable of preventing T cell receptor (TcR)-mediated T cell activation in trans. The ability to trigger a state of nonresponsiveness represents a unique MHC-independent mechanism for blocking antiviral T cell activation and inflammatory cytokine production and is likely an important attribute involved with viral dissemination in the infected host.

4. Gastrointestinal mucosal immunity

Mucosal surfaces constitute a large host-environmental interface that must be protected from pathogenic organisms. The mucosal immune system has evolved as a distinct immune organ functioning independently from its systemic counterpart. The mucosal immune system has the difficult task of mounting protective responses to invading microorganisms while maintaining a state of nonresponsiveness to commensal bacteria and food antigens. The system has unique cellular components and functional aspects that permit it to carry out this dual role.

Intestinal immune system of young rats influenced by cocoa-enriched diet

Gut-associated lymphoid tissue (GALT) maintains mucosal homeostasis by counteracting pathogens and inducing a state of nonresponsiveness when it receives signals from food antigens and commensal bacteria. We report for the first time the influence of continuous cocoa consumption on GALT function in rats postweaning. Weaned Wistar rats were fed cocoa-enriched diets (4% or 10% food intake) for 3 weeks. The function of the primary inductive sites of GALT, such as Peyer's patches (PP) and mesenteric lymph nodes (MLN), was evaluated through an analysis of IgA-secretory ability and lymphocyte composition (T, B and natural killer cells), activation (IL-2 secretion and IL-2 receptor α expression) and proliferation. T-helper effector cell balance was also established based on cytokine profile (interferon γ, IL-4 and IL-10) after mitogen activation. A 10% cocoa intake induced significant changes in PP and MLN lymphocyte composition and function, whereas a 4% cocoa diet did not cause significant modifications in either tissues. Cocoa diet strongly reduced secretory IgA (S-IgA) in the intestinal lumen, although IgA's secretory ability was only slightly decreased in PP. In addition, the 10% cocoa diet increased T-cell-antigen receptor γδ cell proportion in both lymphoid tissues. Thus, cocoa intake modulates intestinal immune responses in young rats, influencing γδ T-cells and S-IgA production.

Decision-making at the surface of the intact or barrier disrupted skin: potential applications for vaccination or therapy.

The skin is a highly accessible organ and constitutes an active immunological site. Both these properties make this surface an attractive route for what promises to be a cost-effective, simple, practical and needle-free delivery of vaccines and immunomodulators. Less obvious is the fact that the state of the skin barrier can influence quantitative and qualitative aspects of antigen-specific immune responses. The everyday decision-making at the skin epithelium concerns the choice between the induction of an immune response and the establishment of a state of non-responsiveness (tolerance). This decision is influenced by various factors such as the dose, the route (intact vs barrier-disrupted skin), the cytokine microenvironment and the nature of the antigenic stimulus. By increasing our understanding of how immune responses are regulated in the epidermis we can envisage the development of immunisation protocols aimed at eliciting a protective immune response or inducing tolerance, with direct applications to preventive or therapeutic vaccination, respectively.

Preventing Intolerance: The Induction of Nonresponsiveness to Dietary and Microbial Antigens in the Intestinal Mucosa

The gut-associated lymphoid tissue (GALT) is constantly exposed to a variety of Ags and must therefore decipher a large number of distinct signals at all times. Responding correctly to each set of signals is crucial. When the GALT receives signals from the intestinal flora or food Ags, it must induce a state of nonresponsiveness (mucosal tolerance). In contrast, when pathogenic bacteria invade the intestinal mucosa, it is necessary to elicit strong T and B cell responses. The GALT is therefore in the position of constantly fighting intolerance to food and the commensal flora while effectively battling infectious microbes. Determining precisely which type of response to generate in each case is key to the prevention of immune dysregulation and tissue damage.

Basic concepts in transplant immunology

Subsequently,spontaneousliverallograftacceptancewasalsoobservedintransplants done in several allogeneic rat strain combina-tions and most allogeneic mouse strain combinations.Moreover, other experimental studies in rodents demon-strated that allogeneic liver transplantation providesimmune“protection”againstsubsequentcardiac,kidney,pancreas, islet, and skin grafts from the same donor.

The influence of oral tolerance on local and systemic immunoregulation in a Murine Model of type I allergy

Rationale Oral tolerance is defined as the induction of a state of systemic immune nonresponsiveness to orally administrated antigen upon subsequent antigen challenge. The influence of high-dose antigen induced oral tolerance on the local and systemic immune response was investigated. Methods BALB/c mice were sensitized to OVA. Oral tolerance was induced with OVA using a special feeding regimen before sensitization. Antigen-induced eosinophil infiltration in the lung tissue, and levels of Th1 and Th2 cytokine production in bronchoalveolar lavage fluid (BAL) of sensitized mice was determined. Serum was used for measurement of allergen-specific antibodies. Peripheral blood cell and spleen cell cultures were performed to determine cytokine production before and after in vitro stimulation (IL-2, IL-4, IL-5, IL-10, TNF-alpha and IFN-gamma). Results Oral administration of OVA in high doses inhibited antigen-induced eosinophil infiltration in the lung in an antigen-specific manner, reducing IL-4 and IL-5 levels and eosinophils in the BAL. In vitro antigen- induced production of IL-2, IL-4, IL-5, IL-10, IFN-gamma was decreased in spleen cells, indicating the induction of both Th1 and Th2 tolerance in vivo. Allergen-specific IgE/IgG1 production was also reduced. Conclusions High-doses of antigens provide oral tolerance, influencing both local and systemic immune responses, inducing not only Th1 but also Th2 cell tolerance in vivo and inhibits allergen-induced eosinophil recruitment in lung tissue.

Modulation of the allergic immune response in BALB/c mice by subcutaneous injection of high doses of the dominant T cell epitope from the major birch pollen allergen Bet v 1.

Several in vitro and in vivo studies indicate that application of high doses of dominant T cell epitopes can induce a state of antigen-specific non-responsiveness (anergy). In the present study, we developed a murine model of an allergic immune response to Bet v 1, the major birch pollen allergen. Mice were sensitized by injection of rBet v 1 and the allergic state was proven by the presence of allergen-specific IgE and positive immediate-type skin tests to Bet v 1. In epitope mapping experiments, an immunodominant T cell epitope of Bet v 1 in BALB/c mice was identified by the use of overlapping peptides. This peptide (BV 139) was subsequently employed for treatment. Two tolerization protocols were used: in one approach, the peptide was administered to naive mice before immunization (group BV139-S), in the second, already sensitized mice were treated (S-BV139). The results demonstrated that administering high doses of the dominant T cell epitope of Bet v 1 profoundly diminished T cell proliferation to the peptide in the BV139-S group, and to the peptide as well as to the whole protein in the S-BV139 group. Skin test reactivity to Bet v 1 was reduced in the BV139-S group. However, no differences in terms of specific antibody production between treated and untreated mice could be observed. This study provides evidence that administration of dominant T cell epitopes can down-regulate the allergen-specific T cell response. Proceeding on the assumption that the T lymphocyte response to allergens is crucial for the induction and maintenance of the allergic disease, a modulation of the immune response to allergens by treatment with T cell epitope peptides could represent a promising concept for immunotherapy in the future.

The B subunit of Escherichia coli heat labile enterotoxin abrogates oral tolerance, promoting predominantly Th2-type immune responses.

Mucosal antigen encounter usually results in a state of systemic non-responsiveness (tolerance). This failure to mount a protective response is a major hurdle to mucosal vaccine development. Hence, the identification of safe and effective mucosal adjuvants promoting protective immunity is of critical importance. The non-toxic B subunit of Escherichia coli heat labile enterotoxin(EtxB) is a potent nasal adjuvant; however, its usefulness following oral delivery is unconfirmed. We used DO11.10 chimeric mice to assess whether EtxB could abrogate tolerance to oral OVA. We show that admixing EtxB with OVA for oral immunization abrogates oral tolerance and results in a weak anti-OVA immune response. Importantly, EtxB profoundly modulated the nature of the response to subsequent parenteral challenge, promoting IgG1 in favor of IgG2a antibodies and depressing IFN-gamma production while elevating TGF-beta secretion. The addition of EtxB promoted T cell division, as assessed by loss of staining with carboxyfluorescein diacetate succinimidyl ester. Enhanced cell division promoted by EtxB was associated with T cell differentiation (increased numbers of CD45RBlow cells) in vivo, although dividing OVA-specific T cells were CD25-. These data suggest that although EtxB is a weak oral adjuvant, it can profoundly modulate the nature of the immune response to admixed antigen.

Modulation of Immunosuppression by DNA Transfer in Transplantation

Acute and chronic rejection of organ transplants continues to be the single most important source of allograft loss, and the induction of chronic immunosuppression leads to significant morbidity and mortality in transplants with long-term allograft function. Thus, induction of donor-specific unresponsiveness to organ allografts remains an elusive goal in clinical transplantation. Successful clinical transplantation depends upon a complex variety of factors. Such factors include the compatibility between the immunogenetic markers of the donor-recipient pairs, particularly the HLA antigens; the balance between the immunoregulatory mediators including cytokines and growth factors; and the adequacy of immunosuppression. Despite the availability of powerful immunosuppressive drugs, allograft survival remains imperfect, and long-term therapy has often been accompanied by numerous side effects. Because the ultimate goal of transplant immunology is the development of a state of nonresponsiveness to the transplanted organ, gene transfer approaches may provide useful strategies for fulfillment of this goal.

Immunologic tolerance.

Immunologic tolerance, an active state of antigen specific non-responsiveness, is important in understanding autoimmune diseases and the potential for transplant acceptance. Recent progress in basic studies and preclinical models involving mixed chimerism, costimulatory blockade, immune deviation, and HLA derived peptides suggest that clinical applications are now possible. The National Institutes of Health and Juvenile Diabetes Research Foundation recently launched an Immune Tolerance Network made up of investigators from around the world to develop clinical trials in immune tolerance in autoimmune disease, transplantation and allergy. This review highlights historical perspectives and recent progress in induction and maintenance of immunologic tolerance relevant to pediatric nephrology.

Rheumatoid arthritis synovial T cells regulate transcription of several genes associated with antigen-induced anergy.

Rheumatoid arthritis (RA) is a chronic, inflammatory synovitis whose pathogenesis may involve autoimmune mechanisms. Anergy is a state of T-cell nonresponsiveness characterized by downregulated IL-2 production. Paradoxically, RA T cells are hyporesponsive and proliferate poorly to antigens and mitogens, thus sharing some characteristics with anergic T cells. We analyzed the molecular basis of anergy in cloned human CD4+ T cells using differential display RT-PCR and subsequently examined the levels of differentially expressed transcripts in RA and, as control, reactive arthritis (ReA) synovium. Several transcriptional events were common to anergic T cells and RA synovium. These included downregulation of CALMODULIN:, which is critical to T-cell activation, and of cellular apoptosis susceptibility protein, which may mediate resistance to apoptosis in RA. Transcription of CALMODULIN: in RA synovium was less than 1% of that in ReA and was lower in RA synovial fluid mononuclear cells than in paired PBMCs. Following anti-TNF-alpha therapy in vivo, RA PBMC CALMODULIN: transcripts increased five- to tenfold. Pharmacological calmodulin blockade in vitro impaired antigen-specific proliferation. These data provide a link between reduced CALMODULIN: transcription and impaired T-cell responsiveness in RA. The identification of transcriptional changes common to anergic and RA synovial T cells should help interpret some of the characteristic RA cellular defects.

Intracutaneous Genetic Immunization with Autologous Melanoma-Associated Antigen Pmel17/gp100 Induces T Cell-Mediated Tumor Protection In Vivo

Using the differentiation antigen Pmel17/gp100 to genetically immunize C57BL/6 mice (H-2(b)), we and colleagues noticed that only mice that had received the human homolog but not animals injected with the murine counterpart were protected against the growth of syngeneic B16 melanoma cells. The goal of this study was to determine whether the state of nonresponsiveness to the autoantigen Pmel17/gp100 can be broken by immunization with a plasmid DNA construct encoding the autologous form of the molecule. A construct containing the murine form of Pmel17 was administered intradermally to DBA/2 mice (H-2(d)), which were then investigated for the presence of Pmel17/gp100-specific immunity. We show that administration of plasmid DNA coding for the autologous melanoma-associated antigen Pmel17/gp100 protects DBA/2 mice against the growth of Pmel17-positive M3 melanoma cells but not against Pmel17-negative M3 melanoma cells or unrelated P815 mastocytoma cells. Cell depletion experiments demonstrated that this protective effect is mediated by T lymphocytes. The notion that Pmel17/gp100 represents the biologically relevant target in this system was supported by the observations (i) that recipients of Pmel17/gp100 DNA mount an antigen-specific cytotoxic T lymphocyte response and (ii) that M3 tumors growing in mice immunized with autologous Pmel17/gp100 had lost expression of this melanoma-associated antigen whereas M3 melanomas appearing in control-vector-treated animals were still Pmel17/gp100-positive. These results indicate that intracutaneous genetic immunization with autologous melanoma-associated antigen Pmel17/gp100 encoding plasmid DNA can lead to protection against melanoma cells as a result of the induction of a melanoma-associated antigen-specific and protective T-cell-mediated immune response. J Invest Dermatol 115:1082-1087 2000