Ubiquitous Self-antigen Research Articles

T cells take aim at a ubiquitous autoantigen in multiple sclerosis.

CD4+ T cells from multiple sclerosis lesions target a ubiquitous self-antigen that is shared by gut commensal bacteria (Planas et al, this issue).

Abstract PR11: Targeted engagement of B cell autoimmunity checkpoints to overcome drug resistance in pediatric Ph-like ALL

Abstract Background: Unlike other cell types, B cells are selected for an intermediate level of signaling strength. Critical survival and proliferation signals emanate from the B cell receptor (BCR): If B-cells fail to express a functional BCR, signaling output is too weak, resulting in “death by neglect”. If the BCR binds to ubiquitous self-antigen, BCR signals are exceedingly strong. Both attenuation below minimum (non-functional BCR; death by neglect) and hyperactivation above maximum (autoreactive BCR) thresholds of signaling strength trigger negative selection and cell death. Rationale: Unlike any other types of cancer, we recently discovered that pre-B acute lymphoblastic leukemia (ALL) cells are bound by the same rules that also govern normal B cell selection. Oncogenic tyrosine kinases in pediatric Ph-like ALL mimic active pre-BCR signaling. The Ph-like group defines the ALL subset with particularly poor clinical outcome. Current therapy approaches are largely focused on the development of more potent tyrosine kinase inhibitors (TKI) to suppress oncogenic signaling. However resistance to TKI is developed invariably. Here, we test the hypothesis that targeting hyperactivation above a maximum threshold will selectively kill Ph-like ALL cells through a mechanism that is functionally equivalent to removal of self-reactive B cells. Results: Ph-like ALL typically originates from pre-B cells that critically depend on survival signals emanating from a functional pre-BCR. Despite their pre-B cell origin, ALL cells lack expression of pre-BCR signaling chains Igα and Igβ, indicating lack of a functional pre-BCR. Reconstitution of Igα and Igβ was indeed sufficient to induce cell death in Ph-like ALL. TKI-treatment, while designed to kill leukemia cells, seemingly paradoxically rescued Ph-like ALL cells in this experimental setting. These findings suggest that Ph-like ALL cells are selectively sensitive to even incremental increases of pre-BCR signaling. Consistent with this concept, patient-derived Ph-like ALL cells express high levels of inhibitory surface receptors PECAM1, CD300A and LAIR1 that recruit and activate phosphatases SHP1 and SHIP1, which terminate pre-BCR signaling. Importantly, high expression levels of these surface receptors were strong predictors of poor outcome of patients with ALL in two clinical trials, including Ph-like ALL patients (COG; P9906). Genetic studies revealed that Pecam1, Cd300a and Lair1 were critical to calibrate pre-BCR signaling strength through recruitment of the inhibitory phosphatases SHP1 and SHIP1. Genetic deletion of Lair1, Ptpn6 or Inpp5d in BCR-ABL1 transformed ALL caused cell death in vitro and in vivo through hyperactivation of pre-BCR signaling. Testing various components of proximal pre-BCR signaling, we found that an incremental increase of SYK tyrosine kinase activity was required and sufficient to induce cell death. Hyperactive SYK was functionally equivalent to acute activation of an autoreactive BCR on ALL cells. Despite oncogenic transformation, this basic mechanism of negative selection was still functional in Ph-like ALL cells. Using chimeric PECAM1, CD300A and LAIR1 receptor decoys and a novel small molecule inhibitor of SHIP1, we demonstrated that pharmacological hyperactivation of pre-BCR signaling and engagement of negative B cell selection represents a promising new strategy to overcome drug-resistance in pediatric Ph-like ALL. Conclusion: These results indicated that inhibitory receptors and downstream phosphatases are critical regulators of pre-BCR signaling strength in Ph-like ALL, and identified targeting hyperactivation of pre-BCR signaling as a potential novel class of therapeutic strategy. This abstract is also presented as Poster B06. Citation Format: Zhengshan Chen, Huimin Geng, Clifford A. Lowell, Stephen P. Hunger, Markus Müschen. Targeted engagement of B cell autoimmunity checkpoints to overcome drug resistance in pediatric Ph-like ALL. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr PR11.

Targeted Activation of B Cell Autoimmunity Checkpoints in Acute Lymphoblastic Leukemia

Background: Unlike other cell types, B cells are selected for an intermediate level of signaling strength. Critical survival and proliferation signals emanate from the B cell receptor (BCR): If B-cells fail to express a functional BCR, signaling output is too weak, resulting in "death by neglect". If the BCR binds to ubiquitous self-antigen, BCR signals are exceedingly strong. Both attenuation below minimum (non-functional BCR; death by neglect) and hyperactivation above maximum (autoreactive BCR) thresholds of signaling strength trigger negative selection and cell death.Rationale: Unlike any other types of cancer, we recently discovered that pre-B acute lymphoblastic leukemia (ALL) cells are bound by the same rules that also govern normal B cell selection. The oncogenic BCR-ABL1 tyrosine kinase mimics active pre-BCR signaling in Ph+ acute lymphoblastic leukemia which defines the ALL subgroup with the worst clinical outcome. Current therapy approaches are largely focused on the development of more potent tyrosine kinase inhibitors (TKI) to suppress oncogenic signaling. However resistance to TKI is developed invariably. Here, we test the hypothesis that targeting hyperactivation above a maximum threshold will selectively kill Ph+ ALL cells through a mechanism that is functionally equivalent to removal of self-reactive B cells.Results: ALL typically originates from pre-B cells that critically depend on survival signals emanating from a functional pre-BCR. Despite their pre-B cell origin, Ph+ ALL cells lack expression of pre-BCR signaling chains Iga and Igb, indicating lack of a functional pre-BCR. Reconstitution of Iga and Igb was indeed sufficient to induce cell death in BCR-ABL1 ALL. TKI-treatment, while designed to kill leukemia cells, seemingly paradoxically rescued Ph+ ALL cells in this experimental setting. These findings suggest that Ph+ ALL are uniquely sensitive to even incremental increases of pre-BCR signaling. Consistent with this concept, patient-derived Ph+ ALL cells express high levels of inhibitory surface receptors PECAM1, CD300A and LAIR1 that recruit and activate phosphatases SHP1 and SHIP1, which terminate pre-BCR signaling. Importantly, high expression levels of these surface receptors were strong predictors of poor outcome of patients with ALL in two clinical trials, including both pediatric and adult ALL patients. Genetic studies revealed that Pecam1, Cd300a and Lair1 were critical to calibrate pre-BCR signaling strength through recruitment of the inhibitory phosphatases SHP1 and SHIP1. Genetic deletion of Lair1, Ptpn6 or Inpp5d in BCR-ABL1 transformed ALL caused cell death in vitro and in vivo through hyperactivation of pre-BCR signaling. Testing various components of proximal pre-BCR signaling, we found that an incremental increase of SYK tyrosine kinase activity was required and sufficient to induce cell death. Hyperactive SYK was functionally equivalent to acute activation of an autoreactive BCR on ALL cells. Despite oncogenic transformation, this basic mechanism of negative selection was still functional in Ph+ ALL cells. Using chimeric PECAM1, CD300A and LAIR1 receptor decoys and a novel small molecule inhibitor of SHIP1, we demonstrated that pharmacological hyperactivation of pre-BCR signaling and engagement of negative B cell selection represents a promising new strategy to overcome drug-resistance in human Ph+ ALL.Conclusion: These results indicated that inhibitory receptors and downstream phosphatases are critical regulators of pre-BCR signaling strength in Ph+ ALL, and identified targeting hyperactivation of pre-BCR signaling as a potential novel class of therapeutic strategy. DisclosuresNo relevant conflicts of interest to declare.

Tolerization against atherosclerosis using heat shock protein 60.

Atherosclerosis is a chronic inflammatory disease of the artery wall, and both innate and adaptive immunity play important roles in the pathogenesis of this disease. In several experimental and human experiments of early atherosclerotic lesions, it has been shown that the first pathogenic event in atherogenesis is intimal infiltration of T cells at predilection sites. These T cells react to heat shock protein 60 (HSP60), which is a ubiquitous self-antigen expressed on the surface of endothelial cells (ECs) together with adhesion molecules in response to classical risk factors for atherosclerosis. When HSP60 is expressed on the EC surface, it can act as a "danger-signal" for both cellular and humoral immune reactions. Acquired by infection or vaccination, beneficial protective immunity to microbial HSP60 and bona fide autoimmunity to biochemically altered autologous HSP60 is present in all humans. Thus, the development of atherosclerosis during aging is paid by the price for lifelong protective preexisting anti-HSP60 immunity by harmful (auto)immune cross-reactive attack on arterial ECs maltreated by atherosclerosis risk factors. This is supported by experiments, which shows that bacterial HSP60 immunization can lead and accelerate experimental atherosclerosis. This review article presents accumulating proof that supports the idea that tolerization with antigenic HSP60 protein or its peptides may arrest or even prevent atherosclerosis by increased production of regulatory T cells and/or anti-inflammatory cytokines. Recent data indicates that HSP60, or more likely some of its derivative peptides, has immunoregulatory functions. Therefore, these peptides may have important potential for being used as diagnostic agents or therapeutic targets.

Differential roles for Bim and Nur77 in thymocyte clonal deletion induced by ubiquitous self-antigen.

Negative selection, primarily mediated through clonal deletion of self-reactive thymocytes, is critical for establishing self-tolerance and preventing autoimmunity. Recent studies suggest that the molecular mechanisms of negative selection differ depending on the thymic compartment and developmental stage at which thymocytes are deleted. Using the physiological HY(cd4) TCR transgenic model of negative selection against ubiquitous self-antigen, we previously found that one of the principal mediators implicated in clonal deletion, Bim, is required for caspase-3 activation but is ultimately dispensable for negative selection. On the basis of these data, we hypothesized that Nur77, another molecule thought to be a key mediator of clonal deletion, could be responsible for Bim-independent deletion. Despite comparable Nur77 induction in thymocytes during negative selection, Bim deficiency resulted in an accumulation of high-affinity-signaled thymocytes as well as impairment in caspase-mediated and caspase-independent cell death. Although these data suggested that Bim may be required for Nur77-mediated cell death, we found that transgenic Nur77 expression was sufficient to induce apoptosis independently of Bim. However, transgenic Nur77-induced apoptosis was significantly inhibited in the context of TCR signaling, suggesting that endogenous Nur77 could be similarly regulated during negative selection. Although Nur77 deficiency alone did not alter positive or negative selection, combined deficiency in Bim and Nur77 impaired clonal deletion efficiency and significantly increased positive selection efficiency. Collectively, these data shed light on the different roles for Bim and Nur77 during ubiquitous Ag-mediated clonal deletion and highlight potential differences from their reported roles in tissue-restricted Ag-mediated clonal deletion.

CD4 Cells Engineered to Express an MHC Class I Restricted TCR Can Rescue CD8 Cells Tolerized to Tumour-Associated Antigens

AbstractAbstract 952 Background:The efficacy of T cell therapies for cancer may be limited when targeting tumour-associated antigens (TAA) which are also self-antigens. Ongoing exposure to TAA on normal cells may lead to tolerance via anergy or exhaustion of antigen-specific T cells. Methods:We have designed a model of tolerance to TAA in which T cell receptor (TCR)-transduced CD8 T cells recognise pMDM2, a TAA that is also a ubiquitous self-antigen. CD8+ T cells were transduced with pMDM2-specific TCR (MDM-CD8) and transferred to sub-lethally irradiated B6 mice that express pMDM2 in the context of MHC Class I (H2-Kb). MDM-CD8 cells are detectable 4 weeks after transfer but show defective in vivo killing of target cells pulsed with MDM2 peptide. We have used this model to determine the mechanism of tolerance and to evaluate whether tolerant CD8+ T cells can be rescued by CD4 help. Results:To determine whether tolerance of MDM-CD8 cells was dependent upon recognition of cognate antigen, we transferred MDM-CD8 cells into mice of a different MHC background (BALB/c) which lack H2-Kb required for presentation of the TCR-recognised MDM2 peptide. When BALB/c MDM-CD8 cells were transferred to BALBc hosts their functions were preserved and they retained efficient antigen-specific cytolysis.To determine whether tolerance could be modified by provision of CD4+ T cell help, we co-transferred MDM-CD8 with transgenic OT-II CD4+ cells. OT-II cells were primed with dendritic cells (DCs) loaded with cognate pOVA323-339 or irrelevant peptide. When activated through their TCR, OT-II cells increased both the frequency of MDM2-specific CD8 cells and their cytotoxic functions, indicating that CD4 help can overcome CD8 tolerance to TAA.Ineffective antigen presentation to CD4 cells and lack of known MHC class II-restricted TAA are major limitations to providing CD4 help in T cell therapy for cancer. We therefore tested whether transfer of the MHC Class I-restricted MDM2 TCR into CD4 cells could provide help upon transfer to antigen-expressing hosts. Co-transfer of MDM2-TCR-transduced CD4 cells with CD8 cells improved antigen-specific killing of target cells when compared to single transfer of either TCR-transduced CD8 or CD4 cells. Conclusion:CD4 cells rendered capable of responding to an MHC class I restricted TAA by TCR transfer can rescue tolerance developing in a CD8 population with the same specificity. This is potentially a novel way to circumvent defective immune responses arising in adoptively transferred effector cells due to prolonged exposure to cognate antigen on normal host cells. Disclosures:Stauss:Cell Medica: Scientific Advisor Other.

Clonal deletion of thymocytes can occur in the cortex with no involvement of the medulla

The thymic medulla is generally held to be a specialized environment for negative selection. However, many self-reactive thymocytes first encounter ubiquitous self-antigens in the cortex. Cortical epithelial cells are vital for positive selection, but whether such cells can also promote negative selection is controversial. We used the HYcd4 model, where T cell receptor for antigen (TCR) expression is appropriately timed and a ubiquitous self-antigen drives clonal deletion in male mice. We demonstrated unambiguously that this deletion event occurs in the thymic cortex. However, the kinetics in vivo indicated that apoptosis was activated asynchronously relative to TCR activation. We found that radioresistant antigen-presenting cells and, specifically, cortical epithelial cells do not efficiently induce apoptosis, although they do cause TCR activation. Rather, thymocytes undergoing clonal deletion were preferentially associated with rare CD11c+ cortical dendritic cells, and elimination of such cells impaired deletion.

Secretory autoantibodies in primary biliary cirrhosis (PBC).

It is unclear how breakdown in immune tolerance to the ubiquitous self-antigen pyruvate dehydrogenase complex (PDC), seen in the autoimmune liver disease PBC, gives rise to tissue damage with such a limited distribution (restricted to the liver and salivary and lachrymal glands). One property shared by these tissues is the ability to export secretory IgA by the process of transcytosis. The aim of this study was to address whether active transcytosis of anti-PDC IgA occurs across epithelial surfaces in PBC, a finding that might implicate mucosal specific immune mechanisms in the pathogenesis of this disease. Parotid saliva was collected from PBC patients (n = 44), normal controls (n = 28) and PBC patients post-liver transplantation (n = 11). IgA and secretory component-positive antibodies specific for human PDC were quantified by ELISA and immunoblotting. PBC patients (but not control subjects) had anti-PDC IgA in their saliva. The strong correlation seen between titres detected using anti-IgA and anti-secretory component antibodies suggests that this is predominantly secretory IgA reaching the saliva by the active process of epithelial transcytosis. Titres of anti-PDC IgA remain high in PBC patients saliva post-liver transplant. Findings from studies of IgA in viral infection models raise the possibility that anti-PDC IgA could, whilst undergoing transcytosis, bind to newly translated PDC components in the cytoplasm of the epithelial cells transporting them out of the cell and inducing metabolic damage. This model would, if correct, help to explain the mechanism and tropism of tissue damage in PBC and the aberrant pattern of expression of PDC on the apical surface of biliary and salivary epithelial cells reported in this disease.

Langerhans' cell histiocytosis: possible association with malignant germ cell tumour.

A rare case of adult onset Langerhans' cell histiocytosis associated with dysgerminoma in a 35 year old Chinese woman is reported. The patient had a history of dysgerminoma of left ovary 15 years previously and had undergone surgery followed by radiotherapy and an uneventful recovery. She presented again in March 1994, this time with a left clavicular mass, which was shown histologically to be Langerhans' cell histiocytosis. The report illustrates the probable association between the two lesions, with some discussion on the underlying pathogenesis.

Antigen-independent, integrin-mediated T cell activation.

The "outside-in" signals produced by the interaction of integrin molecules with the extracellular matrix (ECM) trigger a multitude of cellular events. The vitronectin receptor (VNR), an alpha v beta 3 heterodimer, functions as a costimulatory molecule for the activation of a subset of V gamma 1.1/C gamma 4-bearing gamma/delta T cells, which have been postulated to recognize a ubiquitous self-antigen. We addressed the question of whether stimulation of these T cells requires both engagement of the VNR by ECM proteins and engagement of the TCR by its Ag. We introduced into a TCR- but VNR+ mutant T cell hybridoma, TG40 (derived from 2B4), a chimeric molecule that contains the cytoplasmic tail of the TCR zeta-chain fused to the cytoplasmic and transmembrane region of either human CD8 or human CD25. The transfectants expressing the chimeric molecules secreted IL-2 constitutively when the VNR was engaged with a ligand, e.g., provided by ECM proteins present in FCS. This constitutive cytokine secretion could be blocked with mAb directed against the VNR, with or the peptide RGD, or by growth in serum-free medium. VNR-mediated cell activation also induced the phosphorylation of the zeta-chain. Signaling through the zeta-chain was required, as cells transfected with a chimera containing only a 22 amino-acid long, truncated zeta-chain did not secrete IL-2 constitutively. Thus, we demonstrated that the binding of the VNR to ECM protein in the presence of the zeta-chain is sufficient to induce cytokine secretion by T cells and does not require the recognition of an Ag by the TCR. Such integrin-mediated, Ag-independent activation of T cells may play a critical role in the potentiation of inflammatory responses.