CD52 glycan binds the proinflammatory B box of HMGB1 to engage the Siglec-10 receptor and suppress human T cell function.

CD52, a glycophosphatidylinositol (GPI)-anchored glycoprotein, is released in a soluble form following T cell activation and binds to the Siglec (sialic acid-binding Ig-like lectin)-10 receptor on T cells to suppress their function. We show that binding of CD52-Fc to Siglec-10 and T cell suppression requires the damage-associated molecular pattern (DAMP) protein, high-mobility group box 1 (HMGB1). CD52-Fc bound specifically to the proinflammatory Box B domain of HMGB1, and this in turn promoted binding of the CD52 N-linked glycan, in α-2,3 sialic acid linkage with galactose, to Siglec-10. Suppression of T cell function was blocked by anti-HMGB1 antibody or the antiinflammatory Box A domain of HMGB1. CD52-Fc induced tyrosine phosphorylation of Siglec-10 and was recovered from T cells complexed with HMGB1 and Siglec-10 in association with SHP1 phosphatase and the T cell receptor (TCR). Thus, soluble CD52 exerts a concerted immunosuppressive effect by first sequestering HMGB1 to nullify its proinflammatory Box B, followed by binding to the inhibitory Siglec-10 receptor, triggering recruitment of SHP1 to the intracellular immunoreceptor tyrosine-based inhibitory motif of Siglec-10 and its interaction with the TCR. This mechanism may contribute to immune-inflammatory homeostasis in pathophysiologic states and underscores the potential of soluble CD52 as a therapeutic agent.

CD52 inhibits Toll-like receptor activation of NF-κB and triggers apoptosis to suppress inflammation.

Soluble CD52 is a small glycoprotein that suppresses T-cell activation, but its effect on innate immune cell function is unknown. Here we demonstrate that soluble CD52 inhibits Toll-like receptor and tumor necrosis factor receptor signaling to limit activation of NF-κB and thereby suppress the production of inflammatory cytokines by macrophages, monocytes and dendritic cells. At higher concentrations, soluble CD52 depletes the short-lived pro-survival protein MCL-1, contributing to activation of the BH3-only proteins BAX and BAK to cause intrinsic apoptotic cell death. In vivo, administration of soluble CD52 suppresses lipopolysaccharide (LPS)-induced cytokine secretion and other features of endotoxic shock, whereas genetic deletion of CD52 exacerbates LPS responses. Thus, soluble CD52 exhibits broad immune suppressive effects that signify its potential as an immunotherapeutic agent.

Characterization of the Human Pancreas Side Population as a Potential Reservoir of Adult Stem Cells.

OBJECTIVES: The side population (SP) contains cells with stem cell/progenitor properties. Previously, we observed that the mouse pancreas SP expanded after pancreatic injury. We aimed to characterize the SP in human pancreas as a potential source of stem cells. METHODS: Human organ donor pancreata were fractionated into islets and exocrine tissue, enriched by tissue culture and dispersed into single cells. Cells were phenotyped by flow cytometry, and the SP was defined by efflux of fluorescent dye Hoechst 33342 visualized by ultraviolet excitation. Cells were flow sorted, and their colony-forming potential measured on feeder cells in culture. RESULTS: An SP was identified in islet and exocrine cells from human organ donors: 2 with type 1 diabetes, 3 with type 2 diabetes, and 28 without diabetes. Phenotyping revealed that exocrine SP cells had an epithelial origin, were enriched for carbohydrate antigen 19-9 ductal cells expressing stem cell markers CD133 and CD26, and had greater colony-forming potential than non-SP cells. The exocrine SP was increased in a young adult with type 1 diabetes and ongoing islet autoimmunity. CONCLUSIONS: The pancreatic exocrine SP is a potential reservoir of adult stem/progenitor cells, consistent with previous evidence that such cells are duct-derived and express CD133.

Antigen-based vaccination and prevention of type 1 diabetes.

Insulin-dependent or type 1 diabetes (T1D) is a paradigm for prevention of autoimmune disease: Pancreatic ß-cell autoantigens are defined, at-risk individuals can be identified before the onset of symptoms, and autoimmune diabetes is preventable in rodent models. Intervention in asymptomatic individuals before or after the onset of subclinical islet autoimmunity places a premium on safety, a requirement met only by lifestyle-dietary approaches or autoantigen-based vaccination to induce protective immune tolerance. Insulin is the key driver of autoimmune ß-cell destruction in the nonobese diabetic (NOD) mouse model of T1D and is an early autoimmune target in children at risk for T1D. In the NOD mouse, mucosal administration of insulin induces regulatory T cells that protect against diabetes. The promise of autoantigen-specific vaccination in humans has yet to be realized, but recent trials of oral and nasal insulin vaccination in at-risk humans provide grounds for cautious optimism.

Proinsulin is encoded by an RNA splice variant in human blood myeloid cells.

Genes for peripheral tissue-restricted self-antigens are expressed in thymic and hematopoietic cells. In thymic medullary epithelial cells, self-antigen expression imposes selection on developing autoreactive T cells and regulates susceptibility to autoimmune disease in mouse models. Less is known about the role of self-antigen expression by hematopoietic cells. Here we demonstrate that one of the endocrine self-antigens expressed by human blood myeloid cells, proinsulin, is encoded by an RNA splice variant. The surface expression of immunoreactive proinsulin was significantly decreased after transfection of monocytes with small interfering RNA to proinsulin. Furthermore, analogous to proinsulin transcripts in the thymus, the abundance of the proinsulin RNA splice variant in blood cells corresponded with the length of the variable number of tandem repeats 5' of the proinsulin gene, known to be associated with type 1 diabetes susceptibility. Self-antigen expression by peripheral myeloid cells extends the umbrella of "immunological self" and, by analogy with the thymus, may be implicated in peripheral immune tolerance.

Results of a Phase I study utilizing monocyte-derived dendritic cells pulsed with tumor RNA in children with Stage 4 neuroblastoma.

BACKGROUND: A Phase I study of 11 pediatric patients with newly diagnosed, Stage 4 neuroblastoma was conducted using monocyte-derived dendritic cells (DC) pulsed with tumor RNA to produce antitumor vaccines (DC(RNA)). METHODS: Patients received two courses of induction with carboplatin followed by standard chemotherapy, surgery, radiation, high-dose therapy, stem cell rescue, and DC(RNA) vaccine therapy. RESULTS: The results showed that this method for producing and administering DC(RNA) from a single leukapheresis product was both feasible and safe in this pediatric neuroblastoma population. Two courses of carboplatin maintained lymphocyte counts at normal levels. However, immune function 6 weeks after high-dose chemotherapy and stem cell rescue and prior to receiving DC(RNA) was impaired in all patients tested. There was an alteration in the ratio of CD4-positive and CD80-positive T cells. CD4-positive cell numbers were below normal, whereas CD8-positive cell numbers were above normal for all patients. In addition, CD19-positive cell numbers were below normal for all but one patient. It was found that humoral responses to recall antigens (diphtheria and tetanus) and cellular responses to mitogen and recall antigens were below normal in most patients. Despite this, two of three patients tested showed a tumor-specific humoral immune response to DC(RNA). Among the patients who had measurable disease at the time of DC(RNA) vaccine, none showed any objective tumor response. CONCLUSIONS: DC(RNA) vaccines were both safe and feasible in children with Stage 4 neuroblastoma. Humoral responses to tumor were detected, although remained immunosuppressed at the time of administration, limiting efficacy.

Results of a phase 1 study utilizing monocyte-derived dendritic cells pulsed with tumor RNA in children and young adults with brain cancer.

We conducted a phase 1 study of 9 pediatric patients with recurrent brain tumors using monocyte-derived dendritic cells pulsed with tumor RNA to produce antitumor vaccine (DCRNA) preparations. The objectives of this study included (1) establishing safety and feasibility and (2) measuring changes in general, antigen-specific, and tumor-specific immune responses after DCRNA. Dendritic cells were derived from freshly isolated monocytes after 7 days of culture with IL-4 and granulocyte-macrophage colony-stimulating factor, pulsed with autologous tumor RNA, and then cryopreserved. Patients received at least 3 vaccines, each consisting of an intravenous and an intradermal administration at biweekly intervals. The study showed that this method for producing and administering DCRNA from a single leukapheresis product was both feasible and safe in this pediatric brain tumor population. Immune function at the time of enrollment into the study was impaired in all patients tested. While humoral responses to recall antigens (diphtheria and tetanus) were intact in all patients, cellular responses to mitogen and recall antigens were below normal. Following DCRNA vaccine, 2 of 7 patients showed stable clinical disease and 1 of 7 showed a partial response. Two of 7 patients who were tested showed a tumor-specific immune response to DCRNA. This study showed that DCRNA vaccines are both safe and feasible in children with tumors of the central nervous system with a single leukapheresis.