Eculizumab reduces complement activation, inflammation, endothelial damage, thrombosis, and renal injury markers in aHUS.

Atypical hemolytic uremic syndrome (aHUS) is a genetic, life-threatening disease characterized by uncontrolled complement activation, systemic thrombotic microangiopathy (TMA), and vital organ damage. We evaluated the effect of terminal complement blockade with the anti-C5 monoclonal antibody eculizumab on biomarkers of cellular processes involved in TMA in patients with aHUS longitudinally, during up to 1 year of treatment, compared with in healthy volunteers. Biomarker levels were elevated at baseline in most patients, regardless of mutational status, plasma exchange/infusion use, platelet count, or lactate dehydrogenase or haptoglobin levels. Eculizumab reduced terminal complement activation (C5a and sC5b-9) and renal injury markers (clusterin, cystatin-C, ß2-microglobulin, and liver fatty acid binding protein-1) to healthy volunteer levels and reduced inflammation (soluble tumor necrosis factor receptor-1), coagulation (prothrombin fragment F1+2 and d-dimer), and endothelial damage (thrombomodulin) markers to near-normal levels. Alternative pathway activation (Ba) and endothelial activation markers (soluble vascular cell adhesion molecule-1) decreased but remained elevated, reflecting ongoing complement activation in aHUS despite complete terminal complement blockade. These results highlight links between terminal complement activation and inflammation, endothelial damage, thrombosis, and renal injury and underscore ongoing risk for systemic TMA and progression to organ damage. Further research regarding underlying complement dysregulation is warranted. This trial was registered at www.clinicaltrials.gov as #NCT01194973.

Selective blockade of the inhibitory Fcgamma receptor (FcgammaRIIB) in human dendritic cells and monocytes induces a type I interferon response program.

The ability of dendritic cells (DCs) to activate immunity is linked to their maturation status. In prior studies, we have shown that selective antibody-mediated blockade of inhibitory FcgammaRIIB receptor on human DCs in the presence of activating immunoglobulin (Ig) ligands leads to DC maturation and enhanced immunity to antibody-coated tumor cells. We show that Fcgamma receptor (FcgammaR)-mediated activation of human monocytes and monocyte-derived DCs is associated with a distinct gene expression pattern, including several inflammation-associated chemokines, as well as type 1 interferon (IFN) response genes, including the activation of signal transducer and activator of transcription 1 (STAT1). FcgammaR-mediated STAT1 activation is rapid and requires activating FcgammaRs. However, this IFN response is observed without a detectable increase in the expression of type I IFNs themselves or the need to add exogenous IFNs. Induction of IFN response genes plays an important role in FcgammaR-mediated effects on DCs, as suppression of STAT1 by RNA interference inhibited FcgammaR-mediated DC maturation. These data suggest that the balance of activating/inhibitory FcgammaRs may regulate IFN signaling in myeloid cells. Manipulation of FcgammaR balance on DCs and monocytes may provide a novel approach to regulating IFN-mediated pathways in autoimmunity and human cancer.

Frequent and specific immunity to the embryonal stem cell-associated antigen SOX2 in patients with monoclonal gammopathy.

Specific targets of cellular immunity in human premalignancy are largely unknown. Monoclonal gammopathy of undetermined significance (MGUS) represents a precursor lesion to myeloma (MM). We show that antigenic targets of spontaneous immunity in MGUS differ from MM. MGUS patients frequently mount a humoral and cellular immune response against SOX2, a gene critical for self-renewal in embryonal stem cells. Intranuclear expression of SOX2 marks the clonogenic CD138(-) compartment in MGUS. SOX2 expression is also detected in a proportion of CD138(+) cells in MM patients. However, these patients lack anti-SOX2 immunity. Cellular immunity to SOX2 inhibits the clonogenic growth of MGUS cells in vitro. Detection of anti-SOX2 T cells predicts favorable clinical outcome in patients with asymptomatic plasmaproliferative disorders. Harnessing immunity to antigens expressed by tumor progenitor cells may be critical for prevention and therapy of human cancer.

Enhancement of clonogenicity of human multiple myeloma by dendritic cells.

Infiltration by dendritic cells (DCs) is a common feature of most human tumors. Prior studies evaluating the interaction of DCs with tumors have focused largely on their immunologic properties (for review see Banchereau, J., and R.M. Steinman. 1998. Nature. 392:245-252). In this study, we show that the clonogenicity of several human tumor cell lines and primary tumor cells from myeloma patients is enhanced by their interactions with DCs. Myeloma cells cultured in the presence of DCs have an altered phenotype with an increased proportion of cells lacking terminal plasma cell differentiation marker CD138. DC-tumor interaction also leads to the up-regulation of B cell lymphoma 6 expression in myeloma cells. Effects of DCs on myeloma cells are inhibited by blockade of the receptor activator of NF-kB (RANK)-RANK ligand and B cell-activating factor-APRIL (a proliferation-inducing ligand)-mediated interactions. Together, these data suggest that tumor-DC interactions may directly impact the biology of human tumors, particularly multiple myeloma, and may be a target for therapeutic intervention.

Sustained expansion of NKT cells and antigen-specific T cells after injection of alpha-galactosyl-ceramide loaded mature dendritic cells in cancer patients.

Natural killer T (NKT) cells are distinct glycolipid reactive innate lymphocytes that are implicated in the resistance to pathogens and tumors. Earlier attempts to mobilize NKT cells, specifically, in vivo in humans met with limited success. Here, we evaluated intravenous injection of monocyte-derived mature DCs that were loaded with a synthetic NKT cell ligand, alpha-galactosyl-ceramide (alpha-GalCer; KRN-7000) in five patients who had advanced cancer. Injection of alpha-GalCer-pulsed, but not unpulsed, dendritic cells (DCs) led to >100-fold expansion of several subsets of NKT cells in all patients; these could be detected for up to 6 mo after vaccination. NKT activation was associated with an increase in serum levels of interleukin-12 p40 and IFN-gamma inducible protein-10. In addition, there was an increase in memory CD8+ T cells specific for cytomegalovirus in vivo in response to alpha-GalCer-loaded DCs, but not unpulsed DCs. These data demonstrate the feasibility of sustained expansion of NKT cells in vivo in humans, including patients who have advanced cancer, and suggest that NKT activation might help to boost adaptive T cell immunity in vivo.

Dominant role of CD47-thrombospondin-1 interactions in myeloma-induced fusion of human dendritic cells: implications for bone disease.

Lytic bone disease in myeloma is characterized by an increase in multinucleate osteoclasts in close proximity to tumor cells. However, the nature of osteoclast precursors and the mechanisms underlying multinuclearity are less understood. Here we show that culture of myeloma cell lines as well as primary myeloma cells with human dendritic cells (DCs) but not monocytes or macrophages leads to spontaneous cell-cell fusion, which then leads to the facile formation of multinucleate bone-resorbing giant cells. Osteoclastogenesis is cell contact dependent, leading to up-regulation of thrombospondin-1 (TSP-1) in DCs. Disruption of CD47-TSP-1 interaction by TSP-1-blocking antibodies or down-regulation of CD47 on tumor cells by RNA interference abrogates tumor-induced osteoclast formation. Blockade of CD47-TSP-1 interactions also inhibits receptor activator for nuclear factor kappaB ligand- and macrophage colony-stimulating factor-induced formation of osteoclasts from human monocytes. Further, TSP-1 blockade attenuates hypercalcemia induced by parathyroid hormone in vivo. These data point to a role for CD47-TSP-1 interactions in regulating cell-fusion events involved in human osteoclast formation. They also suggest that DCs, known to be enriched in myeloma tumors, may be direct precursors for tumor-associated osteoclasts. Disruption of CD47-TSP-1 interactions or preventing the recruitment of DCs to tumors may provide novel approaches to therapy of myeloma bone disease and osteoporosis.

Dendritic cells mediate the induction of polyfunctional human IL17-producing cells (Th17-1 cells) enriched in the bone marrow of patients with myeloma.

IL17-producing (Th17) cells are a distinct lineage of T helper cells that regulate immunity and inflammation. The role of antigen-presenting cells in the induction of Th17 cells in humans remains to be fully defined. Here, we show that human dendritic cells (DCs) are efficient inducers of Th17 cells in culture, including antigen-specific Th17 cells. Although most freshly isolated circulating human Th17 cells secrete IL17 alone or with IL2, those induced by DCs are polyfunctional and coexpress IL17 and IFNgamma (Th17-1 cells). The capacity of DCs to expand Th17-1 cells is enhanced upon DC maturation, and mature DCs are superior to monocytes for the expansion of autologous Th17 cells. In myeloma, where tumors are infiltrated by DCs, Th17 cells are enriched in the bone marrow relative to circulation. Bone marrow from patients with myeloma contains a higher proportion of Th17-1 cells compared with the marrow in preneoplastic gammopathy (monoclonal gammopathy of undetermined significance [MGUS]). Uptake of apoptotic but not necrotic myeloma tumor cells by DCs leads to enhanced induction of Th17-1 cells. These data demonstrate the capacity of DCs to induce expansion of polyfunctional IL17-producing T cells in humans, and suggest a role for DCs in the enrichment of Th17-1 cells in the tumor bed.

Role of teichoic acid choline moieties in the virulence of Streptococcus pneumoniae.

In recent reports it was shown that genetically modified choline-free strains of Streptococcus pneumoniae (D39Cho(-)licA64 and D39ChiplicB31) expressing the type II capsular polysaccharide were virtually avirulent in the murine sepsis model, in sharp contrast to the isogenic and highly virulent strains D39Cho(-) and D39Chip, which have retained the choline residues at their surface. We now demonstrate that this choline-associated virulence is independent of Toll-like receptor 2 recognition. Also, despite the lack of virulence, choline-free strains of S. pneumoniae were able to activate splenic dendritic cells, induce secretion of proinflammatory cytokines, and produce specific protective immunity against subsequent challenge. However, after this transient engagement of the immune system the choline-free bacteria were rapidly cleared from the blood, while the isogenic virulent strain D39Cho(-) continued to grow, accompanied by prolonged expression of cytokines, eventually killing the experimental animals. The critical contribution of choline residues to the virulence potential of pneumococci appears to be the role that these amino alcohol residues play in a pneumococcal immune evasion strategy, the mechanism of which is unknown at the present time.

Systemic Alanine Glyoxylate Aminotransferase mRNA Improves Glyoxylate Metabolism in a Mouse Model of Primary Hyperoxaluria Type 1.

Primary Hyperoxaluria Type 1 (PH1) is an autosomal recessive disorder of glyoxylate metabolism. Loss of alanine glyoxylate aminotransferase (AGT) function to convert intermediate metabolite glyoxylate to glycine causes the accumulation and reduction of glyoxylate to glycolate, which eventually is oxidized to oxalate. Excess oxalate in PH1 patients leads to the formation and deposition of calcium oxalate crystals in the kidney and urinary tract. Oxalate crystal deposition causes a decline in renal function, systemic oxalosis, and eventually end-stage renal disease and premature death. mRNA-based therapies are a new class of drugs that work by replacing the missing enzyme. mRNA encoding AGT has the potential to restore normal glyoxylate to glycine metabolism, thus preventing the buildup of calcium oxalate in various organs. Panels of codon-optimized AGT mRNA constructs were screened in vitro and in wild-type mice for the production of a functional AGT enzyme. Two human constructs, wild-type and engineered AGT (RHEAM), were tested in Agxt-/- mice. Repeat dosing in Agxt-/- mice resulted in a 40% reduction in urinary oxalate, suggesting therapeutic benefit. These studies suggest that mRNA encoding AGT led to increased expression and activity of the AGT enzyme in liver that translated into decrease in urinary oxalate levels. Taken together, our data indicate that AGT mRNA may have the potential to be developed into a therapeutic for PH1.

Phase I study of samalizumab in chronic lymphocytic leukemia and multiple myeloma: blockade of the immune checkpoint CD200.

PURPOSE: Samalizumab is a novel recombinant humanized monoclonal antibody that targets CD200, an immunoregulatory cell surface member of the immunoglobulin superfamily that dampens excessive immune responses and maintains self-tolerance. This first-in-human study investigated the therapeutic use of samalizumab as a CD200 immune checkpoint inhibitor in chronic lymphocytic leukemia (CLL) and multiple myeloma (MM). EXPERIMENTAL DESIGN: Twenty-three patients with advanced CLL and 3 patients with MM were enrolled in an open-label phase 1 study (NCT00648739). Patients were assigned sequentially to one of 7 dose level cohorts (50 to 600 mg/m2) in a 3 + 3 study design, receiving a single dose of samalizumab intravenously once every 28 days. Primary endpoints were safety, identification of the maximum tolerated dose (MTD), and pharmacokinetics. Secondary endpoints were samalizumab binding to CD200, pharmacodynamic effects on circulating tumor cells and leukocyte subsets, and clinical responses. RESULTS: Twenty-one patients received > 1 treatment cycle. Adverse events (AEs) were generally mild to moderate in severity. Samalizumab produced dose-dependent decreases in CD200 expression on CLL cells and decreased frequencies of circulating CD200 + CD4+ T cells that were sustained at higher doses. The MTD was not reached. Decreased tumor burden was observed in 14 CLL patients. One CLL patient achieved a durable partial response and 16 patients had stable disease. All MM patients had disease progression. CONCLUSIONS: Samalizumab had a good safety profile and treatment was associated with reduced tumor burden in a majority of patients with advanced CLL. These preliminary positive results support further development of samalizumab as an immune checkpoint inhibitor. TRIAL REGISTRATION: ClinicalTrials.gov, NCT00648739 registered April 1, 2008.

Multiple immuno-regulatory defects in type-1 diabetes.

Susceptibility to immune-mediated diabetes (IMD) in humans and NOD mice involves their inherently defective T cell immunoregulatory abilities. We have followed natural killer (NK) T cell numbers in patients with IMD, both by flow cytometry using mAbs to the characteristic junctions found in the T cell receptors of this cell subtype, and by semiquantitative RT-PCR for the corresponding transcripts. Both before and after clinical onset, the representation of these cells in patients' PBMCs is reduced. We also report low numbers of resting CD4(+) CD25(+) T cells in IMD patients, a subset of T cells shown to have important immunoregulatory functions in abrogating autoimmunities in 3-day thymectomized experimental mice. Whereas a biased Th1 to Th2 cytokine profile has been suggested to underlie the pathogenesis of IMD in both species, we found defective production of IFN-gamma in our patients after in vitro stimulation of their PBMCs by phorbol-myristate acetate and ionomycin and both IFN-gamma and IL-4 deficiencies in V(alpha)24(+) NK T-enriched cells. These data suggest that multiple immunoregulatory T (Treg) cell defects underlie islet cell autoimmunity leading to IMD in humans and that these lesions may be part of a broad T cell defect.

NKT cells and type-1 diabetes and the "hygiene hypothesis" to explain the rising incidence rates.

Immune-mediated (type-1) diabetes (IMD) is a multigenetic disease that is strongly influenced by the environment. Whereas the incidence rates are steadily rising worldwide, less than half of affected identical twins ever become concordant for IMD or even beta-cell autoimmunity. Worldwide, it is the tropical regions of the world that are replete in infectious and parasitic diseases that are the least affected. Repeated efforts to identify the putative inductive agents for beta-cell autoimmunity have proved unrewarding. Rather, we suggest that some environments are less protective than others and argue that it is the fall in incidences of infectious diseases and intestinal parasites that are likely responsible for the rise in autoimmune diseases like IMD in the West. Nonobese diabetic (NOD) mice reared in gnotobiotic environments have only worsened diabetes, while recent studies suggest that multiple defects in immune tolerance to self must be present before IMD can develop in the human or mouse. We speculate herein that the deficiency in natural killer T (NKT) cells in IMD in both species may be both genetic and environmentally influenced, predisposing to pancreatic beta-cell autoimmunity through a dysfunction of immunoregulatory T cells, with defective peripheral control of islet cell protein autoreactive cytotoxic CD8+ T cells. The encouraging results in NOD mice using alpha-galactosylceramide to stimulate NKT cells now warrant trials with this and other glycolipid NKT cell-stimulating agents in humans. Since it has become apparent that autoimmune diseases such as IMD are the result of an underlying immunodeficiency state, we strongly argue that its effective prevention will likely come through the use of immunostimulation and not through side effect-prone immunosuppression.

Bortezomib disrupts tumour-dendritic cell interactions in myeloma and lymphoma: therapeutic implications.

Recent studies have shown that the interactions between tumour and dendritic cells (DCs) promote clonogenic growth of lymphoproliferative tumours, particularly myeloma. The present study showed that the proteasome inhibitor, bortezomib, disrupts this interaction. Targeting the drug to DCs was required for optimal suppression of tumour growth, including primary myeloma tumour progenitors in clonogenic assays. Bortezomib lead to dose-dependent induction of apoptosis in both myeloid and plasmacytoid DCs, and the sensitivity of DCs to bortezomib was comparable with that of tumour cells. These data suggest that disruption of tumour-DC interactions may contribute to the clinical effects of bortezomib.

NKT cell defects in NOD mice suggest therapeutic opportunities.

Recent studies have reported that immunoregulatory NKT cells are defective in NOD mice and that treatment of mice with alpha-galactosylceramide that selectively stimulate NKT cells, is anti-diabetogenic. The objective of this study was to document the natural history of changes in NKT cells in various organs in NOD mice in the period up to the time of diabetes onset so that novel intervention therapies could be devised. We found that NKT cell-specific receptor (NKT-TCR) Valpha14Jalpha281 expressions by quantitative (RealTime) RT-PCR in thymus, spleen and liver of NOD male and female mice were low at 1-3 months of life compared to BALB/c and C57BL/6 mice, albeit a transient spike in levels occurred in female NOD livers at 2 months. Female pancreases showed low levels of these transcripts despite their active and destructive insulitis. In contrast, NOD males exhibited high expression of this invariant TCR in pancreas, where their insulitis was less destructive. A survey of NKT-TCR expressions in a battery of congenic, non-diabetes prone NOD strains indicated that this NKT phenotype was quite variable but higher than diabetes prone NOD. Bone marrow transplantation of NOD females from B6.NOD-H2(g7) donors raised their NKT-TCR expressions. Tuberculin administrations in the forms of BCG and CFA in a manner known to protect NOD mice from diabetes both raised NKT-TCR levels, as did the anti-inflammatory PPAR-gamma agonist rosiglitazone. These findings provide exciting therapeutic avenues to be explored in the treatment of human immune mediated type-1 diabetes where there are similar immunoregulatory lesions.