Anjli Kukreja

Sustained expansion of NKT cells and antigen-specific T cells after injection of α-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, α-galactosyl-ceramide (α-GalCer; KRN-7000) in five patients who had advanced cancer. Injection of α-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-γ inducible protein-10. In addition, there was an increase in memory CD8+ T cells specific for cytomegalovirus in vivo in response to α-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.

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.

Selective blockade of the inhibitory Fcγ receptor (FcγRIIB) 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 FcγRIIB 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 Fcγ receptor (FcγR)–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). FcγR-mediated STAT1 activation is rapid and requires activating FcγRs. 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 FcγR-mediated effects on DCs, as suppression of STAT1 by RNA interference inhibited FcγR-mediated DC maturation. These data suggest that the balance of activating/inhibitory FcγRs may regulate IFN signaling in myeloid cells. Manipulation of FcγR balance on DCs and monocytes may provide a novel approach to regulating IFN-mediated pathways in autoimmunity and human cancer.

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, β2-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.

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.

Role of Teichoic Acid Choline Moieties in the Virulence of Streptococcus pneumoniae

ABSTRACT 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.