Radhakrishnan, Suresh

Abstract Myeloid dendritic cells (mDC) activated with a B7-DC-specific cross-linking IgM Ab (B7-DC XAb) take up and retain Ag and interact with T cell compartments to affect a number of biologic changes that together cause strong antitumor responses and blockade of inflammatory airway disease in animal models. The molecular events mediating the initial responses in mDC remain unclear. In this study we show that B7-DC XAb caused rapid phosphorylation of the adaptor protein DAP12 and intracellular kinases Syk and phospholipase C-γ1. Pretreatment of mDC with the Syk inhibitor piceatannol blocked B7-DC XAb-induced Ag uptake with a concomitant loss of tumor protection in mice. Vaccination with tumor lysate-pulsed wild-type B7-DC XAb-activated mDC, but not TREM-2 knockout XAb-activated mDC, protected mice from lethal melanoma challenge. Multimolecular caps appeared within minutes of B7-DC XAb binding to either human or mouse mDC, and FRET analysis showed that class II, CD80, CD86, and TREM-2 are recruited in tight association on the cell surface. When TREM-2 expression was reduced in wild-type mDC using short hairpin RNA or by using mDC from TREM-2 knockout mice, in vitro DC failed to take up Ag after B7-DC XAb stimulation. These results directly link TREM-2 signaling with one change in the mDC phenotype that occurs in response to this unique Ab. The parallel signaling events observed in both human and mouse mDC support the hypothesis that B7-DC cross-linking may be useful as a therapeutic immune modulator in human patients.

Abstract Cross-linking cell surface molecules with IgM Abs is a specific approach for activating cells in vitro or in vivo. Dendritic cells (DC) activated with a human B7-DC (PD-L2)-specific IgM Ab can induce strong antitumor responses and block inflammatory airway disease in experimental models, yet the Ab-mediated molecular events promoting these responses remain unclear. Analysis of human or mouse DC treated with the B7-DC cross-linking Ab revealed PI3K-dependent phosphorylation of AKT accompanied by mobilization of NF-κB. Ab-activated DC up-regulated expression of cytokine and chemokine genes in an NF-κB-dependent manner. Importantly, PI3K→AKT→NF-κB activation was found to be indispensable for B7-DC cross-linking Ab-mediated protection of DC from cell death caused by cytokine withdrawal. Although other DC activators similarly protect DC from cell death, a synergy between cross-linking B7-DC and ligating RANK was observed. The parallel signaling events induced in human and mouse DC demonstrate that activation of cells using IgM Ab results in a response governed by a common mechanism and support the hypothesis that B7-DC cross-linking using this Ab may provide beneficial therapeutic immune modulation in human patients similar to those seen in animal models.

Abstract Asthma is thought to result from dysregulated Th2-like airway inflammatory responses to the environment. Although the etiology of asthma is not fully understood in humans, clinical and epidemiological evidence suggest a potential link between exposure to environmental fungi, such as Alternaria, and development and/or exacerbation of asthma. The goal of this project was to investigate the mechanisms of airway Th2 responses by using Alternaria as a clinically relevant model for environmental exposure. Airway exposure of naive animals to an experimental Ag, OVA, or a common allergen, short ragweed pollen, induced no or minimal immune responses to these Ags. In contrast, mice developed strong Th2-like immune responses when they were exposed to these Ags in the presence of Alternaria extract. Extracts of other fungi, such as Aspergillus and Candida, showed similar Th2 adjuvant effects, albeit not as potently. Alternaria stimulated bone marrow-derived dendritic cells (DCs) to express MHC class II and costimulatory molecules, including OX40 ligand, in vitro. Importantly, Alternaria inhibited IL-12 production by activated DCs, and DCs exposed to Alternaria enhanced Th2 polarization of CD4+ T cells. Furthermore, adoptive airway transfer of DCs, which had been pulsed with OVA in the presence of Alternaria, showed that the recipient mice had enhanced IgE Ab production and Th2-like airway responses to OVA. Thus, the asthma-related environmental fungus Alternaria produces potent Th2-like adjuvant effects in the airways. Such immunogenic properties of certain environmental fungi may explain their strong relationships with human asthma and allergic diseases.

ABSTRACT Epidemiological studies suggest that an environmental factor (possibly a virus) acquired early in life may trigger multiple sclerosis (MS). The virus may remain dormant in the central nervous system but then becomes activated in adulthood. All existing models of MS are characterized by inflammation or demyelination that follows days after virus infection or antigen inoculation. While investigating the role of CD4 + T cell responses following Theiler's virus infection in mice deficient in STAT4 or STAT6, we discovered a model in which virus infection was followed by demyelination after a very prolonged incubation period. STAT4−/− mice were resistant to demyelination for 180 days after infection, but developed severe demyelination after this time point. Inflammatory cells and up‐regulation of Class I and Class II MHC antigens characterized these lesions. Virus antigen was partially controlled during the early chronic phase of the infection even though viral RNA levels remained high throughout infection. Demyelination correlated with the appearance of virus antigen expression. Bone marrow reconstitution experiments indicated that the mechanism of the late onset demyelination was the result of the STAT4−/− immune system. Thus, virus infection of STAT4−/− mice results in a model that may allow for dissection of the immune events predisposing to late‐onset demyelination in MS.

Abstract A naturally occurring human antibody potentiates dendritic cell function on cross-linking B7-DC (PD-L2), supporting robust T-cell responses in vitro. Moreover, treatment of dendritic cells with B7-DC cross-linking antibody resulted in secretion of interleukin-12, suggesting a TH1 polarization of this response. Here we show an in vivo immunotherapeutic effect of this B7-DC cross-linking antibody using a poorly immunogenic B16 melanoma tumor model. Treatment of mice systemically with antibody at the time of tumor cell engraftment prevented tumor growth in a CD4 and CD8 T-cell-dependent manner. The protective effect of B7-DC cross-linking antibody treatment was independent of endogenous antibody responses. Tumor-specific CTL precursors could be isolated from lymph nodes draining the tumor site in animals treated with B7-DC cross-linking antibody, but not from those treated with isotype control antibodies. The elicited antitumor responses in vivo were specific and long-lasting. More strikingly, treatment of mice with B7-DC cross-linking antibody after the tumors were established in the lungs resulted in protection in a CD8-, perforin-, and granzyme B-dependent fashion. Depletion of natural killer cells did not block the effects of treatment with B7-DC cross-linking antibody. Together, these findings demonstrate that cross-linking B7-DC with the human IgM antibody sHIgM12 can induce a protective immune response against a weakly antigenic experimental tumor and therefore has potential as a novel immunotherapeutic approach for treating cancer.

Dendritic cells (DCs) are classified in two states: immature DCs (iDCs), which perform sentinel functions, sampling for antigen and danger signals, and mature DCs (mDCs), which exhibit enhanced antigen-presenting functions but are no longer capable of acquiring antigen. We now describe DCs with a different activation phenotype: cells having the strong antigen-presenting functions of mDCs and the antigen-acquiring functions of iDCs. We have described an antibody that binds the costimulatory molecule B7-DC and activates DCs. The resulting phenotype is distinct from iDCs or mDCs matured by using Toll-like receptor (TLR) agonists. Ability to take up antigen increases, while expression of B71/2 costimulatory and MHC molecules remains unchanged. DCs matured with TLR agonists and then superactivated through B7-DC exhibit a previously unrecognized phenotype. These DCs recover the ability to take up antigen, which is normally lost after treatment with TLR-3 and TLR-9 agonists, yet retain the high expression of costimulatory and MHC molecules and strong antigen-presenting functions of mDCs. Immunization using TLR agonists and B7-DC XAb (cross-linking antibody) together as adjuvant resulted in substantially increased cytolytic T cell responses, particularly when minimal peptide antigens were used. By stimulating DCs with two distinct activation signals, a previously unrecognized phenotype exhibiting augmented antigen-presenting functions was obtained.

B7-DC molecules are known to function as ligands on antigen-presenting cells (APCs), enhancing T cell activation. In this study, cross-linking B7-DC with the monoclonal antibody sHIgM12 directly potentiates dendritic cell (DC) function by enhancing DC presentation of major histocompatibility complex–peptide complexes, promoting DC survival; and increasing secretion of interleukin (IL)-12p70, a key T helper cell type 1 promoting cytokine. Furthermore, ex vivo treatment of DCs or systemic treatment of mice with sHIgM12 increases the number of transplanted DCs that reach draining lymph nodes and increases the ability of lymph node APCs to activate naive T cells. Systemic administration of the antibody has an equivalent effect on DCs transferred at a distant site. These findings implicate B7-DC expressed on DCs in bidirectional communication. In addition to the established costimulatory and inhibitory functions associated with B7-DC, this molecule can also function as a conduit for extracellular signals to DCs modifying DC functions.