Radhakrishnan, Suresh

Abstract We investigated whether human IgM antibody, B7-DCXAb, which binds to B7-DC and enhances DCs' antigen uptake and retention, migration, and cytokine production, improves the efficacy of allergen immunotherapy. Mice were sensitized by intraperitoneal injection of OVA or house dust mite (HDM) antigens plus alum and then treated by intravenous B7-DCXAb injection intranasal or sublingual administration of antigen. OVA-sensitized mice developed anti-OVA IgE and IgG1, Th2-type splenocyte responses to OVA; marked airway eosinophilic inflammation was observed after intranasal OVA challenge. When these mice were treated by intravenous B7-DCXAb plus intranasal OVA, anti-OVA IgE and IgG1 decreased by 75~95% within 1 week; this decrease persisted for >8 weeks. Anti-OVA IgG2a decreased transiently. Splenocyte production of IL-4, IL-5, and IL-13 in response to OVA was abolished; no increases in IFN-g or IL-17 were noted. In OVA-challenged mice, airway eosinophilia and lung levels of IL-4, IL-5 and IL-13 were reduced by >90%. Immunotherapeutic effects were also observed with intravenous B7-DCXAb plus sublingual OVA, but not with intravenous B7-DCXAb alone. Furthermore, HDM-sensitized mice were treated successfully by intravenous B7-DCXAb plus intranasal HDM. B7-DCXAb injection plus mucosal administration of antigen effectively treats allergic responses and may provide novel strategy for allergen immunotherapy.

Abstract Dendritic cells (DC) are important regulators of T cell immunity. The degree of stimulation, the pattern of co-stimulatory molecules expressed, and the cytokines secreted by DC dictate the nature of the effector and memory cells generated, particularly with respect to their Th1 or Th2 phenotypes. In this report we demonstrate that addition of activated DC to spleen cultures containing established Th2 polarized CD4+ T cells was sufficient to suppress Th2 and to induce Th1 cytokines in a recall response, a phenomenon referred to as phenotype reversal. The ability of activated DC to induce phenotype reversal displayed exquisite antigen specificity. The DC activator B7-DC XAb was more than 10,000-fold more efficient at inducing phenotype reversal than the TLR agonists, CpG-ODN and Gardiquimod. Characterization of the mechanisms governing phenotype reversal revealed the requirement for cognate interaction between the TCR: pMHC complex, the expression of the co-stimulation/adhesion molecule ICAM-1, and secretion of IL-12 and IFNƒ×ƒnby the activated DC. Requirement for the co-stimulation/adhesion molecule SLAM was found to be quantitative. Thus, activation of DC, particularly by cross linking B7-DC, can modulate well established Th2 T cell responses in an antigen specific manner.

Abstract IgM antibodies, as multivalent ligands, have the capacity to activate cells differentially by cross-linking targeted cell surface signaling molecules. For example, hIgM12 (B7DC Xab) activates dendritic cells (DCs), leading to potent immunomodulation, while hIgM22 binds glial cells and causes remyelination in mouse models of multiple sclerosis. Both antibodies activate common signaling intermediates in their respective cellular targets suggesting a common mechanism of action. Elucidation of these shared mechanisms became possible by targeting both antibodies to a common cell type. Our previous studies demonstrate that B7-DC expression is required to activate downstream signaling in DCs by hIgM12. We utilized a phage display system to identify 7mer peptides bound by each IgM and expressed each peptide attached to a surrogate cell surface carrier on retrovirally tranduced bone marrow-derived DCs from B7-DC -/- mice. Transduced cells were treated with hIgM12, hIgM22 or anti-carrier IgM, and subsequent activity was compared against B7-DC +/+ DCs or B7-DC-reconstituted B7-DC -/- DCs. DC activation by each IgM was specific to expression of their conjugate peptide construct. Activation by hIgM22 was indistinguishable from activation by hIgM12, while anti-carrier IgM caused no activation. This indicates that while their target specificity differs, once bound these IgMs activate cells via common mechanisms.

Abstract Dendritic cells (DC) initiate and shape an adaptive immune response. DC can be stimulated with B7-DC XAb, a novel immunomodulatory IgM that crosslinks B7-DC/PDL2 on the DC surface. Previously, we have shown that B7-DC XAb treated DC (DCXAb) reprogram Foxp3+ Tregulatory cells into IFNgamma+, IL-17+ effectors. Adoptive transfer of neoself antigen (OVA)-pulsed DCXAb induces destruction of OVA-bearing pancreatic islets and autoimmune diabetes in RIP-OVA mice. We hypothesized that DC-derived molecules involved in Th1 or Th17 polarization would be important in autoreactive T cell activation by DCXAb. In spite of their ability to induce a CTL response against OVA-bearing targets, we found that DCXAb deficient in either Tbet, IFNgamma (Th1 polarizing) or CD40, IL-6 (Th17 polarizing) fail to cause autoimmunity. In conclusion, both Th1 and Th17 polarizing molecules on DC are needed to stimulate an autoimmune response.

Abstract Mouse CD8+ T cells can be activated to lyse targets expressing self class I molecules more potently than allogeneic targets. Self-reactive CTL express IL-17 and can be enriched following intracellular staining. Such CTL lyse targets in an MHC restricted fashion. This recognition is independent of the bound peptides, as demonstrated by cold-target inhibition studies. Activation of OT-1 transgenic T cells using traditional APC results in peptide specific CTL recognition, stimulation of the same T cells induces peptide independent, yet Kb-restricted recognition, irrespective of whether RAG sufficient or deficient mice were used. Self-reactive CTL efficiently lyse targets expressing wild type Kb or Kb-mutants with amino acid substitutions on the β-strands of the antigen binding groove, but not mutants with substitutions on the α1 and α2 helices, suggesting self MHC recognition is mediated by interactions of the CDR1 and CDR2 regions of the TCR with the α-helices of the class I molecules, and not by bound peptides. The ability of CTL to react in an MHC restricted, yet peptide independent, manner raises important questions about the nature of TCR-MHC interactions. We propose a novel "pre-positive selection" check point in thymic development and in peripheral T cell activation in recruitment of TCR-MHC complexes at the immunological synapse during T cell activation and homeostatic recognition.

We developed a novel model to study peripheral tolerance using mice expressing chicken ovalbumin under a rat insulin promoter. In contrast to previous RIP‐OVA mouse models, we induce autoimmune diabetes by activating the natural T cell repertoire; not by transfer of high affinity TCR T cells. Our treatment, DCXAb + OVA, employs adoptive transfer of dendritic cells (DC) pulsed with neoself antigen OVA and monoclonal therapeutic antibody (MTAb) B7‐DC XAb. This immunomodulatory MTAb crosslinks B7‐DC (PD‐L2) and activates DC. In our model, Tg mice develop severe hyperglycemia and loss of islet beta cells in 7 days. Adoptive transfer of IL‐6 KO DC, a cytokine involved in suppression of Treg, fails to cause diabetes. Also, nondiabetic splenocytes suppress proliferation of diabetogenic T cells, suggesting Treg are involved. DCXAb + OVA induces a specific break in tolerance that is beneficial when targeting tumors. In this light, splenocytes from diabetic mice specifically kill B16‐OVA targets but not B16. DCXAb treatment allows specific activation of the natural T cell repertoire and will help elucidate effectors important in autoimmunity and cancer therapy.

Abstract A human IgM Ab, serum-derived human IgM 12 (sHIgM12), is identified that binds mouse and human dendritic cells (DC), inducing dramatic immunopotentiation following treatment of the mouse DC in vitro. Competition, transfection, and knockout studies identified the ligand on mouse DC as the costimulatory molecule family member B7-DC. Potent T cell responses are stimulated by Ag-pulsed DC treated with the sHIgM12 Ab in vitro and upon adoptive transfer of Ab-treated Ag-pulsed DC into animals. The multivalent structure of pentameric IgM provides the potential for cross-linking cell surface targets, endowing the soluble Abs with biological potential not normally associated with immune function. The ability of the sHIgM12 Ab to potentiate the immune response is dependent on the multimeric structure of IgM, as bivalent monomers do not retain this property. Furthermore, pretreatment of DC with IgM monomers blocks subsequent potentiation by intact IgM pentamers, an indication that cross-linking of B7-DC on the cell surface is critical for potentiation of Ag presentation. These findings imply that, in addition to known costimulatory roles, B7-DC can function as a receptor for signals delivered by cells expressing B7-DC ligands.

Abstract We present a novel immunotherapeutic strategy using a human B7-DC cross-linking Ab that prevents lung inflammation, airway obstruction, and hyperreactivity to allergen in a mouse model of allergic inflammatory airway disease. Dendritic cells (DC) have the ability to skew the immune response toward a Th1 or Th2 polarity. The sHIgM12 Ab functions in vitro by cross-linking the costimulatory family molecule B7-DC (PD-L2) on DC up-regulating IL-12 production, homing to lymph nodes, and T cell-activating potential of these APCs. Using chicken OVA as a model Ag, the administration of sHIgM12 Ab to BALB/c mice blocked lung inflammation, airway pathology, and responsiveness to methacholine, even after animals were presensitized and a Th2-polarized immune response was established. This therapeutic strategy was ineffective in STAT4-deficient animals, indicating that IL-12 production is critical in this system. Moreover, the polarity of the immune response upon in vitro restimulation with Ag is changed in wild-type mice, with a resulting decrease in Th2 cytokines IL-4 and IL-5 and an increase in the immunoregulatory cytokine IL-10. These studies demonstrate that the immune response of hypersensitized responders can be modulated using B7-DC cross-linking Abs, preventing allergic airway disease upon re-exposure to allergen.

Abstract Lymphocyte differentiation from naive CD4+ T cells into mature Th1, Th2, Th17, or T regulatory cell (Treg) phenotypes has been considered end stage in character. In this study, we demonstrate that dendritic cells (DCs) activated with a novel immune modulator B7-DC XAb (DCXAb) can reprogram Tregs into T effector cells. Down-regulation of FoxP3 expression after either in vitro or in vivo Treg-DCXAb interaction is Ag-specific, IL-6-dependent, and results in the functional reprogramming of the mature T cell phenotype. The reprogrammed Tregs cease to express IL-10 and TGFβ, fail to suppress T cell responses, and gain the ability to produce IFN-γ, IL-17, and TNF-α. The ability of IL-6+ DCXAb and the inability of IL-6−/− DCXAb vaccines to protect animals from lethal melanoma suggest that exogenously modulated DC can reprogram host Tregs. In support of this hypothesis and as a test for Ag specificity, transfer of DCXAb into RIP-OVA mice causes a break in immune tolerance, inducing diabetes. Conversely, adoptive transfer of reprogrammed Tregs but not similarly treated CD25− T cells into naive RIP-OVA mice is also sufficient to cause autoimmune diabetes. Yet, treatment of normal mice with B7-DC XAb fails to elicit generalized autoimmunity. The finding that mature Tregs can be reprogrammed into competent effector cells provides new insights into the plasticity of T cell lineage, underscores the importance of DC-T cell interaction in balancing immunity with tolerance, points to Tregs as a reservoir of autoimmune effectors, and defines a new approach for breaking tolerance to self Ags as a strategy for cancer immunotherapy.

Abstract Dendritic cells (DC) are important regulators of T cell immunity. The degree of stimulation, the pattern of costimulatory molecules expressed, and the cytokines secreted by DC dictate the nature of the effector and memory cells generated, particularly with respect to their Th1 or Th2 phenotypes. In this study, we demonstrate that the addition of activated DC to spleen cultures containing established Th2-polarized CD4+ T cells was sufficient to suppress Th2 and induce Th1 cytokines in a recall response, a phenomenon referred to as phenotype reversal. The ability of activated DC to induce phenotype reversal displayed exquisite Ag specificity. The DC activator B7-DC cross-linking Ab (XAb) was >10,000-fold more efficient at inducing phenotype reversal than the TLR agonists CpG-oligodeoxynucleotide and Gardiquimod. Characterization of the mechanisms governing phenotype reversal revealed the requirement for cognate interaction between the TCR:peptide-MHC complex, the expression of the costimulation/adhesion molecule ICAM-1, and secretion of IL-12 and IFN-γ by the activated DC. The requirement for the costimulation/adhesion molecule SLAM (signaling lymphocytic activation molecule) was found to be quantitative. Thus, activation of DC, particularly by crosslinking B7-DC, can modulate well-established Th2 T cell responses in an Ag-specific manner. Because the regulation of mouse and human DC by B7-DC XAb overlaps in several significant ways, immune modulation with B7-DC XAb is a potential strategy for treating Th2-mediated diseases.