Maria-Concetta Veri

Activating and inhibitory IgG Fc receptors on human DCs mediate opposing functions

Human monocyte-derived DCs (moDCs) and circulating conventional DCs coexpress activating (CD32a) and inhibitory (CD32b) isoforms of IgG Fcgamma receptor (FcgammaR) II (CD32). The balance between these divergent receptors establishes a threshold of DC activation and enables immune complexes to mediate opposing effects on DC maturation and function. IFN-gamma most potently favors CD32a expression on immature DCs, whereas soluble antiinflammatory concentrations of monomeric IgG have the opposite effect. Ligation of CD32a leads to DC maturation, increased stimulation of allogeneic T cells, and enhanced secretion of inflammatory cytokines, with the exception of IL-12p70. Coligation of CD32b limits activation through CD32a and hence reduces the immunogenicity of moDCs even for a strong stimulus like alloantigen. Targeting CD32b alone does not mature or activate DCs but rather maintains an immature state. Coexpression of activating and inhibitory FcgammaRs by DCs reveals a homeostatic checkpoint for inducing tolerance or immunity by immune complexes. These findings have important implications for understanding the pathophysiology of immune complex diseases and for optimizing the efficacy of therapeutic mAbs. The data also suggest novel strategies for targeting antigens to the activating or inhibitory FcgammaRs on human DCs to generate either antigen-specific immunity or tolerance.

Monoclonal antibodies capable of discriminating the human inhibitory Fcγ-receptor IIB (CD32B) from the activating Fcγ-receptor IIA (CD32A): biochemical, biological and functional characterization

Human CD32B (FcγRIIB), the low‐affinity inhibitory Fcγ receptor (FcγR), is highly homologous in its extracellular domain to CD32A (FcγRIIA), an activating FcγR. Available monoclonal antibodies (mAb) against the extracellular region of CD32B recognize both receptors. Through immunization of mice transgenic for human CD32A, we generated a set of antibodies specific for the extracellular region of CD32B with no cross‐reactivity with CD32A, as determined by enzyme‐linked immunosorbent assay and surface plasmon resonance with recombinant CD32A and CD32B, and by fluorescence‐activated cell sorting analysis of CD32 transfectants. A high‐affinity mAb, 2B6, was used to explore the expression of CD32B by human peripheral blood leucocytes. While all B lymphocytes expressed CD32B, only a fraction of monocytes and almost no polymorphonuclear cells stained with 2B6. Likewise, natural killer cells, which express CD32C, a third CD32 variant, did not react with 2B6. Immune complexes co‐engage the inhibitory receptor with activating Fcγ receptors, a mechanism that limits cell responses. 2B6 competed for immune complex binding to CD32B as a monomeric Fab, suggesting that it directly recognizes the Fc‐binding region of the receptor. Furthermore, when co‐ligated with an activating receptor, 2B6 triggered CD32B‐mediated inhibitory signalling, resulting in diminished release of inflammatory mediators by FcεRI in an in vitro allergy model or decreased proliferation of human B cells induced by B‐cell receptor stimulation. These antibodies form the basis for the development of investigational tools and therapeutics with multiple potential applications, ranging from adjuvants in FcγR‐mediated responses to the treatment of allergy and autoimmunity.

Therapeutic control of B cell activation via recruitment of Fcγ receptor IIb (CD32B) inhibitory function with a novel bispecific antibody scaffold

OBJECTIVE To exploit the physiologic Fcgamma receptor IIb (CD32B) inhibitory coupling mechanism to control B cell activation by constructing a novel bispecific diabody scaffold, termed a dual-affinity retargeting (DART) molecule, for therapeutic applications. METHODS DART molecules were constructed by pairing an Fv region from a monoclonal antibody (mAb) directed against CD32B with an Fv region from a mAb directed against CD79B, the beta-chain of the invariant signal-transducing dimer of the B cell receptor complex. DART molecules were characterized physicochemically and for their ability to simultaneously bind the target receptors in vitro and in intact cells. The ability of the DART molecules to negatively control B cell activation was determined by calcium mobilization, by tyrosine phosphorylation of signaling molecules, and by proliferation and Ig secretion assays. A DART molecule specific for the mouse ortholog of CD32B and CD79B was also constructed and tested for its ability to inhibit B cell proliferation in vitro and to control disease severity in a collagen-induced arthritis (CIA) model. RESULTS DART molecules were able to specifically bind and coligate their target molecules on the surface of B cells and demonstrated a preferential simultaneous binding to both receptors on the same cell. DART molecules triggered the CD32B-mediated inhibitory signaling pathway in activated B cells, which translated into inhibition of B cell proliferation and Ig secretion. A DART molecule directed against the mouse orthologs was effective in inhibiting the development of CIA in DBA/1 mice. CONCLUSION This innovative bispecific antibody scaffold that simultaneously engages activating and inhibitory receptors enables novel therapeutic approaches for the treatment of rheumatoid arthritis and potentially other autoimmune and inflammatory diseases in humans.

Membrane Raft-Dependent Regulation of Phospholipase Cγ-1 Activation in T Lymphocytes

ABSTRACT Numerous signaling molecules associate with lipid rafts, either constitutively or after engagement of surface receptors. One such molecule, phospholipase Cγ-1 (PLCγ1), translocates from the cytosol to lipid rafts during T-cell receptor (TCR) signaling. To investigate the role played by lipid rafts in the activation of this molecule in T cells, an influenza virus hemagglutinin A (HA)-tagged PLCγ1 was ectopically expressed in Jurkat T cells and targeted to these microdomains by the addition of a dual-acylation signal. Raft-targeted PLCγ1 was constitutively tyrosine phosphorylated and induced constitutive NF-AT-dependent transcription and interleukin-2 secretion in Jurkat cells. Tyrosine phosphorylation of raft-targeted PLCγ1 did not require Zap-70 or the interaction with the adapters Lat and Slp-76, molecules that are necessary for TCR signaling. In contrast, the Src family kinase Lck was required. Coexpression in HEK 293T cells of PLCγ1-HA with Lck or the Tec family kinase Rlk resulted in preferential phosphorylation of raft-targeted PLCγ1 over wild-type PLCγ1. These data show that localization of PLCγ1 in lipid rafts is sufficient for its activation and demonstrate a role for lipid rafts as microdomains that dynamically segregate and integrate PLCγ1 with other signaling components.

Functional Independence and Interdependence of the Src Homology Domains of Phospholipase C-γ1 in B-Cell Receptor Signal Transduction

ABSTRACT B-cell receptor (BCR)-induced activation of phospholipase C-γ1 (PLCγ1) and PLCγ2 is crucial for B-cell function. While several signaling molecules have been implicated in PLCγ activation, the mechanism coupling PLCγ to the BCR remains undefined. The role of PLCγ1 SH2 and SH3 domains at different steps of BCR-induced PLCγ1 activation was examined by reconstitution in a PLCγ-negative B-cell line. PLCγ1 membrane translocation required a functional SH2 N-terminal [SH2(N)] domain, was decreased by mutation of the SH3 domain, but was unaffected by mutation of the SH2(C) domain. Tyrosine phosphorylation did not require the SH2(C) or SH3 domains but depended exclusively on a functional SH2(N) domain, which mediated the association of PLCγ1 with the adapter protein, BLNK. Forcing PLCγ1 to the membrane via a myristoylation signal did not bypass the SH2(N) domain requirement for phosphorylation, indicating that the phosphorylation mediated by this domain is not due to membrane anchoring alone. Mutation of the SH2(N) or the SH2(C) domain abrogated BCR-stimulated phosphoinositide hydrolysis and signaling events, while mutation of the SH3 domain partially decreased signaling. PLCγ1 SH domains, therefore, have interrelated but distinct roles in BCR-induced PLCγ1 activation.

Differential effects of Cbl and 70Z/3 Cbl on T cell receptor-induced phospholipase Cγ-1 activity

We demonstrate that the differential effects Cbl and oncogenic 70Z/3 Cbl have on Ca2+/Ras‐sensitive NF‐AT reporters is partially due to their opposing ability to regulate phospholipase Cγ1 (PLCγ1) activation as demonstrated by analysis of the activation of an NF‐AT reporter construct and PLCγ1‐mediated inositol phospholipid (PI) hydrolysis. Cbl over‐expression resulted in reduced T cell receptor‐induced PI hydrolysis, in the absence of any effect on PLCγ1 tyrosine phosphorylation. In contrast, expression of 70Z/3 Cbl led to an increase in basal and OKT3‐induced PLCγ1 phosphorylation and PI hydrolysis. These data indicate that Cbl and 70Z/3 Cbl differentially regulate PLCγ1 phosphorylation and activation. The implications of these data on the mechanism of Cbl‐mediated signaling regulation are discussed.

A dynamic constitutive and inducible binding of c-Cbl by PLCγ1 SH3 and SH2 domains (negatively) regulates antigen receptor-induced PLCγ1 activation in lymphocytes

We investigated the structural requirements for c-Cbl-mediated inhibition of Ag receptor-induced PLCgamma1 activation. Analysis of site-specific c-Cbl mutants indicated that tyrosine phosphorylation of c-Cbl was required for down-regulation of the PLCgamma1/Ca2+ pathway. Coprecipitation experiments indicated that c-Cbl and PLCgamma1 constitutively interact through a PLCgamma1 SH3 domain-dependent mechanism and that c-Cbl and PLCgamma1 can inducibly interact through the SH2(C) domain of PLCgamma1. Additional data indicate that the SH3 domain of PLCgamma1 binds to both canonical and noncanonical SH3 domain-binding sites in the proline-rich region of c-Cbl. Overexpression of c-Cbl in a PLCgamma-deficient B cell line, P10-14, stably reconstituted with wild-type PLCgamma1 led to a significant decrease in B cell receptor-induced NF-AT-dependent transcription, a PLCgamma- and Ca(2+)-dependent event. In contrast, c-Cbl overexpression in P10-14 cells reconstituted with a PLCgamma1 SH3 domain mutant had little effect on receptor-induced NF-AT activation. These data suggest that c-Cbl-mediated regulation of PLCgamma1 requires an interaction between c-Cbl and PLCgamma1 that is primarily mediated by the SH3 domain of PLCgamma1. The interaction of c-Cbl with PLCgamma1 may negatively effect events required for PLCgamma1 activation.

70Z/3 Cbl induces PLC gamma 1 activation in T lymphocytes via an alternate Lat- and Slp-76-independent signaling mechanism

The oncoprotein 70Z/3 Cbl signals in an autonomous fashion or through blockade of endogenous c-Cbl, a negative regulator of signaling. The mechanism of 70Z/3 Cbl-induced signaling was investigated by comparing the molecular requirements for 70Z/3 Cbl- and TCR-induced phospholipase Cγ1 (PLCγ1) activation. 70Z/3 Cbl-induced PLCγ1 tyrosine phosphorylation required, in addition to the PLCγ1 N-terminal SH2 domain, the C-terminal SH2 and SH3 domains that were dispensable for TCR-induced phosphorylation. Deletion of the leucine zipper of 70Z/3 Cbl did not eliminate 70Z/3 Cbl-induced PLCγ1 phosphorylation, suggesting that blockage of c-Cbl via dimerization with 70Z/3 Cbl cannot fully explain 70Z/3 Cbl activating characteristics. The complete elimination of PLCγ1 phosphorylation required deleting the SH3 domain-binding region of 70Z/3 Cbl, consistent with 70Z/3 Cbl binding the PLCγ1 SH3 domain. 70Z/3 Cbl-induced PLCγ1 phosphorylation required Zap-70, as for the TCR, and the tyrosine kinase binding domain of 70Z/3 Cbl, which binds Zap-70, but did not require PLCγ1 binding to Lat, a crucial interaction in TCR-induced PLCγ1 phosphorylation. Furthermore, 70Z/3 Cbl-induced activation of NFAT, a PLCγ1/Ca2+-dependent transcriptional event, required Zap-70, but was independent of Slp-76, an adapter required for TCR-induced NFAT activation. These results suggest that 70Z/3 Cbl and PLCγ1 form a TCR-, Lat- and Slp-76-independent complex that leads to PLCγ1 phosphorylation and activation.

Abstract 5629: Application of a novel bispecific antibody-based scaffold for optimal redirected T-cell killing of cancer cells

Despite significant progress, improved treatment modalities are required to eradicate cancer. Cytotoxic T lymphocytes are among the most potent cell populations capable of killing target cells and considerable effort has been dedicated to developing strategies aimed at redirecting T cells toward cancer, including the generation of bispecific molecules capable of co-engaging a tumor target together with an activating receptor on T lymphocytes. Limitations of the existing technology, however, including shortcomings in manufacturability, stability, specificity and potency, have often hampered progress in harnessing the power of T cells with bi-specific molecules. We have applied a novel antibody-based scaffold, termed DART (Dual-Affinity Re-Targeting), to redirect T-cell killing toward tumor cells in the absence of cognate interactions. DART molecules are structurally comprised of a covalently linked “diabody” generated through co-expression in mammalian cells of two separate Fv-encoding chains. To evaluate the ability of the platform to redirect T-cell killing, DARTs were designed to target hematological malignancies by comprising a B-cell lineage specificity (CD19) together with T-cell recognition (CD3 or TCR), while a DART comprising specificities for the HER2 target and the TCR was used to evaluate applicability to solid tumors. All three DART molecules assembled efficiently and were evaluated in a series of redirected T-cell killing assays to evaluate potency and mechanistic aspects of cytotoxicity. Both the CD19xCD3 and CD19xTCR DART molecules demonstrated efficient co-ligation of B-cell lymphoma cells with human T cells and mediated equivalently potent redirected T-cell killing against a panel of CD19-positive B-cell lymphoma cell lines but not CD19-ve cell lines. In similar fashion, the TCRxHER2 DART revealed potent T-cell mediated killing of a panel of HER2 expressing cell lines, including those expressing low HER2 levels (1+ by HercepTest), but was inactive against HER2-negative lines. Analyses of the mechanism of T-cell mediated killing by the DART molecules revealed a dependence on the granzyme B/perforin pathways, with potent killing observed during short-term incubation of the effector populations with target cells and at low E:T ratios. Furthermore, activation of human T cells by DART molecules proceeded strictly in a target-dependent manner. Finally, analyses in xenograft models reconstituted with human peripheral blood mononuclear cells, demonstrated efficient tumor regression by TCR-based DART molecules. In conclusion, T-cell-directed DARTs represent a robust and flexible platform to recruit T-cell populations for cancer cell killing and may offer potential application toward tumor eradication in cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5629.