Indirect Recruitment of a CD40 Signaling Pathway in Dendritic Cells by B7-DC Cross-Linking Antibody Modulates T Cell Functions

Suresh Radhakrishnan,Kristina M Bruns,Michael J Hansen,Larry R Pease,Sara J Felts,Rosalyn Cabrera,Virginia P Van Keulen

doi:10.1371/journal.pone.0005373

Abstract

The human IgM B7-DC XAb protects mice from tumors in both therapeutic and prophylactic settings. Its mechanism of action is mediated by its binding to B7-DC/PD-L2 molecules on the surface of dendritic cells (DCs) to induce a multimolecular cap and subsequent activation of signaling cascades that determine a unique combination of DC phenotypes. One such phenotype, the B7-DC XAb-induced antigen accumulation in mTLR-matured DCs, has been linked to signaling through TREM-2, but the signals required for other DC phenotypes critical for the therapeutic effects in animal models remain unclear. Here, FRET and co-immunoprecipitation studies show that CD40 is recruited to the multi-molecular complex by B7-DC XAb. Signals emanating from CD40 are important, as CD40−/− DCs treated with B7-DC XAb (DCXAb) activated DAP12, but failed to activate NFκB, and were not protected from cell death upon cytokine withdrawal or treatment with Vitamin D3. CD40−/− DCXAb also failed to secrete IL-6 and were unable to support the conversion of T regulatory cells into IL-17+ effector T cells in vitro. Importantly, the expression of CD40 was required for the overall ability of B7-DC XAb to induce anti-tumor CTL, to provide protection from a number of tumor types, and for DCXAb to be effective anti-tumor vaccines in vivo. These results indicate that B7-DC XAb modulation of DC phenotypes is through its ability to indirectly recruit common signaling molecules and elements of their endogenous signaling pathways through targeted binding to a cell-specific surface determinant.

Highlights

Generation of a vigorous T cell response is dependent on the activation of the T cells by professional antigen presenting cells and requires stable pMHC:TCR interaction, co-stimulation through CD28 and other membrane molecules, and secretion of T cell growth factors
Since CD40 is a known activator of NFkB, we used Fluorescence Resonance Energy Transfer (FRET) analysis to determine whether CD40 was among the cell surface molecules recruited into the B7-dendritic cells (DCs) XAb-induced cap on DC
CD40 was detected in MHC class II complexes isolated from the DCs stimulated with B7-DC XAb but not from DCs stimulated with control antibody (Fig. 1B)

Summary

Introduction

Generation of a vigorous T cell response is dependent on the activation of the T cells by professional antigen presenting cells and requires stable pMHC:TCR interaction (signal 1), co-stimulation through CD28 and other membrane molecules (signal 2), and secretion of T cell growth factors (signal 3). B7-DC XAb is an IgM antibody from the serum of a patient with Waldenstrom’s macroglobulinemia that binds to the surface of mouse and human DCs [1]. This binding leads to activation of the DCs and an augmentation of a number of phenotypic functions that are distinct from those elicited in DCs activated by TLR ligands or CD40L [2]. The pentameric structure of the IgM antibody is mandatory for the antibody to execute its effect on DCs. Monomers fail to bind and activate the DC and can prevent DC binding by the pentamer [1]. B7-DC XAb activates human DC [3] and has actions in several experimental models of cancer in which results from the use of TLR ligands or TNF family members have been less impressive [4,5]

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Results

Conclusion