Molecular Characterization of ILT3-Triggered Pathways in Suppression of T Cell Immunity

Abstract

Introduction: ILT3 is an inhibitory receptor expressed on antigen presenting cells which inhibits T cell activation and effector function, while mediating the differentiation of CD8 T suppressor cells (Ts) and induction of anergy. We have demonstrated that recombinant ILT3Fc induces tolerance to pancreatic islet allografts in a humanized mouse model, however the molecular mechanisms of CD8 Ts generation and function remain poorly understood. Methods: We have analyzed the transcriptional profile of CD8 T cells primed in the presence or absence of ILT3Fc and identified over 2500 genes in 23 different gene ontology categories significantly up- or down-regulated by ILT3Fc treatment. To further analyze the relevance of individual molecular changes, we used siRNA technology to determine their impact on the acquisition of Ts function. Results: An interesting aspect of this study was the sheer magnitude of the changes induced by a single molecule, ILT3Fc. We demonstrated that this is accomplished through the transcriptional suppression of several microRNA species which in turn regulate hundreds of target genes. ILT3Fc-induced Ts displayed a significant up regulation of zinc finger proteins, most of which act as transcriptional repressors. Among these BCL6 was shown to play a critical role in the differentiation of ILT3Fc-induced Ts. A striking feature of ILT3-treated T cells is their inability to secrete pro-inflammatory cytokines in response to stimuli. The up regulation of BCL6, a known transcriptional repressor of IFNg, IL2 and IL5, is partly responsible for this phenotype, but alone it cannot explain the wide ranging cytokine and chemokine suppression observed in ILT3Fc treated cells. Other factors, such as SOCS1 and the suppression of the TCR signaling pathways, are involved. The down regulation of several cell cycle proteins and the up regulation of cyclin inhibitors underlie the ability of ILT3Fc to induce cell cycle arrest and T cell anergy. Several members of the WNT and transforming growth factor-beta pathways were consistently up regulated, although their exact involvement in the differentiation is CD8 Ts has not been yet elucidated. Conclusion: ILT3Fc treatment during T cell priming results in a vast number of transcriptional changes influencing the ultimate effector fate of these T cells. This data furthers our understanding of T suppressor cell induction and reveals several pathways which may be amenable to iatrogenic manipulation to induce immunological tolerance in transplantation.