Loss of ß2-integrin function results in metabolic reprogramming of dendritic cells, leading to increased dendritic cell functionality and anti-tumor responses.

Dendritic cells (DCs) are the main antigen presenting cells of the immune system and are essential for anti-tumor responses. DC-based immunotherapies are used in cancer treatment, but their functionality is not optimized and their clinical efficacy is currently limited. Approaches to improve DC functionality in anti-tumor immunity are therefore required. We have previously shown that the loss of ß2-integrin-mediated adhesion leads to epigenetic reprogramming of bone marrow-derived DCs (BM-DCs), resulting in an increased expression of costimulatory markers (CD86, CD80, and CD40), cytokines (IL-12) and the chemokine receptor CCR7. We now show that the loss of ß2-integrin-mediated adhesion of BM-DCs also leads to a generally suppressed metabolic profile, with reduced metabolic rate, decreased ROS production, and lowered glucose uptake in cells. The mRNA levels of glycolytic enzymes and glucose transporters were reduced, indicating transcriptional regulation of the metabolic phenotype. Surprisingly, although signaling through a central regulator of immune cell metabolisms, the mechanistic target of rapamycin (mTOR), was increased in BM-DCs with dysfunctional integrins, rapamycin treatment revealed that mTOR signaling was not involved in suppressing DC metabolism. Instead, bioinformatics and functional analyses showed that the Ikaros transcription factor may be involved in regulating the metabolic profile of non-adhesive DCs. Inversely, we found that induction of metabolic stress through treatment of cells with low levels of an inhibitor of glycolysis, 2-deoxyglucose (2DG), led to increased BM-DC activation. Specifically, 2DG treatment led to increased levels of Il-12 and Ccr7 mRNA, increased production of IL-12, increased levels of cell surface CCR7 and increased in vitro migration and T cell activation potential. Furthermore, 2DG treatment led to increased histone methylation in cells (H3K4me3, H3K27me3), indicating metabolic reprogramming. Finally, metabolic stress induced by 2DG treatment led to improved BM-DC-mediated anti-tumor responses in vivo in a melanoma cancer model, B16-OVA. In conclusion, our results indicate a role for ß2-integrin-mediated adhesion in regulating a novel type of metabolic reprogramming of DCs and DC-mediated anti-tumor responses, which may be targeted to enhance DC-mediated anti-tumor responses in cancer immunotherapy.

Cell Adhesion Molecules and Their Roles and Regulation in the Immune and Tumor Microenvironment.

The immune system and cancer have a complex relationship with the immune system playing a dual role in tumor development. The effector cells of the immune system can recognize and kill malignant cells while immune system-mediated inflammation can also promote tumor growth and regulatory cells suppress the anti-tumor responses. In the center of all anti-tumor responses is the ability of the immune cells to migrate to the tumor site and to interact with each other and with the malignant cells. Cell adhesion molecules including receptors of the immunoglobulin superfamily and integrins are of crucial importance in mediating these processes. Particularly integrins play a vital role in regulating all aspects of immune cell function including immune cell trafficking into tissues, effector cell activation and proliferation and the formation of the immunological synapse between immune cells or between immune cell and the target cell both during homeostasis and during inflammation and cancer. In this review we discuss the molecular mechanisms regulating integrin function and the role of integrins and other cell adhesion molecules in immune responses and in the tumor microenvironment. We also describe how malignant cells can utilize cell adhesion molecules to promote tumor growth and metastases and how these molecules could be targeted in cancer immunotherapy.

Beta2-Integrins and Interacting Proteins in Leukocyte Trafficking, Immune Suppression, and Immunodeficiency Disease.

Beta2-integrins are complex leukocyte-specific adhesion molecules that are essential for leukocyte (e.g., neutrophil, lymphocyte) trafficking, as well as for other immunological processes such as neutrophil phagocytosis and ROS production, and T cell activation. Intriguingly, however, they have also been found to negatively regulate cytokine responses, maturation, and migratory responses in myeloid cells such as macrophages and dendritic cells, revealing new, and unexpected roles of these molecules in immunity. Because of their essential role in leukocyte function, a lack of expression or function of beta2-integrins causes rare immunodeficiency syndromes, Leukocyte adhesion deficiency type I, and type III (LAD-I and LAD-III). LAD-I is caused by reduced or lost expression of beta2-integrins, whilst in LAD-III, beta2-integrins are expressed but dysfunctional because a major integrin cytoplasmic regulator, kindlin-3, is mutated. Interestingly, some LAD-related phenotypes such as periodontitis have recently been shown to be due to an uncontrolled inflammatory response rather than to an uncontrolled infection, as was previously thought. This review will focus on the recent advances concerning the regulation and functions of beta2-integrins in leukocyte trafficking, immune suppression, and immune deficiency disease.

Filamin A Is Required for Optimal T Cell Integrin-Mediated Force Transmission, Flow Adhesion, and T Cell Trafficking.

T cells traffic from the bloodstream into tissues to perform their functions in the immune system and are therefore subjected to a range of different mechanical forces. Integrins are essential for T cell trafficking into the tissues, as they mediate firm adhesion between the T cell and the endothelium under shear flow conditions. In addition, integrins are important for the formation of the contact between the T cell and the APC required for T cell activation. The actin-binding protein filamin A (FlnA) provides an important link between the integrin and the actin cytoskeleton. FlnA has been reported to function as an integrin inhibitor by competing with talin. However, its role in regulating integrin-dependent immune functions in vivo is currently poorly understood. In this study, we have investigated the role of FlnA in T cells, using T cell-specific FlnA knockout mice. We report that FlnA is required for the formation of strong integrin-ligand bonds under shear flow and for the generation of integrin-mediated T cell traction forces on ligand-coated hydrogels. Consequently, absence of FlnA leads to a reduction in T cell adhesion to integrin ligands under conditions of shear flow, as well as reduced T cell trafficking into lymph nodes and sites of skin inflammation. In addition, FlnA is not needed for T cell activation in vivo, which occurs in shear-free conditions in lymphoid organs. Our results therefore reveal a role of FlnA in integrin force transmission and T cell trafficking in vivo.

Optimal T Cell Activation and B Cell Antibody Responses In Vivo Require the Interaction between Leukocyte Function-Associated Antigen-1 and Kindlin-3.

Kindlin-3 is an important integrin regulator that is mutated in the rare genetic disorder, leukocyte adhesion deficiency type III, a disorder characterized by defective neutrophil trafficking and platelet function, leading to recurrent bacterial infections and bleeding. Kindlin-3 is also known to regulate T cell adhesion in vitro and trafficking in vivo, but whether the integrin/kindlin interaction regulates T or B cell activation in vivo is unclear. In this study, we used TTT/AAA ß2-integrin knock-in (KI) mice and TCR-transgenic (OT-II) KI mice, in which the integrin/kindlin connection is disrupted, to investigate the role of the integrin/kindlin interaction in T cell activation. We show that basal T cell activation status in these animals in vivo is normal, but they display reduced T cell activation by wild-type Ag-loaded dendritic cells in vitro. In addition, T cell activation in vivo is reduced. We also show that basal Ab levels are normal in TTT/AAA ß2-integrin KI mice, but B cell numbers in lymph nodes and IgG and IgM production after immunization are reduced. In conclusion, we show that the integrin/kindlin interaction is required for trafficking of immune cells, as well as for T cell activation and B cell Ab responses in vivo. These results imply that the immunodeficiency found in leukocyte adhesion deficiency type III patients, in addition to being caused by defects in neutrophil function, may be due, in part, to defects in lymphocyte trafficking and activation.