Targeting PVR (CD155) and its receptors in anti-tumor therapy.

Poliovirus receptor (PVR, CD155) has recently been gaining scientific interest as a therapeutic target in the field of tumor immunology due to its prominent endogenous and immune functions. In contrast to healthy tissues, PVR is expressed at high levels in several human malignancies and seems to have protumorigenic and therapeutically attractive properties that are currently being investigated in the field of recombinant oncolytic virotherapy. More intriguingly, PVR participates in a considerable number of immunoregulatory functions through its interactions with activating and inhibitory immune cell receptors. These functions are often modified in the tumor microenvironment, contributing to tumor immunosuppression. Indeed, increasing evidence supports the rationale for developing strategies targeting these interactions, either in terms of checkpoint therapy (i.e., targeting inhibitory receptors) or in adoptive cell therapy, which targets PVR as a tumor marker.

Talin1 is required for integrin-dependent B lymphocyte homing to lymph nodes and the bone marrow but not for follicular B-cell maturation in the spleen.

Talin1 is a key integrin coactivator. We investigated the roles of this cytoskeletal adaptor and its target integrins in B-cell lymphogenesis, differentiation, migration, and function. Using CD19 Cre-mediated depletion of talin1 selectively in B cells, we found that talin1 was not required for B-cell generation in the bone marrow or for the entry of immature B cells to the white pulp of the spleen. Loss of talin1 also did not affect B-cell maturation into follicular B cells but compromised differentiation of marginal zone B cells. Nevertheless, serum IgM and IgG levels remained normal. Ex vivo analysis of talin1-deficient spleen B cells indicated a necessary role for talin1 in LFA-1 and VLA-4 activation stimulated by canonical agonists, but not in B-cell chemotaxis. Consequently, talin1 null B splenocytes could not enter lymph nodes nor return to the bone marrow. Talin1 deficiency in B cells was also impaired in the humoral response to a T cell-dependent antigen. Collectively, these results indicate that talin1 is not required for follicular B-cell maturation in the spleen or homeostatic humoral immunity but is critical for integrin-dependent B lymphocyte emigration to lymph nodes and optimal immunity against T-dependent antigens.

Follicular shuttling of marginal zone B cells facilitates antigen transport.

The splenic marginal zone is a site of blood flow, and the specialized B cell population that inhabits this compartment has been linked to the capture and follicular delivery of blood-borne antigens. However, the mechanism of this antigen transport has remained unknown. Here we show that marginal zone B cells were not confined to the marginal zone but continuously shuttled between the marginal zone and follicular areas, such that many of the cells visited a follicle every few hours. Migration to the follicle required the chemokine receptor CXCR5, whereas return to the marginal zone was promoted by the sphingosine 1-phosphate receptors S1P1 and S1P3. Treatment with an S1P1 antagonist caused displacement of marginal zone B cells from the marginal zone. Marginal zone-follicle shuttling of marginal zone B cells provides an efficient mechanism for systemic antigen capture and delivery to follicular dendritic cells.

Chemoattractant signals and beta 2 integrin occupancy at apical endothelial contacts combine with shear stress signals to promote transendothelial neutrophil migration.

Lymphocyte transendothelial migration (TEM) is promoted by fluid shear signals and apical endothelial chemokines. Studying the role of these signals in neutrophil migration across differently activated HUVEC in a flow chamber apparatus, we gained new insights into how neutrophils integrate multiple endothelial signals to promote TEM. Neutrophils crossed highly activated HUVEC in a beta(2) integrin-dependent manner but independently of shear. In contrast, neutrophil migration across resting or moderately activated endothelium with low-level beta(2) integrin ligand activity was dramatically augmented by endothelial-presented chemoattractants, conditional to application of physiological shear stresses and intact beta(2) integrins. Shear stress signals were found to stimulate extensive neutrophil invaginations into the apical endothelial interface both before and during TEM. A subset of invaginating neutrophils completed transcellular diapedesis through individual endothelial cells within <1 min. Our results suggest that low-level occupancy of beta(2) integrins by adherent neutrophils can mediate TEM only if properly coupled to stimulatory shear stress and chemoattractant signals transduced at the apical neutrophil-endothelial interface.

Sphingosine 1-phosphate receptor 1 promotes B cell localization in the splenic marginal zone.

The factors directing marginal zone B cells to the splenic marginal zone are not well understood. Here we report that FTY720, a drug that targets sphingosine 1-phosphate (S1P) receptors, induced marginal zone B cell migration into follicles. Marginal zone B cells expressed S1P receptors 1 and 3 (S1P(1) and S1P(3), respectively). Using gene-targeted mice, we show that S1P(1) but not S1P(3) was required for localization in the marginal zone. In mice lacking the chemokine CXCL13, S1P(1)-deficient marginal zone B cells reacquired a marginal zone distribution. Exposure to lipopolysaccharide or antigen caused marginal zone B cells to downregulate S1P(1) and S1P(3) and to migrate into the splenic white pulp. These data suggest that marginal zone B cell localization to the marginal zone depends on responsiveness to the blood lysophospholipid S1P, with S1P(1) signaling overcoming the recruiting activity of CXCL13.

Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on S1P receptor 1.

Adaptive immunity depends on T-cell exit from the thymus and T and B cells travelling between secondary lymphoid organs to survey for antigens. After activation in lymphoid organs, T cells must again return to circulation to reach sites of infection; however, the mechanisms regulating lymphoid organ exit are unknown. An immunosuppressant drug, FTY720, inhibits lymphocyte emigration from lymphoid organs, and phosphorylated FTY720 binds and activates four of the five known sphingosine-1-phosphate (S1P) receptors. However, the role of S1P receptors in normal immune cell trafficking is unclear. Here we show that in mice whose haematopoietic cells lack a single S1P receptor (S1P1; also known as Edg1) there are no T cells in the periphery because mature T cells are unable to exit the thymus. Although B cells are present in peripheral lymphoid organs, they are severely deficient in blood and lymph. Adoptive cell transfer experiments establish an intrinsic requirement for S1P1 in T and B cells for lymphoid organ egress. Furthermore, S1P1-dependent chemotactic responsiveness is strongly upregulated in T-cell development before exit from the thymus, whereas S1P1 is downregulated during peripheral lymphocyte activation, and this is associated with retention in lymphoid organs. We find that FTY720 treatment downregulates S1P1, creating a temporary pharmacological S1P1-null state in lymphocytes, providing an explanation for the mechanism of FTY720-induced lymphocyte sequestration. These findings establish that S1P1 is essential for lymphocyte recirculation and that it regulates egress from both thymus and peripheral lymphoid organs.

A novel genetic leukocyte adhesion deficiency in subsecond triggering of integrin avidity by endothelial chemokines results in impaired leukocyte arrest on vascular endothelium under shear flow.

Leukocyte arrest on vascular endothelium under disruptive shear flow is a multistep process that requires in situ integrin activation on the leukocyte surface by endothelium-displayed chemoattractants, primarily chemokines. A genetic deficiency of leukocyte adhesion to endothelium associated with defective beta2 integrin expression or function (LAD-1) has been described. We now report a novel severe genetic disorder in this multistep process associated with functional defects in multiple leukocyte integrins, reflected in recurrent infections, profound leukocytosis, and a bleeding tendency. This syndrome is associated with an impaired ability of neutrophil and lymphocyte beta1 and beta2 integrins to generate high avidity to their endothelial ligands and arrest cells on vascular endothelium in response to endothelial chemoattractant signals. Patient leukocytes roll normally on endothelial selectins, express intact integrins and G protein-coupled chemokine receptors (GPCR), spread on integrin ligands, and migrate normally along a chemotactic gradient. Activation of beta2 integrins in response to GPCR signals and intrinsic soluble ligand binding properties of the very late activation antigen-4 (VLA-4) integrin are also retained in patient leukocytes. Nevertheless, all integrins fail to generate firm adhesion to immobilized ligands in response to in situ GPCR-mediated activation by chemokines or chemoattractants, a result of a primary defect in integrin rearrangement at ligand-bearing contacts. This syndrome is the first example of a human integrin-activation deficiency associated with defective GPCR stimulation of integrin avidity at subsecond contacts, a key step in leukocyte arrest on vascular endothelium under shear flow.

A real time in vitro assay for studying leukocyte transendothelial migration under physiological flow conditions.

The mechanisms underlying leukocyte migration across endothelial barriers are largely elusive. Most of the current knowledge on transendothelial migration (TEM) of leukocytes has been derived from in vitro modified Boyden chamber transfilter migration assays. In these assays, leukocyte migration towards chemokine gradients constructed across the endothelial barrier is measured under shear-free conditions. These assays do not incorporate the contribution of shear flow to leukocyte adherence and migration across the endothelial barrier. Furthermore, transfilter assays do not reconstitute the physiological distribution of endothelial chemokines shown to be displayed in vivo at high levels on vessel walls. To overcome these two drawbacks, we have recently developed a novel in vitro assay to follow real time leukocyte migration across endothelial barriers under physiological flow conditions. Using this assay, we have found that apically displayed endothelial chemokines could trigger robust lymphocyte TEM through signaling to lymphocyte-expressed G-protein coupled receptors. This migration required continuous exposure of lymphocytes, adherent to the endothelial barrier, to fluid shear, but did not require a chemotactic gradient across the barrier. In the present review, we describe this new flow-based migration assay and discuss future applications for investigating TEM processes of different types of leukocytes across distinct endothelial barriers.