Dual role of ALCAM in neuroinflammation and blood-brain barrier homeostasis.

Activated leukocyte cell adhesion molecule (ALCAM) is a cell adhesion molecule found on blood-brain barrier endothelial cells (BBB-ECs) that was previously shown to be involved in leukocyte transmigration across the endothelium. In the present study, we found that ALCAM knockout (KO) mice developed a more severe myelin oligodendrocyte glycoprotein (MOG)35-55-induced experimental autoimmune encephalomyelitis (EAE). The exacerbated disease was associated with a significant increase in the number of CNS-infiltrating proinflammatory leukocytes compared with WT controls. Passive EAE transfer experiments suggested that the pathophysiology observed in active EAE was linked to the absence of ALCAM on BBB-ECs. In addition, phenotypic characterization of unimmunized ALCAM KO mice revealed a reduced expression of BBB junctional proteins. Further in vivo, in vitro, and molecular analysis confirmed that ALCAM is associated with tight junction molecule assembly at the BBB, explaining the increased permeability of CNS blood vessels in ALCAM KO animals. Collectively, our data point to a biologically important function of ALCAM in maintaining BBB integrity.

P-selectin glycoprotein ligand-1 in T cells.

PURPOSE OF REVIEW: We review P-selectin glycoprotein ligand-1 (PSGL-1) as a selectin and chemokine-binding adhesion molecule. PSGL-1 is widely studied in neutrophils. Here, we focus on T cells, because PSGL-1 was recently described as a major immunomodulatory molecule during viral infection. PSGL-1 also plays a crucial role in T-cell homeostasis by binding to lymphoid chemokines, and can induce tolerance by enhancing the functions of regulatory T cells. RECENT FINDINGS: PSGL-1 was originally described as a leukocyte ligand for P-selectin, but it is actually a ligand for all selectins (P-, L- and E-selectin), binds chemokines, activates integrins and profoundly affects T-cell biology. It has been shown recently that PSGL-1 can modulate T cells during viral infection by acting as a negative regulator for T-cell functions. Absence of PSGL-1 promotes effector CD4 and CD8 T-cell differentiation and prevents T-cell exhaustion. Consistent with this, tumor growth was significantly reduced in PSGL-1-deficient mice because of an enhanced number of effector T cells together with reduced levels of inhibitory receptors that induce T-cell exhaustion. SUMMARY: PSGL-1 is the best-studied selectin ligand and has become a posterchild of versatility in leukocyte adhesion, inflammation and immunology. The direct involvement of PSGL-1 in T-cell biology suggests that it might be a drug target. Indeed, PSGL-1 has been tested in some clinical trials and recently, PSGL-1 blockers were proposed as a potential cotherapy in cancer immunotherapy.

Cell surface levels of endothelial ICAM-1 influence the transcellular or paracellular T-cell diapedesis across the blood-brain barrier.

The extravasation of CD4(+) effector/memory T cells (TEM cells) across the blood-brain barrier (BBB) is a crucial step in the pathogenesis of experimental autoimmune encephalomyelitis (EAE) or multiple sclerosis (MS). Endothelial ICAM-1 and ICAM-2 are essential for CD4(+) TEM cell crawling on the BBB prior to diapedesis. Here, we investigated the influence of cell surface levels of endothelial ICAM-1 in determining the cellular route of CD4(+) TEM -cell diapedesis across cytokine treated primary mouse BBB endothelial cells under physiological flow. Inflammatory conditions, inducing high levels of endothelial ICAM-1, promoted rapid initiation of transcellular diapedesis of CD4(+) T cells across the BBB, while intermediate levels of endothelial ICAM-1 favored paracellular CD4(+) T-cell diapedesis. Importantly, the route of T-cell diapedesis across the BBB was independent of loss of BBB barrier properties. Unexpectedly, a low number of CD4(+) TEM cells was found to cross the inflamed BBB in the absence of endothelial ICAM-1 and ICAM-2 via an obviously alternatively regulated transcellular pathway. In vivo, this translated to the development of ameliorated EAE in ICAM-1(null) //ICAM-2(-/-) C57BL/6J mice. Taken together, our study demonstrates that cell surface levels of endothelial ICAM-1 rather than the inflammatory stimulus or BBB integrity influence the pathway of T-cell diapedesis across the BBB.

Kindlin-3 regulates integrin activation and adhesion reinforcement of effector T cells.

Activated T cells use very late antigen-4/α4ß1 integrin for capture, rolling on, and firm adhesion to endothelial cells, and use leukocyte function-associated antigen-1/αLß2 integrin for subsequent crawling and extravasation. Inhibition of α4ß1 is sufficient to prevent extravasation of activated T cells and is successfully used to combat autoimmune diseases, such as multiple sclerosis. Here we show that effector T cells lacking the integrin activator Kindlin-3 extravasate and induce experimental autoimmune encephalomyelitis in mice immunized with autoantigen. In sharp contrast, adoptively transferred autoreactive T cells from Kindlin-3-deficient mice fail to extravasate into the naïve CNS. Mechanistically, autoreactive Kindlin-3-null T cells extravasate when the CNS is inflamed and the brain microvasculature expresses high levels of integrin ligands. Flow chamber assays under physiological shear conditions confirmed that Kindlin-3-null effector T cells adhere to high concentrations of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1, albeit less efficiently than WT T cells. Although these arrested T cells polarize and start crawling, only few remain firmly adherent over time. Our data demonstrate that the requirement of Kindlin-3 for effector T cells to induce α4ß1 and αLß2 integrin ligand binding and stabilization of integrin-ligand bonds is critical when integrin ligand levels are low, but of less importance when integrin ligand levels are high.

Differential requirement for ROCK in dendritic cell migration within lymphatic capillaries in steady-state and inflammation.

Dendritic cell (DC) migration via lymphatic vessels to draining lymph nodes (dLNs) is crucial for the initiation of adaptive immunity. We imaged this process by intravital microscopy (IVM) in the ear skin of transgenic mice bearing red-fluorescent vasculature and yellow-fluorescent DCs. DCs within lymphatic capillaries were rarely transported by flow, but actively migrated within lymphatics and were significantly faster than in the interstitium. Pharmacologic blockade of the Rho-associated protein kinase (ROCK), which mediates nuclear contraction and de-adhesion from integrin ligands, significantly reduced DC migration from skin to dLNs in steady-state. IVM revealed that ROCK blockade strongly reduced the velocity of interstitial DC migration, but only marginally affected intralymphatic DC migration. By contrast, during tissue inflammation, ROCK blockade profoundly decreased both interstitial and intralymphatic DC migration. Inhibition of intralymphatic migration was paralleled by a strong up-regulation of ICAM-1 in lymphatic endothelium, suggesting that during inflammation ROCK mediates de-adhesion of DC-expressed integrins from lymphatic-expressed ICAM-1. Flow chamber assays confirmed an involvement of lymphatic-expressed ICAM-1 and DC-expressed ROCK in DC crawling on lymphatic endothelium. Overall, our findings further define the role of ROCK in DC migration to dLNs and reveal a differential requirement for ROCK in intralymphatic DC crawling during steady-state and inflammation.

Prognostic implication of methylation-based circulating tumor DNA detection prior to surgery in stage I non-small cell lung cancer.

BACKGROUND: Circulating tumor DNA (ctDNA) positivity at diagnosis, which is associated with worse outcomes in multiple solid tumors including stage I-III non-small cell lung cancer (NSCLC), may have utility to guide (neo)adjuvant therapy. METHODS: In this retrospective study, 260 patients with clinical stage I NSCLC (180 adenocarcinoma, 80 squamous cell carcinoma) were allocated (2:1) to high- and low-risk groups based on relapse versus disease-free status ≤5 years post-surgery. We evaluated the association of preoperative ctDNA detection by a plasma-only targeted methylation-based multi-cancer early detection (MCED) test with NSCLC relapse ≤5 years post-surgery in the overall population, followed by histology-specific subgroup analyses. RESULTS: Across clinical stage I patients, preoperative ctDNA detection did not associate with relapse within 5 years post-surgery. Sub-analyses confined to lung adenocarcinoma suggested a histology-specific association between ctDNA detection and outcome. In this group, ctDNA positivity tended to associate with relapse within 2 years, suggesting prognostic implications of MCED test positivity may be histology- and time-dependent in stage I NSCLC. Preoperative ctDNA detection was associated with upstaging of clinical stage I to pathological stage II-III NSCLC. CONCLUSIONS: Our findings suggest preoperative ctDNA detection in patients with resectable clinical stage I NSCLC using MCED, a pan-cancer screening test developed for use in an asymptomatic population, has no detectable prognostic value for relapse ≤5 years post-surgery. MCED detection may be associated with early adenocarcinoma relapse and increased pathological upstaging rates in stage I NSCLC. However, given the exploratory nature of these findings, independent validation is required.

Arginine 313 of the putative 8th helix mediates Gαq/14 coupling of human CC chemokine receptors CCR2a and CCR2b.

In man, two CC chemokine receptor isoforms, CCR2a and CCR2b, are present that belong to the rhodopsin-like G protein-coupled receptor family, and couple to Gi and Gq family members. The CCR2 receptors are known to regulate canonical functions of chemokines such as directed migration of leukocytes, and to potentially control non-canonical functions such as differentiation, proliferation, and gene transcription of immune and non-immune cells. We recently reported on the activation of phospholipase C isoenzymes and RhoA GTPases by coupling of the two CCR2 receptors to members of the Gq family, in particular Gαq and Gα14. So far little is known about the structural requirements for the CCR2/Gq/14 interaction. Interestingly, the CCR2 receptor isoforms are identical up to arginine 313 (R313) that is part of the putative 8th helix in CCR2 receptors, and the 8th helix has been implicated in the interaction of rhodopsin-like G protein-coupled receptors with Gαq. In the present work we describe that the 8th helix of both CCR2a and CCR2b is critically involved in selectively activating Gαq/14-regulated signaling. Refined analysis using various CCR2a and CCR2b mutants and analyzing their cellular signaling, e.g. ligand-dependent (i) activation of phospholipase C isoenzymes, (ii) stimulation of serum response factor-mediated gene transcription, (iii) activation of mitogen-activated protein kinases, (iv) internalization, and (v) changes in intracellular calcium concentrations, identified arginine 313 within the amino terminal portion of helix 8 to play a role for the agonist-mediated conformational changes and the formation of a Gαq/14 binding surface. We show that R313 determines Gαq/14 protein-dependent but not Gi protein-dependent cellular signaling, and plays no role in Gq/Gi-independent receptor internalization, indicating a role of R313 in biased signaling of CCR2 receptors.

A Single-Step Chemoenzymatic Reaction for the Construction of Antibody-Cell Conjugates.

Employing live cells as therapeutics is a direction of future drug discovery. An easy and robust method to modify the surfaces of cells directly to incorporate novel functionalities is highly desirable. However, genetic methods for cell-surface engineering are laborious and limited by low efficiency for primary cell modification. Here we report a chemoenzymatic approach that exploits a fucosyltransferase to transfer bio-macromolecules, such as an IgG antibody (MW∼ 150 KD), to the glycocalyx on the surfaces of live cells when the antibody is conjugated to the enzyme's natural donor substrate GDP-Fucose. Requiring no genetic modification, this method is fast and biocompatible with little interference to cells' endogenous functions. We applied this method to construct two antibody-cell conjugates (ACCs) using both cell lines and primary cells, and the modified cells exhibited specific tumor targeting and resistance to inhibitory signals produced by tumor cells, respectively. Remarkably, Herceptin-NK-92MI conjugates, a natural killer cell line modified with Herceptin, exhibit enhanced activities to induce the lysis of HER2+ cancer cells both ex vivo and in a human tumor xenograft model. Given the unprecedented substrate tolerance of the fucosyltransferase, this chemoenzymatic method offers a general approach to engineer cells as research tools and for therapeutic applications.

Effector and Regulatory T Cells Roll at High Shear Stress by Inducible Tether and Sling Formation.

The adaptive immune response involves T cell differentiation and migration to sites of inflammation. T cell trafficking is initiated by rolling on inflamed endothelium. Tethers and slings, discovered in neutrophils, facilitate cell rolling at high shear stress. Here, we demonstrate that the ability to form tethers and slings during rolling is highly inducible in T helper 1 (Th1), Th17, and regulatory T (Treg) cells but less in Th2 cells. In vivo, endogenous Treg cells rolled stably in cremaster venules at physiological shear stress. Quantitative dynamic footprinting nanoscopy of Th1, Th17, and Treg cells uncovered the formation of multiple tethers per cell. Human Th1 cells also showed tethers and slings. RNA sequencing (RNA-seq) revealed the induction of cell migration and cytoskeletal genes in sling-forming cells. We conclude that differentiated CD4 T cells stabilize rolling by inducible tether and sling formation. These phenotypic changes approximate the adhesion phenotype of neutrophils and support CD4 T cell access to sites of inflammation.

ALCAM (CD166) is involved in extravasation of monocytes rather than T cells across the blood-brain barrier.

Activated leukocyte cell adhesion molecule (ALCAM) has been proposed to mediate leukocyte migration across the blood-brain barrier (BBB) in multiple sclerosis or experimental autoimmune encephalomyelitis (EAE). Here, we confirmed vascular ALCAM expression in human brain tissue samples in situ and on two different human in vitro BBB models. Antibody-mediated inhibition of ALCAM reduced diapedesis of human CD4+ Th1 but not of Th17 cells across the human BBB in vitro. In accordance to human Th1 cells, mouse Th1 cells showed reduced diapedesis across an ALCAM-/- in vitro BBB model under static but no longer under flow conditions. In contrast to the limited role of ALCAM in T cell extravasation across the BBB, we found a contribution of ALCAM to rolling, adhesion, and diapedesis of human CD14+ monocytes across the human BBB under flow and static conditions. Taken together, our study highlights the potential differences in the CNS expression of ALCAM in mouse and human and supports a prominent role for ALCAM in the multi-step extravasation of monocytes across the BBB.