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.
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.
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•Tethers and slings are inducible in mouse and human differentiated CD4 T cell subsets•Th1, Th17, and Treg cells but few Th2 cells bind selectins and induce tethers and slings•The sling-forming T cells show overexpression of genes controlling cell migration•Tethers and slings allow Th1, Th17, and Treg cells to infiltrate non-lymphoid tissues
Abadier et al. report that profound transcriptomic changes during CD4 T cell differentiation enable effector and regulatory T cells to form tethers and slings, enabling rolling at high shear stress. This inducible phenotype facilitates Th1, Th17, and Treg cell rolling and homing to inflamed peripheral tissues.
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.
Activated T cells use very late antigen-4/α4β1 integrin for capture, rolling on, and firm adhesion to endothelial cells, and use leukocyte function-associated antigen-1/αLβ2 integrin for subsequent ...crawling and extravasation. Inhibition of α4β1 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 α4β1 and αLβ2 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.
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.
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.
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.
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.
BackgroundMEDI1191 is an investigational therapy composed of mRNA encoding interleukin-12 (IL-12p70) in a lipid nanoparticle optimized for intratumoral injection. Previously1 2 we reported that all ...dose levels induce pharmacodynamic (PD) changes such as elevated serum IL-12, increased T cell infiltration and upregulation of immune gene signatures associated with anti-tumor responses. Since the actual dose and drug distribution may be affected by injected lesion size, we hypothesize that a normalized dose (ND) to lesion size will accurately reflect the dose effect and PD changes within tumor microenvironment (TME).MethodsIn a phase 1 dose escalation study (NCT03946800), patients received 0.1–12 µg MEDI1191 sequentially (Part 1A) or concurrently (Part 1B and Part 1D) with intravenous durvalumab (1500 mg). To determine the dose effect on PD changes within the TME, we calculated a normalized dose (µg/mm) for each patient by dividing the first actual injected dose by the longest (for non-lymph node lesions) or shortest (for lymph node lesions) diameter of injected lesions (14–130 mm).ResultsThe first administration induced serum IL-12 levels at all dose levels and the total IL-12 exposure measured by area under the curve was induced in a dose-dependent manner. Patients dosed at 8 and 12 µg had higher normalized dose (0.08–0.75 µg/mm) than patients dosed at 0.1–3 µg (0.002–0.15 µg/mm). High normalized dose (NDhi) elevated serum IL-12 and IFN-γ levels at 24h following injection to a greater extent than low normalized dose (NDlo).To correlate the dose effect with PD changes within the TME, we looked at the immune gene signatures in 14 patients (7 patients in each dose group) with available paired biopsies at screening and day 15. The gene set enrichment analysis revealed an increased enrichment of the hallmark interferon-gamma response pathway in NDhipatients. The average scores of gene signatures such as T cell inflamed, cytotoxic activity and T cell proliferation were induced in NDhipatients, whereas NDlopatients showed decrease or no major changes. Lesions of NDhipatients exhibited an inflammatory phenotype post-treatment. The best anti-tumor PD effect was observed in a partial responder in the NDhi group, which was associated with the largest increase in tumoral CD8 T cell density (6-fold) and PD-L1 (8.5-fold) by immunohistochemistry at injected lesions.ConclusionsIntratumoral injection of mRNA IL-12 can induce an immunostimulatory effect, including elevated anti-tumor gene signatures and T cell infiltration. Evidence of a dose-dependent immune modulation within the TME was observed at injected lesion sites.Trial RegistrationNCT03946800ReferencesEduardo Castañón, Dmitriy Zamarin, Benedito A Carneiro, Thomas Marron, Sandip Pravin Patel, Vivek Subbiah, Inderjit Mehmi, Honey Kumar Oberoi, Anthony El-Khoueiry, Benjamin Ridgway, Nairouz Elgeioushi, Nicholas M Durham, Emily Jennings, Michael Abadier, Paula G Fraenkel, Analia Azaro, Omid Hamid; Abstract CT004: Intratumoral (IT) MEDI1191 + durvalumab (D): Update on the first-in-human study in advanced solid tumors. Cancer Res 15 April 2023;83(8_Supplement):CT004. https://doi.org/10.1158/1538–7445.AM2023-CT004Abadier M, Jennings E, Eyles J, et al. 708 MEDI1191 (IL-12 mRNA) induces peripheral and intratumoral immunostimulatory effect in patients with cutaneous or subcutaneous (C/SC) lesions Journal for ImmunoTherapy of Cancer 2022;10:doi: 10.1136/jitc-2022-SITC2022.0708Ethics ApprovalThe IRB/IEC responsible for each site reviewed and approved the final study protocol, including the final version of the ICF and any other written information and/or materials to be provided to the subjects. Institutional Review Boards, Mount Sinai Health System, New York (Board Number 19–00279).
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Leukocyte recruitment towards the site of injury is a tightly regulated multi‐step process initiated by rolling along the luminal surface of inflamed endothelium in the presence of high ...shear flow. The interaction of endothelial P‐selectin‐to‐P‐selectin glycoprotein ligand (PSGL)‐1 constitutively expressed in leukocytes mediates leukocyte rolling by rapid formation and breakage of selectin‐to‐ligand bonds to balance the hydrodynamic drag forces exerted by blood shear stress. Once pulling forces due to the high shear exceed a critical threshold, tethers and slings become essential to support leukocyte rolling. Slings, recently discovered by our group, are long cell adhesive structures derived from long tethers. Here, we polarized naïve T cells towards distinct lineages of CD4
+
effector T cells (Th1, Th2, Th17 and Treg). Considering the critical role of functional PSGL‐1 for T cell rolling, we tested functional PSGL‐1 by P‐selectin‐Fc binding in flow cytometry. PSGL‐1 in Th1, Th17 and Treg is functional in 70–80% of cells, but only in 10–20% of Th2 cells and 0% of Naïve CD4 T cells. To visualize the dynamic process of T cell rolling, we performed live cell imaging under high shear stress (>5 dyn/cm
2
). Interestingly, the rolling speed of Th2 cells was significantly higher when compared to the other T cell subsets. This was mirrored by observing tethers and slings in Th1, Th17, Treg but to a lesser extent in Th2. Surprisingly, some of the few Th1 or Th17 cells bearing non‐functional PSGL‐1 were able to form tethers and slings, suggesting that functional PSGL‐1 binding to P‐selectin allows for T cell rolling at reduced speed but might not be the sole requirement for tether and sling formation. To identify the molecular and cellular requirements for tether and sling formation, Naïve CD4
+
T cells and neutrophils will be used as negative and positive controls, respectively. By developing gene expression maps we will identify the differentially regulated genes during T cell lineage differentiation. Upregulated genes in Th1, Th17 and Treg, but not in Th2, are excellent candidates for being involved in tether and sling formation.
Support or Funding Information
Swiss National Science Foundation and P01 HL078784/NIH
BackgroundTAK-573, a humanized, anti-CD38, IgG4, monoclonal antibody genetically fused to two attenuated IFNα2b molecules, was designed for targeted delivery of attenuated IFNα2b to CD38 expressing ...(CD38+) cells, utilizing a unique epitope of CD38 that does not compete with current anti-CD38 therapies. Preclinical evaluation of TAK-573 confirmed activation of type I IFN signaling in CD38+ cells inducing direct anti-proliferative effects on multiple myeloma (MM) cells and direct and indirect immune cell activation. Here we provide the preliminary analyses of the pharmacodynamic data currently available from the ongoing Ph I/II TAK-573-1501 clinical study in patients with relapsed/refractory MM (NCT03215030).MethodsPeripheral blood (PB) and bone marrow (BM) aspirates were collected from patients at pre- and post-dose time points for exploratory biomarker analyses. CD38 receptor occupancy (RO) and receptor density (RD) were determined using a 9-color flow cytometry assay. Whole transcriptome sequencing of bulk RNA was performed and analyzed to assess the type I IFN gene signature. Serum samples were analyzed using Olink’s Proximity Extension Assay Immuno-Oncology panel to measure changes in cytokine levels. Mass cytometry-based immunophenotyping was utilized to characterize changes in immune cell prevalence and activation status of cryopreserved cells.ResultsAdministration of TAK-573 resulted in a dose dependent increase in CD38 RO of PB-derived immune cells with saturation detected 4 hours after the end of infusion (EOI) at doses ≥ 0.2 mg/kg. The duration of saturation was dose dependent with doses ≥ 0.75 mg/kg saturating CD38 RO through 24 hours. All dose levels tested resulted in increases in the type I IFN gene signature at 24 hours. Consistent with CD38 being an IFN stimulated gene, TAK-573 treatment resulted in CD38 RD increases most notably on NK cells, but also on other CD38+ cells including MM cells. Circulating levels of IFN-associated cytokines were also elevated, with maximal induction 4 hours after the EOI. CD8+ T-cells in BM showed increased CD69 expression in 7 of 9 patients analyzed, 3 of whom also showed increases in both IFNγ and granzyme B positivity suggesting TAK-573 treatment results in increased BM cytolytic CD8+ T-cells, in a subset of patients.Abstract 357 Figure 1Proposed Mechanism of Action of TAK-573ConclusionsThese preliminary biomarker data indicate that TAK-573 is a pharmacologically active molecule that mediates its effect through IFNAR pathway modulation. Additional data are being collected to further refine the mechanism of action (Image 1), which will inform the recommended phase 2 dose and optimal schedule of administration for the development of TAK-573.Trial RegistrationClinicalTrials. gov: NCT03215030Ethics ApprovalThe TAK-573-1501 study is approved by WIRB-Copernicus Group, University of Nebraska Medical Center, Dana Farber Cancer Institute and Advarra IRBs.
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