CD56/NCAM mediates cell migration of human NK cells by promoting integrin-mediated adhesion turnover.

Natural killer (NK) cells patrol tissue to mediate lysis of virally infected and tumorigenic cells. Human NK cells are typically identified by their expression of neural cell adhesion molecule (NCAM, CD56), yet despite its ubiquitous expression on NK cells, CD56 remains a poorly understood protein on immune cells. CD56 has been previously demonstrated to play roles in NK cell cytotoxic function and cell migration. Specifically, CD56-deficient NK cells have impaired cell migration on stromal cells and CD56 is localized to the uropod of NK cells migrating on stroma. Here, we show that CD56 is required for NK cell migration on ICAM-1 and is required for the establishment of persistent cell polarity and unidirectional actin flow. The intracellular domain of CD56 (NCAM-140) is required for its function and the loss of CD56 leads to enlarged actin foci and sequestration of phosphorylated Pyk2 accompanied by increased size and frequency of activated LFA-1 clusters. Together, these data identify a role for CD56 in regulating human NK cell migration through modulation of actin dynamics and integrin turnover.

Piezo1 mechanosensing regulates integrin-dependent chemotactic migration in human T cells.

T cells are crucial for efficient antigen-specific immune responses and thus their migration within the body, to inflamed tissues from circulating blood or to secondary lymphoid organs, plays a very critical role. T cell extravasation in inflamed tissues depends on chemotactic cues and interaction between endothelial adhesion molecules and cellular integrins. A migrating T cell is expected to sense diverse external and membrane-intrinsic mechano-physical cues, but molecular mechanisms of such mechanosensing in cell migration are not established. We explored if the professional mechanosensor Piezo1 plays any role during integrin-dependent chemotaxis of human T cells. We found that deficiency of Piezo1 in human T cells interfered with integrin-dependent cellular motility on ICAM-1-coated surface. Piezo1 recruitment at the leading edge of moving T cells is dependent on and follows focal adhesion formation at the leading edge and local increase in membrane tension upon chemokine receptor activation. Piezo1 recruitment and activation, followed by calcium influx and calpain activation, in turn, are crucial for the integrin LFA1 (CD11a/CD18) recruitment at the leading edge of the chemotactic human T cells. Thus, we find that Piezo1 activation in response to local mechanical cues constitutes a membrane-intrinsic component of the 'outside-in' signaling in human T cells, migrating in response to chemokines, that mediates integrin recruitment to the leading edge.

ICAMs in Immunity, Intercellular Adhesion and Communication.

Interactions among leukocytes and leukocytes with immune-associated auxiliary cells represent an essential feature of the immune response that requires the involvement of cell adhesion molecules (CAMs). In the immune system, CAMs include a wide range of members pertaining to different structural and functional families involved in cell development, activation, differentiation and migration. Among them, ß2 integrins (LFA-1, Mac-1, p150,95 and αDß2) are predominantly involved in homotypic and heterotypic leukocyte adhesion. ß2 integrins bind to intercellular (I)CAMs, actin cytoskeleton-linked receptors belonging to immunoglobulin superfamily (IgSF)-CAMs expressed by leukocytes and vascular endothelial cells, enabling leukocyte activation and transendothelial migration. ß2 integrins have long been viewed as the most important ICAMs partners, propagating intracellular signalling from ß2 integrin-ICAM adhesion receptor interaction. In this review, we present previous evidence from pioneering studies and more recent findings supporting an important role for ICAMs in signal transduction. We also discuss the contribution of immune ICAMs (ICAM-1, -2, and -3) to reciprocal cell signalling and function in processes in which ß2 integrins supposedly take the lead, paying particular attention to T cell activation, differentiation and migration.

CAR-mediated targeting of NK cells overcomes tumor immune escape caused by ICAM-1 downregulation.

BACKGROUND: The antitumor activity of natural killer (NK) cells can be enhanced by specific targeting with therapeutic antibodies that trigger antibody-dependent cell-mediated cytotoxicity (ADCC) or by genetic engineering to express chimeric antigen receptors (CARs). Despite antibody or CAR targeting, some tumors remain resistant towards NK cell attack. While the importance of ICAM-1/LFA-1 interaction for natural cytotoxicity of NK cells is known, its impact on ADCC induced by the ErbB2 (HER2)-specific antibody trastuzumab and ErbB2-CAR-mediated NK cell cytotoxicity against breast cancer cells has not been investigated. METHODS: Here we used NK-92 cells expressing high-affinity Fc receptor FcγRIIIa in combination with trastuzumab or ErbB2-CAR engineered NK-92 cells (NK-92/5.28.z) as well as primary human NK cells combined with trastuzumab or modified with the ErbB2-CAR and tested cytotoxicity against cancer cells varying in ICAM-1 expression or alternatively blocked LFA-1 on NK cells. Furthermore, we specifically stimulated Fc receptor, CAR and/or LFA-1 to study their crosstalk at the immunological synapse and their contribution to degranulation and intracellular signaling in antibody-targeted or CAR-targeted NK cells. RESULTS: Blockade of LFA-1 or absence of ICAM-1 significantly reduced cell killing and cytokine release during trastuzumab-mediated ADCC against ErbB2-positive breast cancer cells, but not so in CAR-targeted NK cells. Pretreatment with 5-aza-2'-deoxycytidine induced ICAM-1 upregulation and reversed NK cell resistance in ADCC. Trastuzumab alone did not sufficiently activate NK cells and required additional LFA-1 co-stimulation, while activation of the ErbB2-CAR in CAR-NK cells induced efficient degranulation independent of LFA-1. Total internal reflection fluorescence single molecule imaging revealed that CAR-NK cells formed an irregular immunological synapse with tumor cells that excluded ICAM-1, while trastuzumab formed typical peripheral supramolecular activation cluster (pSMAC) structures. Mechanistically, the absence of ICAM-1 did not affect cell-cell adhesion during ADCC, but rather resulted in decreased signaling via Pyk2 and ERK1/2, which was intrinsically provided by CAR-mediated targeting. Furthermore, while stimulation of the inhibitory NK cell checkpoint molecule NKG2A markedly reduced FcγRIIIa/LFA-1-mediated degranulation, retargeting by CAR was only marginally affected. CONCLUSIONS: Downregulation of ICAM-1 on breast cancer cells is a critical escape mechanism from trastuzumab-triggered ADCC. In contrast, CAR-NK cells are able to overcome cancer cell resistance caused by ICAM-1 reduction, highlighting the potential of CAR-NK cells in cancer immunotherapy.

LFA-1 nanoclusters integrate TCR stimulation strength to tune T-cell cytotoxic activity.

T-cell cytotoxic function relies on the cooperation between the highly specific but poorly adhesive T-cell receptor (TCR) and the integrin LFA-1. How LFA-1-mediated adhesion may scale with TCR stimulation strength is ill-defined. Here, we show that LFA-1 conformation activation scales with TCR stimulation to calibrate human T-cell cytotoxicity. Super-resolution microscopy analysis reveals that >1000 LFA-1 nanoclusters provide a discretized platform at the immunological synapse to translate TCR engagement and density of the LFA-1 ligand ICAM-1 into graded adhesion. Indeed, the number of high-affinity conformation LFA-1 nanoclusters increases as a function of TCR triggering strength. Blockade of LFA-1 conformational activation impairs adhesion to target cells and killing. However, it occurs at a lower TCR stimulation threshold than lytic granule exocytosis implying that it licenses, rather than directly controls, the killing decision. We conclude that the organization of LFA-1 into nanoclusters provides a calibrated system to adjust T-cell killing to the antigen stimulation strength.

Endoplasmic Reticulum Protein 72 Regulates Integrin Mac-1 Activity to Influence Neutrophil Recruitment.

BACKGROUND: Integrins mediate the adhesion, crawling, and migration of neutrophils during vascular inflammation. Thiol exchange is important in the regulation of integrin functions. ERp72 (endoplasmic reticulum-resident protein 72) is a member of the thiol isomerase family responsible for the catalysis of disulfide rearrangement. However, the role of ERp72 in the regulation of Mac-1 (integrin αMß2) on neutrophils remains elusive. METHODS: Intravital microscopy of the cremaster microcirculation was performed to determine in vivo neutrophil movement. Static adhesion, flow chamber, and flow cytometry were used to evaluate in vitro integrin functions. Confocal fluorescent microscopy and coimmunoprecipitation were utilized to characterize the interactions between ERp72 and Mac-1 on neutrophil surface. Cell-impermeable probes and mass spectrometry were used to label reactive thiols and identify target disulfide bonds during redox exchange. Biomembrane force probe was performed to quantitatively measure the binding affinity of Mac-1. A murine model of acute lung injury induced by lipopolysaccharide was utilized to evaluate neutrophil-associated vasculopathy. RESULTS: ERp72-deficient neutrophils exhibited increased rolling but decreased adhesion/crawling on inflamed venules in vivo and defective static adhesion in vitro. The defect was due to defective activation of integrin Mac-1 but not LFA-1 (lymphocyte function-associated antigen-1) using blocking or epitope-specific antibodies. ERp72 interacted with Mac-1 in lipid rafts on neutrophil surface leading to the reduction of the C654-C711 disulfide bond in the αM subunit that is critical for Mac-1 activation. Recombinant ERp72, via its catalytic motifs, increased the binding affinity of Mac-1 with ICAM-1 (intercellular adhesion molecule-1) and rescued the defective adhesion of ERp72-deficient neutrophils both in vitro and in vivo. Deletion of ERp72 in the bone marrow inhibited neutrophil infiltration, ameliorated tissue damage, and increased survival during murine acute lung injury. CONCLUSIONS: Extracellular ERp72 regulates integrin Mac-1 activity by catalyzing disulfide rearrangement on the αM subunit and may be a novel target for the treatment of neutrophil-associated vasculopathy.

LFA-1 regulated by IL-2/STAT5 pathway boosts antitumor function of intratumoral CD8+ T cells for improving anti-PD-1 antibody therapy.

Anti-PD-1 antibody therapy has achieved success in tumor treatment; however, the duration of its clinical benefits are typically short. The functional state of intratumoral CD8+ T cells substantially affects the efficacy of anti-PD-1 antibody therapy. Understanding how intratumoral CD8+ T cells change will contribute to the improvement in anti-PD-1 antibody therapy. In this study, we found that tumor growth was not arrested after the late administration of anti-PD-1 antibody and that the antitumor function of CD8+ T cells decreased with tumor progression. The results of the RNA sequencing of CD8+ T cells infiltrating the tumor site on days 7 and 14 showed that the cell adhesion molecule Lymphocyte Function-associated Antigen-1 (LFA-1) participates in regulating the antitumor function of CD8+ T cells and that decreased LFA-1 expression in intratumoral CD8+ T cells is associated with tumor progression. By analyzing the Gene Expression Omnibus (GEO) database and our results, we found that the antitumor function of intratumoral CD8+ T cells with high LFA-1 expression was stronger. The formation of immune synapses is impaired in Itgal-si CD8+ T cells, resulting in decreased anti-tumor function. LFA-1 expression in intratumoral CD8+ T cells is regulated by the IL-2/STAT5 pathway. The combination of IL-2 and anti-PD-1 antibody effectively enhanced LFA-1 expression and the antitumor function of intratumoral CD8+ T cells. The adoptive transfer of OT-1 T cells overexpressing LFA-1, STAT5A, or STAT5B resulted in higher antitumor function, deferred tumor growth, and prolonged survival. These findings indicate that LFA-1-mediated immune synapse acts as a regulator of the antitumor function of intratumoral CD8+ T cells, which can be applied to improve anti-PD-1 antibody therapy.

Engineered extracellular vesicles expressing ICAM-1: A promising targeted delivery system for T cell modifications.

BACKGROUND: Extracellular vesicles (EVs) are natural nano-carriers that possess the required crucial features of an ideal biomolecular delivery system. However, using unmodified EVs may have some limitations such as low accumulation in target sites. Studies have established that engineering EVs against different cell surface markers can overcome most of these hurdles. METHODS: In this study, engineered EVs expressing ICAM-1/LAMP2b fusion protein on their surfaces were produced and isolated. The uptake of isolated targeted and non-targeted EVs was evaluated by imaging and flow cytometry. To assess the ability of targeted EVs to be applied as a safe carrier, pAAVS1-Puro-GFP plasmids were encapsulated into EVs by electroporation. RESULTS: The HEKT 293 cell line was successfully modified permanently by a lentiviral vector to express ICAM-1 on the surface of the derived EVs. The ELISA and western blot tests established the binding affinity of targeted EVs for recombinant LFA-1 with a remarkable difference from non-targeted EVs. Furthermore, flow cytometry results revealed noteworthy differences in the binding of LFA-1-positive, non-targeted EVs, and targeted EVs to LFA-1-negative cells. Finally, imaging and flow cytometry indicated that newly produced EVs could efficiently interact with T cells and functionally deliver loaded plasmids to them. CONCLUSION: These LFA-1-targeted EVs were able to interact with T cells as their recipient cells. They can be utilized as an ideal delivery system to transfer various biomolecules to T cells, facilitating immunotherapies or other cell-based treatments.

Isolation and characterization of a novel single-chain variable fragment (scFv) against Lymphocyte function-associated antigen-1 (LFA-1) using phage display method.

Lymphocyte function-associated antigene-1 (LFA-1) is a well-described integrin found on lymphocytes and other leukocytes, which is known to be overexpressed in leukemias and lymphomas. This receptor plays a significant role in immune responses such as T-cell activation, leukocyte cell-cell interactions, and trafficking of leukocyte populations. Subsequently, binders of LFA-1 emerge as potential candidates for cancer and autoimmune therapy. This study used the phage display technique to construct and characterize a high-affinity single-chain fragment variable (scFv) antibody against LFA-1. After expression, purification, dialysis, and concentration of the recombinant LFA-1 protein, four female BALB/c mice were immunized, splenocyte's mRNA was extracted, and cDNA was synthesized. A scFv library was constructed by linking the amplified VH/Vκ fragments through a 72-bp linker using SOEing PCR. Next, the scFv gene fragments were cloned into the pComb-3XSS phagemid vector; thus, the phage library was developed. The selection process involved three rounds of phage-bio-panning, polyclonal, and monoclonal phage ELISA. AF17 was chosen and characterized among the positive clones through SDS-PAGE, Western blotting, indirect ELISA, and in-silico analyses. The results of the study showed the successful construction of a high-affinity scFv library against LFA-1. The accuracy of the AF17 production and its ability to bind to the LFA-1 were confirmed through SDS-PAGE, Western blot, and ELISA. This study highlights the potential application of the high-affinity AF17 against LFA-1 for targeting T lymphocytes for therapeutic purposes.

WAVE2 Regulates Actin-Dependent Processes Induced by the B Cell Antigen Receptor and Integrins.

B cell antigen receptor (BCR) signaling induces actin cytoskeleton remodeling by stimulating actin severing, actin polymerization, and the nucleation of branched actin networks via the Arp2/3 complex. This enables B cells to spread on antigen-bearing surfaces in order to increase antigen encounters and to form an immune synapse (IS) when interacting with antigen-presenting cells (APCs). Although the WASp, N-WASp, and WAVE nucleation-promoting factors activate the Arp2/3 complex, the role of WAVE2 in B cells has not been directly assessed. We now show that both WAVE2 and the Arp2/3 complex localize to the peripheral ring of branched F-actin when B cells spread on immobilized anti-Ig antibodies. The siRNA-mediated depletion of WAVE2 reduced and delayed B cell spreading on immobilized anti-Ig, and this was associated with a thinner peripheral F-actin ring and reduced actin retrograde flow compared to control cells. Depleting WAVE2 also impaired integrin-mediated B cell spreading on fibronectin and the LFA-1-induced formation of actomyosin arcs. Actin retrograde flow amplifies BCR signaling at the IS, and we found that depleting WAVE2 reduced microcluster-based BCR signaling and signal amplification at the IS, as well as B cell activation in response to antigen-bearing cells. Hence, WAVE2 contributes to multiple actin-dependent processes in B lymphocytes.

Evaluation of cell adhesion molecules (LFA-1 and L-selectin) in ankylosing spondylitis patients after treatment with ß-D-mannuronic acid (M2000).

Background & objectives: To examine ß-D-mannuronic acid (M2000) effects on L-selectin shedding and leucocyte function-associated antigen-1 (LFA-1) expression as mechanisms of action of this drug in patients with ankylosing spondylitis (AS). Methods: To investigate the molecular consequences of ß-D-mannuronic acid on L-selectin shedding, flow cytometry method was used. Furthermore, the effect of it on LFA-1 gene expression was analyzed by using quantitative real time (qRT)-PCR technique. Results: The LFA-1 expression in patients with AS was higher than controls (P=0.046). The LFA-1 expression after 12 wk therapy with ß-D-mannuronic acid was meaningfully decreased (P=0.01). After 12 wk treatment with ß-D-mannuronic acid, the frequency of CD62L-expressing CD4+ T cells in patients with AS, was not considerably altered, compared to the patients before therapy (P=0.5). Furthermore, after 12 wk therapy with ß-D-mannuronic acid, L-selectin expression levels on CD4+ T-cells in patients with AS, were not remarkably changed, compared to the expression levels of these in patients before treatment (P=0.2). Interpretation & conclusions: The results of this study for the first time showed that ß-D-mannuronic acid can affect events of adhesion cascade in patients with AS. Moreover, ß-D-mannuronic acid presented as an acceptable benefit to AS patients and could aid in the process of disease management.

CD11a regulates hematopoietic stem and progenitor cells.

Integrin αLß2 (CD11a/CD18, CD11a) is a critical leukocyte adhesion molecule in leukocyte arrest and immunological synapse formation. However, its role in the bone marrow has not been investigated in depth. Here we showed that CD11a was expressed on all subsets of hematopoietic stem and progenitor cells (HPSCs). CD11a deficiency enhanced HSPCs activity under lipopolysaccharide (LPS) stimulation as demonstrated by a higher HSPC cell count along with an increase in cell proliferation. However, our mixed chimera experiment did not support that this phenotype was driven in a cell-intrinsic manner. Rather we found that the production of IL-27, a major cytokine that drives HSPC proliferation, was significantly upregulated both in vivo and in vitro. This adds a novel role of CD11a biology.

LFA-1 knockout inhibited the tumor growth and is correlated with treg cells.

Cancer immunotherapy has been proven to be clinically effective in multiple types of cancers. Lymphocyte function-associated antigen 1 (LFA-1), a member of the integrin family of adhesion molecules, is expressed mainly on αß T cells. LFA-1 is associated with tumor immune responses, but its exact mechanism remains unknown. Here, two kinds of mice tumor model of LFA-1 knockout (LFA-1-/-) mice bearing subcutaneous tumor and Apc Min/+;LFA-1-/- mice were used to confirm that LFA-1 knockout resulted in inhibition of tumor growth. Furthermore, it also demonstrated that the numbers of regulatory T cells (Treg cells) in the spleen, blood, mesenteric lymph nodes were decreased in LFA-1-/- mice, and the numbers of Treg cells in mesenteric lymph nodes were also decreased in Apc Min/+;LFA-1-/- mice compared with Apc Min/+ mice. LFA-1 inhibitor (BIRT377) was administered to subcutaneous tumor-bearing LFA-1+/+ mice, and the results showed that the tumor growth was inhibited and the number of Treg cells was reduced. The analysis of TIMER tumor database indicated that LFA-1 expression is positively associated with Treg cells and TNM stage. Conclusively, this suggests that LFA-1 knockout would inhibit tumor growth and is correlated with Treg cells. LFA-1 may be one potential target for cancer immunotherapy. Video Abstract.

IL-1ß stimulated human umbilical cord mesenchymal stem cells ameliorate rheumatoid arthritis via inducing apoptosis of fibroblast-like synoviocytes.

Rheumatoid arthritis (RA) is characterized by synovial proliferation and lymphocyte accumulation leading to progressive damage of the periarticular bone and the articular cartilage. The hyperplasia of the synovial intima lining mainly consists of fibroblast-like synoviocytes-rheumatoid arthritis (HFLS-RA) which exhibit apoptosis-resistance, hyper-proliferation, and high invasiveness. The therapeutic efficacy of mesenchymal stem cells (MSCs) treatment in RA has been shown to be due to its immuno-regulatory ability. However, the exact factors and mechanisms involved in MSCs treatment in RA remain unclear. In this study, TRAIL receptor-Death receptor 4 (DR4), DR5, and LFA-1 ligand-intercellular adhesion molecule-1 (ICAM-1) were upregulated in IL-1ß-stimulated HFLS-RA. We demonstrated that the total cell number of IL-1ß-stimulated hUCMSCs adhering to IL-1ß-stimulated HFLA-RA increased via LFA-1/ICAM-1 interaction. Direct co-culture of IL-1ß-stimulated hUCMSCs with IL-1ß-stimulated HFLS-RA increased the apoptosis of HFLS-RA. RA symptoms in the CIA mouse model improved after administration of IL-1ß-stimulated hUCMSCs. In conclusion, IL-1ß-stimulated hUCMSCs adhering to HFLS-RA occurred via LFA-1/ICAM-1 interaction, apoptosis of HFLS-RA was induced via TRAIL/DR4, DR5 contact, and RA symptoms and inflammation were significantly improved in a CIA mouse model. The results of this study suggest that IL-1ß-stimulated hUCMSCs have therapeutic potential in RA treatment.

LFA-1/ICAM-1 Adhesion Pathway Mediates the Homeostatic Migration of Lymphocytes from Peripheral Tissues into Lymph Nodes through Lymphatic Vessels.

Lymphocyte function-associated antigen-1 (LFA-1) and its endothelial ligand intercellular adhesion molecule-1 (ICAM-1) are important for the migration of lymphocytes from blood vessels into lymph nodes. However, it is largely unknown whether these molecules mediate the homeostatic migration of lymphocytes from peripheral tissues into lymph nodes through lymphatic vessels. In this study, we find that, in naive mice, ICAM-1 is expressed on the sinus endothelia of lymph nodes, but not on the lymphatic vessels of peripheral tissues. In in vivo lymphocyte migration assays, memory CD4+ T cells migrated to lymph nodes from peripheral tissues much more efficiently than from blood vessels, as compared to naive CD4+ T cells. Moreover, ICAM-1 deficiency in host mice significantly inhibited the migration of adoptively transferred wild-type donor lymphocytes from peripheral tissues, but not from blood vessels, into lymph nodes. The migration of LFA-1-deficient donor lymphocytes from peripheral tissues into the lymph nodes of wild-type host mice was also significantly reduced as compared to wild-type donor lymphocytes. Furthermore, the number of memory T cells in lymph nodes was significantly reduced in the absence of ICAM-1 or LFA-1. Thus, our study extends the functions of the LFA-1/ICAM-1 adhesion pathway, indicating its novel role in controlling the homeostatic migration of lymphocytes from peripheral tissues into lymph nodes and maintaining memory T cellularity in lymph nodes.

Islet beta-cells and intercellular adhesion molecule-1 (ICAM-1): Integrating immune responses that influence autoimmunity and graft rejection.

Type 1 diabetes (T1D) develops due to autoimmune targeting of the pancreatic islet ß-cells. Clinical symptoms arise from reduced insulin in circulation. The molecular events and interactions between discrete immune cell populations, infiltration of such leukocytes into pancreatic and islet tissue, and selective targeting of the islet ß-cells during autoimmunity and graft rejection are not entirely understood. One protein central to antigen presentation, priming of immune cells, trafficking of leukocytes, and vital for leukocyte effector function is the intercellular adhesion molecule-1 (ICAM-1). The gene encoding ICAM-1 is transcriptionally regulated and rapidly responsive (i.e., within hours) to pro-inflammatory cytokines. ICAM-1 is a transmembrane protein that can be glycosylated; its presence on the cell surface provides co-stimulatory functions for immune cell activation and stabilization of cell-cell contacts. ICAM-1 interacts with the ß2-integrins, CD11a/CD18 (LFA-1) and CD11b/CD18 (Mac-1), which are present on discrete immune cell populations. A whole-body ICAM-1 deletion protects NOD mice from diabetes onset, strongly implicating this protein in autoimmune responses. Since several different cell types express ICAM-1, its biology is fundamentally essential for various physiological and pathological outcomes. Herein, we review the role of ICAM-1 during both autoimmunity and islet graft rejection to understand the mechanism(s) leading to islet ß-cell death and dysfunction that results in insufficient circulating quantities of insulin to control glucose homeostasis.

Abnormal generation of IL-17A represses tumor infiltration of stem-like exhausted CD8+ T cells to demote the antitumor immunity.

BACKGROUND: Variated anti-cancer therapies are combined with immune checkpoint blockades (ICBs) for improving ICB therapeutic efficacy. Occurrence of tissue damage is common that triggers multiple inflammatory cytokine generation. Gastrointestinal organs are the commonly affected. We investigated the impact of acute colitis on tumor infiltration of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) for controlling tumor growth and responding to antibody against PD-1 (anti-PD-1). METHODS: Several tumor cell lines were inoculated into syngeneic mice subcutaneously or intra-hepatically. When tumor mass formed, activated CTLs were intravenously transferred into the tumor-bearing mice, that were given the drinking water containing 2% dextran sulfate sodium (DSS) for acute colitis induction. Tumor growth, infiltration of two exhausted CTL subsets, and the CTL interaction with tumor vascular endothelium were examined. RESULTS: Acute colitis dampened CTL-mediated antitumor effects, correlating with IL-17A elevation in the inflamed intestine. In the tumor bed, stem-like exhausted CTLs, which were defined as PD-1+Slamf6+Tim3-, expressed higher IL-17A receptor heterodimers and lower leukocyte function-associated antigen-1 (LFA-1) than terminally exhausted CTLs did, that were defined as PD-1+Slamf6-Tim3+. IL-17A stimulation reduced LFA-1 surface expression on stem-like exhausted CTLs and the counterpart ICAM-1 (intracellular adhesion molecule-1) on tumor vascular endothelium. IL-17A stimulation suppressed the extravasation across tumor vascular endothelium and self-renewal of stem-like, not the terminally exhausted CTLs. Administration of anti-IL-17A neutralizing antibody to the colitis mice restored the CTL tumor infiltration and enhanced anti-PD-1 treatment efficacy against tumors. In 33 hepatocellular carcinoma patients being treated with anti-PD-1 plus antibody against vascular endothelial growth factor, disease progression of 15 patients, that exhibited serum IL-17A increase 24 h post-therapy as compared to pre-therapy level, was poorer than that of 18 patients that exhibited serum IL-17A no-increase. CONCLUSIONS: Abnormal generation of IL-17A mainly repressed tumor infiltration of stem-like exhausted CTLs. ICB-based immunotherapeutic efficacy could be upgraded with administration of anti-IL-17A, when treatment-related IL-17A elevation occurred due to tissue damage, such as acute colitis.

Magnesium enhances the graft-versus-tumor effect of donor lymphocytic infusion on hematologic malignancies.

Donor lymphocyte infusion (DLI) cures relapsed hematologic malignancies after allogeneic hematopoietic stem cell transplantation through the graft-versus-tumor (GVT) effect. Although the important role of magnesium in enhancing immunity has been mentioned in studies, limited clinical data have explored how magnesium affects the efficacy of DLI. Besides, although laboratory data demonstrate that magnesium can enhance CD8+ T cells effector function, whether magnesium regulates the tumor killing effect of peripheral blood mononuclear cells (PBMCs) remains to be explored. Here, for the retrospective study, we collected clinical data of relapsed patients receiving DLI and explored the relationship between different serum magnesium levels and patient outcomes. For in vitro studies, we investigated the effect of magnesium on the cytotoxicity of DLI cells which were PBMCs and preliminarily explored the mechanism. Eighty-one patients were enrolled in this study. It was found that the high post-DLI magnesium level was significantly associated with a higher incidence of complete remission (CR) or partial remission (CR/PR) and a higher possibility of survival. The magnesium level after DLI was an independent risk factor of overall survival. In vitro studies proved that increased magnesium enhanced the cytotoxic function of PBMCs on hematologic malignancies. Besides, magnesium modulated LFA-1 headpiece opening. When blocking the integrin-ligand interaction between LFA-1 and ICAM-1, the regulation effect of magnesium on PBMCs was weakened. Therefore, it was possible that magnesium regulated PBMCs effector function by stimulating LFA-1. These results show that serum magnesium levels affect immunological responses mediated by donor lymphocytes in hematologic malignancies.

Polylysine-Containing Hydrogel Formulation of Fuzapladib, Inhibitor of Leukocyte-Function Associated Antigen-1 (LFA-1) Activation, for Sustained Release.

The aim of the present study was to develop an injectable hydrogel (HG) formulation of fuzapladib sodium (FZP), an animal drug for acute pancreatitis (AP), with the use of polyethyleneoxide (PEO) and polylysine (pLys), a cationic polymer. A mixture of pLys and FZP was added to PEO to prepare an HG formulation, and the formulation was optimized by release test and viscosity measurements. Circular dichroism (CD) and infrared absorption (IR) spectral analyses were applied to clarify the intermolecular interactions between FZP and pLys. The pharmacokinetic behavior of FZP was evaluated after a subcutaneous administration of FZP samples (2.0 mg-FZP/kg) to rats. Although the immediate release of FZP was observed for the HG formulation, the addition of pLys at a 20-fold amount of FZP or higher led to the sustained release of FZP. Considering release behavior, the concentration of pLys was optimized as 100-fold that of FZP in the HG formulation. CD and IR spectroscopic analyses of FZP and/or pLys demonstrated an intermolecular interaction between FZP and pLys, as evidenced by the slight spectral transition. After a subcutaneous administration of HG formulation containing pLys to rats, compared with FZP alone, significant differences were observed in the pharmacokinetic behavior with a decrease of Cmax from 2.3 to 0.9 mg/mL and slower elimination kinetics. HG formulation using pLys might be a viable dosage option for FZP for the treatment of AP in animals.

Tumor-derived GDF-15 blocks LFA-1 dependent T cell recruitment and suppresses responses to anti-PD-1 treatment.

Immune checkpoint blockade therapy is beneficial and even curative for some cancer patients. However, the majority don't respond to immune therapy. Across different tumor types, pre-existing T cell infiltrates predict response to checkpoint-based immunotherapy. Based on in vitro pharmacological studies, mouse models and analyses of human melanoma patients, we show that the cytokine GDF-15 impairs LFA-1/ß2-integrin-mediated adhesion of T cells to activated endothelial cells, which is a pre-requisite of T cell extravasation. In melanoma patients, GDF-15 serum levels strongly correlate with failure of PD-1-based immune checkpoint blockade therapy. Neutralization of GDF-15 improves both T cell trafficking and therapy efficiency in murine tumor models. Thus GDF-15, beside its known role in cancer-related anorexia and cachexia, emerges as a regulator of T cell extravasation into the tumor microenvironment, which provides an even stronger rationale for therapeutic anti-GDF-15 antibody development.