Lymphocyte Adhesion Molecules Research Articles

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

ABSTRACT 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.

Nicotinamide enhances natural killer cell function and yields remissions in patients with non-Hodgkin lymphoma.

Allogeneic natural killer (NK) cell adoptive transfer has shown the potential to induce remissions in relapsed or refractory leukemias and lymphomas, but strategies to enhance NK cell survival and function are needed to improve clinical efficacy. Here, we demonstrated that NK cells cultured ex vivo with interleukin-15 (IL-15) and nicotinamide (NAM) exhibited stable induction of l-selectin (CD62L), a lymphocyte adhesion molecule important for lymph node homing. High frequencies of CD62L were associated with elevated transcription factor forkhead box O1 (FOXO1), and NAM promoted the stability of FOXO1 by preventing proteasomal degradation. NK cells cultured with NAM exhibited metabolic changes associated with elevated glucose flux and protection against oxidative stress. NK cells incubated with NAM also displayed enhanced cytotoxicity and inflammatory cytokine production and preferentially persisted in xenogeneic adoptive transfer experiments. We also conducted a first-in-human phase 1 clinical trial testing adoptive transfer of NK cells expanded ex vivo with IL-15 and NAM (GDA-201) combined with monoclonal antibodies in patients with relapsed or refractory non-Hodgkin lymphoma (NHL) and multiple myeloma (MM) (NCT03019666). Cellular therapy with GDA-201 and rituximab was well tolerated and yielded an overall response rate of 74% in 19 patients with advanced NHL. Thirteen patients had a complete response, and 1 patient had a partial response. GDA-201 cells were detected for up to 14 days in blood, bone marrow, and tumor tissues and maintained a favorable metabolic profile. The safety and efficacy of GDA-201 in this study support further development as a cancer therapy.

Choroid plexus imaging to track neuroinflammation - a translational model for mouse and human studies.

Choroid plexus imaging to track neuroinflammation - a translational model for mouse and human studies.

Cancer immunotherapies transition endothelial cells into HEVs that generate TCF1+ T lymphocyte niches through a feed-forward loop.

The lack of Tcell infiltrates is a major obstacle to effective immunotherapy in cancer. Conversely, the formation of tumor-associated tertiary-lymphoid-like structures (TA-TLLSs), which are the local site of humoral and cellular immune responses against cancers, is associated with good prognosis, and they have recently been detected in immune checkpoint blockade (ICB)-responding patients. However, how these lymphoid aggregates develop remains poorly understood. By employing single-cell transcriptomics, endothelial fate mapping, and functional multiplex immune profiling, we demonstrate that antiangiogenic immune-modulating therapies evoke transdifferentiation of postcapillary venules into inflamed high-endothelial venules (HEVs) via lymphotoxin/lymphotoxin beta receptor (LT/LTβR) signaling. In turn, tumor HEVs boost intratumoral lymphocyte influx and foster permissive lymphocyte niches for PD1- and PD1+TCF1+ CD8 Tcell progenitors that differentiate into GrzB+PD1+ CD8T effector cells. Tumor-HEVs require continuous CD8 and NK cell-derived signals revealing that tumor HEV maintenance is actively sculpted by the adaptive immune system through a feed-forward loop.

Formyl peptide receptors are involved in CTX-induced impairment of lymphocyte functions

Crotoxin (CTX) is a neurotoxin that is isolated from the venom of Crotalus durissus terrificus, which displays immunomodulatory, anti-inflammatory, and anti-tumoral effects. Previous research has demonstrated that CTX promotes the adherence of leukocytes to the endothelial cells in blood microcirculation and the high endothelial venules of lymph nodes, which reduces the number of blood cells and lymphocytes. Studies have also shown that these effects are mediated by lipoxygenase-derived mediators. However, the exact lipoxygenase-derived eicosanoid involved in the CTX effect on lymphocytes is yet to be characterized. As CTX stimulates lipoxin-derived mediators from macrophages and lymphocyte effector functions could be modulated by activating formyl peptide receptors, we aimed to investigate whether these receptors were involved in CTX-induced redistribution and functions of lymphocytes in rats. We used male Wistar rats treated with CTX to demonstrate that Boc2 (butoxycarbonyl-Phe-Leu-Phe-Leu-Phe), an antagonist of formyl peptide receptors, prevented CTX-induced decrease in the number of circulating lymphocytes and increased the expression of the lymphocyte adhesion molecule LFA1. CTX reduced the T and B lymphocyte functions, such as lymphocyte proliferation in response to the mitogen Concanavalin A and antibody production in response to BSA immunization, respectively, which was prevented by the administration of Boc2. Importantly, mesenteric lymph node lymphocytes from CTX-treated rats showed an increased release of 15-epi-LXA4. These results indicate that formyl peptide receptors mediate CTX-induced redistribution of lymphocytes and that 15-epi-LXA4 is a key mediator of the immunosuppressive effects of CTX.

Plasminogen and plasmin can bind to human T cells and generate truncated CCL21 that increases dendritic cell chemotactic responses

Plasmin is a broad-spectrum protease and therefore needs to be tightly regulated. Active plasmin is formed from plasminogen, which is found in high concentrations in the blood and is converted by the plasminogen activators. In the circulation, high levels of α2-antiplasmin rapidly and efficiently inhibit plasmin activity. Certain myeloid immune cells have been shown to bind plasmin and plasminogen on their cell surface via proteins that bind to the plasmin(ogen) kringle domains. Our earlier work showed that T cells can activate plasmin but that they do not themselves express plasminogen. Here, we demonstrate that T cells express several known plasminogen receptors and that they bind plasminogen on their cell surface. We show T cell–bound plasminogen was converted to plasmin by plasminogen activators upon T cell activation. To examine functional consequences of plasmin generation by activated T cells, we investigated its effect on the chemokine, C-C motif chemokine ligand 21 (CCL21). Video microscopy and Western blotting confirmed that plasmin bound by human T cells cleaves CCL21 and increases the chemotactic response of monocyte-derived dendritic cells toward higher CCL21 concentrations along the concentration gradient by increasing their directional migration and track straightness. These results demonstrate how migrating T cells and potentially other activated immune cells may co-opt a powerful proteolytic system from the plasma toward immune processes in the peripheral tissues, where α2-antiplasmin is more likely to be absent. We propose that plasminogen bound to migrating immune cells may strongly modulate chemokine responses in peripheral tissues.

Immunobiology, preliminary safety, and efficacy of CPI-006, an anti-CD73 antibody with immune modulating activity, in a phase 1 trial in advanced cancers.

2505 Background: CPI-006 inhibits CD73, a nucelotidase that converts AMP to adenosine and functions as a lymphocyte adhesion molecule. CPI-006 is a humanized IgG1 FcγR binding-deficient antibody that binds to CD73+ T and B lymphocytes leading to activation of B cells and expression of CD69. This study investigates the immunobiology, safety, and efficacy of CPI-006 monotherapy and in combination with CPI-444, an adenosine A2A receptor (A2AR) antagonist (NCT03454451). Methods: Patients with relapsed solid tumors were treated in a 3 + 3 escalation study with 1, 3, 6 or 12 mg/kg CPI-006 (Q3w IV infusion) monotherapy or in combination with CPI-444 (100 mg, PO, BID). Flow cytometry was performed on blood samples for lymphocyte subset analysis and receptor occupancy. Results: 17 patients were enrolled; 11 monotherapy and 6 combination. CPI-006 was associated with Grade 1 infusion reactions occuring within 30 minutes of the first infusion and were eliminated by premedication with non-steroidals. No DLTs with monotherapy or combination therapy were seen. Receptor occupancy on peripheral lymphocytes was maintained for the full dosing interval at 12 mg/kg. Pharmacodynamic effects suggesting immune modulation were observed within 1 hr of infusion at all dose levels and included a decrease in peripheral blood CD73pos B cells (mean reduction 86%, p < 0.05), increased CD73neg CD4 T cells (mean increase 37%, p < 0.01), and decreased CD8 T cells (mean reduction 20%, p < 0.01) compared to baseline. Overall, CD4:CD8 ratios were increased. Tumor regression was observed in a prostate cancer patient after 5 cycles of monotherapy at 6 mg/kg; peripheral B cells partially returned by the second cycle and reached a new homeostatic level through subsequent cycles. No change in serum immunoglobulins were observed. Conclusions: CPI-006 induces a rapid lymphocyte redistribution, including a transient reduction of circulating CD73pos B cells suggesting redistribution to lymphoid tissues, and an increased CD4:CD8 ratio, consistent with increased TH effector/memory cells in the blood. The treatment has been well-tolerated, and there is early evidence of anti-tumor activity of CPI-006 monotherapy. Clinical trial information: NCT03454451.

Microglia prevent peripheral immune cell invasion and promote an anti-inflammatory environment in the brain of APP-PS1 transgenic mice

BackgroundUndoubtedly, neuroinflammation is a major contributor to Alzheimer’s disease (AD) progression. Neuroinflammation is characterized by the activity of brain resident glial cells, in particular microglia, but also by peripheral immune cells, which infiltrate the brain at certain stages of disease progression. The specific role of microglia in shaping AD pathology is still controversially discussed. Moreover, a possible role of microglia in the interaction and recruitment of peripheral immune cells has so far been completely ignored.MethodsWe ablated microglia cells in 12-month-old WT and APP-PS1 transgenic mice for 4 weeks using the CSF1R inhibitor PLX5622 and analyzed its consequences to AD pathology and in particular to peripheral immune cell infiltration.ResultsPLX5622 treatment successfully reduced microglia numbers. Interestingly, it uncovered a treatment-resistant macrophage population (Iba1+/TMEM119−). These cells strongly expressed the phagocytosis marker CD68 and the lymphocyte activation, homing, and adhesion molecule CD44, specifically at sites of amyloid-beta plaques in the brains of APP-PS1 mice. In consequence, ablation of microglia significantly raised the number of CD3+/CD8+ T-cells and reduced the expression of anti-inflammatory genes in the brains of APP-PS1 mice.ConclusionWe conclude that in neurodegenerative conditions, chronically activated microglia might limit CD3+/CD8+ T-cell recruitment to the brain and that local macrophages connect innate with adaptive immune responses. Investigating the role of peripheral immune cells, their interaction with microglia, and understanding the link between innate and adaptive immune responses in the brain might be a future directive in treating AD pathology.

Effect of irradiation-induced intercellular adhesion molecule-1 expression on natural killer cell-mediated cytotoxicity toward human cancer cells

Background aimsIrradiation enhances the adhesion between natural killer (NK) cells and target cells by up-regulating intercellular adhesion molecule-1 (ICAM-1) on target cells. Therefore, we investigated the effect of irradiation-induced ICAM-1 expression on human cancer cells on NK cell–mediated cytotoxicity. MethodsExpression levels of ICAM-1 on the target cell surface before and after irradiation of six human cancer cell lines (HL60, SKBR-3, T47D, HCT-116, U937 and U251) were analyzed by flow cytometry. Ex vivo expansion of NK cells from human peripheral blood mononuclear cells was performed by co-culture with irradiated K562 cells. The related adhesion molecule lymphocyte function–associated antigen 1 (LFA-1) on NK cells was analyzed by flow cytometry. An enzyme-linked immunosorbent assay was used to detect interferon-γ (IFN-γ), and WST-8 assays were performed to check NK cell cytotoxicity. Finally, blocking assays were performed using monoclonal antibodies against ICAM-1 or LFA-1. ResultsLFA-1 expression increased on NK cells after expansion (P < 0.001). The expression of ICAM-1 was significantly upregulated by irradiation after 24 h in various cell lines, including HL60 (P < 0.001), SKBR-3 (P < 0.001), T47D (P < 0.001) and U937 (P < 0.001), although the level of expression depended on the cell line. ICAM-1 expression was extremely low before and after irradiation in U251 cells. NK cell–mediated cytotoxicity increased after irradiation of HL60 (P < 0.001), SKBR-3 (P < 0.001), T47D (P = 0.003), and U937 (P = 0.004) cells, in which ICAM-1 expression was significantly increased after irradiation. IFN-γ production by NK cells in response to HL60 (P < 0.001) and T47D (P = 0.011) cells significantly increased after irradiation. NK cell–mediated cytotoxicity against irradiated SKBR-3 (P < 0.001) and irradiated T47D cells (P = 0.035) significantly decreased after blocking of ICAM-1. Blocking of LFA-1 on NK cells resulted in reduced cytotoxicity against irradiated HL60 (P < 0.001) and irradiated SKBR-3 (P < 0.001). ConclusionsIrradiation upregulates ICAM-1 expression on the surface of human cancer cells and enhances activated NK cell–mediated cytotoxicity. Therefore, irradiation combined with NK cell therapy may improve the antitumor effects of NK cells.

Altered levels of soluble CD18 may associate immune mechanisms with outcome in sepsis.

The pathogenesis of sepsis involves a dual inflammatory response, with a hyperinflammatory phase followed by, or in combination with, a hypoinflammatory phase. The adhesion molecules lymphocyte function-associated antigen (LFA-1) (CD11a/CD18) and macrophage-1 (Mac-1) (CD11b/CD18) support leucocyte adhesion to intercellular adhesion molecules and phagocytosis through complement opsonization, both processes relevant to the immune response during sepsis. Here, we investigate the role of soluble (s)CD18 in sepsis with emphasis on sCD18 as a mechanistic biomarker of immune reactions and outcome of sepsis. sCD18 levels were measured in 15 septic and 15 critically ill non-septic patients. Fifteen healthy volunteers served as controls. CD18 shedding from human mononuclear cells was increased in vitro by several proinflammatory mediators relevant in sepsis. sCD18 inhibited cell adhesion to the complement fragment iC3b, which is a ligand for CD11b/CD18, also known as Mac-1 or complement receptor 3. Serum sCD18 levels in sepsis non-survivors displayed two distinct peaks permitting a partitioning into two groups, namely sCD18 'high' and sCD18 'low', with median levels of sCD18 at 2158 mU/ml [interquartile range (IQR) 2093-2811 mU/ml] and 488 mU/ml (IQR 360-617 mU/ml), respectively, at the day of intensive care unit admission. Serum sCD18 levels partitioned sepsis non-survivors into one group of 'high' sCD18 and low CRP and another group with 'low' sCD18 and high C-reactive protein. Together with the mechanistic data generated in vitro, we suggest the partitioning in sCD18 to reflect a compensatory anti-inflammatory response syndrome and hyperinflammation, respectively, manifested as part of sepsis.

Dynamic Patterns of Clonal Evolution in Tumor Vasculature Underlie Alterations in Lymphocyte-Endothelial Recognition to Foster Tumor Immune Escape.

Although tumor blood vessels have been a major therapeutic target for cancer chemotherapy, little is known regarding the stepwise development of the tumor microenvironment. Here, we use a multicolor Cre-dependent marker system to trace clonality within the tumor microenvironment to show that tumor blood vessels follow a pattern of dynamic clonal evolution. In an advanced melanoma tumor microenvironment, the vast majority of tumor vasculature clones are derived from a common precursor. Quantitative lineage analysis reveals founder clones diminish in frequency and are replaced by subclones as tumors evolve. These tumor-specific blood vessels are characterized by a developmental switch to a more invasive and immunologically silent phenotype. Gene expression profiling and pathway analysis reveals selection for traits promoting upregulation of alternative angiogenic programs such as unregulated HGF-MET signaling and enhanced autocrine signaling through VEGF and PDGF. Furthermore, we show a developmental switch in the expression of functionally significant primary lymphocyte adhesion molecules on tumor endothelium, such as the loss in expression of the mucosal addressin MAdCAM-1, whose counter receptor a4β7 on lymphocytes controls lymphocyte homing. Changes in adhesive properties on tumor endothelial subclones are accompanied by decreases in expression of lymphocyte chemokines CXCL16, CXCL13, CXCL12, CXCL9, CXCL10, and CCL19. These evolutionary patterns in the expressed genetic program within tumor endothelium will have both a quantitative and functional impact on lymphocyte distribution that may well influence tumor immune function and underlie escape mechanisms from current antiangiogenic pharmacotherapies.

Clinical value of detection of immune index and inflammatory reaction changes in patients with autoimmune disease.

Previous studies have shown a close correlation between the generation of B cell autoantibodies and imbalances in T lymphocyte subpopulations and the occurrence of disease. In this study, we have analyzed the effects of abnormal expression of CD4+CD25+-regulatory T cells, T lymphocyte subpopulations, immunoglobulins, complement factors, inflammatory factors, and adhesion molecules in the peripheral blood on the occurrence and development of autoimmune disease. Eighty patients with autoimmune disease were randomly (equally) divided into active-stage and stable-stage disease groups (according to pre-defined criteria). Fifty healthy people were recruited to the control group. The above-mentioned indices were detected by flow cytometry, immunity transmission turbidity, and enzyme-linked immunosorbent assay. We observed an obvious decrease in the CD4+CD25+- regulatory T cell, CD4+ cell, CD4+/CD8+ cell, NK cell, C3, and C4 expression in all three groups; however, this decrease was statistically significant in the active-stage group (P < 0.05). Alternately, we observed a significant increase in the expression of CD8+ cells, immunoglobulin (Ig) A, IgG, IgM, tumor necrosis factor-α, interleukin (IL)-10, IL-17, interferon-g, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin expression in the active-stage group (P < 0.05). Therefore, inflammatory reactions and immune dysfunction occurs during the active-stage of autoimmune disease, and detection of the immune indices and inflammatory and adhesion factors could help evaluate the immune stage in these patients, providing an experimental basis for the determination of disease progression and clinical treatment.

EAACI IG Biologicals task force paper on the use of biologic agents in allergic disorders

Biologic agents (also termed biologicals or biologics) are therapeutics that are synthesized by living organisms and directed against a specific determinant, for example, a cytokine or receptor. In inflammatory and autoimmune diseases, biologicals have revolutionized the treatment of several immune-mediated disorders. Biologicals have also been tested in allergic disorders. These include agents targeting IgE; T helper 2 (Th2)-type and Th2-promoting cytokines, including interleukin-4 (IL-4), IL-5, IL-9, IL-13, IL-31, and thymic stromal lymphopoietin (TSLP); pro-inflammatory cytokines, such as IL-1β, IL-12, IL-17A, IL-17F, IL-23, and tumor necrosis factor (TNF); chemokine receptor CCR4; and lymphocyte surface and adhesion molecules, including CD2, CD11a, CD20, CD25, CD52, and OX40 ligand. In this task force paper of the Interest Group on Biologicals of the European Academy of Allergy and Clinical Immunology, we review biologicals that are currently available or tested for the use in various allergic and urticarial pathologies, by providing an overview on their state of development, area of use, adverse events, and future research directions.

Interleukin-7 and anti-programmed cell death 1 antibody have differing effects to reverse sepsis-induced immunosuppression.

Sepsis remains a major cause of morbidity and mortality in most intensive care units. Protracted sepsis can evolve into a state of profound immunosuppression characterized by secondary infections, frequently with opportunistic-type pathogens. Immunoadjuvant therapy is currently being evaluated as a novel treatment for patients with sepsis. Two of the most promising immunoadjuvants are interleukin-7 (IL-7) and anti-programmed cell death 1 antibody (anti-PD-1). Both IL-7 and anti-PD-1 have been reported to boost host immunity and improve outcomes in patients with viral infections and cancer. The purpose of this study was to define the immunological mechanisms of action of IL-7 and anti-PD-1 in the two-hit sepsis model of cecal ligation and puncture followed by Candida albicans. In addition, we examined whether combined treatment with IL-7 and anti-PD-1 provided any additive beneficial effects in reversing immune dysfunction. The present findings demonstrated that IL-7 and anti-PD-1 had differing effects on innate and adaptive immune functions. Compared with anti-PD-1, IL-7 increased lymphocyte proliferation; expression of lymphocyte adhesion molecules, lymphocyte function-associated antigen 1, and very late antigen-4; interferon-γ production; and CD28 expression on splenic CD8 T cells. In contrast, anti-PD-1 seemed to have a greater effect on major histocompatibility complex class II expression on splenic macrophages and dendritic cells than IL-7. Combined treatment with IL-7 and anti-PD-1 produced additive effects on CD28 expression, lymphocyte proliferation, and splenic secretion of interferon-γ. In conclusion, the present study shows differences in immunomodulatory actions between IL-7 and anti-PD-1 and provides a potential rationale for combining IL-7 and anti-PD-1 in the therapy of sepsis.

Food for Thought: The Effects of Nutritional Support on Outcomes in Hospitalized Elderly Patients and the Critically Ill

The provision of adequate nutritional support appears to be essential for critically ill patients and other groups of patients at high risk for having malnutrition. In this review paper we describe our recent research regarding the amount of energy to be provided, how this should be assessed, and the beneficial effects of specialized nutritional support. We have shown that repeated measurements of energy expenditure using indirect calorimetry capture the dynamic energy changes characteristic of hospitalized patients. The provision of energy according to these measurements was associated with lower hospital mortality in critically ill patients when compared to patients receiving a fixed energy intake. A similar study performed in geriatric patients revealed a significant reduction in the number of infections when energy was provided according to repeated measurements. We have also shown that a diet enriched with eicosapentaenoic acid and gamma-linolenic acid improved oxygenation and lung dynamics and decreased ventilation duration in ICU patients with acute lung injury and ARDS. A similarly enriched diet together with micronutrients resulted in significantly less progression of existing pressure ulcers in ICU patients compared to an isonitrogenous, nutrient-sufficient formula. This may be related to an increase in the percentage of positive lymphocyte and granulocyte adhesion molecules.

Inflammatory activation and recovering BKV-specific immunity correlate with self-limited BKV replication after renal transplantation

As BKV-associated nephropathy has emerged as an important cause of allograft failure, it has been of major importance to find immune mechanisms suitable to identify kidney transplant recipients (KTRs) at increased risk of BKV replication. We monitored 29 KTRs with seven measurements during the first year post-transplantation. BKV-specific T cells directed to 5 BKV proteins were analyzed in an interferon-γ ELISPOT assay. BKV-specific antibodies were measured using an ELISA. The extent of immunosuppression and inflammatory activation were quantified by measures of immune function including lymphocyte subpopulations, IP-10, and adhesion molecule serum levels. All 5 BKV-specific T cells increased significantly from diagnosis to resolution of BKV replication (P<0.001). While antistructural T cells were significantly higher in KTRs with BKV replication (P<0.05), no differences were observed for antismall t- and large T-antigen-directed T cells (P>0.05). Interestingly, 65% of KTRs without BKV replication showed transient appearance of antismall t- and large T-antigen-directed T cells. Although no significant differences were observed for T-cell subpopulations and adhesion molecules, IP-10 levels increased significantly during BKV replication (P<0.05). Assessment of BKV-specific T cells identifies recovering BKV-specific immunity in KTRs with BKV replication and suggests their protective ability in KTRs without BKV replication. Increases in IP-10 levels stress the importance of infiltrating inflammatory leukocytes in the regulation of BKV replication and point to inflammatory activation in the pathogenesis of BKV replication.

Characterization Of a Novel In Vitro Circulation System Designed To Model The Migration Of Primary CLL Cells Across The Vascular Endothelium

There is growing evidence that lymphocyte trafficking contributes to the clinical course of chronic lymphocytic leukemia (CLL). However, no in vitro models exist to quantify migration from the peripheral vasculature or establish the key molecular events that drive this process. We have therefore established and characterized a novel dynamic in vitro model in which CLL cells are placed under shear forces equivalent to those experienced in the capillary beds and are made to flow through capillary-like hollow fibers lined with endothelial cells (HUVECs). Electronic microscopy demonstrated that HUVEC cells subjected to a shear force of 10 dynes/cm2 flattened against the wall of the hollow fibers giving confluent coverage and allowing formation of tight junctions in our system. Shear force was subsequently reduced to levels found in capillary beds (5 dynes/cm2) and CLL cells were introduced into the hollow fibers (106 CLL cells/ml). CLL cells circulated under these conditions increased their expression of selectin CD62L (associated with lymphocyte homing), CXCR4 (SDF-1 receptor) (both P<0.0001), lymphocyte adhesion molecule CD49d and the regulator of B cell receptor signaling, CD5 (both P=0.003) when compared to static cultures. We then demonstrated that CLL cells could migrate though the HUVEC-lined hollow fibers into the ‘extravascular' space (EVS) and that this was an active process as 2mm fluorescent polystyrene beads were unable to move from the circulating compartment of the system to the EVS. CLL migration out of circulation into the EVS increased over time with a mean percentage migration of 1.37% ± 2.32% after 48h (n=22). Furthermore, CLL cells that migrated had significantly higher expression of CD49d (P=0.02), MMP-9 (breaks down extracellular matrix, P=0.004), the signaling molecule and activation marker CD38 (P=0.009), the co-stimulatory molecule CD80 (P=0.04) and the early activation marker CD69 (P=0.04) when compared with CLL cells that remained in the circulation. Similar results were also seen using HBMEC-lined hollow fibers suggesting that these effects were not HUVEC specific. Addition of an SDF-1 chemokine gradient from the EVS towards the circulating compartment resulted in significantly increased migration of CLL cells into the EVS after just 1h (P=0.04, n=8). Importantly, the degree of migration observed was strongly correlated with CD49d expression (r2=0.47, P=0.01) and in keeping with previously published data we showed a strong correlation between CD49, CD38 and MMP-9. Given these findings, we investigated the effects on migration of the CD49d blocking antibody, Natalizumab. This resulted in a significantly decreased migration (P=0.01) confirming that CD49d plays a critical role in CLL cell migration. Taken together, we have developed a dynamic in vitro model system of the peripheral vasculature. Using this unique model, we have identified some of the key pathophysiological events that drive CLL cell migration in contrast to normal cell migration and have shown the utility of our dynamic model to further understand the biology of CLL and as a novel potential drug testing platform. Disclosures:No relevant conflicts of interest to declare.

Metal ions activate vascular endothelial cells and increase lymphocyte chemotaxis and binding

Metal on metal articulations in hip arthroplasty offer advantages, including lower volumetric wear compared to conventional metalonpolyethylene bearings, and increased resistance to dislocation. Reports described early failures, with histologic features similar to a Type IV immune response. Mechanisms by which metal wear products cause this reaction are not completely understood. We hypothesized a mechanism through direct activation of endothelial cells (ECs) by metal ions, resulting in both vasculitis and accumulation of lymphocytes without prior immune sensitization. Effects of metal ions were evaluated using human ECs in culture. Alterations in chemotactic proteins IL8 and MCP1 were assessed, as was upregulation of the adhesion molecule ICAM-1 and lymphocyte binding to ECs. Cobalt increased secretion of IL8 and MCP1 significantly, and upregulated the expression of ICAM-1 in ECs compared to stimulation by chromium and controls. Binding of lymphocytes to ECs and transEC migration were both significantly increased by cobalt but not chromium. These findings suggest that cobalt contributes more to the activation of ECs and lymphocyte binding than chromium without an allergic response. Some of the adverse tissue reactions to implants with components made of cobalt-chromium-molybdenium alloys may be due in part to activation of the endothelium by metal ions.

Dimethyl Fumarate Modulation of Immune and Antioxidant Responses: Application to HIV Therapy

The persistence of chronic immune activation and oxidative stress in human immunodeficiency virus (HIV)-infected, antiretroviral drug-treated individuals are major obstacles to fully preventing HIV disease progression. The immune modulator and antioxidant dimethyl fumarate (DMF) is effective in treating immune-mediated diseases and it also has potential applications to limiting HIV disease progression. Among the relevant effects of DMF and its active metabolite monomethyl fumarate (MMF) are induction of a Th1 to Th2 lymphocyte shift, inhibition of pro-inflammatory cytokine signaling, inhibition of NF-κB nuclear translocation, inhibition of dendritic cell maturation, suppression of lymphocyte and endothelial cell adhesion molecule expression, and induction of the Nrf2-dependent antioxidant response element (ARE) and effector genes. Associated with these effects are reduced lymphocyte and monocyte infiltration into psoriatic skin lesions in humans and immune-mediated demyelinating brain lesions in rodents, which confirms potent systemic and central nervous system (CNS) effects. In addition, DMF and MMF limit HIV infection in macrophages in vitro, albeit by unknown mechanisms. Finally, DMF and MMF also suppress neurotoxin production from HIV-infected macrophages, which drives CNS neurodegeneration. Thus, DMF might protect against systemic and CNS complications in HIV infection through its effective suppression of immune activation, oxidative stress, HIV replication, and macrophage-associated neuronal injury.

Measurement of Interferon-Gamma Induced Protein 10 in Serum: A Risk Assessment Approach for BKV-Associated Nephropathy

Since BKV-associated nephropathy has emerged as a major cause of allograft failure, it has been of importance to identify biomarkers to differentiate between kidney transplant recipients (KTRs) in three sets of clinical situations: (1) KTRs with an increased risk for BKV-reactivation, (2) KTRs with BKV-reactivation and an increased risk for BKV-associated nephropathy, (3) KTRs with BKV-associated nephropathy and an increased risk for progression to allograft failure. We monitored 10 KTRs with prolonged BKV-reactivation/BKV-associated nephropathy, 12 KTRs with self-limited BKV-reactivation, and 17 KTRs without BKV-reactivation in a longitudinal analysis. The extent of immunosuppression was quantified by measures of immune function including lymphocyte subpopulations, interferon-gamma induced protein 10 (IP-10), and adhesion molecules. BKV-specific T-cells were analyzed in an interferon-gamma Elispot assay. BKV-specific antibodies were measured in an ELISA. Although no differences were observed for lymphocyte subpopulations and adhesion molecules, KTRs with self-limited BKV-reactivation showed significantly higher IP-10 levels at the time of diagnosis (p< 0.001) and after recovery from BKV-reactivation (p=0.011) compared to KTRs with prolonged BKV-reactivation and BKV-associated nephropathy. While KTRs with BKV-associated nephropathy didn't show a significant increase of IP-10 levels from diagnosis to recovery from BKV-reactivation (p=0.468), BKV-specific T-cells increased significantly after therapeutic interventions. A cutoff level of 180pg/mL was able to identify KTRs with a risk for BKV-associated nephropathy at diagnosis of BKV-reactivation with a sensitivity of 92.3% and a specificity of 88.9%. Although anti-nonstructural T-cells are suggested as a marker of protection from BKV-reactivation, our data don't provide a characteristic profile to identify KTRs with an increased risk for BKV-reactivation. Assessment of IP-10 differentiates KTRs with an increased risk to progress to BKV-associated nephropathy from KTRs with self-limited BKV-reactivation as soon as at diagnosis of BKV-reactivation. Despite consistently low IP-10, assessment of BKV-specific T-cells identifies recovering BKV-specific immunity in KTRs with BKV-associated nephropathy.