Surface CD4 Expression Research Articles

All-Trans Retinoic Acid (ATRA) up-Regulates Cell Surface CD38 Expression Which Promotes Pro-Tumoral Mitochondrial Trafficking from Stromal Cells to Multiple Myeloma

BackgroundMultiple myeloma (MM) is malignancy highly reliant on its microenvironment. In this study, we investigated whether mitochondrial transfer occurred between bone marrow stromal cells (BMSC) and malignant plasma cells. We then used our observations as a platform to investigate the mechanisms controlling pro-tumoral mitochondrial transfer with a view to identifying druggable targets.MethodsPrimary MM cells were obtained from patients' bone marrow after informed consent and under approval from the United Kingdom Health Research Authority. Animal experiments were conducted under approvals from the UK Home Office and the University of East Anglia Animal Welfare and Ethics Review Board. Primary BMSC were also obtained from patient bone marrow, using adherence and characterised using flow cytometry. Mitochondrial transfer was assessed using two methods; a MitoTracker Green based staining of the BMSC (in-vitro), rLV.EF1.AcGFP-Mem9 labelling of the MM plasma membrane with MitoTracker CMXRos staining of the BMSC (in-vitro) and an in vivo MM NSG xenograft model. CD38 expression on MM cells was tested after ATRA treatment, using RT-qPCR and flow cytometry. Mitochondrial transfer levels were assessed when CD38 was over expressed using ATRA or inhibited using lentivirus targeted shRNA.ResultsWe report that mitochondria are transferred from BMSC to MM cells. First, we cultured MM cells on MitoTracker Green labelled BMSC and found increased MitoTracker Green fluorescence in the MM cells. We then transduced MM with rLV.EF1.AcGFP-Mem9 lentivirus and stained BMSC with MitoTracker CMXRos and used wide field microscopy to show MM derived tunnelling nanotubles (TNT) formed between MM cells and BMSC, with red mitochondria located within the GFP-tagged TNT. Next, we engrafted the MM cell lines MM1S and U266 into NSG mouse, after isolation we detected the presence of mouse mitochondrial DNA in the purified MM population. Together, these data show that mitochondria are transferred from BMSC to MM cells.We next analysed OXPHOS levels in MM cells grown on BMSC, using the seahorse extracellular flux assay. We found that the MM cells had increased levels of OXPHOS after culture with BMSC, which was also the case for MM cell lines analysed after isolation from NSG mice, showing the micro-environment of MM can alter the metabolism of the malignant cell. To examine whether the mitochondrial transfer process was controlled by CD38, we knocked down CD38 in MM cells using lentiviral targeted shRNA. We found reduced levels of mitochondrial transfer in CD38KD MM cells, with a consequent reduction of OXPHOS in the malignant cells.Finally, as ATRA has previously been shown to increase CD38 expression in AML, we next quantified CD38 mRNA and surface glycoprotein level on malignant plasma cells with and without ATRA treatment. We found ATRA increased CD38 expression at the mRNA and protein levels and this resulted in an increase in mitochondrial transfer from BMSC to MM cells.ConclusionHere we show that CD38 mediated mitochondrial transfer in the MM micro-environment forms part of the malignant phenotype of multiple myeloma. This finding develops our understanding of the mechanisms which underpin the efficacy of CD38 directed therapy in MM. DisclosuresNo relevant conflicts of interest to declare.

CAR antigens beyond recognition.

Orlando et al. and Ruella, Xu, Barrett et al. identified distinct mechanisms of resistance to anti-CD19 chimeric antigen receptor (CAR) T cell therapy in relapsed leukaemia patients, based on loss of CD19 surface expression.

Features of immunoregulation in patients with pulmonary tuberculosis with blood eosinophilia

The aimof the investigation was to determine the characteristics of the immune response regulation for pulmonary tuberculosis (TB) and to analyze the role of regulatory T cells in the immunopathogenesis of TB with eosinophilia in the blood, depending on the clinical form of the disease and sensitivity of Micobacterium tuberculosis to anti-TB drugs.Materials and methods.157 patients who were initially diagnosed with infiltrative and disseminated TB were examined. The material of the study was venous blood and culture of mononuclear leukocytes isolated from venous blood. The content of interleukin (IL) 4, IL-10 and transforming factor beta (TGFβ) in culture suspensions of mononuclear leukocytes in vitro and IL-5 in the blood was determined by enzyme-linked immunosorbent assay (ELISA) test. The expression of surface molecules CD4, CD20, CD25 and intracellular transcription factor Foxp3 by lymphocytes of the blood was evaluated by flow cytometry. The obtained results were analyzed by statistical methods.Results.It is shown that excessive generation of regulatory T cells in patients with TB is associated with eosinophilia of the blood and imbalance of immune response regulation mechanisms. In TB with eosinophilia, an increase in the number of Foxp3-positive regulatory T cells in the blood is combined with in vitro hypersecretion of anti-inflammatory cytokines TGFβ, IL-10, IL-4 and an increase in the content of CD20+ B lymphocytes and IL-5 in the blood. These changes are most pronounced in the disseminated form of TB in combination with drug resistance.Conclusion.Characteristics of immunoregulation at TB with blood eosinophilia are associated with activation of immunosuppression mechanisms and polarization of immune response towards Th2-dependent pathway.

Indolent hematodermic CD4+ T-cell lymphoma lacking surface CD3 expression: Structural and functional deficiency of T-cell receptor/CD3 complex

The expression of surface CD3 (sCD3) is often missing in T-cell neoplasms. We present two patients with indolent cutaneous T-cell lymphoma (CTCL) who had a clonal leukemic fraction of sCD3-, cCD3+, sCD4+ T-cell cells with less cell atypia. The cutaneous lesion of case 1 (60s, female) showed epidermotropic and perivascular infiltration of cCD3+, CD4+ T-cells, the features of which were consistent with those of mycosis fungoides, while in case 2 (50s, male), the diagnosis of CTCL was not confirmed histologically. In both cases, clonality analysis with T-cell receptor (TCR) gene rearrangement detected the same T-cell clones in the cutaneous lesions and leukemic cells. We further examined subclones of the neoplastic T cells infiltrating in the skin and blood, and the expression of TCR/ CD3 complex. Flow cytometric analysis revealed that the leukemic T-cells lost TCR/CD3 complex on the cell surface. Of the same T-cell clones, the CD62L+ subclone was mainly present in the blood, while the CD62L- one was infiltrating in the skin. Induction of activation markers such as CD69 and CD25 following CD3/CD28-stimulation was markedly diminished in the neoplastic T-cells, whereas PMA/ionomycin-mediated activation was normal. In conclusions, our observations indicate that TCR/CD3 complex is missing in the neoplastic cells, and the presence of CD62L+ and CD62L- subclones may account for the hematodermic involvement. We attribute our patients’ indolent clinical course partly to the deficiency of cell activation via TCR/CD3 complex. Based on our results, we propose a diagnosis of ‘indolent hematodermic TCR/CD3 complex-deficient lymphoma’ to our patients.

Dibutyl phthalate modulates phenotype of granulocytes in human blood in response to inflammatory stimuli

BackgroundPhthalates are plasticizers used in many common commercial products. They are ubiquitous environmental contaminants and epidemiological studies suggest that phthalate exposure is associated with development or worsening of airway diseases. Dibutyl phthalate (DBP) is a type of phthalate, found in high concentrations in indoor air, which appears to have a high inflammatory potential. In vitro studies on innate immune cells like macrophages have shown a reduction in phagocytic and antigen-presenting capacity and decreased production of stimuli-induced cytokines after DBP exposure. ObjectiveWe aimed to assess how DBP may alter the in vitro cellular and humoral innate immune response to inflammatory stimuli in blood innate immune cells. MethodsHuman whole blood was stimulated with inflammatory stimuli (lipopolysaccharide (LPS), resiquimod (R848) and phorbol 12-myristate 13-acetate (PMA)) in the presence or absence of DBP. The expression of surface markers CD16, CD24, CD69 and CD14 on granulocytes and monocytes was quantified by flow cytometry analysis. The release of TNFα, IFNγ, IL8 and IL10 cytokines was measured by ELISA. ResultsThe presence of DBP reduced the inflammatory stimuli-induced expression of CD24 on neutrophils and eosinophils and CD69 on activated eosinophils, whereas expression of CD16 on neutrophils was increased. DBP also had a dampening effect on the release of pro-inflammatory mediators TNFα and IFNγ in response to the inflammatory stimuli. ConclusionsThese responses may reflect an immunosuppressive effect of DBP through impairment of immune cell function.

Dynamics of CD25 Positive Myeloid Derived Suppressor Cells in Solid Organ Transplanted Patients and Relationship with Rejection

Backgound Myeloid derived suppressor cells (MDSC) are immature cells with immunosuppressive capacities. Three MDSC subsets are currently defined: monocytic, early stage, and polymorphonuclear MDSC (M-MDSC, eMDSC and PMN-MDSC respectively). While MDSC increase in cancer and chronic infections and associate with poor prognosis, their role in transplantation (Tx) is unknown. Changes in MDSC in transplant patients could provide biomarkers of graft evolution, and they could be useful as immunosuppressive therapy and/or to stimulate the allograft tolerance. Methods Peripheral blood MDSC were identified in cohorts of kidney (n=164) and liver (n=30) recipients and healthy volunteers (HV) as CD33+CD11b+HLA-DRlo/-. We characterized CD14+CD15- (M-MDSC) and CD14-CD15- (eMDSC) subsets. Suppression assays and measurement of surface CD25 expression (MFI) on MDSC were performed. Results Renal recipients MDSC were able to suppress CD4 and CD8 T cell proliferation in vitro. MDSC % were similar in pre-transplant, kidney transplant recipients (KTR), than in HV. However, MDSC and M-MDSC increased, mainly at 7 and 14 days post-transplant (3.48% and 3.85% vs 1.14%; 2.47% and 2.48% vs 0.24% p≤0,001 vs pre-transplant). The increase of MDSC in KTR with basiliximab as induction therapy was lower than in patients induced with thymoglobulin or without induction, and eMDSC were particularly decreased in basiliximab-treated patients (vs no induction, p≤0.05; vs thymoglobulin, p≤0.05). We confirmed that eMDSC expressed CD25, target of basiliximab. Pre-Tx, liver transplant recipients (LTR) had higher % of CD25+ eMDSC and higher CD25 MFI than KTR and HV. In LTR and KTR without basiliximab, % and MFI of CD25 in eMDSC increased after transplant. In KTR cohort, 7% of patients suffered acute rejection (AR). Absolute numbers of MDSC at 7 days post-transplant (measured before the rejection event) were significantly lower in AR than in non-rejectors (25.84 cells/uL vs 53.18 cells/uL respectively, p≤0,05). Conclusions MDSC and M-MDSC increase post-Tx except in patients receiving basiliximab. MDSC were lower in AR patients and low MDSC counts significantly preceded rejection. Transplant recipients eMDSC express CD25 which upregulates after Tx. Whether eMDSC express a complete and functional IL-2R is under investigation.

Overcoming Resistance of Human Non-Hodgkin's Lymphoma to CD19-CAR CTL Therapy by Celecoxib and Histone Deacetylase Inhibitors.

Patients with B-cell non-Hodgkin’s lymphoma (B-NHL) who fail to respond to first-line treatment regimens or develop resistance, exhibit poor prognosis. This signifies the need to develop alternative treatment strategies. CD19-chimeric antigen receptor (CAR) T cell-redirected immunotherapy is an attractive and novel option, which has shown encouraging outcomes in phase I clinical trials of relapsed/refractory NHL. However, the underlying mechanisms of, and approaches to overcome, acquired anti-CD19CAR CD8+ T cells (CTL)-resistance in NHL remain elusive. CD19CAR transduced primary human CTLs kill CD19+ human NHLs in a CD19- and caspase-dependent manner, mainly via the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) apoptotic pathway. To understand the dynamics of the development of resistance, we analyzed several anti-CD19CAR CTL-resistant NHL sublines (R-NHL) derived by serial exposure of sensitive parental lines to excessive numbers of anti-CD19CAR CTLs followed by a limiting dilution analysis. The R-NHLs retained surface CD19 expression and were efficiently recognized by CD19CAR CTLs. However, R-NHLs developed cross-resistance to CD19CAR transduced human primary CTLs and the Jurkat human T cell line, activated Jurkat, and lymphokine activated killer (LAK) cells, suggesting the acquisition of resistance is independent of CD19-loss and might be due to aberrant apoptotic machinery. We hypothesize that the R-NHL refractoriness to CD19CAR CTL killing could be partially rescued by small molecule sensitizers with apoptotic-gene regulatory effects. Chromatin modifiers and Celecoxib partially reversed the resistance of R-NHL cells to the cytotoxic effects of anti-CD19CAR CTLs and rhTRAIL. These in vitro results, though they require further examination, may provide a rational biological basis for combination treatment in the management of CD19CAR CTL-based therapy of NHL.

Inflammatory mediator ultra-low-molecular-weight hyaluronan triggers necrosis of B-precursor leukemia cells with high surface CD44 expression

Acute lymphoblastic leukemia (ALL) with mixed lineage leukemia (MLL) gene rearrangements (MLL+ALL) has a dismal prognosis and is characterized by high surface CD44 expression. Known that CD44 has the specific binding sites for a natural ligand hyaluronan (HA), we investigated biological effects of HA with different molecular sizes on MLL+ALL cell lines, and found that the addition of ultra-low-molecular-weight (ULMW)-HA strongly suppressed their thymidine uptakes. The MLL+ALL cell line lacking surface CD44 expression established by genome editing showed no suppression of thymidine uptake. Surface CD44-high B-precursor ALL cell lines other than MLL+, but not T-ALL cell lines, were also suppressed in their thymidine uptakes. The inhibition of thymidine uptakes was because of induction of cell death, but dead cells lacked features of apoptosis on cytospin smears and flow cytometric analysis. The cell death was neither blocked by pan-caspase inhibitor nor autophagy inhibitor, but was completely blocked by necrosis inhibitor necrostatin-1. Necrotic cell death was further supported by a marked release of a high-mobility protein group B1 and morphological changes on transmission electron microscopy. Elevation of intracellular reactive oxygen species production suggested a role for inducing this necrotic cell death. ULMW-HA-triggered cell death was similarly demonstrated in surface CD44-high primary B-precursor leukemia cells. Assuming that ULMW-HA is abundantly secreted at the site of infection and inflammation, this study sheds light on understanding the mechanism of a transient inflammation-associated remission of leukemia. Further, the CD44-targeting may become an effective approach in future for the treatment of refractory B-precursor ALL by its capability of predominantly eradicating CD44-high leukemia-initiating cells.

Target cell killing effects of CD20 targeting chimeric antigen receptor T cells derived from the type II anti-CD20 antibody.

e14548 Background: Chimeric Antigen Receptor T cells (CAR-Ts) targeting CD19 have shown very promising clinical outcomes in treatment of B-cell linage hematological malignancies. However, many patients with relapsed diseases were found to have down-regulated/loss of CD19 surface expression after CD19 CAR-T therapy. To solve this issue of CD19 single-targeting escape, we explored the application of another B-cell antigen, CD20, for targeted CAR-T therapy. Methods: We constructed four CD20 targeting CARs (all with 4-1BB co-stimulatory signaling) base on single-chain variable fragments (scFV) derived from four well-studied CD20 specific antibodies: Leu16, Rituximab, Obinutuzumab, and Ofatumumab. Leu16, Rituximab, and Obinutuzumab belong to the type I anti-CD20 antibody family and appear to bind to different epitopes located on the large loop of CD20, whereas Ofatumumab is the type II anti-CD20 antibody which has been shown to interact with the hydrophobic residues on the small loop surrounding a deep binding cleft. Results: All four CAR-T cells can specifically recognized CD20 positive target cells in our pre-clinical studies. They all showed up-regulated antigen-specific cell activation and high level of IFN-g release upon CD20 stimulation, and CAR-T20-Ofatumumab cells appeared to have significantly higher cell activation and more than 2-fold increase in IFN-g release compared to the other three CAR-T20 cells with their scFVs deriving from type I anti-CD20 antibodies. CAR-T20-Ofatumumab cells also showed higher degranulation upon stimulation, and it displayed ~50% of increase in ability to kill CD20 positive cells in cytotoxicity assays. Conclusions: Our data suggested that CAR-T20-Ofatumumab has better in vitro function and appears to be a CAR superior to those derived from other three antibodies. A possible explanation for this observation is that Ofatumumab interacts with the hydrophobic residues on the small loop, which is very close to cell membrane and confers more extensive binding to the small loop with striking slow off-rate. Our results suggest that CAR-Ts targeting CD20 with the scFVs from the type II anti-CD20 antibody may have superior cell killing effects.

N-glycan branching inhibits pro-inflammatory B cell activity by controlling TLR4 and BCR signaling.

Abstract N-glycosylation is a critical regulator of T cell function. The branching and number of N-glycans on cell surface glycoproteins drive binding to galectins, forming a galectin-glycoprotein lattice that controls T cell growth, differentiation and development by regulating receptor clustering, distribution and endocytosis. N-glycan branching is ~1.5–2 fold higher in B cells compared to T cells, yet effects on B cell function are unknown. Here we report that branching regulates pro-inflammatory signaling via Toll Like Receptor – 4 (TLR4) and the B cell receptor (BCR). Mechanisms controlling pro-inflammatory (cell surface) versus anti-inflammatory (endosomal) TLR4 signaling in B cells is poorly understood. Loss of branching in B cells de-couples LPS induced proliferative from pro-inflammatory responses by inhibiting TLR4 endocytosis. Similar de-coupling is observed with BCR signaling, where loss of branching inhibits BCR driven proliferation and Lyn/Syk/PLCγ2/Ca2+ signaling via reduced CD19 surface expression while concurrently driving pro-inflammatory effector functions and PI3K/Akt/mTOR activation. Together, these data reveal N-glycan branching as a critical negative regulator of TLR4 and BCR triggered pro-inflammatory B cell responses.

Melanocortin 1 Receptor Signaling Regulates Cholesterol Transport in Macrophages.

The melanocortin 1 receptor (MC1-R) is expressed by monocytes and macrophages, where it exerts anti-inflammatory actions on stimulation with its natural ligand α-melanocyte-stimulating hormone. The present study was designed to investigate the specific role of MC1-R in the context of atherosclerosis and possible regulatory pathways of MC1-R beyond anti-inflammation. Human and mouse atherosclerotic samples and primary mouse macrophages were used to study the regulatory functions of MC1-R. The impact of pharmacological MC1-R activation on atherosclerosis was assessed in apolipoprotein E-deficient mice. Characterization of human and mouse atherosclerotic plaques revealed that MC1-R expression localizes in lesional macrophages and is significantly associated with the ATP-binding cassette transporters ABCA1 and ABCG1, which are responsible for initiating reverse cholesterol transport. Using bone marrow-derived macrophages, we observed that α-melanocyte-stimulating hormone and selective MC1-R agonists similarly promoted cholesterol efflux, which is a counterregulatory mechanism against foam cell formation. Mechanistically, MC1-R activation upregulated the levels of ABCA1 and ABCG1. These effects were accompanied by a reduction in cell surface CD36 expression and in cholesterol uptake, further protecting macrophages from excessive lipid accumulation. Conversely, macrophages deficient in functional MC1-R displayed a phenotype with impaired efflux and enhanced uptake of cholesterol. Pharmacological targeting of MC1-R in atherosclerotic apolipoprotein E-deficient mice reduced plasma cholesterol levels and aortic CD36 expression and increased plaque ABCG1 expression and signs of plaque stability. Our findings identify a novel role for MC1-R in macrophage cholesterol transport. Activation of MC1-R confers protection against macrophage foam cell formation through a dual mechanism: It prevents cholesterol uptake while concomitantly promoting ABCA1- and ABCG1-mediated reverse cholesterol transport.

Flow cytometric single cell analysis reveals heterogeneity between adipose depots

ABSTRACTUnderstanding adipose tissue heterogeneity is hindered by the paucity of methods to analyze mature adipocytes at the single cell level. Here, we report a system for analyzing live adipocytes from different adipose depots in the adult mouse. Single cell suspensions of buoyant adipocytes were separated from the stromal vascular fraction and analyzed by flow cytometry. Compared to other lipophilic dyes, Nile Red uptake effectively distinguished adipocyte populations. Nile Red fluorescence increased with adipocyte size and granularity and could be combined with MitoTracker® Deep Red or fluorescent antibody labeling to further dissect adipose populations. Epicardial adipocytes exhibited the least mitochondrial membrane depolarization and highest fatty-acid translocase CD36 surface expression. In contrast, brown adipocytes showed low surface CD36 expression. Pregnancy resulted in reduced mitochondrial membrane depolarisation and increased CD36 surface expression in brown and epicardial adipocyte populations respectively. Our protocol revealed unreported heterogeneity between adipose depots and highlights the utility of flow cytometry for screening adipocytes at the single cell level.

Selection of single chain antibody fragments binding to the extracellular domain of 4-1BB receptor by phage display technology.

The 4-1BB is a surface glycoprotein that pertains to the tumor necrosis factor-receptor family. There is compelling evidence suggesting important roles for 4-1BB in the immune response, including cell activation and proliferation and also cytokine induction. Because of encouraging results of different agonistic monoclonal antibodies against 4-1BB in the treatment of cancer, infectious, and autoimmune diseases, 4-1BB has been suggested as an attractive target for immunotherapy. In this study, single chain variable fragment phage display libraries, Tomlinson I+J, were screened against specific synthetic oligopeptides (peptides I and II) designed from 4-1BB extracellular domain. Five rounds of panning led to selection of four 4-1BB specific single chain variable fragments (PI.12, PI.42, PII.16, and PII.29) which showed specific reaction to relevant peptides in phage enzyme-linked immunosorbent assay. The selected clones were successfully expressed in Escherichia coli Rosetta-gami 2, and their expression was confirmed by western blot analysis. Enzyme-linked immunosorbent assay experiments indicated that these antibodies were able to specifically recognize 4-1BB without any cross-reactivity with other antigens. Flow cytometry analysis demonstrated an acceptable specific binding of the single chain variable fragments to 4-1BB expressed on CCRF-CEM cells, while no binding was observed with an irrelevant antibody. Anti-4-1BB single chain variable fragments enhanced surface CD69 expression and interleukin-2 production in stimulated CCRF-CEM cells which confirmed the agonistic effect of the selected single chain variable fragments. The data from this study have provided a rationale for further experiments involving the biological functions of anti-4-1BB single chain variable fragments in future studies.

Inactivation of Rab11a GTPase in Macrophages Facilitates Phagocytosis of Apoptotic Neutrophils.

The timely and efficient clearance of apoptotic neutrophils by macrophages (efferocytosis) is required for the resolution of inflammation and tissue repair, but the regulatory mechanisms remain unclear. In this study, we investigated the role of the small GTPase Ras-related protein in brain (Rab)11a in regulating efferocytosis, and on this basis the resolution of inflammatory lung injury. We observed that apoptotic neutrophil feeding induced a rapid loss of Rab11a activity in bone marrow-derived macrophages and found that depletion of Rab11a in macrophages by small interfering RNA dramatically increased the phagocytosis of apoptotic neutrophils compared with control cells. Additionally, overexpression of wild-type Rab11a inhibited macrophage efferocytosis, whereas overexpression of dominant-negative Rab11a (Rab11a S25N) increased the clearance of apoptotic neutrophils. Rab11a knockdown also increased the surface level of CD36 in macrophages, but it reduced cell surface expression of a disintegrin and metalloproteinase (ADAM) 17. Depletion of ADAM17 rescued the decreased surface CD36 expression found in macrophages overexpressing wild-type Rab11a. Also, blockade of CD36 abolished the augmented efferocytosis seen in Rab11a-depleted macrophages. In mice challenged with endotoxin, intratracheal instillation of Rab11a-depleted macrophages reduced neutrophil count in bronchoalveolar lavage fluid, increased the number of macrophages containing apoptotic neutrophils, and prevented inflammatory lung injury. Thus, Rab11a inactivation in macrophages as a result of apoptotic cell binding initiates phagocytosis of apoptotic neutrophils via the modulation of ADAM17-mediated CD36 cell surface expression. Our results raise the possibility that inhibition of Rab11a activity in macrophages is a promising strategy for activating the resolution of inflammatory lung injury.

Abstract WP292: Insulin Resistance is Associated with a Reduction of Leukocyte Surface CD36 Expression and an Increase of Soluble CD36

Introduction: Diabetes is a major risk factor for ischemic stroke. Evidence suggests that the deficiency in CD36 is linked to insulin resistance in humans, yet patients with type 2 diabetes often display elevated plasma level of soluble CD36 (sCD36). However, cell type specific expression of CD36 is not well understood in the context of type 2 diabetes. Hypothesis: We hypothesize that insulin resistance is correlated with a reduction in the surface expression of CD36 among inflammatory cells, coincident with an increase in sCD36. Methods: Middle-aged db/db and db/+ mice (30-50 weeks-old) were subjected to left distal middle cerebral artery occlusion (dMCAO) or sham surgery. One, 3, or 7 days after stroke, leukocytes from blood and left hemisphere of the brain were analyzed by flow cytometry for cell type specific expression of CD36. The level of sCD36 in platelet free plasma was determined by ELISA. PPAR-γ agonist pioglitazone was administered at 2.5 mg/kg/day by oral gavage. Oral glucose tolerance test was performed at baseline and following two weeks of drug treatment. Both form of CD36 was also measured in 3-week-old mice without stroke. Results: Infiltrating neutrophils in brain peaked at 1 day after dMCAO, in contrast to a delayed but sustained increase of macrophages from 3 to 7 days post stroke. The adult db/db mice had a lower leukocyte surface expression of CD36 in both blood and brain, but had a higher level of sCD36 compared to age-matched db/+ mice whether at baseline or after stroke. The sCD36 is unlikely to exist in the form of extracellular vesicles (EV) since there were very few CD36(+) EVs detected in either type of mice. Prior to the development of obesity, 3-week-old db/db mice had similar leukocyte CD36 surface expression and plasma level of sCD36 compared to juvenile db/+ mice. Pioglitazone improved glucose sensitivity and enhanced macrophage CD36 surface expression in db/db mice after stroke. Conclusions: The reduced surface expression of CD36 on leukocytes and increased sCD36 was directly correlated with insulin resistance. Ongoing studies will determine the molecular event induced by obesity that triggers the cleavage of CD36 and how it affects macrophage function in the setting of diabetes.

IFITM3 Is a Central Regulator of Lipid Raft Signaling and Essential for CD19 Surface Expression and PI3K Signaling in Human B Cell Malignancies

Background & Hypothesis: IFITM3 (Interferon-induced transmembrane protein 3) was identified as interferon-inducible molecule in the context of viral infection. Endosomal membrane localized IFITM3 appear to prevent fusion events of intraluminal viral particles to the endosomal membrane through accumulation of cholesterol which makes membrane more rigid. We recently found that IFITM3 is a dual-pass transmembrane protein expressed on B cell lineage ALL cells. Thereby IFITM3 is associated with known B cell co-receptors including CD19, CD81 and CD21. While the significance of this association was unknown, we found that IFITM3 is required for surface expression of the B cell antigen CD19. Although immunotherapy approaches based on CD19-specific engineered chimeric antigen receptors (CART19) have achieved spectacular successes in eliminating pre-B ALL cells based on surface expression of CD19 (Grupp et al., 2013), in some cases, CD19-specific engineered chimeric antigen receptors (CART19) treatment was followed by ALL relapse developing from clones that lacked CD19 surface expression.Results: Studying IFITM3 mRNA levels in B cell lineage ALL cells at the time of diagnosis in clinical trials for childhood (COG P9906) and adult ALL (ECOG E2993), we found that higher than median expression levels of IFITM3 predicted shorter overall and relapse-free survival (P=0.014). In addition, higher than median IFITM3 mRNA levels at the time of diagnosis were associated with a higher risk of ALL relapse and positive MRD status at the end of induction chemotherapy.To study the function of Ifitm3 in a model for human pre-B ALL, pre-B cells from Ifitm3-/- mice were transformed with BCR-ABL1 or oncogenic NRAS. Strikingly, deletion of IFITM3 resulted in loss of CD19 expression on the surface of normal and leukemic pre-B cells. Besides loss of surface expression, loss of Ifitm3 also caused impaired phosphorylation of CD19-Y513, which mediates downstream activation of PI3K signaling in both normal and malignant B cells. These changes were paralleled by G0/1 cell cycle arrest (P<0.001), loss of colony formation capacity (P=0.0004) and increased propensity to apoptosis. In vivo transplant setting, Ifitm3-/- pre-B ALL cells failed to initiate fetal leukemia in transplant-recipient mice.In mechanistic study, we identified type II transmembrane topology for IFITM3 at plasma membrane with extracellular C and intracellular N terminus which interacted with CD19, LYN, SYK, PI3K and AKT. Disruption of endocytic motif (20YEML23) by substitution of Tyr20 to Phe caused accumulation of IFITM3 at plasma membrane and led to constitutive activation of CD19/PI3K-AKT signaling. In addition to the gain-of-function mutants, extracellularly exposed C terminus was further stimulated by agonistic antibodies against IFITM3, which triggers CD19/PI3K-AKT signaling, intracellular calcium mobilization, homotypic cellular aggregation and massively increased proliferation of pre-B ALL cells. Through Filipin based cholesterol staining, we found Ifitm3-/- pre-B cells have low levels of cholesterol at plasma membrane, which causes disruption of lipid rafts formation with decreased levels of ganglioside GM1. Thereby, Inadequate CD19/BCR co-receptor signaling caused by disruption of lipid rafts homeostasis by Ifitm3 deficiency results in critical developmental defects of peritoneal B1 cell compartment in vivo.Conclusion: These findings identify novel role of the IFITM3 surface receptor maintaining lipid rafts and CD19 surface expression. IFITM3-dependent lipid raft stability and CD19 surface expression were essential for normal and oncogenic PI3K signaling. IFITM3 is a central mediator of CD19 surface trafficking and mediates the proliferation and survival signaling via PI3K in normal pre-B cells and various subtypes of human pre-B ALL. DisclosuresNo relevant conflicts of interest to declare.

Harnessing the Tumor Microenvironment for the Treatment of Double Hit Lymphoma

Double Hit Lymphomas (DHL) constitute 6-9% of all newly-diagnosed Diffuse Large B-cell Lymphomas (DLBCL) and are defined by MYC translocation in concert with BCL2 (80%) or BCL6 (20%) rearrangement. DHL is characterized by poor upfront response to standard chemoimmunotherapies. The development of more effective treatment for DHL has been limited by the lack of models that accurately recapitulate the biology of this disease. To faithfully model bona fide human DHL, we generated two patient-derived xenografts (PDXs) in NSG mice from patients with DHL who relapsed after or were refractory to R-CHOP. Both PDXs had low/negative expression of surface CD20, in line with previous reports of DHL, and high surface expression of CD52. Importantly, these features recapitulated the original tumor phenotypes. We reasoned that immunologic approaches that target DHLs may help overcome their resistance to cytotoxic chemotherapy. Given the surface expression of CD52 on both PDXs, we treated xenografted mice with the anti-CD52 antibody Alemtuzumab (2x5mg/kg) when >2% tumor was detected in peripheral blood, a time corresponding to marked splenomegaly and ~30% bone marrow involvement. In both models, Alemtuzumab markedly reduced tumor involvement in the peripheral blood and spleen; however, the bone marrow was largely refractory to Alemtuzumab treatment. This is in line with previous results (Pallasch et al. Cell 2014) who also observed bone marrow-specific resistance to Alemtuzumab using a humanized transgenic model of DHL. In the latter model, Alemtuzumab resistance was successfully overcome by high-dose cyclophosphamide (CTX, 100mg/kg), which induced a tumor secretory response that induced macrophage-mediated phagocytosis of tumor cells. Thus, we treated both PDX models with vehicle, CTX 100mg/kg, Alemtuzumab or CTX+Alemtuzumab. While CTX monotherapy was able to partially reduce bone marrow tumor burden, in both PDXs, a single dose of CTX+Alemtuzumab was highly synergistic and eradicated nearly all bone marrow disease (Figure 1), which resulted in significantly extend murine survival compared to either CTX or Alemtuzumab alone alone (p<0.01). In contrast, high-dose Doxorubicin (DOX, 5mg/kg) had minimal effect on bone marrow DHL and DOX+Alemtuzumab was not synergistic. Strikingly, CD11b+F4/80+CD11c- macrophages comprised 20-40% of bone marrow cellularity 8 days after CTX treatment of xengorafted mice. DHL cells isolated from treated mice were next examined for changes in surface protein expression. At 16 hours after treatment, bone marrow DHL cells in mice receiving CTX (but not those receiving DOX or vehicle) had significantly reduced levels of surface CD47, the “don’t eat me” signal that suppresses macrophage phagocytosis. By 48 hours after treatment, bone marrow DHL cells in mice receiving CTX (but not those receiving DOX or vehicle) had significantly increased expression of the pro-phagocytic proteins CD31 and Calreticulin. Next, we performed cytokine array analysis on bone marrow from mice engrafted with DHL PDXs at 16 hours after treatment. CTX-treated (but not vehicle- or DOX-treated) bone marrow had significantly increased levels of human IL-16 and CCL4, cytokines known to promote monocyte recruitment, as well as VEGF, PDGF and TNFα. Ex-vivo phagocytosis assays using murine bone marrow-derived macrophages confirmed that recombinant human VEGF and TNFα each enhanced macrophage phagocytosis of Alemtuzumab coated Raji lymphoma cells. Findings from RNA-seq studies on tumor cells and macrophages from the spleen and bone marrow as well as therapy induced changes in these respective niches will be further discussed at the meeting. In summary, we have demonstrated that high-dose CTX induces a phenotypic and secretory response from human DHL cells in vivo that promotes phagocytosis of Alemtuzumab-opsinized cells. As a result, we are initiating a clinical trial of this combination in patients with relapsed/refractory DHL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

CD19 Epitope Escape after 4SCAR19 T Cell Therapy Resulted in Re-Establishment of Chemo-Sensitivity in Adult B-Cell Acute Lymphocytic Leukemia Patients

Background: CD19-targeted chimeric antigen receptor (CAR) T cell treatment has emerged as a highly effective therapy in patients with refractory B cell acute lymphoblastic leukemia (B-ALL). Despite obvious successes, there have been documented relapses in which CAR T cells are still present but the leukemia cells have lost CD19 surface epitopes. In this study, we aim to investigate these patients and characterize their leukemic cell clones.Methods: As part of a multi-center trial, this single-arm, phase I trial enrolled 10 patients between March 2015 and June 2016 based on the following criteria: poor physical condition, chemotherapy resistance, and lack of suitable donor. Following salvage chemotherapy, peripheral lymphocytes were collected from leukapheresis, and T cells were transduced with a 4th generation, safety-engineered, CD19scFv/CD28/CD137/CD27/ CD3zeta lentivector. The pre-infusion lymphodepleting chemotherapy conditioning is cyclophosphamide 800mg/m2 x 3 days, followed by infusions at a dose of 2.13 (range from 0.42 - 5.9) x106 4SCAR19 T cells per kg body weight . Patients who achieved complete remission (CR) but later relapsed received chemotherapy of VDP (vincristine 1.4mg/m2 d1,8,15,22, daunomycin 40mg/m2 d1,8,15,22, prednison 40mg/m2d1-28) regimen. CD19-negative relapsed patients were assessed by surface immunophenotyping, cytoplasm immunophenotyping ,bone marrow biopsy and immunohistochemistry staining, T cell receptor(TCR) and immunoglobulin heavy chain (IgH) rearrangement analysis.Results and Discussion: Total 10 patients received 4SCAR19 treatment and 7 achieved CR. Following CR, 5 relapsed, including 3 CD19 positive and 2 CD19 negative relapses. Surface marker analysis showed that the CD19-negative relapsed leukemia cells displayed altered surface immunophenotype different from the original CD19-positive leukemia cells. Comparing the leukemia cells of the two CD19-negative relapsed patients by immunophenotype analysis for CD19,CD10,CD34,and CD79a, the CD10 surface expression in one of the two CD19-negative leukemia cells was down-regulated from 90% to 10%, but cytoplasmic CD10 expression remained at 90% which was confirmed by immunohistochemical staining of bone marrow biopsy. The CD10 expression profile, however, did not alter in the other CD19-negative leukemia cells. The loss of surface CD19 expression was coupled with the loss of cytoplasm CD19 as determined by intracellular flow cytometry and immunohistochemical staining; however, the nuclear PAX-5 expression was positive. Another B-ALL index ,the IgH rearrangement was examined by multiplex PCR and their original clonal identify was confirmed. It is thus conceivable that the CD19 negative relapsed leukemia cells were associated with escape variants rather than selection of de novo leukemia clones following 4SCAR19 therapy. From the diverse immune phenotype of the CD19 negative relapsed leukemia cells, it should be feasible to select another surface epitope(s) as target(s) for CAR T therapy. Importantly, after CAR T therapy, the two CD19-negative relapsed patients responded to chemotherapy and achieved CR again. However, the three CD19-positive relapsed patients experienced inferior chemotherapy outcomes. These results suggest that chemo-sensitivity could be re-established after CD19-negative relapse following 4SCAR19 therapy. Other studies have found that CD19 mutations in CAR T-treated leukemic cells could result in the loss of PI3K and SFTK signaling, and further, altered signaling pathways such as p53 and PTEN mutations have been shown to contribute to chemo-resistance of leukemic cells. The forced pathway alteration in the CD19-negative leukemic clones after CAR T treatment could involve one of these pathways, and further investigation is warranted. DisclosuresNo relevant conflicts of interest to declare.

Rituximab Preferentially Eliminates BCR Signaling Proficient Chronic Lymphocytic Leukemia B Cells In Vivo

The use of anti-CD20 antibody rituximab has significantly improved the outcome of patients with chronic lymphocytic leukemia (CLL). Rituximab has been shown to act through several mechanisms including antibody-dependent cell cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), direct induction of apoptosis, and sensitization to chemotherapy. However, the exact contribution of each of these mechanisms to the clinical efficacy of rituximab in vivo and the exact mechanism of its action remain unclear. Importantly, the levels of cell surface CD20 expression were shown to associate with the efficacy of rituximab.We and others have described that CLL cells that have recently exited the lymph node microenvironment to the peripheral blood express lower cell surface levels of the chemokine receptor CXCR4 and higher levels of the activation marker CD5 (Callisano et al., 2011). We observed that these CLL cells (CXCR4dimCD5bright) have also higher cell surface CD20 expression (~2-fold; P<0.0001) compared to CLL cells from the same patient circulating in the blood stream for a longer time (CXCR4brightCD5dim), and we showed that CD20 is up-regulated by stromal cell interactions through the binding of SDF-1α to CXCR4 (P<0.001). We further hypothesized that higher levels of CD20 on CXCR4dimCD5bright cells will make them the primary target for rituximab in vivo. To investigate this, we obtained peripheral blood samples from CLL patients (N=17) before rituximab administration (day 0) and 24 hours (day 1) after rituximab administration (375 mg/m2, single agent). This showed that rituximab primarily and nearly completely eliminates the CXCR4dimCD5bright subpopulation (8.3 % pre-rituximab vs. 2.1 % post-rituximab, P<0.0001), which harbors the highest levels of CD20 (P<0.005).It has been previously suggested that the CD20 plays a direct role in microenvironmental interactions and especially in B cell receptor signaling (BCR; Uchida et al., 2004). Therefore, we investigated the BCR signaling propensity of CXCR4dimCD5bright subpopulation. We have observed that CXCR4dimCD5bright CLL cells have higher surface immunoglobulin (Ig) expression than CXCR4brightCD5dim cells from the same patient (~2-fold, P<0.005). This was also coupled with higher responsiveness of CXCR4dimCD5bright cells to BCR crosslinking with anti-IgM as measured by calcium flux (P=0.005). Moreover, CXCR4dimCD5bright cells also had higher levels of CD19 (1.8-fold, P<0.0001), which is an important docking molecule in PI3K signaling, and its higher levels lower the threshold for BCR signaling activation.Altogether, this study shows that rituximab primarily and effectively eliminates, at least in the short term, the BCR signaling proficient CLL B cells. It is likely that one of the mechanisms of rituximab action is the indirect inhibition of BCR signaling by eliminating CLL cells with strongly activated BCR pathway. These observations might have important implications for combinatorial therapies with BCR signaling inhibitors such as ibrutinib or idelalisib.This work was supported by: the Ministry of Education, Youth and Sports of the Czech Republic under the project CEITEC 2020 (LQ1601); the European Union's Horizon 2020 research and innovation programme under grant agreement No. 692298; Czech Science Foundation (project No. 16-13334Y); the Ministry of Health of the Czech Republic, grant No. 16-29622A. All rights reserved. This work was financed from the SoMoPro II Programme (project No. 4SGA8684), co-financed by European Union and the South-Moravian Region. This publication reflects only the author's views and the Union is not liable for any use that may be made of the information contained therein. This work was supported by the Ministry of Health of the Czech Republic - conceptual development of research organization (FNBr, 65269705, Sup 3/16), MUNI/A/1028/2015, and G.P. is a city of Ostrava scholarship holder. contact: marek.mraz@email.cz DisclosuresNo relevant conflicts of interest to declare.

Increased CD69 Expression on Peripheral Eosinophils from Patients with Food Protein-Induced Enterocolitis Syndrome

Background: Food protein-induced enterocolitis syndrome (FPIES) is an uncommon, non-IgE-mediated food allergy. We recently described a significant increase in fecal eosinophil-derived neurotoxin (EDN) after ingestion of the causative food. However, little is known about the activation status of circulating eosinophils in patients with an acute FPIES reaction. Methods: Surface CD69 expression was assessed by flow cytometry on peripheral eosinophils from 5 patients with FPIES before and after ingestion of the causative food. Fecal EDN was measured by enzyme-linked immunosorbent assay. Results: No eosinophil activation was observed before ingestion; however, a significant increase in CD69 expression on eosinophils after an acute FIPES reaction was demonstrated in all of the patients. There was no significant change in absolute eosinophil counts in the peripheral blood. The levels of fecal EDN increased on the day after ingestion of the causative food in all patients. Conclusion: These results suggest that circulating eosinophils as well as eosinophils in the intestinal mucosal tissue are activated in acute FPIES reactions and might be associated with systemic immune events in FPIES.