Surface Expression Of CD86 Research Articles

Mechanism and therapeutic implications of pomalidomide-induced immune surface marker upregulation in EBV-positive lymphomas

Epstein-Barr virus (EBV) downregulates immune surface markers to avoid immune recognition. Pomalidomide (Pom) was previously shown to increase immune surface marker expression in EBV-infected tumor cells. We explored the mechanism by which Pom leads to these effects in EBV-infected cells. Pom increased B7-2/CD86 mRNA, protein, and surface expression in EBV-infected cells but this was virtually eliminated in EBV-infected cells made resistant to Pom-induced cytostatic effects. This indicates that Pom initiates the upregulation of these markers by interacting with its target, cereblon. Interestingly, Pom increased the proinflammatory cytokines IP-10 and MIP-1∝/β in EBV infected cells, supporting a possible role for the phosphoinositide 3-kinase (PI3K)/AKT pathway in Pom’s effects. Idelalisib, an inhibitor of the delta subunit of PI3 Kinase, blocked AKT-Ser phosphorylation and Pom-induced B7-2 surface expression. PU.1 is a downstream target for AKT that is expressed in EBV-infected cells. Pom treatment led to an increase in PU.1 binding to the B7-2 promoter based on ChIP analysis. Thus, our data indicates Pom acts through cereblon leading to degradation of Ikaros and activation of the PI3K/AKT/PU.1 pathway resulting in upregulation of B7-2 mRNA and protein expression. The increased immune recognition in addition to the increases in proinflammatory cytokines upon Pom treatment suggests Pom may be useful in the treatment of EBV-positive lymphomas.

Effects of ruxolitinib on murine regulatory T cells are immune-context dependent

Aplastic anemia is a bone marrow failure (BMF) disorder characterized by pancytopenia and hypocellular marrow from an immune-mediated etiology. Regulatory T cells (Tregs) prevent autoimmunity by suppressing autoreactive T cells. We recently demonstrated the efficacy of ruxolitinib (RUX), a JAK 1/2 inhibitor, in attenuating murine BMF. Herein, we investigated the changes of Tregs in the context of RUX treatment for murine BMF. Tregs are conventionally identified by surface expression of CD4 and CD25, in addition to intracellular transcription factor FoxP3. RUX promoted the expansion of Tregs in BMF mice defined by increased expression of FoxP3 in CD4 T cells but suppressed expression of activation marker CD25 in CD4 and CD8 T cells. In this context, CD25 is no longer a reliable surface marker for Tregs. We observed strong co-expression of FoxP3 with surface marker GITR instead of CD25 in RUX-treated BMF mice. Fluorescence-activated cell sorting (FACS)-sorted CD4+GITRhi cells showed high FoxP3 expression and intact suppressive function in vitro, suggesting GITR to be a surrogate marker for Tregs. In contrast to its expansive effect on Tregs in BMF, RUX suppressed Tregs in normal and sublethal irradiation conditions, indicating that the effects of RUX on Tregs are immune-context dependent.

Electron transport chain and mTOR inhibition synergistically decrease CD40 signaling and counteract venetoclax resistance in chronic lymphocytic leukemia.

CD40 signaling upregulates BCL-XL and MCL-1 expression in the chronic lymphocytic leukemia (CLL) lymph node microenvironment, affording resistance to the BCL-2 inhibitor, venetoclax. Venetoclax resistance in the therapeutic setting and after long-term laboratory selection has been linked to metabolic alterations, but the underlying mechanism(s) are unknown. We aimed here to discover how CD40 stimulation as a model for tumor microenvironment-mediated metabolic changes, affects venetoclax sensitivity/resistance. CD40 stimulation increased oxidative phosphorylation and glycolysis, but only inhibition of oxidative phosphorylation countered venetoclax resistance. Furthermore, blocking mitochondrial import of pyruvate, glutamine or fatty acids affected CLL metabolism, but did not prevent CD40-mediated resistance to venetoclax. In contrast, inhibition of the electron transport chain (ETC) at complex I, III or V attenuated CLL activation and ATP production, and downregulated MCL-1 and BCL-XL, correlating with reduced CD40 surface expression. Moreover, ETC inhibition equaled mTOR1/2 but not mTOR1 inhibition alone for venetoclax resistance, and all three pathways were linked to control of general protein translation. In line with this, ETC plus mTOR inhibition synergistically counteracted venetoclax resistance. These findings link oxidative CLL metabolism to CD40 expression and cellular signaling, and may hold clinical potential.

Binding of respiratory syncytial virus particles to platelets does not result in their degranulation in vitro.

Respiratory syncytial virus (RSV) is a major cause of severe respiratory infection in infants and the elderly. The mechanisms behind severe RSV disease are incompletely understood, but a dysregulated immune response probably plays an important role. Platelets are increasingly being recognized as immune cells and are involved in the pathology of several viruses. The release of chemokines from platelets upon activation may attract, for example, neutrophils to the site of infection, which is a hallmark of RSV pathology. In addition, since RSV infections are sometimes associated with cardiovascular events and platelets express several known RSV receptors, we investigated the effect of RSV exposure on platelet degranulation. Washed human platelets were incubated with sucrose-purified RSV particles. P-selectin and CD63 surface expression and CCL5 secretion were measured to assess platelet degranulation. We found that platelets bind and internalize RSV particles, but this does not result in degranulation. Our results suggest that platelets do not play a direct role in RSV pathology by releasing chemokines to attract inflammatory cells.

#3777 ACTIVATION OF HYPOXIA-INDUCIBLE FACTORS IN ERYTHROID PROGENITOR CELLS RESULTS IN DEFECTIVE ERYTHROPOIESIS

Abstract Background and Aims Hypoxia-inducible factor prolyl hydroxylase inhibitor (HIF-PHI) activates HIF in renal pericytes and fibroblasts to promote erythropoietin production and erythropoiesis. However, the effects of HIF stabilization in the erythroid lineage are not clear. We aim to study the effects of erythroid lineage-specific stabilization of HIF on erythropoiesis by preclinical models. Method EporGFP-Cre/+;VhlF/F mice were used to achieve erythroid lineage-specific stabilization of HIF. Surface expression of TER-119, CD44, and forward scatter (FSC) were used to define erythroblast, reticulocyte, and red blood cell in the bone marrow. Expression of propidium iodide and surface annexin V were used to define apoptosis. Stress erythropoiesis was induced by subcutaneous administration of phenylhydrazine. Results Compared with littermate VhlF/F mice, EporGFP-Cre/+;VhlF/F mice had decreased hematocrit, decreased percentage of erythroblasts in the bone marrow, and increased apoptosis of erythroblasts in the bone marrow under steady state. These phenotypes of defective erythropoiesis were normalized in mice harboring concomitant Vhl, Hif1a, and Hif2a deletion (EporGFP-Cre/+;VhlF/F;Hif1aF/F;Hif2aF/Fmice). Although macrophages in the bone marrow also express Epor, macrophage-specific Vhl deletion in either Tg(Csf1r-CreESR1);VhlF/F or Lyz2Cre/+;VhlF/F mice did not result in defective erythropoiesis. During stress erythropoiesis, compared with littermate VhlF/F mice, EporGFP-Cre/+;VhlF/F mice had similar hematocrit, lower percentage of erythroblast in the bone marrow, and increased percentage of erythroblast in the spleen. Conclusion Vhl deletion in erythroid progenitor cells impairs erythropoiesis in murine bone marrow in the steady state. The phenotypes of defective erythropoiesis were partially reversed during stress erythropoiesis.

Increased expression of tyrosine protein phosphatase (CD45) on the surface of human blood granulocytes under the influence of the plague microbe vaccine strain <I>Yersinia pestis</I> EV NIIEG <I>ex vivo</I> and <I>in vivo</I>

Tyrosine protein phosphatase (common leukocyte antigen CD45) regulates FcᵧR-mediated cell signaling and secretory function of neutrophilic granulocytes (NG) when interacting with antigen-antibody immune complexes. The aim of the work is to study changes in the expression of CD45 on the surface of human granulocytes in ex vivo modeling of bacteremia by live cells of the plague microbe vaccine strain Yersinia pestis EV NIIEG and to evaluate the priming effect of the live plague vaccine (LPV) in vivo in terms of this parameter. The expression density of CD45 on NG was determined by flow cytometry in conventional units of fluorescence intensity (MFI) after staining the cells with the CD45-FITC labeled mouse antibody reagent (Backman Coulter, USA) during immunophenotyping of blood leukocytes according to the Lyse/No Wash protocol. In donors not previously vaccinated against plague (group 1), the value of the indicator was assessed before and 30 min, 2 h, 6 h after the addition of Y pestis EV cells to whole blood at a dose of 108 mc/ml, as well as 1 month and 6 months after the primary anti-plague vaccination. In individuals who had previously been repeatedly vaccinated with LPV in the territory of the natural plague focus (group 2), CD45 expression on blood granulocytes was determined one year after the last annual vaccination, and then 1 month and 6 months after revaccination. Getting into human blood, living cells of the vaccine strain Y pestis EV of the plague microbe induced a change in the NC phenotype already after 30 minutes, associated with a 3.5-fold increase in the surface expression of CD45, which remained at an elevated level for 6 hours. The studied indicator depended ex vivo on the degree of resistance of plague microbes to phagocytosis and killing of NG. Plague vaccination had a similar stimulating effect on human peripheral blood NG in vivo. Under the influence of HPV, CD45 expression increased on blood NG in groups 1 and 2 one month after vaccination, and the changes persisted in volunteers for 6 months. The experimental data obtained in the work may reflect the result of NG priming with lipopolysaccharide and other Y. pestis antigens. The registered functional activation of NG by expression of tyrosine protein phosphatase probably indicates the formation of “immune memory” at the level of innate immunity cells under the influence of LPV, the functioning of which explains the development of a faster and more intense antigen-specific immune response to repeated introduction of the plague vaccine into the body.

In vitro and in vivo evaluation of cerium oxide nanoparticles in respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is the most common cause of viral bronchiolitis among children worldwide, yet there is no vaccine for RSV disease. This study investigates the potential of cube and sphere-shaped cerium oxide nanoparticles (CNP) to modulate reactive oxygen (ROS) and nitrogen (RNS) species and immune cell phenotypes in the presence of RSV infection in vitro and in vivo. Cube and sphere-shaped CNP were synthesized by hydrothermal and ultrasonication methods, respectively. Physico-chemical characterization confirmed the shape of sphere and cube CNP and effect of various parameters on their particle size distribution and zeta potential. In vitro results revealed that sphere and cube CNP differentially modulated ROS and RNS levels in J774 macrophages. Specifically, cube CNP significantly reduced RSV-induced ROS levels without affecting RNS levels while sphere CNP increased RSV-induced RNS levels with minimal effect on ROS levels. Cube CNP drove an M1 phenotype in RSV-infected macrophages in vitro by increasing macrophage surface expression of CD80 and CD86 with a concomitant increase in TNFα and IL-12p70, while simultaneously decreasing M2 CD206 expression. Intranasal administration of sphere and cube-CNP were well-tolerated with no observed toxicity in BALB/c mice. Notably, cube CNP preferentially accumulated in murine alveolar macrophages and induced their activation, avoiding enhanced uptake and activation of other inflammatory cells such as neutrophils, which are associated with RSV-mediated inflammation. In conclusion, we report that sphere and cube CNP modulate macrophage polarization and innate cellular responses during RSV infection.

Evidence that platelets from transfusion-dependent β-thalassemia patients induce T cell activation

A hypercoagulable state leading to increased risk for thrombotic events represents one of the most common complications observed in transfusion-dependent β-thalassemia (TDT) patients. TDT patients have increased frequencies of circulating activated platelets. However, there is no information so far if platelets from TDT patients can activate T cells. In the present study we showed that T cells treated with platelets from TDT patients showed significant increased surface expression of CD69 compared to the T cells treated with platelets from healthy individuals. Patients with splenectomy showed increased T cell activation compared to patients with intact spleen. No T cell activation was observed following incubation with plasma alone, nor with platelets from healthy subjects. The percentages of regulatory T cells (Tregs) were also examined. TDT patients showed statistically significant increased percentages of Tregs compared to healthy controls. Additionally, we observed a positive statistically significant correlation between the percentages of Tregs and the platelet-induced activated T cells in patients who were not treated with aspirin. TDT patients showed increased levels of sP-selectin, suPAR and GDF-15, molecules implicated in platelet activation. We show that platelets from TDT patients can activate T cells in vitro. This activation correlates with markers of platelet activation and increased numbers of Tregs, perhaps in an effort to eliminate immune dysregulation, conceivably secondary to platelet activation.

Targeting Wnt/β-catenin signaling using XAV939 nanoparticles in tumor microenvironment-conditioned macrophages promote immunogenicity

The aberrant activation of Wnt/β-catenin signaling in tumor cells and immune cells in the tumor microenvironment (TME) promotes malignant transformation, metastasis, immune evasion, and resistance to cancer treatments. The increased Wnt ligand expression in TME activates β-catenin signaling in antigen (Ag)-presenting cells (APCs) and regulates anti-tumor immunity. Previously, we showed that activation of Wnt/β-catenin signaling in dendritic cells (DCs) promotes induction of regulatory T cell responses over anti-tumor CD4+ and CD8+ effector T cell responses and promotes tumor progression. In addition to DCs, tumor-associated macrophages (TAMs) also serve as APCs and regulate anti-tumor immunity. However, the role of β-catenin activation and its effect on TAM immunogenicity in TME is largely undefined. In this study, we investigated whether inhibiting β-catenin in TME-conditioned macrophages promotes immunogenicity. Using nanoparticle formulation of XAV939 (XAV-Np), a tankyrase inhibitor that promotes β-catenin degradation, we performed in vitro macrophage co-culture assays with melanoma cells (MC) or melanoma cell supernatants (MCS) to investigate the effect on macrophage immunogenicity. We show that XAV-Np-treatment of macrophages conditioned with MC or MCS significantly upregulates the cell surface expression of CD80 and CD86 and suppresses the expression of PD-L1 and CD206 compared to MC or MCS-conditioned macrophages treated with control nanoparticle (Con-Np). Further, XAV-Np-treated macrophages conditioned with MC or MCS significantly increased IL-6 and TNF-α production, with reduced IL-10 production compared to Con-Np-treated macrophages. Moreover, the co-culture of MC and XAV-Np-treated macrophages with T cells resulted in increased CD8+ T cell proliferation compared to Con-Np-treated macrophages. These data suggest that targeted β-catenin inhibition in TAMs represents a promising therapeutic approach to promote anti-tumor immunity.

Importance of circulating monocyte subpopulations and Fcγ receptors in immune thrombocytopenia.

Monocytes have been linked to the pathogenesis of immune thrombocytopenia (ITP) because of their role in autoantibody-mediated platelet phagocytosis. However, monocytes constitute unique populations with major differences in expression for surface Fcγ receptors (FcγRs). Thus, we evaluated monocytes in whole blood samples from patients with newly diagnosed and chronic ITP. Monocyte subpopulations were identified phenotypically by flow cytometry and defined according to the surface expression of CD14 (lipopolysaccharide receptor) and of CD16 (low-affinity Fcγ receptor III) into classical (CLM), intermediate (INTM) and nonclassical (non-CLM) monocytes. We also examined the expression of FcγRI/CD64 and FcγRIII/CD16 by monocyte subpopulations. Newly diagnosed patients showed a decrease in non-CLM, expressed as a relative percentage of total monocytes compared with controls and chronic ITP patients. Both non-CLM and INTM of newly diagnosed patients closely correlated with platelet count. These monocyte subpopulations showed significantly enhanced CD64 expression in newly diagnosed patients. On the contrary, patients with chronic ITP presented higher non-CLM in percentage than controls and concomitant lower CLM and total monocytes, in percentage and number. The expression of CD64 was increased by all monocyte subpopulations, CLM, INTM, and non-CLM in chronic patients. In conclusion, differences in monocyte subpopulations, together with enhanced expression of FcγRI/CD64 are evident in patients with ITP.

Investigating tissue-resident memory T cell differentiation of CAR T cells reveals an unexpected role for CD69

Abstract Adoptive cell therapy using chimeric antigen receptor-expressing (CAR) CD8+ T cells has revolutionized the treatment of liquid tumors; however, CAR T cells have had more limited success against solid tumors. CD8+ tissue-resident memory T cells (Trm), which are characterized by surface expression of CD69 and CD103 and a unique transcription factor circuit, are found within solid tumors and associated with improved tumor control. However, it is unclear whether CAR T cells adopt Trm features in the tumor microenvironment and how enhancing Trm differentiation of CAR T cells might impact their efficacy against solid tumors. Using the murine tumor model MC38, we identified both CD69+CD103- and CD69+CD103+ subsets that express Trm signature genes. To examine if CAR T cells follow a similar differentiation pattern, human carcinoembryonic antigen (hCEA)-specific CAR T cells with either a CD28 or 4-1BB costimulatory domain were transferred into hosts bearing MC38-hCEA tumors. We found the CD28 domain drove expansion of the CAR T population while delaying Trm marker expression compared to 4-1BB and non-CAR T cells. The persistence of a CD69-CD103-subset of CD28 CAR T cells strongly correlated with the number of hCEA-CD28 CAR T cells within the tumor. CD69-CD103- hCEA-CD28 CAR T cells were more proliferative and expressed lower levels of surface markers associated with dysfunction, including PD-1 and LAG3. We hypothesized CD69 could negatively regulate T cell proliferation and function; indeed, deletion of CD69 in hCEA-CD28 CAR T cells enhanced their proliferation and accumulation within tumors. In summary, hCEA-CD28 CAR signaling delayed Trm differentiation and CD69 expression in solid tumors resulting in prolonged T cell proliferation. NIH 7R01AI153096

Deletion of protein kinase D1 in myeloid lineage cells suppresses development of IFNγ +IL-17A +CD4 T cells and ameliorates hypersensitivity pneumonitis

Abstract Hypersensitivity pneumonitis (HP) is an extrinsic allergic alveolitis characterized by inflammation of the interstitium, bronchioles, and alveoli of the lung that leads to granuloma formation and ultimately irreversible fibrosis. Previous studies found that exposure to HP inciting antigen S. rectivirgula(SR) leads to the activation of protein kinase D1 (PKD1) in the mouse lung in vivoand that PKD1 contributes to the acute pulmonary inflammation, IL-17A expression in the lungs, and development of HP caused by repeated exposure to SR. In the present study, we investigated whether PKD1 in myeloid cells affects the pathogenic course of the SR-induced HP. Compared to control PKD1-sufficient mice, mice with deficiency of PKD1 in myeloid cells (PKD1mKO) showed significantly suppressed expression of IL-6, TNFα, IFNγ, CCL2, CCL3, CCL4, CXCL1, CXCL2, and CXCL10 and neutrophilic alveolitis after single intranasal exposure to SR. Substantially reduced levels of alveolitis and granuloma formation were observed in the PKD1mKO mice repeatedly exposed to SR. Surface expression of MHC-II and CD86 on myeloid cells, and expression of IFNγ, IL-17A, CCL20, CXCL9, and CXCL10 in lungs were significantly reduced in the PKD1mKO mice repeatedly exposed to SR. In addition, surface expression of CD69, CXCR3, and CCR6 on CD4 T cells and frequency of IFNγ +IL-17A +CD4 T cells in the lungs were significantly reduced in PKD1mKO mice repeatedly exposed to SR. Our results demonstrate PKD1 in myeloid lineage cells plays an essential role in the initial proinflammatory responses and neutrophil influx in the lung after exposed to SR and contributes to the development of IFNγ +IL-17A +CD4 T cells and HP caused by repeated exposure to SR. Supported by grants from NIH (R01 AR064723, R01 AR069010) and UTHSC.

CD25 recruits inhibitory phosphatases for feedback control of B-cell receptor signaling

Abstract Successful initiation of the B-cell receptor (BCR) signaling, and subsequent antigen-encounter in germinal centers are both marked by sharp increases of CD25 surface-expression. Likewise, oncogenic signaling in B-cell leukemia (B-ALL) and lymphoma induces CD25-surface expression. While CD25 is known as an IL2-receptor chain on T- and NK-cells, the significance of its expression on B-cells was unclear. We discovered that, rather than functioning as an IL2-receptor chain, CD25 expressed on B-cells feedback regulates BCR-signaling or its oncogenic mimics. Recapitulating phenotypes of genetic ablation of PKCD, SHIP1 and SHP1, conditional CD25-deletion suppressed early B-cell development but induced hyperactivation of antigen-experienced B-cells and autoimmunity. Additionally, B-cell-specific CD25 deletion resulted in formation of spontaneous germinal centers and expansion of autoreactive B cells. Biochemical and interactome studies revealed that BCR-signaling induced PKCd-dependent CD25-phosphorylation of the CD25 cytoplasmic tail (S268) to recruit inhibitory phosphatases to the BCR complex. Upon CD25-deletion, SHIP1 and SHP1 were no longer recruited and activated to limit duration and strength of BCR-signaling. Loss of phosphatase-function, autonomous BCR-signaling and Ca 2+-oscillations induced anergy and negative selection during early B-cell development, as opposed to proliferation and autoantibody production in antigen-experienced B-cells. These findings highlight the previously unrecognized role of CD25 in assembling inhibitory phosphatases to prevent chronic BCR signaling and safeguard B cell tolerance.

Abstract 896: Investigating tissue-resident memory T cell differentiation of CAR T cells reveals an unexpected role for CD69

Abstract Adoptive cell therapy using chimeric antigen receptor-expressing (CAR) CD8+ T cells has revolutionized the treatment of liquid tumors; however, CAR T cells have had more limited success against solid tumors. The tumor microenvironment can impair T cell function within solid tumors through a variety of mechanisms, including nutrient deprivation, hypoxia, and recruitment of immuno-suppressive cells. CD8+ tissue-resident memory T cells (Trm), which are characterized by surface expression of CD69 and CD103 and a unique transcription factor circuit, are found within solid tumors, important for tumor surveillance, and responsive to checkpoint inhibitor blockade. However, it is unclear whether CAR T cells adopt Trm features in the tumor microenvironment and how the nature of the CAR costimulatory domain could impact Trm differentiation and efficacy against solid tumors. To investigate Trm differentiation in solid tumors, we utilized murine the MC38 syngeneic graft tumor model and found three populations of tumor-infiltrating CD8+ T lymphocytes: CD69-CD103-, CD69+CD103- and CD69+CD103+. CD69+ populations downregulated circulating T cells genes and expressed Trm signature genes often found in non-tumor Trm cells. To examine if tumor infiltrating CAR T cells follow a similar differentiation pattern, human carcinoembryonic antigen (hCEA)-specific CAR T cells with either a CD28 or 4-1BB costimulatory domain were transferred into hosts bearing MC38-hCEA tumors. hCEA-CD28 CAR T cells accumulated in the tumor, while hCEA-41BB CAR T cells and T cells without a CAR were present at much lower numbers. hCEA-CD28 CAR T cells displayed delayed Trm differentiation with an increased proportion of CD69-CD103- cells when compared to hCEA-41BB CAR T cells or T cells without a CAR. While hCEA-CD28 CAR signaling drove T cell accumulation, there was significant heterogeneity in the degree of T cell expansion, and the size of CD69-CD103- population correlated strongly with the number of hCEA-CD28 CAR T cells within the tumor. CD69-CD103- hCEA-CD28 CAR T cells were more proliferative and expressed lower levels of surface markers associated with dysfunction, including PD-1 and LAG3. These observations suggested CD69 could be a negative regulator of T cell proliferation and function. Indeed, deletion of CD69 in hCEA-CD28 CAR T cells further enhanced proliferation, which resulted in greater numbers of CAR T cells in tumor. In summary, hCEA-CD28 CAR signaling prolonged T cell proliferation in solid tumors by delaying Trm differentiation and the upregulation of CD69, whose expression impairs T cell proliferation. Citation Format: Menglin Cheng, Tessa Bergsbaken. Investigating tissue-resident memory T cell differentiation of CAR T cells reveals an unexpected role for CD69 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 896.

Abstract 2900: To develop a next generation NK therapy with deep and durable response by engineered CD16 enhancing NK cytotoxicity

Abstract Introduction: Allogeneic NK cell-based therapies have been demonstrated to elicit clinical efficacy against hematopoietic malignancies. Unlike T-cell therapies, allogeneic NK cells do not cause toxicities such as serious cytokine release syndrome, neurotoxicity, or graft-vs-host-disease, and are less expensive. We believe NK cell-based therapies provide an important platform for “off-shelf” allogeneic cell therapies, so we designed a panel of arming strategies to enhance NK cytotoxicity and elicit deep and durable response. CD16a is a transmembrane protein that co-localizes with CD3ζ and FcεRIγ on NK cells and binds to the Fc portion of IgG antibodies. Upon ligation, it induces a potent series of signals resulting in cytokine production and cytotoxic effector activity via ADCC. Here we use the term CD16 to refer to CD16a on NK cells. CD16 is a potent activating receptor and is necessary for NK cell cytotoxic effector, so engineered CD16 is the cornerstone of NK enhancing strategy. CD16 displays a functional allelic dimorphism generating allotypes with either a phenylalanine (F) or a valine (V) residue at amino acid position 158. CD16VV is associated with higher ADCC activity and clinical response than CD16VF and CD16FF. However, only 15% of the population is CD16VV that maximizes ADCC. Several studies reported that tumor microenvironment may cause the shedding of CD16 which suppresses ADCC though ADAM17-mediated proteolytic cleavage upon NK cell activation. Therefore, selective inhibition of ADAM17 can reduce the shedding of CD16 in NK cells. Method: We designed a library of CD16 point mutations in the cleavage region of ADAM17 to screen for ADAM17-resistant CD16 mutations to prevent CD16 shedding and improve the efficacy of NK therapy. NK92 cells lacking expression of endogenous CD16 were transduced to express wild type CD16 or CD16 mutations, then activated with PMA. The surface expression of CD16 on these cells was then examined using flow cytometry. Result: We found 3 mutations effectively blocked CD16 cleavage in cell-based assays, while wild-type CD16 and other CD16 mutations underwent a marked down-regulation of expression. We also demonstrated these 3 mutations were resistant to cleavage when expressed in the human primary NK cells and did not disrupt IgG binding. Conclusion: We developed a non-cleavable CD16 with high affinity to Fc receptor, which can be expressed in engineered NK cells to develop a next generation NK therapy to elicit more potent and durable cytotoxic response. Citation Format: Cuiqing Yang, Fuwei Jiang, Yifang Wang, Tingting Liu, Qingyang Wang, Qin Wang, Gang Ye, Renhong Tang, Zhuoxiao Cao. To develop a next generation NK therapy with deep and durable response by engineered CD16 enhancing NK cytotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2900.

Abstract 6384: ATG-034, an LILRB4 antagonist antibody, reinvigorates dendritic cells and prevents tumor progression

Abstract Background: Dendritic cells (DCs) are vital for initiating antigen-specific T cell-dependent antitumor immune responses. However, emerging evidence reveals that local DCs within the tumor microenvironment (TME) are tolerized to facilitate immune evasion. Reprogramming of DCs is recognized as a promising strategy for tumor immunotherapy. Leukocyte immunoglobulin-like receptor B4 (LILRB4) is an inhibitory receptor belonging to the LILR family which is mainly expressed on normal myeloid cells and myeloid-derived malignant cells. It is upregulated on tolerogenic DCs (tolDCs), which exhibit low expression levels of costimulatory molecules and resistance to DC maturation. Targeting LILRB4 to reprogram tolDCs has been reported to be a promising strategy for cancer treatment. Here we report the preclinical development of an LILRB4 antagonist antibody, ATG-034. Method: The protein-based and cell-based binding affinity of ATG-034 were measured using SPR, ELISA and FACS analysis. Fc receptor (FcR) stimulation assay was used to evaluate the ability of ATG-034 to block the interaction of LILRB4 with its ligand fibronectin. ATG-034-mediated enhancement of antigen presentation and co-stimulation ability of DCs was determined by FACS analysis for the change of surface expression of HLA-DR, HLA-ABC, CD86, and CD206. The mixed lymphocyte reaction (MLR) assay was used to assess the immunomodulatory potential of ATG-034. The in vivo antitumor efficacy of ATG-034 was evaluated in a radiation therapy-resistant murine Lewis lung carcinoma (LLC) syngeneic model. Results: ATG-034 binds to LILRB4 protein with a single-digit nM affinity. It potently reversed the fibronectin-mediated inhibition of FcR-driven TNF-α cytokine production. TolDCs were reprogrammed by ATG-034 to immunogenic DCs with significant upregulation of the expression of HLA-DR, HLA-ABC, CD86 and downregulation of the expression of CD206. Furthermore, ATG-034 remarkably reinvigorated tolDCs for priming T cell activation with noticeable upregulation of the expression of CD25 and the secretion of IFN-γ, enhancing antitumor immunity. In addition, 10 mg/kg ATG-034 significantly inhibited the LLC tumor growth in vivo with a TGI of 40.28%, while a benchmark antibody only demonstrated a 24.43% TGI. Conclusion: Our data show that ATG-034 reprograms tolDC, enhancing anti-tumor immunity, and demonstrates potent in vivo anti-tumor efficacy. Therefore, ATG-034 may be a promising strategy for the treatment of cancer. Citation Format: Ao Sun, Enlin Zheng, Huili Cao, Mengshi Cao, Suya Bai, Peng Chen, Linjie Tian, Jay Mei, Bo Shan, Bing Hou. ATG-034, an LILRB4 antagonist antibody, reinvigorates dendritic cells and prevents tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6384.

Adiponectin Enhances Fatty Acid Signaling in Human Taste Cells by Increasing Surface Expression of CD36.

Adiponectin, a key metabolic hormone, is secreted into the circulation by fat cells where it enhances insulin sensitivity and stimulates glucose and fatty acid metabolism. Adiponectin receptors are highly expressed in the taste system; however, their effects and mechanisms of action in the modulation of gustatory function remain unclear. We utilized an immortalized human fungiform taste cell line (HuFF) to investigate the effect of AdipoRon, an adiponectin receptor agonist, on fatty acid-induced calcium responses. We showed that the fat taste receptors (CD36 and GPR120) and taste signaling molecules (Gα-gust, PLCβ2, and TRPM5) were expressed in HuFF cells. Calcium imaging studies showed that linoleic acid induced a dose-dependent calcium response in HuFF cells, and it was significantly reduced by the antagonists of CD36, GPR120, PLCβ2, and TRPM5. AdipoRon administration enhanced HuFF cell responses to fatty acids but not to a mixture of sweet, bitter, and umami tastants. This enhancement was inhibited by an irreversible CD36 antagonist and by an AMPK inhibitor but was not affected by a GPR120 antagonist. AdipoRon increased the phosphorylation of AMPK and the translocation of CD36 to the cell surface, which was eliminated by blocking AMPK. These results indicate that AdipoRon acts to increase cell surface CD36 in HuFF cells to selectively enhance their responses to fatty acids. This, in turn, is consistent with the ability of adiponectin receptor activity to alter taste cues associated with dietary fat intake.

KIT D816V Mast Cells Derived from Induced Pluripotent Stem Cells Recapitulate Systemic Mastocytosis Transcriptional Profile.

Mast cells (MCs) represent a population of hematopoietic cells with a key role in innate and adaptive immunity and are well known for their detrimental role in allergic responses. Yet, MCs occur in low abundance, which hampers their detailed molecular analysis. Here, we capitalized on the potential of induced pluripotent stem (iPS) cells to give rise to all cells in the body and established a novel and robust protocol for human iPS cell differentiation toward MCs. Relying on a panel of systemic mastocytosis (SM) patient-specific iPS cell lines carrying the KIT D816V mutation, we generated functional MCs that recapitulate SM disease features: increased number of MCs, abnormal maturation kinetics and activated phenotype, CD25 and CD30 surface expression and a transcriptional signature characterized by upregulated expression of innate and inflammatory response genes. Therefore, human iPS cell-derived MCs are a reliable, inexhaustible, and close-to-human tool for disease modeling and pharmacological screening to explore novel MC therapeutics.

ATP and its metabolite adenosine cooperatively upregulate the antigen-presenting molecules on dendritic cells leading to IFN-γ production by T cells

Dendritic cells (DCs) present foreign antigens to T cells via the major histocompatibility complex (MHC), thereby inducing acquired immune responses. ATP accumulates at sites of inflammation or in tumor tissues, which triggers local inflammatory responses. However, it remains to be clarified how ATP modulates the functions of DCs. In this study, we investigated the effects of extracellular ATP on mouse bone marrow-derived dendritic cells (BMDCs) as well as the potential for subsequent T cell activation. We found that high concentrations of ATP (1mM) upregulated the cell surface expression levels of MHC-I, MHC-II, and co-stimulatory molecules CD80 and CD86 but not those of co-inhibitory molecules PD-L1 and PD-L2 in BMDCs. Increased surface expression of MHC-I, MHC-II, CD80, and CD86 was inhibited by a pan-P2 receptor antagonist. In addition, the upregulation of MHC-I and MHC-II expression was inhibited by an adenosine P1 receptor antagonist and by inhibitors of CD39 and CD73, which metabolize ATP to adenosine. These results suggest that adenosine is required for the ATP-induced upregulation of MHC-I and MHC-II. In the mixed leukocyte reaction assay, ATP-stimulated BMDCs activated CD4 and CD8T cells and induced interferon-γ (IFN-γ) production by these T cells. Collectively, these results suggest that high concentrations of extracellular ATP upregulate the expression of antigen-presenting and co-stimulatory molecules but not that of co-inhibitory molecules in BMDCs. Cooperative stimulation of ATP and its metabolite adenosine was required for the upregulation of MHC-I and MHC-II. These ATP-stimulated BMDCs induced the activation of IFN-γ-producing T cells upon antigen presentation.

The fusion oncogene VAV1-MYO1F triggers aberrant T-cell receptor signaling in vivo and drives peripheral T-cell lymphoma in mice.

VAV1-MYO1F is a recently identified gain-of-function fusion protein of the proto-oncogene Vav guanine nucleotide exchange factor 1 (VAV1) that is recurrently detected in T-cell non-Hodgkin's lymphoma (T-NHL) patients. However, the pathophysiological functions of VAV1-MYO1F in lymphomagenesis are insufficiently defined. Therefore, we generated transgenic mouse models to conditionally express VAV1-MYO1F in T-cells in vivo. We demonstrate that VAV1-MYO1F triggers cell autonomous activation of T-cell signaling with an activation of the ERK, JNK, and AKT pathways. VAV1-MYO1F expression induces a T-cell activation phenotype with high surface expression of CD25, ICOS, CD44, PD-1, and decreased CD62L as well as aberrant T-cell differentiation, proliferation, and neoplastic transformation. Consequently, the VAV1-MYO1F expressing T-cells induce a malignant T lymphoproliferative disease with 100% penetrance in vivo that mimics key aspects of human peripheral T-cell lymphoma. These results demonstrate that the human T-cell oncogene VAV1-MYO1F is sufficient to trigger oncogenic T-cell signaling and neoplastic transformation, and moreover, it provides a new clinically relevant mouse model to explore the pathogenesis of and treatment concepts for human T-cell lymphoma.