Surface Expression Of CD86 Research Articles

Innate cell markers that predict anti-HIV neutralizing antibody titers in vaccinated macaques

SummaryGiven the time and resources invested in clinical trials, innovative prediction methods are needed to decrease late-stage failure in vaccine development. We identify combinations of early innate responses that predict neutralizing antibody (nAb) responses induced in HIV-Env SOSIP immunized cynomolgus macaques using various routes of vaccine injection and adjuvants. We analyze blood myeloid cells before and 24 h after each immunization by mass cytometry using a three-step clustering, and we discriminate unique vaccine signatures based on HLA-DR, CD39, CD86, CD11b, CD45, CD64, CD14, CD32, CD11c, CD123, CD4, CD16, and CADM1 surface expression. Various combinations of these markers characterize cell families positively associated with nAb production, whereas CADM1-expressing cells are negatively associated (p < 0.05). Our results demonstrate that monitoring immune signatures during early vaccine development could assist in identifying biomarkers that predict vaccine immunogenicity.

P06.07.A Natural killer cells lyse glioblastoma stem cells and increase their sensitivity to chemotherapy

Abstract Background Glioblastoma is the most common and lethal brain tumor in the adult population and immunotherapy is playing an increasingly central role in the treatment of many cancers. Nevertheless, the search for effective immunotherapeutic approaches for glioblastoma patients continues. In this study, we aimed to explore the therapeutic potential of allogeneic highly activated super-charged natural killer (NK) cells in glioblastoma. Material and Methods Chromium release- and calcein release-based cytotoxicity assays, ELISA, ELISPOT, and multiplex cytokine assays were used to determine NK cell cytotoxicity against glioblastoma stem cells (GSCs) and secretion of cytokines. Cell surface marker expression using flow cytometry and cell growth in vitro and in vivo were measured to determine GSC phenotype. NK cell killing and penetration in 3D were measured using confocal microscopy of GSC tumorospheres. Results Super-charged NK cells efficiently lysed patient-derived GSCs in 2D and 3D models potentially reversing the immunosuppression observed in patients. NK-cells secreted IFN-γ, upregulated GSC surface expression of CD54 and MHC class I and increased sensitivity of GSCs to chemotherapeutic drugs. Co-localization of NK cells with GBM cells in perivascular niches in glioblastoma tissues and their direct contact with GSCs in tumorospheres suggests their ability to infiltrate glioblastoma tumors and target GSCs. Conclusion Allogeneic super-charged NK cells appear to be a potential therapeutic approach for glioblastoma by selectively killing therapy-resistant cancer stem cell population, increasing their immune-related surface markers and enhancing their sensitivity to chemotherapy. Due to GSC heterogeneity and plasticity personalized immunotherapeutic strategies should be developed to effectively target glioblastomas.

MARCH1 Controls an Exhaustion-like Program of Effector CD4+ T Cells Promoting Allergic Airway Inflammation.

Persistent antigenic signaling leads to T cell exhaustion, a dysfunctional state arising in many chronic infections and cancers. Little is known concerning mechanisms limiting exhaustion in immune-stimulatory diseases such as asthma. We report that membrane-associated RING-CH1 (MARCH1), the ubiquitin ligase that mediates surface turnover of MHC class II (MHCII) and CD86 in professional APCs, plays an essential role in restraining an exhaustion-like program of effector CD4+ T cells in a mouse model of asthma. Mice lacking MARCH1 or the ubiquitin acceptor sites of MHCII and CD86 exhibited increased MHCII and CD86 surface expression on lung APCs, and this increase promoted enhanced expression of immune-inhibitory receptors by effector CD4+ T cells and inhibited their proliferation. Remarkably, ablation of MARCH1 in mice with established asthma reduced airway infiltration of eosinophils and Th2 cells. Thus, MARCH1 controls an exhaustion-like program of effector CD4+ T cells during allergic airway inflammation and may serve as a therapeutic target for asthma.

CRISPR/Cas9-medaited knockout of endogenous T-cell receptor in Jurkat cells and generation of NY-ESO-1-specific T cells: An in vitro study

Adoptive transfer of T-cell receptor (TCR)-engineered T cells has been successful in mediating favorable clinical outcomes. TCR-engineered T cells can be applied for targeting cancers whose associated antigens are intracellular and presented through major histocompatibility complexes (MHC). The mispairing of the exogenous TCR chains with the endogenous TCR chains leads to functionally impaired TCR-engineered T cells. The CRISPR/Cas9 genome-editing system can be utilized for the knockout of the endogenous TCR in T cells before introducing the exogenous TCR chains. In this study, we used the lentiviral delivery of CRISPR/Cas9 for disrupting the expression of the endogenous TCR in the Jurkat cell line. Next, an exogenous TCR targeting human leukocyte antigen (HLA)-A*0201-restricted New York esophageal squamous cell carcinoma 1 (NY-ESO-1) peptide was transduced into the TCR-knockout (KO) Jurkat cells. Further, we assessed lentiviral transduction efficacy using tetramer assay and evaluated the functionality of the NY-ESO-1-specific TCR-engineered T cells by quantifying the cell surface expression of CD69 upon co-cultivation with peptide-pulsed T2 cells. We successfully knocked out the endogenous TCR in ∼40% of the Jurkat cells. TCR-KO cells were selected and subjected to express NY-ESO-1-specific TCRs using lentiviral vectors. Flow cytometry analysis confirmed that up to 55% of the cells expressed the transgenic TCR on their surface. The functionality assay demonstrated that >90% of the engineered cells expressed CD69 when co-cultured with peptide-pulsed T2 cells. Conclusively, we developed a pipeline to engineer Jurkat cells using the state-of-the-art technique CRISPR/Cas9 and generated TCR-engineered cells that can become activated by a tumor-specific antigen.

KIR2DS2 Expression Identifies NK Cells With Enhanced Anticancer Activity.

NK cells are promising cellular therapeutics against hematological and solid malignancies. Immunogenetic studies have identified that various activating killer cell Ig-like receptors (KIRs) are associated with cancer outcomes. Specifically, KIR2DS2 has been associated with reduced incidence of relapse following transplant in hematological malignancies and improved outcomes in solid tumors, but the mechanism remains obscure. Therefore, we investigated how KIR2DS2 expression impacts NK cell function. Using a novel flow cytometry panel, we show that human NK cells with high KIR2DS2 expression have enhanced spontaneous activation against malignant B cell lines, liver cancer cell lines, and primary chronic lymphocytic leukemia cells. Surface expression of CD16 was increased on KIR2DS2high NK cells, and, accordingly, KIR2DS2high NK cells had increased activation against lymphoma cells coated with the clinically relevant anti-CD20 Abs rituximab and obinutuzumab. Bulk RNA sequencing revealed that KIR2DS2high NK cells have upregulation of NK-mediated cytotoxicity, translation, and FCGR gene pathways. We developed a novel single-cell RNA-sequencing technique to identify KIR2DS2+ NK cells, and this confirmed that KIR2DS2 is associated with enhanced NK cell-mediated cytotoxicity. This study provides evidence that KIR2DS2 marks a population of NK cells primed for anticancer activity and indicates that KIR2DS2 is an attractive target for NK-based therapeutic strategies.

Peptidyl arginine deiminase expression and macrophage polarization following stimulation with citrullinated and malondialdehyde-acetaldehyde modified fibrinogen

ObjectivePost-translational modifications of extracellular matrix proteins such as fibrinogen may lead to tolerance loss and have been implicated in rheumatoid arthritis (RA) pathogenesis. The purpose of this study was to determine whether fibrinogen (FIB) modified with citrulline (CIT), malondialdehyde-acetaldehyde (MAA) or both leads to altered macrophage polarization, peptidyl arginine deiminase (PAD) expression, or production of citrullinated proteins. MethodsPMA-treated U-937 cells (M0 cells) were stimulated with MAA, CIT or MAA-CIT modified FIB. Macrophage (M1/M2) phenotypes were evaluated by flow cytometry, RT-PCR, and ELISA. PAD enzyme expression and protein citrullination was evaluated using RT-PCR and Western Blot. ResultsFlow cytometry revealed that M0 macrophages stimulated with FIB-MAA-CIT resulted in mixed M1/M2 phenotypes as demonstrated by cell surface expression and mRNA levels of CD14, CD192, CD163, and CD206 (p < 0.001 vs. others), and the release of IL-18, IP-10, CCL22, and IL-13 (p < 0.001 vs. others). While FIB-MAA treated M0 cells demonstrated a mixed M1/M2 phenotype, cytokine and cell surface markers differed from FIB-MAA-CIT. Finally, M0 cells treated with FIB-CIT demonstrated markers and cytokines consistent with only the M1-like phenotype. Exposure of M0 cells to FIB-MAA-CIT (at 48 h) and FIB-MAA (at 24 h) led to increased mRNA expression and protein expression of PAD2 (p < 0.001) with increased protein citrullination. ConclusionThese findings suggest that MAA-modification and citrullination of FIB, in isolation or combination, yield specific effects on macrophage polarization, PAD expression and citrullination that ultimately may induce inflammatory and fibrotic responses associated with RA.

Resveratrol and ω-3 PUFAs Promote Human Macrophage Differentiation and Function.

Monocytes differentiate into M1 and M2 macrophages, which are classically activated by microbial products such as LPS or IFN-γ and interleukins (e.g., the anti-inflammatory and Th2 promoting IL-4), respectively. The contribution of nutrients or nutrient-based substances such as ω-3 polyunsaturated fatty acids (ω-3 PUFAs) and resveratrol (Res) on the differentiation and function of M1 and M2 macrophages was evaluated. THP-1 cells and peripheral blood mononuclear cells (PBMCs) were differentiated into M1 and M2 cells and activated with LPS/IFN-γ or IL-4/IL-13. Macrophage lineage specific surface determinants (e.g., CD11b, CD11c, CD14, CD206, CD209, CD274, HLA-DR, CCR7, CCR2) were analysed by cytofluorometry. Res and ω-3 PUFAs altered CD14, CD206, CD274 and HL-DR surface expression patterns in M1 and M2 macrophages differentiated from PBMC. LPS/IFN-γ or IL-14/IL-13 activated macrophages subpopulations, which secreted cytokines and chemokines as measured by multiplex ELISA. Res and ω-3 PUFA reduced IL-1β, IL-6, TNF-α, CXCL10/IP-10, CCL13/MCP-4 and CCL20/MIP-3α in LPS/IFN-γ activated human leukaemia THP-1 cells, which is indicative of a dampening effect on M1 macrophages. However, Res increased M1 prototypic cytokines such as IL-1β or IL-6 in macrophages derived from PBMCs and also modified the expression of IL-12p70. Collectively, Res and ω-3 PUFAs distinctly promoted the differentiation and function of M1 and M2 macrophages. We conclude that these substances strengthen the macrophage-mediated effects on the innate and adaptive immune response.

P612: CHRONIC LYMPHOCYTIC LEUKEMIA (CLL)-DERIVED T-CELLS HAVE DISTURBED FATTY ACID METABOLISM, POSSIBLY CONTRIBUTING TO T-CELL DYSFUNCTION

Background: T-cells from CLL patients are dysfunctional and display reduced activation, proliferation and cytotoxicity upon in-vitro activation. Whilst the role of glucose metabolism in activated T cells is extensively studied, the contribution of other energy sources is less clear. Fatty acid (FA) and lipid uptake in T cells involves the scavenger membrane transporter CD36. CD36-mediated lipid transport has been identified to (metabolically) modulate intratumoral T cells (both Treg and cytotoxic CD8+ T cells). However, lipid metabolism and a potential role for CD36 has not been studied in the context of T cell dysfunction in CLL Aims: To examine the role of FA metabolism and the fatty acid transporter CD36 in acquired T-cell dysfunction in CLL. Methods: Untreated CLL patients with a white blood cell count (WBC) greater than 20×109 cells/L and, as control, age-matched healthy donors (HD) were studied. T cells were analyzed by flow cytometry, confocal microscopy or extracellular flux analysis (Seahorse), either directly after thawing or after a 2 day culture with or without αCD3/αCD28 antibodies. Results: Analysis of CD36+ vs CD36- CD4 and CD8 T-cells from HD revealed that CD36+ cells had higher long-chain fatty acid (LCFA) and glucose uptake, and higher expression of the glucose transporter GLUT-1 and the rate-limiting mitochondrial FA transporter CPT1α (Fig. 1A). The cells expressing CD36 were also more likely to increase their CD25 expression and mitochondrial mass upon stimulation, indicating a metabolic and functional advantage potentially conferred by the surface expression of CD36. The absolute amount and percentage of T cells (CD4 and CD8) expressing FA transporter CD36 was lower in CLL T-cells as compared to HD (Fig. 1B). The levels of LCFA uptake and expression of CPT1α were comparable, indicating that the lower amount of CD36 might successfully cater to the FA demands of resting CLL T cells(Fig. 1C). However, while stimulation of HD T-cells markedly upregulated CD36 abundance, LCFA uptake and CPT1α, concomitantly with the activation marker CD25, CLL-derived T-cells failed to mount a similar response (Fig. 1D). This lack of induction of CD36 expression was also observed in CLL-derived T cells that increased CD25, which implies that this response is independent of T-cell activation. The transcription factors peroxisome proliferator-activated receptors (PPAR) α and γ are important regulators of FA metabolism. We observed reduced expression of these transcription factors in resting CLL T cells. However, distinct from CD36, CLL T cells were indistinguishable from HD T-cells in their ability to increase levels of both PPARα and PPARγ upon stimulation, indicating that alternative mechanisms might be implicated in the regulation of CD36 and FA metabolism in CLL T cells. Image:Summary/Conclusion: Collectively, our results show that disturbances in FA metabolism are present on multiple levels in CLL T cells and that lack of LCFA uptake into CLL T-cells could represent a limiting step in FA metabolism. We also highlight CD36+ cells as a metabolically distinct T-cell subtype that is largely absent in CLL T-cells, independently of their activation status. We are currently investigating strategies to increase CD36 expression and FA metabolism in general in CLL T cells, which could ameliorate CLL T cell dysfunction and provide targets to improve autologous T cell therapies.

Co-Culture of THP-1 Cells and Normal Human Epidermal Keratinocytes (NHEK) for Modified Human Cell Line Activation Test (h-CLAT)

To improve the accuracy of skin sensitization prediction of chemicals by conventional alternative methods using cells, it is important to reproduce the environment of skin in vitro, such as the crosstalk between keratinocytes and dendritic cells (DCs). We developed a skin sensitization test system based on the markers and criteria of the human cell line activation test (h-CLAT), which combines THP-1 cells as a surrogate for DCs and keratinized normal human epidermal keratinocytes (NHEK). After exposure to chemicals via keratinized NHEK, the cell surface expression of CD54 and CD86 on THP-1 was measured by flow cytometry. This co-culture system evaluated 2,4-dinitrochlorobenzene (DNCB), a typical sensitizer, as positive, lactic acid (LA), a non-sensitizer, as negative, and isoeugenol (IE), a prohapten that requires biological activation to acquire skin sensitization, as positive. However, the expression levels of CD54 and CD86 in DNCB-treated THP-1 were lower than those in normal h-CLAT. Therefore, we investigated the effects of the medium and secretion by NHEK cells on THP-1 cells. CD54 and CD86 expression was enhanced in monocultured THP-1 in the medium for keratinized NHEK and in the conditioned medium of keratinized NHEK. The increase in CD54 and CD86 by changes in the medium type was higher than that by the NHEK secretion; therefore, it was found that the medium composition has a large effect on the evaluation index among the experimental parameters in the co-culture system. It is necessary to find the optimal medium for immunotoxicity assessment in the co-culture system.

Abstract 1353: Immunomodulatory effects of valproic acid in combination with cisplatin and cetuximab in head and neck squamous cell carcinoma

Abstract INTRODUCTION. Development of immune-check-point inhibitors (ICI) has greatly changed the treatment of recurrent or metastatic (R/M) head and neck squamous cell carcinoma (HNSCC) demonstrating durable antitumor response and overall survival (OS) improvement. However, more than 70% of patients do not respond to ICI and, recently, phase III Checkmate 651 trial did not demonstrate OS improvement of first-line ICI vs standard EXTREME regimen (cetuximab (CX) plus 5-fluorouracil/cisplatin (CDDP) in R/M HNSCC patients (ESMO2021-LBA36). We have previously showed in vitro and in vivo models of HNSCC the synergistic antitumor interaction of CDDP/CX combined with the antiepileptic valproic acid (VPA), a histone deacetylase inhibitor (HDACi) with anticancer and immunomodulatory properties (Iannelli F., 2020). Here we investigate the immunomodulatory effects of VPA/CDDP/CX combination, focusing on the induction of "immunogenic cell death" (ICD)-like features. METHODS. ICD-related damage-associated molecular patterns (DAMPs) calreticulin (CARL), ATP, HMGB1 and Annexin-A1 (ANXA1) expression was evaluated by western blot, immunofluorescence and/or bioluminescent assays on HNSCC Cal27 and osteosarcoma U2OS cells. Maturation, migration, cytokines release, and phagocytic capacity of dendritic cells (IL4_DCs) was evaluated incubating IL4-DCs with conditioned medium from untreated/treated Cal27 cells and/or in co-culture IL4-DCs/Cal27 assays by ELISA, cytofluorimetric assays and confocal microscopy. RESULTS. VPA/CDDP/CX combination induced in both U2OS and Cal27 cells, synergistic pre-apoptotic exposure of CARL along with HMGB1, ANXA1 and ATP release as compared with single drugs or doublet combination. Moreover, the triple combination increases IL4-DCs migration towards pretreated Cal27 cells as well as the rate of IL4-DCs phagocytosis of apoptotic bodies released by Cal27 dying cells. Triple combination also induces phenotypical maturation of DCs as shown by the increased surface expression of CD83 and CD86. Furthermore, conditioned medium from VPA/CDDP/CX treated Cal27 cells, induced increased release of immune-stimulating cytokines and chemokines IL2, IL1β, TNFα, MIP-1α and G-CSF by IL4-DCs as well as the impairment of immunosuppressive cytokines release IL-10 and VEGF, by VPA/CDDP/CX treated Cal27, co-cultured with IL4-DCs. In vivo experiments in immunocompetent syngeneic mouse model using HNSCC AT-84 cells transduced with human-EGFR are ongoing. CONCLUSION. Overall, we demonstrated that VPA/CDDP/CX is able to enhance the immunogenicity of HNSCC model by inducing ICD. This mechanistic hypothesis will be tested by assessing ICD biomarkers on samples of patients enrolled in the V-CHANCE phase 2 clinical trial evaluating the CDDP/CX associated with VPA in R/M SCCHN patients and recently concluded (NCT02624128). Citation Format: Federica Iannelli, Andrea Ilaria Zotti, Maria Serena Roca, Laura Grumetti, tania Moccia, carlo vitagliano, susan costantini, francesca capone, francesca collina, Lucia Gabriele, stefania parlato, Giulia Romagnoli, Oliver Kepp, Guido Kroemer, Elena Di Gennaro, Alfredo Budillon. Immunomodulatory effects of valproic acid in combination with cisplatin and cetuximab in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1353.

Abstract 5473: Isoform-selective HDAC inhibition up-regulates CD26 expression on multiple myeloma cells and augments cytotoxic efficacy by humanized monoclonal antibody

Abstract Objective: CD26, a 110-kDa transmembrane glycoprotein, which is expressed on several tumor cells including malignant lymphoma, has been implicated in tumorigenesis, whereas, its roles in plasma cell malignancies remain elusive. Recently, we have identified that CD26 is uniformly and intensely expressed in osteoclasts, while its expression in plasma cells of patients with multiple myeloma (MM) reveals heterogenous. Decreased expression levels of CD26 in MM cells are one of the mechanisms underlying resistance to humanized anti-CD26 monoclonal antibody (mAb) therapy. In the present study, we clarify the impact of epigenetic modification by HDAC inhibition (HDACi) with isoform-selective or broad inhibitors on the regulation of CD26 in MM cells and its mechanisms, thereby affecting the performance of humanized CD26mAb. Methods and Results: Immunostaining of bone marrow tissues of MM showed that CD26pos/CD138pos plasma cells were detected in several patients but not in others. So, first, we investigated the impact of HDACi on CD26 expression of MM cells. Although the cell surface expression of CD26 was relatively low or not detected on 5 MM cell lines (KMS26, KMS27, KMS28, KMS11, RPMI8226), the increased expression in CD26 levels was detectable within 24 h of the treatment with HDAC1i; FK228, HDAC3i; BG45, MS-275, RG2833 or HDAC6i; nexturastat A, tubastatin A, ACY-1215 as well as broad HDACi; LBH-589, SAHA. It increased further and maximum increase was observed at 72 h of the treatment by each HDACi. Then, subsequent removal of HDACi resulted in a decline of CD26 expression on MM cells to pre-treated levels. In addition, the levels of CD26mRNA were concomitantly enhanced, which was paralleled with an increase in the induction of CD26 protein in MM cells. Next, we examined the effect of HDACi on the viability of CD26neg MM cells in the absence or presence of CD26mAb. The mAb alone did not induce lysis of CD26neg MM cell lines at any doses, whereas, combining HDACi plus CD26mAb (10 μg/ml) synergistically facilitated lysis of CD26neg MM cells via direct effects as well as NK cell-mediated ADCC by mAb. Of note, HDACi plus CD26mAb in combination readily augmented lysis of CD26neg cell populations, refractory to HDACi or mAb alone. Furthermore, to elucidate the mechanisms of CD26 up-regulation in MM cells by HDACi, we performed chromatin immunoprecipitation assay on CD26 gene promoter. Each HDACi increased acetylation of histone 3 lysine 27 (H3K27Ac), concomitant with enhanced binding of Sp1 at the 5’ flanking region of the CD26 gene containing Sp1 binding sites in CD26neg MM cells, which is suggestive of transactivation of CD26 gene by HDACi targeting HDAC1, 3 or 6. Conclusion: Combination with isoform-selective HDACi not only shows anti-MM activity but supports as immunopotentiators by sensitizing CD26neg MM cells to CD26mAb and augment its cytotoxicity against MM cells. Citation Format: Hiroko Nishida, Mari Fujiwara, Mutsumi Hayashi, Michiie Sakamoto. Isoform-selective HDAC inhibition up-regulates CD26 expression on multiple myeloma cells and augments cytotoxic efficacy by humanized monoclonal antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5473.

Abstract 562: AsCas12a gene-edited iPSC-derived NK cells constitutively expressing CD16 and membrane-bound IL-15 demonstrate prolonged persistence and robust anti-tumor activities in a solid tumor mouse model

Abstract Current cell and gene therapy medicines for oncology have reshaped how cancer is treated. Chimeric antigen receptor (CAR)-T cells have demonstrated that cell therapy can achieve durable remissions in hematologic malignancies. CAR-T cell therapies, however, have limited efficacy in solid tumors and are associated with severe toxicity, highlighting the need for safer and more efficacious novel cell therapies. With their intrinsic tumor killing capacity, few treatment-related toxicities, and the ability to be given to patients off-the shelf, natural killer (NK) cells are an attractive alternative therapy option to CAR-T cells. While most NK cell therapies are produced from healthy donor cells, deriving NK cells from induced pluripotent stem cells (iPSCs) has the unique advantage that a clone with any desired edits can be generated. We aim to leverage our iPSC platform in combination with our proprietary gene editing technologies to create highly differentiated off-the-shelf treatments for solid tumors. Using our proprietary engineered AsCas12a, we generated double knocked-in (DKI) iPSC clones in which a bicistronic cargo encoding CD16 and a membrane-bound IL-15 (mbIL-15) was knocked into the GAPDH locus to increase the effector function and persistence of iNKs. Constitutive surface expression of CD16 and mbIL-15 by the DKI iNKs was demonstrated. DKI iNKs showed significantly increased natural and antibody dependent cellular cytotoxicity when compared to wild type (WT) iNKs in a SKOV3 tumor spheroid assay in vitro. Furthermore, in the absence of exogeneous cytokines, DKI iNKs persistence in vitro was dramatically improved over WT iNKs. The anti-tumor efficacy of the DKI iNKs in vivo was evaluated using a SKOV3 ovarian cancer model. Tumor bearing mice were treated with WT or DKI iNKs intraperitoneally in combination with trastuzumab or treated with trastuzumab alone. No exogenous cytokines were administered. DKI iNKs combined with a single dose or multiple doses of trastuzumab exerted greater tumor control compared to WT iNKs with trastuzumab, or trastuzumab alone. A single dose of DKI iNKs combined with three doses of trastuzumab induced tumor clearance in 6 out of 8 mice and significantly prolonged survival. Importantly, DKI iNKs were detected in the peritoneum of the treated animals for greater than 3 months, demonstrating that the mbIL-15 maintained iNK survival for a prolonged period of time in the absence of exogeneous cytokine support. In summary, knocking-in CD16 and mbIL-15 to the GAPDH locus of iPSCs dramatically increased the persistence of the DKI iNKs which exhibited robust anti-tumor activities in a solid tumor mouse model. These data demonstrate that our platform enables the development of off-the-shelf iNK cell medicines that may be highly effective for treating solid tumors. Citation Format: Alexander G. Allen, Samia Q. Khan, Kaitlyn M. Izzo, Mrunali Jagdale, Alexandra Gerew, Nadire R. Cochran, Jared Getgano, Stephen Sherman, Laura Blaha, Mark Shearman, Kate Zhang, Kai-Hsin Chang. AsCas12a gene-edited iPSC-derived NK cells constitutively expressing CD16 and membrane-bound IL-15 demonstrate prolonged persistence and robust anti-tumor activities in a solid tumor mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 562.

Single-cell RNA sequencing reveals immunological rewiring at the maternal-fetal interface following asymptomatic/mild SARS-CoV-2 infection.

While severe coronavirus 2019 (COVID-19) is associated with immune activation at the maternal-fetal interface, responses to asymptomatic/mild severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection during pregnancy remain unknown. Here, we assess immunological adaptations in blood and term decidua in response to asymptomatic/mild disease in pregnant women. We report attenuated antigen presentation and type I interferon (IFN) signaling pathways, loss of tissue-resident decidual macrophages, and upregulated cytokine/chemokine signaling in monocyte-derived decidual macrophages. Furthermore, we describe increased frequencies of activated tissue-resident T cells and decreased abundance of regulatory T cells with infection while frequencies of cytotoxic CD4/CD8 T cells are increased in the blood. In contrast to decidual macrophages, type I IFN signaling is higher in decidual T cells. Finally, infection leads to a narrowing of T cell receptor diversity in both blood and decidua. Collectively, these observations indicate that asymptomatic/mild COVID-19 during pregnancy results in remodeling of the immunological landscape of the maternal-fetal interface, with a potential for long-term adverse outcomes for the offspring.

Synthetic cannabinoid WIN 55,212–2 inhibits growth and induces cell death of oral and pancreatic stem-like/poorly differentiated tumor cells

We report here that synthetic cannabinoid WIN 55,212–2 inhibits tumor cell proliferation and induces cell death of oral and pancreatic tumor cells, and the effect is much more pronounced on stem-like/poorly differentiated OSCSCs and MP2 cells when compared to well-differentiated OSCCs, and PL-12 tumor cells. In addition, WIN 55,212-2 decreases cell surface expression of CD44, CD54, MHC class I and PD-L1 on oral and pancreatic tumor cells with the exception of PD-L1 expression on well-differentiated PL-12 pancreatic tumor cells which exhibits an increase in the expression rather than a decrease. Overall, we demonstrate that WIN 55,212-2 has an increased targeting activity against cancer stem cells/poorly differentiated oral and pancreatic tumor cells when compared to well-differentiated tumor cells, and furthermore, such differences in function do not correlate with the levels of CB1 and CB2 receptor expression on tumor cells, suggesting it's function either through post-receptor mediated activation and/or yet-to-be identified novel receptors. Intraperitoneal (IP) delivery of WIN 55-212-2 in humanized BLT mice is found to impart an activating potential for NK cells demonstrating increased NK cell mediated cytotoxicity and secretion of IFN-γ in our preliminary experiments. These results not only suggest a direct targeting of CSCs/poorly differentiated tumors by WIN 55-212-2 but also by indirect targeting of such tumors through the activation and increased functions of NK cells.

Pharmacological inhibition of the mTOR pathway alters phenotype and cytokine expression in bovine monocyte-derived dendritic cells

Epidemiological studies have long demonstrated the association of nutrient status and immune dysfunction in dairy cows. Postpartum dairy cows experiencing a nutrient deficit show a propensity for increased inflammatory response, decreased pathogen clearance, and increased incidence of infectious disease. Studies in cows and other species show that the nutrient sensing mechanistic target of rapamycin (mTOR) signaling pathway could be one potential causal pathway connecting the deficit in nutrient availability and the heightened inflammatory response. Our objective was to investigate the effects of pharmacological mTOR pathway inhibition on phenotype and cytokine expression of bovine monocyte derived dendritic cells (moDC). We differentiated CD14+ monocytes from dairy cows (n = 14) into moDC in the presence or absence of first- or second-generation mTOR inhibitor rapamycin and PP242 (both 100 nM), respectively. On day seven cells were matured with E. coli lipopolysaccharide (LPS, 100 ng/mL) or left unstimulated to represent naïve moDC. Surface expression of CD14, CD40, CD80, and MHCII was measured via flow cytometry. We measured mRNA expression of IL10, IL12A, IL12B, and TNFα by rt-qPCR, and protein concentrations of IL-10 and TFN-α in cell culture supernatants with a bead-based multiplex assay. Cultures from ten cows successfully developed the moDC phenotype in culture without inhibitors, defined as increased surface expression of CD40, CD80, and MHCII compared with naïve moDC. Only data from these cows were considered for the results on effects of mTOR inhibitors. In naïve and mature moDC mTOR inhibition increased MHCII expression compared to controls. In mature moDC, in addition to MHCII, CD80 expression was increased compared with untreated LPS-stimulated controls. Expression of IL12A mRNA was upregulated in mature, mTOR inhibited moDC compared with untreated controls. In cell culture supernatants mTOR inhibition reduced IL-10 and increased TNF-α concentrations in naïve and mature moDCs compared with untreated controls. Overall rapamycin had a more consistent effect on altering phenotype and cytokine expression of moDC than PP242. In summary we observed an increased expression of co-stimulatory molecules and antigen presentation potential in mature moDC differentiated under mTOR inhibition, and a cytokine pattern that would potentially favor a Th1 type response. This study provides novel data indicating a role for mTOR signaling in bovine moDC phenotype and mediator profile. This proof-of-concept study demonstrates the role of the mTOR pathway in shaping the bovine immune response and may help to provide mechanistic insight and opportunities for modulation of the immune response during the nutrient deficit of early lactation.

RHAMM expression tunes the response of breast cancer cell lines to hyaluronan

Hyaluronan (HA) synthesis and degradation are altered during carcinogenesis leading to an increased HA content in the tumor microenvironment, which correlates with poor prognosis and treatment outcomes. The main HA receptors, CD44 and RHAMM, are also overexpressed in tumors where they activate anti-apoptotic, proliferative, invasive, and migration signaling pathways. Herein, we used a unidirectional HA gradient to investigate in a high-throughput fashion the bi-directional communication between HA and breast cancer cell lines with different surface expression of CD44 and RHAMM. We found that the expression of CD44 and RHAMM depends on the HA density: the expression of these receptors is promoted at higher HA density and RHAMM is more sensitive to these changes when compared to CD44. Blocking either CD44 or RHAMM revealed different functions on binding and recognizing HA and a compensatory expression between these two receptors that maintains protumorigenic effectors such as cortactin. Statement of significanceWe show that the expression of main hyaluronan (HA) receptors CD44 and RHAMM is enhanced in a HA concentration-dependent manner. Blocking activity experiments with either RHAMM or CD44 reveal the redundancy of these two receptors towards HA recognition and activation/recruitment of protumorigenic molecular effector, cortactin. These experiments also demonstrate that cells with overexpressed RHAMM are more sensitive to HA density than CD44 positive cells. The reported results are important for the development of therapies that target the hyaluronan signaling in the tumor microenvironment.

Infiltrating natural killer cells bind, lyse and increase chemotherapy efficacy in glioblastoma stem-like tumorospheres

Glioblastomas remain the most lethal primary brain tumors. Natural killer (NK) cell-based therapy is a promising immunotherapeutic strategy in the treatment of glioblastomas, since these cells can select and lyse therapy-resistant glioblastoma stem-like cells (GSLCs). Immunotherapy with super-charged NK cells has a potential as antitumor approach since we found their efficiency to kill patient-derived GSLCs in 2D and 3D models, potentially reversing the immunosuppression also seen in the patients. In addition to their potent cytotoxicity, NK cells secrete IFN-γ, upregulate GSLC surface expression of CD54 and MHC class I and increase sensitivity of GSLCs to chemotherapeutic drugs. Moreover, NK cell localization in peri-vascular regions in glioblastoma tissues and their close contact with GSLCs in tumorospheres suggests their ability to infiltrate glioblastoma tumors and target GSLCs. Due to GSLC heterogeneity and plasticity in regards to their stage of differentiation personalized immunotherapeutic strategies should be designed to effectively target glioblastomas.

High proportion of inflammatory CD62Llow eosinophils in blood and nasal polyps of severe asthma patients.

In mice models, eosinophils have been divided into different subpopulations with distinct phenotypes and functions, based on CD62L and CD101 patterns of membrane expression. Limited data are available in humans. To investigate eosinophils subpopulations in peripheral blood (PB) and nasal polyp tissue (NP) from severe eosinophilic asthma (SEA) patients plus concomitant chronic rhinosinusitis with nasal polyps (CRSwNP). We recruited 23 SEA patients (14 with CRSwNP); as controls, we enrolled 15 non-severe asthma patients, 15 allergic rhinitis patients without asthma and 15healthy donors. Eosinophils were isolated from PB and NP and analysed by FACS. Eotaxin-3 and eotaxin-1mRNA expression in NP tissue was also evaluated. A significantly higher percentage of circulating CD62Llow cells was observed in SEA, as compared with controls, expressing higher levels of CCR3, CD69 and lower levels of CD125 (IL-5R), CRTH2, CD86 and CD28 in comparison with CD62Lbright cells. In NP, eosinophils showed a high proportion of CD62Llow phenotype, significantly greater than that observed in PB. Surface expression of IL-3R, IL-5R, CD69 and CD86 was significantly higher in CD62Llow eosinophils from NP than in those from blood. Moreover, eotaxin-3mRNA expression positively correlated with the percentage of CD62Llow cells in NP. Two different eosinophil subphenotypes can be identified in blood and NP of SEA patients, with a preferential accumulation of CD62Llow inflammatory cells in NP.

Lysophosphatidylcholine induces azurophil granule translocation via Rho/Rho kinase/F-actin polymerization in human neutrophils.

Translocation of azurophil granules is pivotal for bactericidal activity of neutrophils, the first-line defense cells against pathogens. Previously, we reported that lysophosphatidylcholine (LPC), an endogenous lipid, enhances bactericidal activity of human neutrophils via increasing translocation of azurophil granules. However, the precise mechanism of LPC-induced azurophil granule translocation was not fully understood. Treatment of neutrophil with LPC significantly increased CD63 (an azurophil granule marker) surface expression. Interestingly, cytochalasin B, an inhibitor of action polymerization, blocked LPC-induced CD63 surface expression. LPC increased F-actin polymerization. LPC-induced CD63 surface expression was inhibited by both a Rho specific inhibitor, Tat-C3 exoenzyme, and a Rho kinase (ROCK) inhibitor, Y27632 which also inhibited LPC-induced F-actin polymerization. LPC induced Rho-GTP activation. NSC23766, a Rac inhibitor, however, did not affect LPC-induced CD63 surface expression. Theses results suggest a novel regulatory mechanism for azurophil granule translocation where LPC induces translocation of azurophil granules via Rho/ROCK/F-actin polymerization pathway.

PDZ Proteins SCRIB and DLG1 Regulate Myeloma Cell Surface CD86 Expression, Growth, and Survival.

These findings demonstrate the importance of proper trafficking of CD86 to the cell surface in myeloma cell survival and may provide a new therapeutic target in this disease.