Cell Surface Expression Of CD4 Research Articles

PROTAC-mediated degradation of HIV-1 Nef efficiently restores cell-surface CD4 and MHC-I expression and blocks HIV-1 replication

The HIV-1 Nef accessory factor enhances the viral life cycle invivo, promotes immune escape of HIV-infected cells, and represents an attractive antiretroviral drug target. However, Nef lacks enzymatic activity and an active site, complicating traditional occupancy-based drug development. Here we describe the development of proteolysis targeting chimeras (PROTACs) for the targeted degradation of Nef. Nef-binding compounds, based on an existing hydroxypyrazole core, were coupled to ligands for ubiquitin E3 ligases via flexible linkers. The resulting bivalent PROTACs induced formation of a ternary complex between Nef and the cereblon E3 ubiquitin ligase thalidomide-binding domain invitro and triggered Nef degradation in a Tcell expression system. Nef-directed PROTACs efficiently rescued Nef-mediated MHC-I and CD4 downregulation in Tcells and suppressed HIV-1 replication in donor PBMCs. Targeted degradation is anticipated to reverse all HIV-1 Nef functions and may help restore adaptive immune responses against HIV-1 reservoir cells invivo.

Cystatin from the helminth Ascaris lumbricoides upregulates mevalonate and cholesterol biosynthesis pathways and immunomodulatory genes in human monocyte-derived dendritic cells.

Ascaris lumbricoides cystatin (Al-CPI) prevents the development of allergic airway inflammation and dextran-induced colitis in mice models. It has been suggested that helminth-derived cystatins inhibit cathepsins in dendritic cells (DC), but their immunomodulatory mechanisms are unclear. We aimed to analyze the transcriptional profile of human monocyte-derived DC (moDC) upon stimulation with Al-CPI to elucidate target genes and pathways of parasite immunomodulation. moDC were generated from peripheral blood monocytes from six healthy human donors of Denmark, stimulated with 1 µM of Al-CPI, and cultured for 5 hours at 37°C. RNA was sequenced using TrueSeq RNA libraries and the NextSeq 550 v2.5 (75 cycles) sequencing kit (Illumina, Inc). After QC, reads were aligned to the human GRCh38 genome using Spliced Transcripts Alignment to a Reference (STAR) software. Differential expression was calculated by DESEq2 and expressed in fold changes (FC). Cell surface markers and cytokine production by moDC were evaluated by flow cytometry. Compared to unstimulated cells, Al-CPI stimulated moDC showed differential expression of 444 transcripts (|FC| ≥1.3). The top significant differences were in Kruppel-like factor 10 (KLF10, FC 3.3, PBH = 3 x 10-136), palladin (FC 2, PBH = 3 x 10-41), and the low-density lipoprotein receptor (LDLR, FC 2.6, PBH = 5 x 10-41). Upregulated genes were enriched in regulation of cholesterol biosynthesis by sterol regulatory element-binding proteins (SREBP) signaling pathways and immune pathways. Several genes in the cholesterol biosynthetic pathway showed significantly increased expression upon Al-CPI stimulation, even in the presence of lipopolysaccharide (LPS). Regarding the pathway of negative regulation of immune response, we found a significant decrease in the cell surface expression of CD86, HLA-DR, and PD-L1 upon stimulation with 1 µM Al-CPI. Al-CPI modifies the transcriptome of moDC, increasing several transcripts encoding enzymes involved in cholesterol biosynthesis and SREBP signaling. Moreover, Al-CPI target several transcripts in the TNF-alpha signaling pathway influencing cytokine release by moDC. In addition, mRNA levels of genes encoding KLF10 and other members of the TGF beta and the IL-10 families were also modified by Al-CPI stimulation. The regulation of the mevalonate pathway and cholesterol biosynthesis suggests new mechanisms involved in DC responses to helminth immunomodulatory molecules.

Emedastine Inhibits Th1 and Th2 Cell Differentiation Mediated by Mast Cells.

We have previously clarified that emedastine, a second-generation antihistamine drug, inhibits T helper 1(Th1)/Th2 cell differentiation mediated by Langerhans cells (LCs). In addition, although we have recently found that mast cells also function as antigen-presenting cells (APCs) and induce Th1/Th2 cell differentiation, any influence of emedastine on this function remained unclear. Here we investigated the influence of emedastine on Th1/Th2 cell differentiation via mast cells. Mast cells were obtained by long-term culture of murine splenocytes in medium supplemented with tumor necrosis factor (TNF)-α. The mast cells were then incubated in the presence or absence of emedastine, and cultured with naïve CD4+ T cells in the presence of ovalbumin (OVA) peptide. Five days later, Th cells in the culture were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin, and Th1/Th2 cytokine production was examined by enzyme-linked immunosorbent assay. When mast cells treated with emedastine were used as APCs, production of interferon (IFN)-γ and interleukin (IL)-4 from activated Th cells was significantly suppressed. This suppression was associated with inhibition of CD86 expression on mast cells, and mast cells treated with emedastine were shown to obstruct the differentiation of both Th1 and Th2 cells by down-regulating their cell surface expression of CD86. The present data provide additional information that topical application of emedastine to the lesional skin of patients with atopic dermatitis (AD) would reduce not only LC- but also mast cell-mediated skin inflammation.

Overexpression and Downregulation of HOTAIR Influence the Stemness Markers in Gastric Cancer Cell Lines

Background: Several long non-coding RNAs are implicated in the development and metastasis of cancer. The non-coding RNA HOTAIR is known to play a significant role in the progression of various cancers. Objectives: This study aimed to assess the impact of HOTAIR dysregulation on stemness markers in AGS and MKN45 gastric cancer cell lines. Methods: Downregulation and upregulation of HOTAIR were induced using small interfering RNA (siRNA) and a HOTAIR overexpression vector, respectively. The surface stemness markers CD24 and CD44 were analyzed using flow cytometry. Additionally, the effects on the expression of two stemness markers, NANOG and P21, were investigated using Quantitative Reverse Transcription PCR (qRT-PCR). Results: Flow cytometry analysis showed that HOTAIR modulates the cell-surface expression of CD44 and CD24 in gastric cancer cells. Furthermore, dysregulation of HOTAIR significantly affected the expression of the genes P21 and NANOG. Conclusions: Our studies suggest that HOTAIR may regulate the stemness characteristics of gastric cancer cell lines.

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.

OMIP-089: Cattle T-cell phenotyping by an 8-color panel.

This multiplex staining panel was developed to differentiate cattle T cells into conventional (CD4 and CD8) and unconventional (γδ-TCR) subsets as well as their stage of differentiation and activation. The combination of CD45RO and CD62L allows the identification of naïve (TNaïve ), central memory (TCM ), effector memory (TEM ) and terminal effector (TTE ) T cells. Activated cattle T cells (TAV ) can be identified by the cell surface expression of CD25. This panel was developed using cryopreserved cattle peripheral blood mononuclear cells (PBMCs) and tested on fresh as well as stimulated PBMCs. Therefore, this 8-color, 10-parameter flow cytometry panel simultaneously identifies cattle TNaïve , TAV , TCM , TEM , TTE and γδ-TCR cells. This panel will improve our ability to examine T-cell response to pathogens and vaccines in cattle including the potential to identify previously undescribed subpopulations. Furthermore, this panel can be readily optimized for other bovid species as many of these reagents are likely to cross react.

Novel modular chimeric antigen receptor spacer for T cells derived from signal regulatory protein alpha Ig-like domains

Background: T cells equipped with chimeric antigen receptors (CAR) have shown remarkable efficacy in targeting B lineage malignancies. Improvement of the CAR structure is needed, however, with a view to developing flexibly modifiable spacers that are inert in interactions with unwanted cells. Specifically, binding to cells carrying receptors for IgG’s crystallizable fragment (FcR), that recognize IgG-derived domains in CARs is to be avoided.Methods: Two novel CARs targeting the CD19 antigen where the IgG1-CH2 and -CH3 domains were replaced with Ig-like domains from signal-regulatory protein α (SIRPα) were designed in silico. An IgG1-based CAR and a CAR lacking both SIRPα and IgG1 domains were used as comparators. The phenotype and memory phenotype of the expanded cells were analyzed by flow cytometry, and CAR T cell activation and cytotoxic efficacy were assessed in co-culture experiments in response to CD19+ target cells. Unwanted interactions with FcR-expressing myeloid cells were interrogated in co-culture assays with THP-1 monocytic cells.Results: T cells carrying the novel SIRPα-based CARs enacted potent in vitro cytotoxicity against CD19 positive B-lineage leukemia cells, comparable to traditional IgG1-based CAR T cells. Co-culture of IgG1-based CAR T cells with FcR-expressing THP-1 monocytic cells led to prominent cell surface expression of CD69 on T cells together with production of Interleukin (IL)-2 and Interferon-γ, and production of IL-1β, indicating activation of the T cells and monocytes, respectively. Longer co-culture led to killing of the monocytes. No signs of T cell nor monocyte activation were detected in co-cultures of SIRPα-based CAR T cells with THP-1 cells. Arming T cells with the SIRPα-based CARs favored differentiation towards CD4+ phenotype during expansion, while the effects on memory phenotype of the T cells were equivalent between the SIRPα- and IgG1-based CARs. In a pilot experiment, T cells modified with one of the SIRPα-based CARs showed dose dependent leukemia cell control.Conclusion: The novel SIRPα based spacers offer a suitable backbone for developing chimeric antigen receptors that evade the off-target binding to FcR while the cells retain a favorable memory phenotype and efficient cytotoxicity, establishing a promising candidate for future in vivo and clinical testing.

High Dimensional Mapping of Bone Marrow Immune Characteristics in Immune Thrombocytopenia

Introduction Immune thrombocytopenia (ITP) is an acquired autoimmune disease that is characterized by increased antibody-mediated platelet destruction, decreased megakaryopoiesis and impaired thrombopoiesis. An abnormal bone marrow microenvironment has been proposed to play an essential role in the progression of ITP [Stem Cells Dev, 2017]. Cytometry by Time of Flight (CyTOF) is a new analytic strategy that provides single-cell proteomics for enhanced cellular analysis, and describes the components of immune cell populations and the characteristics of new biomarkers. Here, we aim to provide a high-dimensional overview and summary of the changes in cell subsets and functions in the bone marrow of patients with ITP using CyTOF. Methods A total of 40 patients diagnosed with ITP (21 men and 19 women; 23-71 years of age, median 47 years) were enrolled between July and October 2021. Based on the onset and progression of the disease, the ITP patients were classified into 3 subgroups. Group ITP-N1 included 15 newly diagnosed treatment-naïve patients ≤3 months after diagnosis, Group ITP-N2 included 10 patients with an ITP diagnosis of > 3 months who had not received any standard treatment (>3-12 months after diagnosis), and Group ITP-R included 15 patients who had received standard treatment but relapsed following remission. The healthy control (HC) group consisted of 10 donors who underwent hematopoietic stem cell transplantation. CyTOF panels were designed using 42 parameters that were measured at the single-cell level in the bone marrow of the ITP patients and HCs. The plasma levels of immune checkpoint regulators, such as PD-1, PD-L1, CTLA-4, TIM-3, LAG-3 and Galectin-9, were measured using a multianalyte flow assay kit (BioLegend). Results The platelet counts of enrolled ITP patients ranged from 1 to 96×109/L, with a median count of 25×109/L. Using CyTOF, the immune cells in the bone marrow were classified into 7 major subsets, including B cells, γδT cells, CD4+ T cells, CD8+ T cells, natural killer (NK) cells, dendritic cells (DCs), and monocytes. The proportions of DCs and monocytes were significantly different between the ITP patients and HCs. The proportion of monocytes was observed to increase significantly in the bone marrow of the ITP patients. The proportion of the pDC subset of DCs showed a trend of being reduced in the ITP patients, while the proportion of mDCs was increased significantly in the ITP patients. NK cells can be divided into 2 subsets according to the expression of CD56, and these subsets exhibit different cytokine production patterns and cytotoxic functions. The proportion of CD56low NK cells was elevated and the proportion of CD56bright NK cells decreased significantly in ITP-R patients compared to HCs; similar results were not observed in the ITP-N1 or ITP-N2 groups. NK cells in bone marrow may play an important role in the relapse of ITP. We further investigated the functional status of T cells in the ITP patients using a CyTOF panel. The results showed that the proportions of both CD4+ naïve and CD8+ naïve T cells were decreased in the bone marrow of the ITP patients. In particular, the proportion of CD8+ effector T cells was increased significantly in the ITP patients. Next, we analyzed coinhibitory and costimulatory receptor expression in each T-cell subpopulation. CTLA4 expression was observed to be reduced in CD8+ effector T cells and CD4+ Tfh cells. In addition to the changes in coinhibitory receptor expression, costimulatory molecules such as CD28 and ICOS were found to be expressed at higher levels in CD4+ central memory T cells. Combined with the soluble regulators in plasma, we found that the B7.2 levels in plasma was positively correlated with the cell surface expression of CD28 and negatively correlated with the expression of CTLA4 in T cells. Conclusions This study provides a comprehensive overview of the human bone marrow immune characteristics of patients with ITP. Using CyTOF to conduct single-cell analyses, we mapped myeloid and T-cell subset phenotypes in particular and focused on the expression of coinhibitory molecules such as CTLA4. The different proportions of bone marrow immune cells and patterns of immune checkpoint molecule expression in different stages of ITP may provide novel insights into disease etiology and offer promising therapeutic targets for patients with ITP. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CD25 As Feedback Regulator of Oncogenic TCR Signaling in T-Cell Lymphoma

Introduction: CD25, also known as interleukin-2 receptor α chain (IL-2Rα), forms a heterotrimer with IL-2Rβ and common γ chain, and mediates IL-2 signaling. IL-2 signaling is essential for T- and NK-cell survival and proliferation. Additionally, CD25 is required for development of thymic regulatory T cells (Treg) and their suppressive function. Interestingly, CD25 is upregulated in a variety of lymphoid and myeloid malignancies, which do not depend on IL-2 signaling, suggesting IL-2-independent roles of CD25. Here, we seek to investigate whether CD25 has previously unrecognized functions in T cells and T-cell lymphomas. Results: Our previous study in B cells has discovered the cytoplasmic tail of CD25 as an essential structural element for site-specific recruitment of inhibitory phosphatases. CD25-mediated shuttling of inhibitory phosphatases balanced strength of oncogenic B-cell receptor (BCR)-signaling and was essential for cell-survival in B-cell malignancies. Interestingly, we found that CD25 expressed on B cells was monomeric and showed dynamic recruitment to BCR or its oncogenic mimics within lipid rafts. Studying gene expression data across different hematopoietic lineages, we found CD25 was strongly induced in T cell receptor (TCR)-activated T cells, as well as in T-cell malignancies that harbor oncogenic TCR-mimics, such as PTCL. Our mechanistic studies discovered that the serine residue 268 (S268) in the cytoplasmic tail of CD25 was a principal substrate of protein kinase C (PKC), and that S268 phosphorylation by PKC was critical for cell surface expression of CD25. The proximity-based labeling assays (Bio-ID) revealed that CD25-S268 but not its phospho-dead mutant recruits PKC and its adapter RACK1, which scaffold the inhibitory phosphatases SHIP1 and SHP1 for site-specific activation in lipid rafts. CRISPR-mediated knock-in of CD25-S268A that abrogates the formation of inhibitory phosphatase complexes in primary human T cells enabled us to specifically interrogate the functional relevance of CD25-S268 in the regulation of TCR signaling. By using single stranded homology-directed repair templates (ssHDRT) that contain a Cas9 shuttling sequence, we were able to achieve 70% knock-in efficiency in primary human T cells (Figure 1). Signaling studies with these cells revealed that S268A mutation significantly augmented TCR signaling strength. Additionally, S268A mutation resulted in acute inactivation of inhibitory phosphatases including SHP1. Consistent with these results, CD25 shuttling to TCR complex was promptly induced upon TCR stimulation and was mitigated by S268 mutation. Furthermore, immunophenotyping showed that loss of S268 phosphorylation resulted in increased expression of T-cell activation markers such as CD44 and CD69. Based on the ex vivo results, we generated a transgenic mouse model with germline knock-in of CD25-S268A, which would allow us to study S268-specific roles in CD25-mediated feedback regulation of TCR and oncogenic signaling in vivo. Conclusions: The role of CD25 in mediating IL-2 signaling has been extensively studied. However, increasing evidence suggests that CD25 plays a more sophisticated role in TCR signaling and T-lymphomagenesis. Here we discovered a previously unrecognized function of CD25 as a feedback regulator of TCR signaling. TCR activation or oncogenic TCR-mimics induce CD25-mediated recruitment of inhibitory phosphatases in lipid rafts to mitigate TCR signaling or curb excessive oncogenic signaling. Figure 1. CRISPR-mediated knock-out of CD25 alone, or followed by knock-in of CD25-S268-GFP or CD25-A268-GFP, in primary human T cells. The proportion of CD25-expressing GFP+ cells are analyzed by flow cytometry on day 4 and day 30 post CRISPR. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Trial in Progress: AB-101, an Allogeneic, Non-Engineered, Cord Blood-Derived Natural Killer (NK) Cell Therapy, As Monotherapy or in Combination with Rituximab in Patients with Relapsed or Refractory Non-Hodgkin Lymphoma

Background: Natural Killer (NK) cells are known to eliminate tumors directly or via antibody-dependent cellular cytotoxicity (ADCC) which is dependent on the maturation, activation and FcγRIIIA (CD16) phenotype of NK cells. Many cancer patients have low NK cell number and dysfunctional innate immunity, which has been correlated with outcomes to monoclonal antibody (mAb) therapy. AB-101 is an allogeneic, non-genetically modified cord blood-derived NK cell therapy, developed to potentially enhance patients' innate anti-tumor activity in combination with approved oncology products with known ADCC mechanism of action. AB-101 has been optimized for combination with mAbs through the novel AlloNK™ proprietary large scale manufacturing process utilizing donor cord blood units. The cord blood unit is selected for key attributes, including an immunoglobulin-like receptor B (KIR-B) haplotype and the natural high-affinity variant of CD16 (158V/V polymorphism). The AlloNK™ process results in an activated and mature NK cell product with increased cell surface expression of CD16, NKG2D, NKp30/44/46, and DNAM-1, without any requirement for genetic engineering. Additionally, the process generates sufficient NK cells to treat hundreds to thousands of patients from a single cord blood unit and produces a consistently active NK cell product, with little donor-to-donor variability. The combination of AB-101 and rituximab has demonstrated strong cytotoxic activity in preclinical studies; the combination led to improved survival over rituximab or NK cells alone in xenograft mouse models. As a cryopreserved, "off-the-shelf", and infusion-ready product, AB-101 is being evaluated for the treatment of hematologic malignancies in combination with standard of care antibody therapies. Study Design, Treatment and Methods: AB-101-01 is a first in human, Phase 1/2, multi-center, open-label study to evaluate the safety and anti-tumor activity of AB-101 as monotherapy and in combination with rituximab in patients with relapsed or refractory (R/R) non-Hodgkin lymphoma (NHL) of B-cell origin or CLL. This study has 3 parts: Phase 1, 1b, and 2. Phase 1 will evaluate 2 dose levels as monotherapy or in combination with rituximab using the 3+3 dose escalation method. Phase 1b will enroll up to 45 patients in combination with rituximab to determine the recommended phase 2 dose (RP2D). Phase 2 will enroll an additional 15 patients at the RP2D. AB-101 will be given at 1 or 4 billion cells per dose. All enrolled patients will receive at least 1 treatment cycle of 4 doses of AB-101, administered once weekly. Prior to receiving the first dose of AB-101, patients will receive 3 consecutive days of lymphodepleting chemotherapy consisting of fludarabine (30 mg/m2/day) and cyclophosphamide (250 or 500 mg/m2/day). After each AB-101 infusion, patients will receive interleukin-2 (IL-2) at 1×106 IU/m2/dose or 6×106 IU/dose to potentiate NK cell activation and persistence. If given, 375 mg/m2 of rituximab is dosed on the second day of lymphodepletion and with the second and fourth dose of AB-101. Patients who have a complete or partial response or maintain stable disease with the first cycle of the combination may receive 3 additional cycles such that cycle 2 is given with lymphodepletion and cycles 3 and 4 do not include lymphodepletion. Eligible patients must have a diagnosis of B-cell NHL or CLL and must have received at least 2 prior lines of therapy, including an anti-CD20 antibody therapy. Treatment with prior CD19 CAR T-cell therapy is permitted. Patients must be at least 18 years of age, have ECOG PS of 0 or 1, and have measurable disease per Lugano 2014 at the time of enrollment. The primary objectives of each phase are as follows: in Phase 1, the evaluation of the safety of 1 and 4 billion cells/dose of AB-101 as monotherapy or in combination with rituximab through the incidence of AEs and DLTs; in Phase 1b, determination of the RP2D based on safety and antitumor response to AB-101 with rituximab; and in Phase 2, further characterization of the efficacy profile. The study is currently enrolling at 10 U.S. centers. (ClinicalTrials.gov Identifier: NCT04673617)

Bioengineered immunomodulatory organelle targeted nanozymes for photodynamic immunometabolic therapy.

Intelligent nanomedicines integrated with stimuli-responsive components enable on-demand customizable treatment options which would improve therapeutic outcome and reduce systemic toxicity. In this work, we explore the synergistic therapeutic potential of photodynamic therapy and immunometabolic modulation to achieve tumour regression and to trigger an adaptive immunity to prevent tumour recurrence. The therapeutic potential of the fabricated Bioengineered Immunomodulatory Organelle targeted Nanozymes (BIONs) was tested on 3D printed mini-brains which could effectively recapitulate the biologically relevant interactions between glioblastoma cells and macrophages. In the presence of glioblastoma organotypic brain slices, activated BIONs upregulated the cell surface expression of CD86, a costimulatory molecule and CD83, maturation marker, on monocyte derived dendritic cells, suggesting its ability to elicit a strong immune response. Furthermore, the antigen pulsed dendritic cells by chemotaxis and transendothelial migration readily relocate into the draining lymph node where they present the antigenic cargo to enable the proliferation of T lymphocytes. The stealth and tunable catalytic activity of BIONs prevent ROS mediated diseases such as acute kidney injury by providing environment dependent protection without compromising on its promising anti-cancer activity.

An expanded population of CD8dim T cells with features of mitochondrial dysfunction and senescence is associated with persistent HIV-associated Kaposi's sarcoma under ART.

HIV-associated Kaposi’s sarcoma (KS), which is caused by Kaposi’s sarcoma-associated herpesvirus, usually arises in the context of uncontrolled HIV replication and immunosuppression. However, disease occasionally occurs in individuals with durable HIV viral suppression and CD4 T cell recovery under antiretroviral therapy (ART). The underlying mechanisms associated with this phenomenon are unclear. Suppression of viral infections can be mediated by CD8 T cells, which detect infected cells via their T cell receptor and the CD8 coreceptor. However, CD8 T cells exhibit signs of functional exhaustion in untreated HIV infection that may not be fully reversed under ART. To investigate whether KS under ART was associated with phenotypic and functional perturbations of CD8 T cells, we performed a cross-sectional study comparing HIV-infected individuals with persistent KS under effective ART (HIV+ KS+) to HIV-infected individuals receiving effective ART with no documented history of KS (HIV+ KSneg). A subset of T cells with low cell surface expression of CD8 (“CD8dim T cells”) was expanded in HIV+ KS+ compared with HIV+ KSneg participants. Relative to CD8bright T cells, CD8dim T cells exhibited signs of senescence (CD57) and mitochondrial alterations (PGC-1α, MitoTracker) ex vivo. Mitochondrial activity (MitoTracker) was also reduced in proliferating CD8dim T cells. These findings indicate that an expanded CD8dim T cell population displaying features of senescence and mitochondrial dysfunction is associated with KS disease under ART. CD8 coreceptor down-modulation may be symptomatic of ongoing disease.

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.

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.

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.

Myeloid-Derived Suppressive Cell Expansion Promotes Melanoma Growth and Autoimmunity by Inhibiting CD40/IL27 Regulation in Macrophages.

The relationship between cancer and autoimmunity is complex. However, the incidence of solid tumors such as melanoma has increased significantly among patients with previous or newly diagnosed systemic autoimmune disease (AID). At the same time, immune checkpoint blockade (ICB) therapy of cancer induces de novo autoinflammation and exacerbates underlying AID, even without evident antitumor responses. Recently, systemic lupus erythematosus (SLE) activity was found to drive myeloid-derived suppressor cell (MDSC) formation in patients, a known barrier to healthy immune surveillance and successful cancer immunotherapy. Cross-talk between MDSCs and macrophages generally drives immune suppressive activity in the tumor microenvironment. However, it remains unclear how peripheral pregenerated MDSC under chronic inflammatory conditions modulates global macrophage immune functions and the impact it could have on existing tumors and underlying lupus nephritis. Here we show that pathogenic expansion of SLE-generated MDSCs by melanoma drives global macrophage polarization and simultaneously impacts the severity of lupus nephritis and tumor progression in SLE-prone mice. Molecular and functional data showed that MDSCs interact with autoimmune macrophages and inhibit cell surface expression of CD40 and the production of IL27. Moreover, low CD40/IL27 signaling in tumors correlated with high tumor-associated macrophage infiltration and ICB therapy resistance both in murine and human melanoma exhibiting active IFNγ signatures. These results suggest that preventing global macrophage reprogramming induced by MDSC-mediated inhibition of CD40/IL27 signaling provides a precision melanoma immunotherapy strategy, supporting an original and advantageous approach to treat solid tumors within established autoimmune landscapes. SIGNIFICANCE: Myeloid-derived suppressor cells induce macrophage reprogramming by suppressing CD40/IL27 signaling to drive melanoma progression, simultaneously affecting underlying autoimmune disease and facilitating resistance to immunotherapy within preexisting autoimmune landscapes.

Germinal Center B Cells Derived from TET2-Mutated Clonal Hematopoiesis Provide a Microenviromental Niche for Tumor Cells in Angioimmunoblastic T-Cell Lymphoma

Germinal Center B Cells Derived from TET2-Mutated Clonal Hematopoiesis Provide a Microenviromental Niche for Tumor Cells in Angioimmunoblastic T-Cell Lymphoma

CD73 Inhibition Overcomes Immunosuppression and Triggers Autologous T-Cell Mediated Multiple Myeloma Cell Lysis in the Bone Marrow Milieu

CD73 Inhibition Overcomes Immunosuppression and Triggers Autologous T-Cell Mediated Multiple Myeloma Cell Lysis in the Bone Marrow Milieu

Cerdulatinib Pharmacodynamics and Relationships to Tumor Response Following Oral Dosing in Patients with Relapsed/Refractory B Cell Malignancies: Results from a Phase I Dose Escalation Study

Preclinical studies suggest that the spleen tyrosine kinase (SYK) and Janus kinase (JAK) family members can contribute to both tumor intrinsic and microenvironment-derived survival signals. A dual inhibitor of these kinases (cerdulatinib) was therefore developed, and a phase I dose-escalation study was completed to understand tolerability and efficacy, and their relationship to pharmacokinetics (PK) and pharmacodynamics (PD). Here, we report on PK/PD relationships and associations with tumor response in patients from the phase I dose-escalation portion of the clinical trial (NCT01994382).Repeat CT scans were obtained from 6 patients with chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL), 12 patients with follicular lymphoma (FL), 12 patients with diffuse large B cell lymphoma, and 6 patients with mantle cell lymphoma. Meaningful clinical responses were observed in patients with CLL/SLL and FL, as previously reported (Hamlin et al, 2016); hence, the correlation between tumor response and PD markers was examined in these patient populations.Complete inhibition of SYK and JAK signaling pathways was achieved in whole blood of dosed patients at tolerated exposures. The level of target inhibition in blood correlated with serum cerdulatinib concentration, and the concentrations at which 50% inhibition was acheived (IC50) against B cell antigen receptor (BCR), interleukin (IL)-2, IL-4, and IL-6 signaling pathways ranged from 0.22-1.08 μM, depending on the phosphorylation event measured. Significant correlations were observed between inhibition of both SYK and JAK signaling pathways and tumor response, most notably the BCR and IL-4 signaling pathways. Serum markers of inflammation were also reduced following cerdulatinib administration, several of which significantly correlated with tumor response, including β-2 microglobulin, C-reactive protein, interferon γ-induced protein 10 (IP10), and macrophage inflammatory protein 3β (MIP3β). Of note, lymphocytosis was observed in CLL, SLL, and FL patients. Evidence of diminished tumor cell surface expression of CD69 and CD86, diminished CD5 expression (a negative regulator of BCR signaling), and enhanced C-X-C chemokine receptor type 4 (CXCR4) expression were observed in 2 patients with CLL, consistent with suppression of BCR and IL-4 signaling.Fresh tumor samples were obtained from the peripheral blood of 6 CLL patients treated with cerdulatinib, as well as archival tumor biopsies obtained from 4 FL patients and 1 MCL patient, which were submitted for next-generation sequencing. Clinical activity was observed in CLL patients bearing mutations to NOTCH1, ATM, p53, and KRAS, which are among the most frequently observed mutations in this disease. One of the responding patients bore a 17p deletion. Importantly, 2 patients with ibrutinib-relapsed CLL who progressed within the first cycle of therapy with cerdulatinib had 3 mutations in common: p53, EP300, and BTK (C481S). Genetic correlates in FL were more limited, with data on 4 patients, 3 of whom had evidence of tumor reduction and 1 which progressed. The 2 best responding patients with whom we have genetic information bore mutations to ZMYM3, KMT2D, FAT4, BCL2, BCL6, CD79B, and STAT6 . Lastly, strong clinical activity was observed in a transformed FL patient who presented with increased MYC, BCL2, and BCL6 expression by immunohistochemistry (“triple-hit” lymphoma).These data demonstrate the ability of cerdulatinib to potently and selectively inhibit SYK and JAK signaling at tolerated exposures in patients with relapsed/refractory B cell malignancies. Moreover, the extent of target inhibition, as well as suppression of inflammation, correlated with tumor response. Cerdulatinib is currently under investigation as part of a phase IIa study to further evaluate efficacy and tolerability in this population. DisclosuresCoffey:Portola Pharmaceuticals, Inc.: Employment, Equity Ownership. Feng:Portola Pharmaceuticals, Inc.: Employment, Equity Ownership. Betz:Portola Pharmaceuticals, Inc: Employment, Equity Ownership. Pandey:Portola Pharmaceuticals, Inc.: Employment, Equity Ownership. Birrell:Portola Pharmaceuticals, Inc.: Employment, Equity Ownership. Leeds:Portola Pharmaceuticals, Inc.: Employment, Equity Ownership. Der:Portola Pharmaceuticals, Inc.: Employment, Equity Ownership. Kadri:Portola Pharmacueticals, Inc.: Research Funding. Lu:Portola Pharmacueticals, Inc.: Research Funding. Segal:Portola Pharmacueticals, Inc.: Research Funding. Wang:Karyopharm: Research Funding; Portola: Research Funding. Michelson:Portola Pharmacueticals, Inc.: Employment. Curnutte:Portola Pharmaceuticals, Inc.: Employment, Equity Ownership. Conley:Portola Pharmaceuticals, Inc.: Employment, Equity Ownership, Patents & Royalties.

Results from an Ongoing Phase 1 Study Indicate ACDT-301 (Camidanlumab Tesirine) Is Well-Tolerated in Patients with Relapsed or Refractory CD25-Positive Acute Leukemia

Introduction: There is a significant need for improved therapeutics for older patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Five-year survival rates dip sharply as patient age increases, falling to ~40-45% for patients with ALL over 40 years of age and ~10% for adults with AML over 65 years of age. Cell surface expression of CD25 on AML and ALL blast cells is associated with adverse outcomes, including induction failure, relapse, and shorter overall survival. ADCT-301 (camidanlumab tesirine) is an antibody drug conjugate composed of a human CD25-targeting monoclonal antibody conjugated to a pyrrolobenzodiazepine dimer cytotoxin. Camidanlumab tesirine (Cami-T) has demonstrated anti-tumor efficacy in mouse xenograft models of CD25-expressing hematologic malignancies. We present interim data from a Phase 1 study of Cami-T treatment in patients with relapsed or refractory (R/R) CD25-positive acute leukemia.Methods: A Phase 1, open-label, dose-escalation (part 1) and expansion (part 2) multicenter, US study is enrolling patients with R/R CD25-positive acute leukemia. The primary objective is to assess the tolerability and safety of Cami-T and determine the maximum tolerated dose (MTD). Investigator-assessed anti-tumor activity, pharmacokinetics (PK), and the presence of anti-drug antibodies are also being evaluated. Eligible patients are ≥18 years of age, have CD25-positive AML or ALL without extramedullary disease, have failed or are intolerant to established therapy, and have an ECOG performance status of 0-2. CD25-positive AML or ALL is defined as CD25 expression on ≥5% of leukemic cells within bone marrow aspirate or biopsy. In Part 1, patients are assigned to treatment using a 3+3 dose-escalation design, based on assessment of dose-limiting toxicities (DLTs) during Cycle 1, to determine the MTD. Cami-T is given as an intravenous infusion on Day 1 of each cycle. The first cohort started treatment on a 21-day (q3w) cycle at a starting dose of 3 μg/kg. Dose frequency in subsequent cohorts may increase to once-weekly (qw) in the absence of DLTs. Part 2 will further evaluate safety, tolerability, PK and clinical activity at the dose recommended from Part 1.Results: As of June 26, 2017, 29 patients (20 male, 9 female) with AML have been treated with Cami-T (median 2 cycles, range: 1-7). The median age is 67 years [range 22-82] and patients had received a median of 3 (range: 1-9) previous chemotherapies. Five (17%) patients had undergone prior allogeneic stem cell transplantation. No DLTs were observed up to the highest evaluated q3w dose of 92 µg/kg. After switching to weekly dosing, one DLT was reported in the 30 μg/kg dose group. A total of 250 treatment-emergent adverse events (TEAEs) were reported in 28/29 (97%) patients. The most common TEAEs were fatigue and nausea (both n=7) followed by diarrhea, pneumonia and pyrexia (all n=5; Table). Grade ≥3 TEAEs were reported by 22/29 (76%) patients, of which fatigue (n=4), febrile neutropenia (n=4) and pneumonia (n=3) were the most common. Five deaths from TEAEs were recorded (one case each of blood disorder, cardiac arrest and pneumonia and two cases of disease progression). Two patients experienced infusion-related reactions. Three patients experienced TEAEs leading to a dose delay or reduction (one case each of skin rash, pericarditis and supraventricular tachycardia), and one patient each in the 52 μg/kg q3w and 30 μg/kg qw dose groups discontinued treatment due to Grade 3 skin rash. Transient CD25+ blast clearance in 2 patients who received 1 and 7 cycles, respectively, of Cami-T 32 μg/kg q3w, was observed, supporting an on-target activity of Cami-T. No responses or remissions have been obtained to date. PK data show increasing Cami-T total and PBD-conjugated antibody exposure with dose. Rapid systemic clearance of the drug with levels below limits of quantification suggest that q3w dosing may be insufficient for therapeutic efficacy, prompting exploration of a qw schedule.Conclusions:In this ongoing Phase 1 study in patients with CD25-positive R/R AML, single-agent Cami-T has shown an acceptable safety profile thus far. Additional patients with AML and ALL are being enrolled and dose escalation will continue until the MTD is identified, with further results expected later this year.Study sponsored by ADC Therapeutics. https://clinicaltrials.gov/ct2/show/NCT02588092 [Display omitted] DisclosuresGoldberg:Pfizer: Research Funding; ADC Therapeutics: Research Funding; Genentech: Research Funding. Solh:ADC Therapeutics: Research Funding; Amgen: Speakers Bureau; Celgene: Speakers Bureau. Ungar:ADC Therapeutics: Employment, Other: Stock option interest. Rizzieri:Erytech: Research Funding; Shire: Research Funding. Walter:ADC Therapeutics: Research Funding; Aptevo Therapeutics: Research Funding. Spira:ADC Therapeutics: Research Funding. Chung:ADC Therapeutics: Research Funding. Stock:Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy. He:ADC Therapeutics: Employment, Other: Potential equity interest. Boni:ADC Therapeutics: Employment, Other: Potential equity interest. Atallah:ADC Therapeutics: Research Funding.