Zeta Chain Research Articles

The therapeutic effect of Yinqiaosan decoction against influenza A virus infection by regulating T cell receptor signaling pathway

BackgroundYinqiaosan decoction (YQSD), a traditional Chinese medicinal recipe, has been employed to treat influenza in China for approximately 300 years. ObjectiveOur study aimed to explore the mechanisms of YQSD against influenza via in vivo and in vitro experimental studies. Study designand methods UHPLC-Q-TOF-MS/MS was utilized to examine the substances of the YQSD. The chemical components of YQSD detected by UHPLC-Q-TOF-MS/MS were used for network pharmacology analysis. The antiviral effect of YQSD in vivo was investigated. The potential mechanisms of YQSD in combating influenza, which were predicted from network pharmacology analysis, were validated in vitro. ResultsBy use of UHPLC-Q-TOF-MS/MS, 97 compounds were identified from YQSD. Network pharmacology analysis revealed that the therapeutic effect of YQSD against influenza may be associated with the regulation of T cell receptors (TCR) and Phosphoinositide 3-Kinase (PI3K)- protein kinase B (Akt) signaling pathways. Treatment with YQSD significantly prolonged the mean survival time of the mice and reduced lung injury due to the influenza A virus in vivo. It was discovered that YQSD efficiently inhibited the expression of inflammation-related cytokines. Moreover, YQSD has been found to significantly reduce the expression levels of cluster of differentiation 3 (CD3), monocyte chemoattractant protein-1 (MCP-1), and H1N1 virus nucleoprotein (NP), and prevent the decrease of epithelial cadherin (E-cadherin) protein. In addition, YQSD can inhibit the phosphorylation of the zeta chain of T cell receptor-associated protein kinase 70 (ZAP70) and PI3K proteins in vitro. ConclusionThe capacity of YQSD to suppress viral multiplication and inflammatory response by modulating T cell immunity may explain its effect against influenza viral pneumonia, which may involve the regulation of TCR and PI3K signaling pathways.

Identification and Functional Investigation of Hub Genes Associated with Follicular Lymphoma.

Follicular lymphoma (FL), the most common type of indolent lymphoma, originates from germinal center B cells within the lymphoid follicle. However, the underlying mechanisms of this disease remain unclear. This study aimed to identify the potential hub genes for FL and evaluate their functional roles in clinical applications. Microarray data and clinical characteristics of patients with FL were obtained from the Gene Expression Omnibus database. Differential expression analysis and weighted gene co-expression network analysis (WGCNA) were employed to explore hub genes for FL. Functional enrichment analysis was performed to investigate the potential roles of these hub genes in FL. Mendelian randomization (MR) analysis was performed to verify the causal effect of the top genes on FL risk. In addition, gene set enrichment analysis (GSEA) and immune cell analysis were performed to elucidate the involved mechanisms of the crucial genes in FL. A total of 1363 differentially expressed genes and 157 central genes were identified by differential expression analysis and WGCNA, respectively, resulting in 117 overlapping genes considered as hub genes for FL. Functional enrichment analysis revealed significant correlations between immune-related pathways and FL. MR analysis revealed a significant association only between zeta chain of T-cell receptor-associated protein kinase 70 (ZAP70) and FL risk, with no significance observed for the other top genes. GSEA and immune cell analysis suggested that ZAP70 may be involved in the development and progression of FL through immune-related pathways. By integrating bioinformatics and MR analyses, ZAP70 was successfully identified and validated as a promising FL biomarker. Functional investigations indicated a significant correlation between immune-related pathways and FL. These findings have important implications for the identification of targets for the diagnosis and treatment of FL and provide valuable insights into the molecular mechanisms underlying FL.

CCR1-Targeting CAR T Cells for Acute Myeloid Leukemia

BACKGROUND Chimeric antigen receptor (CAR) T cell therapy is highly effective in patients with B cell malignancies and multiple myeloma, but initial clinical trials with CARs targeting acute myeloid leukemia (AML) have shown less successful results. Challenges inherent to using CAR T cells for AML include lack of a leukemia-specific target antigen due to heterogeneous antigen expression and potential on-target/off-tumor toxicity due to co-expression of target antigens on normal myeloid cells. C-C chemokine receptor 1 (CCR1, also known as CD191), a G protein-coupled receptor that binds to members of the C-C chemokine family, is a promising AML-associated antigen that is reported to be expressed on over 75% of AML samples and minimally expressed on hematopoietic stem/progenitor cells and T cells. The present study aimed to explore the potential of CCR1 as a novel CAR target antigen using a syngeneic mouse model that allows investigation of both efficacy and toxicity of the CAR T cells. METHODS AND RESULTS We developed a CAR based on the single chain variable fragment (scFv) KM5907 targeting both mouse and human CCR1. We constructed a second-generation CCR1-CAR containing a CD28 hinge, a CD28 transmembrane domain, a CD3 zeta signaling domain, and a CD28 co-stimulatory domain. We mutated the immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3 zeta chain to retain only a single active membrane-proximal (1XX) ITAM, based on a prior study showing that the 1XX ITAM format enhanced CAR T cell activity by reducing T cell exhaustion. Primary murine T cells were retrovirally transduced and CCR1-CAR expression was assessed by flow cytometry. CCR1-CAR T cells promoted effective elimination of BM185 leukemia cells overexpressing murine CCR1 in vitro. Similarly, primary human T cells transduced with the CCR1-CAR exhibited potent cytotoxicity against C1498 leukemia cells overexpressing human CCR1 in vitro. To evaluate anti-leukemia activity of the CCR1-CAR T cells in vivo, BALB/c mice were sublethally irradiated and engrafted with 1x10 5 BM185 leukemia cells modified to overexpress murine CCR1. Two days later, mice were treated with 2x10 6 CCR1-CAR T cells and monitored for weight changes and survival. Growth of BM185 leukemia cells (expressing firefly luciferase) and expansion of CAR T cells (expressing gaussia luciferase) were assessed via two-color bioluminescence imaging (BLI). On day 6, mice were bled retro-orbitally and serum was analyzed for cytokine levels. The CCR1-CAR T cells demonstrated potent anti-leukemia activity and T cell expansion in vivo, promoting improved overall survival compared with control mice that received only tumor cells (Figure 1). CCR1-CAR T cell activity was accompanied by mild, self-limited toxicity, characterized by transient weight loss (Figure 2), but not significant elevation of serum levels of cytokines associated with cytokine release syndrome (CRS) such as IL-6 and TNF-α. BLI of the CCR1-CAR T cells revealed enhanced trafficking into the bone marrow and spleen followed by proliferation in irradiated mice even in the absence of tumor, suggesting that CCR1-CAR T cells are stimulated by CCR1 antigen expressed on non-leukemic host cells in addition to leukemia cells. CONCLUSIONS In summary, CCR1-CAR T cells represent a promising therapeutic strategy for AML that to our knowledge has not yet been evaluated in preclinical or clinical studies. We generated CCR1-CAR T cells that demonstrated potent anti-leukemia activity in vitro and in vivo in a syngeneic mouse model. Clinical signs of self-limited toxicity were not associated with elevated levels of inflammatory cytokines and might be caused by an on-target/off-tumor effect, which warrants further investigations to better characterize the safety profile of CCR1-CAR T cells. The CCR1-CAR T cells also displayed potent cytotoxicity in vitro via human CCR1, which will facilitate clinical translation of the findings. A CCR1-CAR may also offer benefit in other hematologic malignancies with reported CCR1 expression such as multiple myeloma or Hodgkin's lymphoma.

Novel Peptide-Based Plectin-1 CAR T-Cells Target Specifically Pancreatic Cancer Cells

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is one of the most devastating cancers, and therapies to treat this malignancy have not evolved rapidly to fulfill the need of affected patients. PDAC, like many solid malignancies, is considered a “cold immunological tumor” where the lack of targetable tumor-associated antigens has hampered the development of successful immunotherapies. Despite newer treatment approaches, PDAC patients with advanced disease have a dismal prognosis. Therefore, there is an urgent need to develop new therapies. Plectin-1 is a high molecular weight protein (∼500 kDa) that participates in cytoskeleton network organization linking intermediate filaments to microtubules and microfilaments. Plectin-1 is aberrantly expressed in the PDAC cellular membrane, contributes to PDAC pathogenesis, and confers a poor prognosis. Plectin-1-targeting peptides (PTP) in mice and humans have been successfully explored for their use in PDAC imaging and targeted payload delivery (small molecules, radioisotopes, and chemotherapy). We sought to take advantage of the specific binding of PTP and develop a peptide-based plectin-1 chimeric antigen receptor (CAR), and test T-cell transduced with this construct in vitro against PDAC cell lines. Methods: Using in silico tools, we optimized different pepto-CAR constructs and selected one that showed the highest levels of cytotoxicity against plectin-1-expressing PDAC cell lines. A myc tag was incorporated in the hinge region for flow cytometry or western blot detection. The intracellular signaling domain of these CAR sequences included 4-1BB and the zeta chain of the CD3 molecule ( Figure 1A). Plasmids containing the CAR sequence were packaged on lentivirus particles using HEK293T cells. Purified plectin-1 CAR lentiviruses were used to transduce T-cells derived from human peripheral blood T-cells obtained and purified from healthy donor whole blood. The expanded CAR T-cells were used for cytotoxic experiments against different cell lines. Results: Expression of plectin-1 CAR was confirmed by flow cytometry using an anti-myc tag antibody ( Figure 1B). Plectin-1 expression was analyzed in different pancreatic cancer cell lines, and the BxPC-3 cells showed the highest expression level ( Figure 1B). Using a luciferase-based assay, we plated 50,000 BxPC-3 cells per well in duplicates with different effector-to-target (E:T) ratios. After 24 hours, luciferin was added, and the plate was read using a luminometer. As a negative control for effector cells, we use T-cells without a CAR ( Figure 1B), and for the target cells, we use the plectin-1 negative lung cancer cell line A549 ( Figure 1B). Plectin-1 CAR T-cells effectively killed BxPC-3 cells at different E:T ratios in a specific manner compared with controls using unmodified T-cells. Furthermore, no cytotoxicity was observed against plectin-1 negative A549 cells ( Figure 1A). Conclusion: Our results show that a novel peptide-based CAR construct design against plectin-1 can induce specific cytotoxicity on plectin-1 expressing PDAC cell line BxPC-3. Furthermore, these data demonstrate the feasibility of using a CAR with a peptide-based antigen binding domain instead of the traditional antibody-derived scFv. This novel peptide-based CAR design offers alternatives for cellular therapy bioengineering. Moreover, our results can significantly impact immune-oncology therapeutic applications, particularly in challenging diseases like pancreatic cancer. Figure 1 . A) In the above section, the Plectin-1 CAR construct is depicted. Cytotoxicity of unmodified T-cells and Plectin-1 CAR T-cells against BxPC-3 and A549 is shown below. B) Expression of Plectin-1 CAR in unmodified T-cells and Plectin-1 CAR T-cells is shown on the left. The expression of plectin-1 antigen on the surface of BxPC-3 cells and A549 cells is shown on the right. PTP: plectin-1 targeting peptide.

Gene expression changes in COVID-19 patients impact pathways related to circadian rhythm, phosphatidylinositol signaling, cytokine storm, and platelet aggregation

The emergence of SARS-CoV-2 and the resulting COVID-19 disease pandemic had a grave impact on human society causing severely ill patients were to be placed on mechanical ventilators. The severity of the disease depended on such factors as age and the presence of pre-existing conditions. Although with the earnest effort, vaccines have been developed against the disease, there is still a long way to go to completely eradicate COVID-19. Currently, there are concerns about the complications associated with COVID-19 patients and associated conditions. This research aimed to find correlation between gene expression profiles of COVID-19 patients and factors such as age, severity, and mechanical ventilators. To do this, we probe COVID-19 patient data for dysregulated genes and pathways in severe disease. The relevant data was selected from the Gene Expression Omnibus (GEO) datasets GSE152418, GSE171110 and GSE157103. We identified differentially expressed genes (DEGs) between defined groups: COVID-19 patients and control individuals, severe ill and moderately ill COVID-19 patients, young (<40 years) and old (>70 years), and patients who require versus do not require mechanical ventilation. Analyses of over-represented pathways among DEGs and identification of enriched gene sets among cohorts provided further etiological insights. The impact of DEGs on protein-protein interactions (PPIs) between products of these genes were evaluated. The results highlight pathways related to immune response as well as inflammation, cancer, circadian clock, and PI signaling in COVID-19 patients. Key genes regulating circadian clock and PI signaling were downregulated, however, various genes and proteins regulating inflammation and cancer were upregulated in the COVID-19 patients. The product of the up-regulated gene, FLT3, which promotes phosphorylation of STAT5A/STAT5β, resulting in increased cytokine production is connected to VAV1 and TET proteins which play a role in cytokine production. Among old patients, besides pathways related to inflammation and the immune system, pathways such as platelet activation, signaling and aggregation as well as extracellular matrix organization were affected by gene expression dysregulation. In patients requiring mechanical ventilation, pathways impacted include PD-1 signaling, TCR signaling, translocation of ZAP-70 to immunological synapse, and phosphorylation of CD3 and TCR zeta chains. These observations shed insights into relevant genes and pathways impacting COVID-19 patients.

Buffered formic acid and a monoglyceride blend improve performance and modulate gut bacteria and immunity gene expression in broilers under necrotic enteritis challenge

Due to the removal of antibiotics from animal feed, alternatives have been sought to control necrotic enteritis (NE) in broilers. The current study investigated the effects of buffered formic acid (Amasil NA) and monoglycerides of short- and medium-chain fatty acids (Balangut LS P) on the performance and gut health of broilers challenged with NE. A total of 816 as-hatched 1-d-old chicks (Cobb 500) were randomly assigned to 6 treatments with 8 replicates. Treatments were: T1) nonchallenged control; T2) NE challenged control; T3) Amasil NA (challenge plus Amasil NA, 0.3% throughout all phases); T4) Balangut LS P (challenge plus Balangut LS P, 0.5%, 0.3%, and 0.2% in the starter, grower and finisher phases, respectively; T5) Combined (challenge plus combination of T3 and T4); T6) Antibiotic (challenge plus Zn bacitracin, 0.05 % throughout all phases). Birds were orally gavaged with live Eimeria vaccine species (d 9) and with Clostridium perfringens (d 14 and 15). On d 16, birds were sampled to evaluate gut permeability, microbiota, and mRNA abundance in the jejunum. The data were analyzed in JMP software using one-way ANOVA with Tukey's test to separate means, and Kruskal-Wallis test was used for non-normally-distributed parameters. Results showed that Balangut LS P decreased (P<0.05) feed conversion ratio compared to nonchallenged ones at the end of the study. Balangut LS P reduced (P < 0.05) the level of cecal Bacteriods compared to nonchallenged group, whereas Amasil NA shifted the levels of ileal Bifidobacteria, Enterobacteriaceae, and Lactobacillus towards nonchallenged control (P > 0.05). NE challenge upregulated (P < 0.001) the expression of IL-21R, zeta chain of T cell receptor (ZAP70), and dual specificity phosphatase 4 (DUSP4) compared to nonchallenged birds, whereas Balangut LS P showed an intermediate (P > 0.05) expression pattern of these genes towards nonchallenged and antibiotic groups. In conclusion, combination of Balangut LS P and Amasil NA has the potential to be used as an additive to improve the performance and gut health of broiler chickens, especially under challenging conditions such as NE infections.

Abstract 889: The efficacy and safety of PD-L1-specific CAR-T in advanced gastric cancer

Abstract [Purpose] Heterogeneity and the tumor microenvironment are major obstacles to overcoming incurable advanced gastric cancer (AGC). Although Epstein-Barr virus-positive and microsatellite instability subtypes can be detected early and properly treated, they are rare in AGC. In this regard, recent combinational therapy of immune checkpoint inhibitors (ICIs) such as PD-1/PD-L1 inhibitors and other various therapeutics has been tried, but there are clinically limited doses and drug resistance. We hypothesized that CAR-T targeting PD-L1, the common tumor-associated antigen (TAA), which expresses in most advanced cancer cells, would make a significant breakthrough in curing a solid tumor. This study aimed to evaluate the killing effects of anti-PD-L1 CAR-T against gastric cancer cell lines. Removal of PD-L1-expressing cells in the AGC TME will improve the clinical outcomes of current therapies. [Results] We constructed a second-generation PD-L1 CAR lentiviral vector targeting PD-L1, which has an intermediate affinity with PD-L1 in solid tumor TME. As the secondary signaling motif, CD28 was chosen and tested along with the CD3 zeta chain. The effector CAR-T, named Vax-CAR-T, specifically recognized the PD-L1-expressing AGC cell line, SNU638, and exhibited rapid and robust cytotoxic activity in vitro. When compared to untransduced T cells, the level of the CD25 activation marker was higher in Vax-CAR-T cells. Besides, the inhibitory markers of Tim3 and LAG3 were expressed even higher than the untransduced. Despite high Tim3 and LAG3 expression in the final manufacturing process, Vax-CAR-T cells significantly suppressed tumor growth in both bigger (&amp;gt;95mm3) and smaller (≤50mm3) AGC xenograft NSG mouse models. More importantly, Vax-CAR-T-treated mice have well tolerated the CAR-T therapy with no significant adverse events during the CRS monitoring period, 2 weeks after Vax-CAR-T injection and have gradually recovered weight gains 5 days after CAR-T cell injection. [Conclusion] We believe that a new autologous CAR-T targeting PD-L1 could be a promising new tool for removing heterogeneous solid tumors in TME, which would be a significant step forward in improving current conventional and immunotherapeutic strategies. Citation Format: Hye-Seong Park, Eun-Ji Choi, Jae-In Yu, Kyung-Sik Lee, Yeo-Jin Lim, Se-Ryeong Choi, Yoonjoo Choi, Bum Chan Park, Jae Eun Park, Mihwa Kim, Je-Jung Lee, Joon Haeng Rhee. The efficacy and safety of PD-L1-specific CAR-T in advanced gastric cancer [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 889.

Abstract 4074: Cellular immunotherapy targeting kappa myeloma antigen for the treatment of multiple myeloma

Abstract Multiple myeloma (MM), the second most common blood cancer, is characterized by the accumulation of malignant plasma cells in the bone marrow. Chimeric Antigen Receptor (CAR)-T cell therapy has recently entered the standard of care for relapsed and refractory MM, following the recent FDA-approval of two CAR-T cell products, ide-cel® and cilta-cel®, which target the B cell maturation antigen (BCMA). However, despite impressive response rates, most patients relapse within 1-3 years. Kappa (κ) myeloma antigen (KMA) is a tumour specific membrane associated protein expressed on malignant plasma cells in patients with kappa light-chain restricted (κ-type) MM. KMA is absent on normal plasma cells and haematopoietic stem cells, making it an attractive and alternative target antigen for CAR-T cell therapy for MM. The monoclonal antibody, KappaMab (MDX-1097), binds to a conformational epitope on KMA, and has been assessed in phase I, IIa and IIb clinical trials in relapse refractory myeloma patients (1). Here, we have engineered a lentiviral vector encoding a second-generation CAR expressing a scFv from MDX-1097, fused to a 4-1BB co-stimulatory domain and CD3 zeta chain. We successfully generated human anti-KMA CAR-T cells with high and stable CAR expression and a predominately memory T cell phenotype. The CAR-T cells selectively killed KMA-expressing tumour lines, secreted interferon-gamma upon target recognition, and demonstrated potent anti-tumour activity in a xenograft model. Anti-KMA CAR-T cell therapy therefore represents a novel and potent treatment ready to enter a phase I clinical trial for patients with myeloma. (1) Spencer et al. Blood Cancer Journal (2019) 9:58 Citation Format: Jessica Li, Nicole Haynes, Katherine Cummins, Kavitha Gowrishankar, Kenneth Micklewaithe, Halley Hilton, Rosanne Dunn, Jane Oliaro, Simon Harrison. Cellular immunotherapy targeting kappa myeloma antigen for the treatment of multiple myeloma. [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 4074.

The signaling and the metabolic differences of various CAR T cell designs

Chimeric antigen receptor (CAR) T cell therapy is introduced as an effective, rapidly evolving therapeutic to treat cancer, especially cancers derived from hematological cells, such as B cells. CAR T cell gene constructs combine a tumor-targeting device coupled to the T cell receptor (TCR) zeta chain domain with different signaling domains such as domains derived from CD28 or 4-1BB (CD137). The incorporation of each specific co-stimulatory domain targets the immunometabolic pathways of CAR T cells as well as other signaling pathways. Defining the immunometabolic and signaling pathways by which CAR T cells become and remain active, survive, and eliminate their targets may represent a huge step forward in this relatively young research field as the CAR gene can be tailored to gain optimal function also for solid tumors with elaborate immunosuppression and protective stroma. There is a close relationship between different signaling domains applied in CAR T cells, and difficult to evaluate the benefit from different tested CAR gene constructs. In this review, we attempt to collect the latest findings regarding the CAR T cell signaling pathways that affect immunometabolic pathways.

Enteric Toll-like receptor 7 stimulation causes acute exacerbation in lupus-susceptible mice.

Autoimmune diseases are often accompanied by acute exacerbation. However, the mechanism underlying systemic lupus erythematosus (SLE) flares remains unclear. We investigated whether short-term enteric Toll-like receptor 7 (TLR7) stimulation can exacerbate SLE using B6SKG mice, which spontaneously develop SLE due to a mutation in the zeta‒chain‒associated protein kinase 70 (Zap70) gene. Imiquimod (IMQ) or phosphate-buffered saline (PBS) were orally administered on B6WT and B6SKG mice every other day for 2weeks. SLE exacerbation was assessed via fluorescent immunohistochemical staining of glomeruli for IgG and C3, hematoxylin and eosin staining of kidneys, and enzyme-linked immunosorbent assay for antinuclear antibody (ANA). Flow cytometry was used to evaluate germinal center B cells (GCBs), plasma cells, follicular helper T cells (Tfhs), regulatory T cells (Tregs), effector T cells (Th1s and Th17s), plasmacytoid dendritic cells (pDCs), conventional dendritic cells (cDCs), and macrophages (Mφs) in spleens. Oral administration of IMQ every other day for 2weeks resulted in exacerbation of splenomegaly, increased IgG and C3 deposition in glomeruli, and increased ANA production in the B6SKG IMQ (SKG-IMQ) group compared to the B6SKG PBS (SKG-PBS) group; the percentages of GCBs, plasma cells, Tfhs, Th1s, pDCs, and Mφs were also increased in the SKG-IMQ group. Splenomegaly, IgG, and C3 deposition in glomeruli, and the percentages of GCBs, plasma cells, Tfhs, and Th1s were enhanced in SKG-IMQ mice compared with B6SKG mice topically treated with IMQ (SKG-ear-IMQ). Oral TLR7 stimulation in a Zap70 genetic mutation background can cause acute exacerbations of SLE. Key Points • The mechanism of SLE flares is not well understood. • We have created a model that causes short-term SLE exacerbations in mice with a genetic background. • IMQ administered orally causes more SLE in mice than transdermally.

Identification of Novel Kinases of Tau Using Fluorescence Complementation Mass Spectrometry (FCMS)

Hyperphosphorylation of the microtubule-associated protein Tau is a major hallmark of Alzheimer's disease and other tauopathies. Understanding the protein kinases that phosphorylate Tau is critical for the development of new drugs that target Tau phosphorylation. At present, the repertoire of the Tau kinases remains incomplete, and methods to uncover novel upstream protein kinases are still limited. Here, we apply our newly developed proteomic strategy, fluorescence complementation mass spectrometry, to identify novel kinase candidates of Tau. By constructing Tau- and kinase-fluorescent fragment library, we detected 59 Tau-associated kinases, including 23 known kinases of Tau and 36 novel candidate kinases. In the validation phase using invitro phosphorylation, among 15 candidate kinases we attempted to purify and test, four candidate kinases, OXSR1 (oxidative-stress responsive gene 1), DAPK2 (death-associated protein kinase 2), CSK (C-terminal SRC kinase), and ZAP70 (zeta chain of T-cell receptor-associated protein kinase 70), displayed the ability to phosphorylate Tau in time-course experiments. Furthermore, coexpression of these four kinases along with Tau increased the phosphorylation of Tau in human neuroglioma H4 cells. We demonstrate that fluorescence complementation mass spectrometry is a powerful proteomic strategy to systematically identify potential kinases that can phosphorylate Tau in cells. Our discovery of new candidate kinases of Tau can present new opportunities for developing Alzheimer's disease therapeutic strategies.

Abstract 6189: Development of an anti hepatocellular carcinoma CAR T strategy targeting PDL1

Abstract [Purpose] Immunosuppressive tumor microenvironment (TME) is the major hurdle to cancer immunotherapy. Advanced hepatocellular carcinoma (aHCC) is one of the representative cold tumors with immunosuppressive TME and presents obvious correlation between immune check point expression, such as programmed cell death ligand 1 (PD-L1), and poorer prognosis. In aHCC patients, cancer cells and stellate cells in TME appeared to express high level of PD-L1, which should suppress activities of antitumor immune cells. In this context, the combination of PD-1/PD-L1 inhibitors and other therapeutic modalities are tried to treat aHCC. The chimeric antigen receptor (CAR) T cell therapy is a potent way of eradicating cells expressing targetable antigens. Removal of PD-L1 expressing cells in the aHCC TME will improve the clinical outcomes of current therapies against aHCC. In the present study, we developed an autologous anti-PD-L1 CAR T strategy and show high efficacy of tumor suppression using an HCC xenograft model. [Result] We constructed second generation CAR lentiviral vector expressing an unique anti-PD-L1 scFv manifesting moderated affinity. The scFv was specifically generated by phage display technique using human B cell-derived naïve cDNA library, which targets PD-L1 with intermediate affinity in solid tumor TME. As the secondary signaling motif, CD28 was chosen and tested along with CD3 zeta chain. The effector CAR T, named VPC1, cells specifically recognized PD-L1 expressing HCC cells and exhibited rapid and robust cytotoxic activity in 2D and 3D culture experiments. In an HCC xenograft NSG mouse model, VPC1 CAR T cells significantly suppressed the tumor growth. More importantly, VPC-1-treated animals have well tolerated the CAR T therapy with no significant adverse manifestations during one month observation period and exhibited gradual weight gains since 3 days after CAR T cell injection. [Conclusion] We hereby report that a new autologous PD-L1 CAR-T therapeutic is capable of eradicating solid tumors expressing PD-L1, which would significantly potentiate efficacies of current conventional and immunotherapeutic strategies. Citation Format: Kyung-Sik Lee, Hye-Seong Park, Guk-Yeol Park, Gi-Chan Lee, Eun-Ji Choi, Jae-in Yu, Seung-Joo Yang, Mihwa Kim, Yoonjoo Choi, Je-Jung Lee, Joon Haeng Rhee, Bum Chan Park, Jae Eun Park. Development of an anti hepatocellular carcinoma CAR T strategy targeting PDL1 [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 6189.

ZAP70 Activation Compensates for Loss of Class IA PI3K Isoforms Through Activation of the JAK–STAT3 Pathway

Tyrosine kinases have crucial functions in cell signaling and proliferation. The phosphatidylinositol 3-kinase (PI3K) pathway is frequently deregulated in human cancer and is an essential regulator of cellular proliferation. We aimed to determine which tyrosine kinases contribute to resistance elicited by PI3K silencing and inhibition. To mimic catalytic inactivation of p110α/β, specific p110α (BYL719) and p110β (KIN193) inhibitors were used in addition to genetic knock-out in in vitro assays. Cell viability was assessed using crystal violet staining, whereas cellular transformation ability was analyzed by soft-agar growth assays. Activated zeta chain of T-cell receptor-associated protein kinase 70 (ZAP70) generated resistance to PI3K inhibition. This resistance was via activation of the Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) axis. We demonstrated that activated ZAP70 has a high transforming capability associated with the formation of malignant phenotype in untransformed cells and has the potential to be a tumor-initiating factor in cancer cells. ZAP70 may be a potent driver of proliferation and transformation in untransformed cells and is implicated in resistance to PI3K inhibitors in cancer cells.

CD247, a Potential T Cell-Derived Disease Severity and Prognostic Biomarker in Patients With Idiopathic Pulmonary Fibrosis.

BackgroundIdiopathic pulmonary fibrosis (IPF) has high mortality worldwide. The CD247 molecule (CD247, as known as T-cell surface glycoprotein CD3 zeta chain) has been reported as a susceptibility locus in systemic sclerosis, but its correlation with IPF remains unclear.MethodsDatasets were acquired by researching the Gene Expression Omnibus (GEO). CD247 was identified as the hub gene associated with percent predicted diffusion capacity of the lung for carbon monoxide (Dlco% predicted) and prognosis according to Pearson correlation, logistic regression, and survival analysis.ResultsCD247 is significantly downregulated in patients with IPF compared with controls in both blood and lung tissue samples. Moreover, CD247 is significantly positively associated with Dlco% predicted in blood and lung tissue samples. Patients with low-expression CD247 had shorter transplant-free survival (TFS) time and more composite end-point events (CEP, death, or decline in FVC >10% over a 6-month period) compared with patients with high-expression CD247 (blood). Moreover, in the follow-up 1st, 3rd, 6th, and 12th months, low expression of CD247 was still the risk factor of CEP in the GSE93606 dataset (blood). Thirteen genes were found to interact with CD247 according to the protein–protein interaction network, and the 14 genes including CD247 were associated with the functions of T cells and natural killer (NK) cells such as PD-L1 expression and PD-1 checkpoint pathway and NK cell-mediated cytotoxicity. Furthermore, we also found that a low expression of CD247 might be associated with a lower activity of TIL (tumor-infiltrating lymphocytes), checkpoint, and cytolytic activity and a higher activity of macrophages and neutrophils.ConclusionThese results imply that CD247 may be a potential T cell-derived disease severity and prognostic biomarker for IPF.

The NLPHL Tumor Microenvironment Is Markedly Enriched in the Tigit and PD-1 Signalling Axes Compared to Classical Hodgkin Lymphoma

The NLPHL Tumor Microenvironment Is Markedly Enriched in the Tigit and PD-1 Signalling Axes Compared to Classical Hodgkin Lymphoma

Aryl Hydrocarbon Receptor (AHR) Gene Expression in AML Is Associated with FLT3-ITD+ AML and HLA-E Induced Immune Resistance Reversed By Ik-364

Background: The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor critical for cellular metabolism, stem cell differentiation and immune control. HLA-E is a non-classical HLA molecule that is critical for NK cell activation. HLA-E is expressed on some AML blasts but genomic characterization is limited. IK-364, a tool compound generated by Ikena Oncology, is an AHR inhibitor in preclinical investigation. We set out to identify the genomic signatures associated with activation of the AHR pathway in AML samples utilizing the Beat AML dataset.Methods: Whole bone marrow or peripheral blood samples were procured from AML patients under IRB-approved protocol (eIRB# 4422). Utilizing the Beat AML database, all samples were examined based on RNA AHR expression levels (RPKM) and correlated with genomic expression. High vs. low AHR expression was based on 90 th and 10 th percentiles. Pathway enrichment from upregulated genes was identified and compared between high vs. low AHR expression. Single cell RNA sequencing by 10x genomics Chromium system by the Massively Parallel Sequencing Shared Resource at OHSU was performed with previously frozen FLT3 positive AML samples (4 high AHR and 4 low AHR). Immunophenotyping was conducted by flow cytometry. The AHR antagonist IK-364 was utilized to study the in vitro effects of AHR modulation. Cytotoxicity was measured by flow cytometric detection of caspase-3/7 and SytoxAAD activation.Results: The highest and lowest 10% expression levels of AHR in all samples in Beat AML were evaluated for mutation profiles. Ranking the top 18 mutations based on frequency we found an enrichment of FLT3 mutations in the high AHR expression group compared to the lower expression group (Figure 1A). ELN risk did not correlate with AHR expression however FAB classification M0/M1 and M4/M5 monocytic subtypes had higher AHR expression than M3 FAB subtype (Figure 2A). AHR gene expression was highly correlated with FLT3-ITD mutations (p=0.0007) (Figure 2B) and HLA-E expression (p=<0.0001) (Figure 3A). High AHR and high HLA-E joint expression in AML was associated with pathway upregulation in translocation of ZAP70 to immunological synapse, upregulation of HLA class II antigen presentation, phosphorylation of CD3 and TCR zeta chain and PD-1 signaling (p-value-1.11E-16), which are critical for innate and adaptive immune responses. AHR low and HLA-E low AML samples showed pathway enrichment for down-regulated genes including Hedgehog on state and neurexins and neuroligins (p value-0.001 and 7.61E-04 respectively). Genes upregulated in AHR high samples included CD14, CD86, HLA-DR, LILRB4, MAFB which are associated with monocytic phenotype. AHR high FLT3+ AML samples had higher protein expression of HLA-E on CD45+ blasts (p=0.03) compared to AHR low FLT3+ AML samples. Pretreatment of FLT3+ AML primary patient samples with IK-364 for 48 hours downregulates HLA-E on blasts (p=0.007) and, in a FLT3 positive AML cell line, Molm14, enhanced susceptibility to NK-cell-mediated killing (Figure 3B, 3C).Discussion: Overall, we found that high AHR gene expression in AML correlates with FLT3-ITD expression and HLA-E as well as monocytic subtype. Pathway enrichment highlight the important role of AHR in immune regulation. Furthermore, AHR antagonism by IK-364, downregulates HLA-E on AML blasts and augments NK cell mediated killing of a mutant FLT3-ITD positive AML cell line. Our results highlight a continued interest in targeting the AHR pathway to augment immune-based therapies in AML.uture studies are underway to determine the mechanism behind this critical immune regulation. [Display omitted] DisclosuresSaultz: IKENA: Research Funding. Lind: IKENA: Research Funding. Tyner: Seattle Genetics: Research Funding; Constellation: Research Funding; Agios: Research Funding; Incyte: Research Funding; Petra: Research Funding; Gilead: Research Funding; Takeda: Research Funding; Janssen: Research Funding; Genentech: Research Funding; Array: Research Funding; Schrodinger: Research Funding; Astrazeneca: Research Funding. Kosaka: IKENA: Research Funding. McGovern: IKENA: Current Employment. Wang: IKENA: Current Employment. Sanchez-Martin: IKENA: Current Employment.

Identification of invariant chain CD74 as a functional receptor of tissue inhibitor of metalloproteinases-1 (TIMP-1)

Multifunctionality of tissue inhibitor of metalloproteinases-1 (TIMP-1) comprising antiproteolytic as well as cytokinic activity has been attributed to its N-terminal and C-terminal domains, respectively. The molecular basis of the emerging proinflammatory cytokinic activity of TIMP-1 is still not completely understood. The cytokine receptor invariant chain (CD74) is involved in many inflammation-associated diseases and is highly expressed by immune cells. CD74 triggers zeta chain–associated protein kinase-70 (ZAP-70) signaling–associated activation upon interaction with its only known ligand, the macrophage migration inhibitory factor. Here, we demonstrate TIMP-1–CD74 interaction by coimmunoprecipitation and confocal microscopy in cells engineered to overexpress CD74. In silico docking in HADDOCK predicted regions of the N-terminal domain of TIMP-1 (N-TIMP-1) to interact with CD74. This was experimentally confirmed by confocal microscopy demonstrating that recombinant N-TIMP-1 lacking the entire C-terminal domain was sufficient to bind CD74. Interaction of TIMP-1 with endogenously expressed CD74 was demonstrated in the Namalwa B lymphoma cell line by dot blot binding assays as well as confocal microscopy. Functionally, we demonstrated that TIMP-1–CD74 interaction triggered intracellular ZAP-70 activation. N-TIMP-1 was sufficient to induce ZAP-70 activation and interference with the cytokine-binding site of CD74 using a synthetic peptide–abrogated TIMP-1-mediated ZAP-70 activation. Altogether, we here identified CD74 as a receptor and mediator of cytokinic TIMP-1 activity and revealed TIMP-1 as moonlighting protein harboring both cytokinic and antiproteolytic activity within its N-terminal domain. Recognition of this functional TIMP-1–CD74 interaction may shed new light on clinical attempts to therapeutically target ligand-induced CD74 activity in cancer and other inflammatory diseases.

Targeting Primary Pre-B Cell Acute Lymphoblastic Leukemia and CD19-Negative Relapses Using Trivalent CAR T Cells

B-Acute Lymphoblastic Leukemia (B-ALL) is the most common malignancy in children, and limited treatment options exist for patients with relapsed or refractory disease. Cellular immunotherapy, specifically chimeric antigen receptor (CAR) T cells targeting CD19, have demonstrated remarkable efficacy in treating B-ALL. However, recent reports show that up to 40% of patients who relapse after CD19 CAR T cell therapy have CD19-negative disease, justifying a need to expand CAR T cell therapy for B-ALL to include additional tumor-associated antigens.Here, we hypothesize that targeting three distinct leukemia antigens including CD19, CD20, and CD22 will improve B-ALL therapy outcomes and control disease progression during CD19-negative relapse.We designed two trivalent CAR T cell products with exodomains derived from single chain variable fragments (ScFv) targeting CD19 (FMC63 ScFv), CD20 (Rituximab ScFv), and CD22 (m971 ScFv). Using viral 2A intervening sequences for near equal expression, the first T cell product expresses the three CARs individually on the surface of a single T cell (TriCAR), and the second T cell product expresses a traditional single CAR targeting CD19 and a second bi-specific CAR targeting CD20 and CD22 through a tandem arrangement (SideCAR). All CARs are fused to the intracellular signaling domains of the co-stimulatory molecule 4-1BB and the T-cell receptor zeta (ζ) chain (2nd generation). Donor T cells were successfully engineered to express the CARs using a retroviral system and the surface expression of these CAR molecules was confirmed by flow cytometry. Using a target expression validated panel of patient derived B-ALL cells (US7 CD19/CD20/CD22 +++/++/++, LAX-56 +++/+/+, TXL-2 +++/++/+++), we observed that TriCAR and SideCAR T cells killed ALL cells more robustly than CD19 CAR T cells at low effector to target (E:T) ratios. TriCAR and SideCAR T cells secreted similar levels of IFN-γ/TNF-α when compared to CD19 CAR T cells suggesting a safety profile similar to the CD19 CAR T cells, but with enhanced killing. Further, we tested the efficacy of TriCAR and SideCAR T cells against primary CD19-negative relapsed bone marrow samples and CRISPR CD19 knockouts of the three primary ALL samples. Using these models of CD19 escape we demonstrated that trivalent CAR T cells effectively mitigated CD19 negative relapse, producing IFN-γ and TNF-α and killing CD19-negative primary ALL, while CD19 CAR T cells remained ineffective. Finally, we interrogated immune synapse (IS) microcluster formation and actin dynamics during interactions between CAR T cells and primary B-ALL cells by quantitative imaging flow cytometry. All CAR T cells exhibited higher actin polymerization compared to non-transduced T cell controls. TriCAR T cells formed significantly higher number of IS microclusters with different morphologies of actin polymerization compared to CD19 CAR T cells, suggesting distinct remodeling and dynamics of T cell polarization and immunoactivity, when interacting with TXL-2 primary B-ALL cells. TriCAR T cells formed IS microclusters, while CD19 CAR T cells failed to form IS with CD19 knockout TXL-2 cells.In conclusion, trivalent CAR T cells are effective at targeting primary ALL cells with varying antigen profiles and at mitigating CD19-negative relapse. This strategy has the potential for use as a front-line therapy for primary ALL as well as a salvage therapy for patients with CD19-negative disease relapse. DisclosuresNo relevant conflicts of interest to declare.

A Role for Human Renal Tubular Epithelial Cells in Direct Allo-Recognition by CD4+ T-Cells and the Effect of Ischemia-Reperfusion.

Direct allorecognition is the earliest and most potent immune response against a kidney allograft. Currently, it is thought that passenger donor professional antigen-presenting cells (APCs) are responsible. Further, many studies support that graft ischemia-reperfusion injury increases the probability of acute rejection. We evaluated the possible role of primary human proximal renal tubular epithelial cells (RPTECs) in direct allorecognition by CD4+ T-cells and the effect of anoxia-reoxygenation. In cell culture, we detected that RPTECs express all the required molecules for CD4+ T-cell activation (HLA-DR, CD80, and ICAM-1). Anoxia-reoxygenation decreased HLA-DR and CD80 but increased ICAM-1. Following this, RPTECs were co-cultured with alloreactive CD4+ T-cells. In T-cells, zeta chain phosphorylation and c-Myc increased, indicating activation of T-cell receptor and co-stimulation signal transduction pathways, respectively. T-cell proliferation assessed with bromodeoxyuridine assay and with the marker Ki-67 increased. Previous culture of RPTECs under anoxia raised all the above parameters in T-cells. FOXP3 remained unaffected in all cases, signifying that proliferating T-cells were not differentiated towards a regulatory phenotype. Our results support that direct allorecognition may be mediated by RPTECs even in the absence of donor-derived professional APCs. Also, ischemia-reperfusion injury of the graft may enhance the above capacity of RPTECs, increasing the possibility of acute rejection.

Packed red blood cells inhibit T-cell activation via ROS-dependent signaling pathways

Numerous observations indicate that red blood cells (RBCs) affect T-cell activation and proliferation. We have studied effects of packed RBCs (PRBCs) on T-cell receptor (TCR) signaling and the molecular mechanisms whereby (P)RBCs modulate T-cell activation. In line with previous reports, PRBCs attenuated the expression of T-cell activation markers CD25 and CD69 upon costimulation via CD3/CD28. In addition, T-cell proliferation and cytokine expression were markedly reduced when T-cells were stimulated in the presence of PRBCs. Inhibitory activity of PRBCs required direct cell–cell contact and intact PRBCs. The production of activation-induced cellular reactive oxygen species, which act as second messengers in T-cells, was completely abrogated to levels of unstimulated T-cells in the presence of PRBCs. Phosphorylation of the TCR-related zeta chain and thus proximal TCR signal transduction was unaffected by PRBCs, ruling out mechanisms based on secreted factors and steric interaction restrictions. In large part, downstream signaling events requiring reactive oxygen species for full functionality were affected, as confirmed by an untargeted MS-based phosphoproteomics approach. PRBCs inhibited T-cell activation more efficiently than treatment with 1 mM of the antioxidant N-acetyl cysteine. Taken together, our data imply that inflammation-related radical reactions are modulated by PRBCs. These immunomodulating effects may be responsible for clinical observations associated with transfusion of PRBCs.