T-cell Pathways Research Articles

Advances in ligand-based surface engineering strategies for fine-tuning Tcell mechanotransduction toward efficient immunotherapy.

T cell-based immunotherapy has recently emerged as a promising strategy to treat cancer, requiring the activation of antigen-directed cytotoxicity to eliminate cancer cells. Mechanical signaling, although often overshadowed by its biochemical counterpart, plays a crucial role in Tcell anticancer responses, from activation to cytolytic killing. Rapid advancements in the fields of chemistry, biomaterials, and micro/nanoengineering offer an interdisciplinary approach to incorporating mechano- and immunomodulatory ligands, including but not limited to synthetic peptides, small molecules, cytokines, and artificial antigens, onto the biomaterial-based platforms to modulate mechanotransducive processes in Tcells. The surface engineering of these immunomodulatory ligands with optimization of ligand density, geometrical arrangement, and mobility has been proven to better mimic the natural ligation between immunoreceptors and ligands to directly enhance or inhibit mechanotransduction pathways in Tcells, through triggering upstream mechanosensitive channels, adhesion molecules, cytoskeletal components, or downstream mechanoimmunological regulators. Despite its tremendous potential, current research on this new biomaterial surface engineering approach for mechanomodulatory Tcell activation and effector functions remains in a nascent stage. This review highlights the recent progress in this new direction, focusing on achievements in mechanomodulatory ligand-based surface engineering strategies and underlying principles, and outlooks the further research in the rapidly evolving field of Tcell mechanotransduction engineering for efficient immunotherapy.

Economic evaluation of anti-CD19 CAR T-cell pathway for large B-cell lymphomas in the real-life setting: the experience of an Italian hub center in the first three years of activity

Poor literature report actual and detailed costs of chimeric antigen receptor (CAR) T-cell pathway in a real-life setting. We retrospectively collect data for all patients with relapsed/refractory aggressive large B-cell lymphoma who underwent leukapheresis between August 2019 and August 2022. All costs and medical resource consumption accountability were calculated on an intention-to-treat (ITT) basis, starting from leukapheresis to the time when the patient (infused or not) exited the CAR T-cell pathway for any reason. Eighty patients were addressed to leukapheresis and 59 were finally infused. After excluding CAR-T product cost, the main driver of higher costs were hospitalizations followed by the examinations/procedures and other drugs, respectively 43.9%, 26.3% and 25.4% of the total. Regarding costs of drugs and medications other than CAR T products, the most expensive items are those referred to AEs, both infective and extra-infective within 30 days from infusion, that account for 63% of the total. Density plot of cost analyses did not show any statistically significant difference with respect to the years of leukapheresis or infusion. To achieve finally 59/80 infused patients the per capita patients without CAR-T products results 74,000 euros. This analysis covers a growing concern on health systems, the burden of expenses related to CAR T-cell therapy, which appears to provide significant clinical benefit despite its high cost, thus making economic evaluations highly relevant. The relevance of this study should be also viewed in light of continuously evolving indications for this therapy.

LKB1 prevents ILC2 exhaustion to enhance antitumor immunity

Group 2 innate lymphoid cells (ILC2s) play crucial roles in mediating allergic inflammation. Recent studies also indicate their involvement in regulating tumor immunity. The tumor suppressor liver kinase B1 (LKB1) inactivating mutations are associated with a variety of human cancers; however, the role of LKB1 in ILC2 function and ILC2-mediated tumor immunity remains unknown. Here, we show that ablation of LKB1 in ILC2s results in an exhausted-like phenotype, which promotes the development of lung melanoma metastasis. Mechanistically, LKB1 deficiency leads to a marked increase in the expression of programmed cell death protein-1 (PD-1) in ILC2s through the activation of the nuclear factor of activated Tcell pathway. Blockade of PD-1 can restore the effector functions of LKB1-deficient ILC2s, leading to enhanced antitumor immune responses invivo. Together, our results reveal that LKB1 acts to restrain the exhausted state of ILC2 to maintain immune homeostasis and antitumor immunity.

RNA-sequencing of Human Kidney Allografts and Delineation of T-Cell Genes, Gene Sets, and Pathways Associated With Acute T Cell-mediated Rejection.

Delineation of T-cell genes, gene sets, pathways, and T-cell subtypes associated with acute T cell-mediated rejection (TCMR) may improve its management. We performed bulk RNA-sequencing of 34 kidney allograft biopsies (16 Banff TCMR and 18 no rejection [NR] biopsies) from 34 adult recipients of human kidneys. Computational analysis was performed to determine the differential intragraft expression of T-cell genes at the level of single-gene, gene set, and pathways. T-cell signaling pathway gene sets for plenary T-cell activation were overrepresented in TCMR biopsies compared with NR biopsies. Heightened expression of T-cell signaling genes was validated using external TCMR biopsies. Pro- and anti-inflammatory immune gene sets were enriched, and metabolism gene sets were depleted in TCMR biopsies compared with NR biopsies. Gene signatures of regulatory T cells, Th1 cells, Th2 cells, Th17 cells, T follicular helper cells, CD4 tissue-resident memory T cells, and CD8 tissue-resident memory T cells were enriched in TCMR biopsies compared with NR biopsies. T-cell exhaustion and anergy were also molecular attributes of TCMR. Gene sets associated with antigen processing and presentation, and leukocyte transendothelial migration were overexpressed in TCMR biopsies compared with NR biopsies. Cellular deconvolution of graft infiltrating cells by gene expression patterns identified CD8 T cell to be the most abundant T-cell subtype infiltrating the allograft during TCMR. Our delineation of intragraft T-cell gene expression patterns, in addition to yielding new biological insights, may help prioritize T-cell genes and T-cell subtypes for therapeutic targeting.

Abstract 3927: Metabolic inhibition of BATF2 dampens type-I interferon-mediated immune sensing of cancer

Abstract Background: The type-I interferon (IFN-I) system appeared before adaptive immunity during evolution as the primary defense system for metazoans. Initiating cancers frequently disable this first line of defense before establishing T-cell exhaustion programs. However, the molecules linking IFN-I, danger signals, and unique metabolic conditions in the tumor microenvironment (TME) remain to be discovered. Methods: We performed centered log-ratio transformation on the cluster composition data using the R-package ALDEx2 to compare the clusters in single-cell data sets. IFN-I signatures were quantified using qPCR and ELISA. ChIP was performed to examine the H3K27me3 epigenetic marker on the promoters of BATF2 and IFN-I genes in bone marrow-derived macrophage (BMDM). For the cigarette smoke carcinogen-induced head and neck squamous cell carcinoma (HNSCC) model, experimental mice were fed with water containing 50 μg/mL of 4-NQO for 16 weeks and then switched to regular feeding water. We started monitoring 10 weeks post-treatment for the number, location, and diameter of the lesions. TME analysis was performed using single-cell RNA-Seq and multispectral imaging. Results: We analyzed three large independent cohorts of patients and found that BATF2 strongly correlated with the immunogenicity of HNSCC, with a stronger correlation coefficient than STING. We generated Batf2−/− BMDM and found that Batf2 deficiency rendered cells insensitive to STING agonists. However, the dsRNA-induced IFN-I activation was not affected by Batf2 deletion. BATF2 interacted with STING, promoting its phosphorylation and oligomerization. Glutamine is enriched in the TME. We found that glutamine inhibited the BATF2-STING pathway by increasing a repressive epigenetic marker, H3K27me3, on the promoters of BATF2 and IFNB1. The expression of BATF2 in cancer cells resulted in IFN-I- and γδ T-cell-dependent tumor rejection. Single-cell immune profiling showed that Batf2-expressing tumors contained significantly expanded Cxcl10+ myeloid cells and γδ T-cells. Utilizing the 4-NQO-induced HNSCC model, we found that BATF2 limited suppressive myeloid cells and expanded effector cells in the TME, serving as a potent tumor suppressor. Conclusion: BATF2 is a glutamine-responsive tumor suppressor that limits tumor initiation by engaging the STING-γδ T-cell pathway. Acknowledgments: This work was supported by NIH grants R01 DE026728, U01 DE029255, R01 DE031951, and U01 DE033330 Citation Format: Wang Gong, Hülya Taner, Yuesong Wu, Zaiye Li, Wanqing Cheng, Christopher Donnelly, Felipe Nör, Yumin He, Zackary Fitzsimonds, Jianqian Li, Haitao Wen, Steven Chinn, Yuying Xie, James Moon, Yu Leo Lei. Metabolic inhibition of BATF2 dampens type-I interferon-mediated immune sensing of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3927.

AIM high: Epigenetic modulation and immune stimulation in bladder cancer.

648 Background: Immune-checkpoint inhibitors (ICI) have emerged as valuable treatment options for patients with bladder cancer, particularly those who are ineligible for other therapies or have experienced disease progression on treatment. Response rates are only 20-30%, highlighting the need for biomarkers to predict treatment response and strategies to overcome resistance. Epigenetic modulators including the DNA methyltransferase (DNMT) inhibitor 5-azacitidine (AZA) and histone deacetylase inhibitors (HDACi) may represent co-targeting therapies in combination with ICI to enhance antitumor immunity. AZA has been demonstrated to increase the expression of a panel of immunomodulatory genes termed the “AZA Immune gene panel (AIM)” in breast, colorectal, and ovarian cancer cell lines. We hypothesized that bladder cancer with low AIM expression can be therapeutically converted by epigenetic modulation to “AIM-high” for future combination with ICI. Methods: Five human bladder cancer cell lines were selected: SCaBER, SW780, T24, RT4 and one developed at our institution (CoCaB1). The Cancer Cell Line Encyclopedia (CCLE) was used to screen the four included lines for intrinsic AIM expression, which were predicted to be AIM-high (n=1) and AIM-low (n=3). A cell viability assay was performed to screen for AZA and HDACi toxicity. Cells were treated with drug or vehicle for 72 hours and harvested at day 6, 14, and 21. RNA was extracted for RNA sequencing (RNA-seq) analyses. For analysis of AIM gene expression in clinical specimens, RNA-seq data was obtained from primary (n=6) and metastatic (n=83) bladder cancer specimens for 20 patients participating in the University of Washington Bladder Cancer Rapid Autopsy Program. Results: RNA-seq analyses of 5 cell lines demonstrated increased expression (>log 2-fold change, p<0.05) of a subset of AIM genes involved in inflammation, interferon, cytokine/chemokine signaling, and cancer testis antigens (CTA, a family of immunogenic proteins) with AZA treatment. Co-treatment with HDACi showed additional upregulation of AIM genes. Gene set enrichment analyses uncovered enrichment in T-cell pathway activation. In metastatic bladder cancer tissues, interferon, cytokine/chemokine, inflammation, and CTA AIM gene set categories were upregulated in a subset of patients. AIM gene enrichment displayed a patient-dependent pattern and was consistent across metastatic sites within a patient. Conclusions: Epigenetic priming therapy increases the expression of immunomodulatory and T-cell related genes in bladder cancer cell lines with low baseline AIM expression. Additional correlative studies in the rapid autopsy series will further determine clinical- and treatment-related factors contributing to intrinsic immune-related gene expression. Building on this paradigm, future in vivo and clinical studies could lead to novel combination therapies for patients with bladder cancer.

The glycoimmune checkpoint receptor Siglec-7 interacts with T-cell ligands and regulates T-cell activation

Siglec-7 (sialic acid-binding immunoglobulin-like lectin 7) is a glycan-binding immune receptor that is emerging as a significant target of interest for cancer immunotherapy. The physiological ligands that bind Siglec-7, however, remain incompletely defined. In this study, we characterized the expression of Siglec-7 ligands on peripheral immune cell subsets and assessed whether Siglec-7 functionally regulates interactions between immune cells. We found that disialyl core 1 O-glycans are the major immune ligands for Siglec-7 and that these ligands are particularly highly expressed on naïve T-cells. Densely glycosylated sialomucins are the primary carriers of these glycans, in particular a glycoform of the cell-surface marker CD43. Biosynthesis of Siglec-7-binding glycans is dynamically controlled on different immune cell subsets through a genetic circuit involving the glycosyltransferase GCNT1. Siglec-7 blockade was found to increase activation of both primary T-cells and antigen-presenting dendritic cells invitro, indicating that Siglec-7 binds T-cell glycans to regulate intraimmune signaling. Finally, we present evidence that Siglec-7 directly activates signaling pathways in T-cells, suggesting a new biological function for this receptor. These studies conclusively demonstrate the existence of a novel Siglec-7-mediated signaling axis that physiologically regulates T-cell activity. Going forward, our findings have significant implications for the design and implementation of therapies targeting immunoregulatory Siglec receptors.

Reduced Proliferation and Cell Death of Acute Myeloid Leukemia Cell Lines after Acalabrutinib Treatment

Background Acute myeloid leukemia (AML) still remains a significant challenge, with intensive chemotherapy treatments showing limited efficacy and lack of druggable targets in contrast to other hematologic malignancies. Exosomes, nanomolecules derived from cells reflect the parent cell's state, present opportunities for disease biomarker discovery and targeted therapy. Proteomic analysis of AML-derived exosomes may identify druggable targets despite limited prior research. In this study we investigated the potential of exosomes as a tool for target discovery in AML. Methods We conducted a proteomic analysis of AML-derived exosomes from HL-60, KG-1, and THP-1 cell lines, using fibroblast and mesenchymal stem cells as controls. Exosomes of each cell line were isolated, and digested via filter-aided sample preparation (FASP), followed by isobaric tagging using tandem mass tag (TMT) reagents. The samples were then fractionated using high-pH reversed-phase liquid chromatography and analyzed using Q-Exactive Orbitrap Hybrid Mass Spectrometry. Proteomic data were evaluated using SEQUEST in Proteome Discoverer 2.1 with a false discovery rate set to 1%. Differentially expressed proteins (DEPs) were identified and visualized in an interaction network and was analyzed for the gene ontology biological process (GOBP) using DAVID software. Protein-protein interactions were analyzed using the STRING software. Based on proteomic analysis, we identified the BTK-related pathway as potential druggable target for AML cells. AML cells were incubated with ACT, a BTK inhibitor, in various exposure time and density. Cell viability and proliferation rate was assessed using CCK-8 assay. BTK activity and signaling were assessed via Western blot analysis and immunocytochemistry of AML-derived exosomes. Cell death was evaluated using Annexin-V & PI apoptosis kit and FACS analysis. Statistical analysis was performed to ascertain significance. Results Proteomics of exosomes from three AML cell types revealed distinct protein profiles via LC-MS, with 2450 unique proteins identified (Figure 1A). For KG1, HL60, and THP1, the number of up-regulated DEPs were 438, 434, and 408, and the number of down-regulated DEPs was 126, 118, and 89 (fold change>1.5 or <0.67, P<0.05). Gene ontology analysis identified four main categories of biological processes, including cell migration, T-cell pathways, cell damage, and vesicle formation pathways. Investigation into protein-protein interactions using the STRING software allowed selection of tyrosine kinases SYK and LYN as biomarker candidates. Both kinases regulate BTK, a critical molecule in B-cell antigen receptor signaling. SYK and LYN were found to be enriched in exosomes, promoting phosphorylation of BTK in recipient cells. (Figure 1B) Cell viability experiments with AML cell lines revealed reduced viability upon treatment with ACT. Viability decreased with increased exposure time and density, with the highest effects observed at 1,000nM ACT concentration for 48 hours (Figure 2A). This was accompanied by decreased cell numbers and proliferation rate which was significant in the HL-60 and KG-1 cell lines (Figure 2B). Western blot analysis demonstrated significant decrease of the BTK downstream signaling (P-BTK, PKC and p-ERK) by acalabrutinib treatment (1,000nM) at 48 hours (Figure 2C). Proliferative markers, such as PCNA, and Cyclin D1 were also significantly decreased. LYN and SYK was also decreased in the exosomes of HL-60 and THP-1 cell line. The inhibition of P-BTK and Cyclin D1 by ACT was further confirmed through Immunofluorescence assay. Finally, ACT was shown to promote cell death in AML cell lines, as evidenced by an apoptosis assay, with the most significant increases observed in cells treated exposed to 1,000nM ACT for 72 h (Figure 2D). Conclusion Overall, the findings of this study revealed that acalabrutinib reduced proliferation and induced cell death in AML cells suggesting the potential of BTK inhibitors in AML treatment. This was explained through proteomic analysis of AML-derived exosomes. However, further studies are required to validate the clinical use of exosomes as biomarkers and druggable target.

Establishment of comorbidity target pools and prediction of drugs candidate for multiple sclerosis and autoimmune thyroid diseases based on GWAS and transcriptome data.

Clinical observation has revealed that multiple sclerosis (MS) and autoimmune thyroid disease (AITD) are strongly correlated. The aim of this study was to explore the shared molecular causes of MS and AITD, and to conduct drug rearrangement on this basis, search for comorbidity drugs and feasible drugs for mutual reference between the two diseases. Based on genome-wide association study (GWAS) data and transcriptome data, susceptibility genes and differentially expressed genes related to MS and AITD were identified by bioinformatics analysis. Pathway enrichment, gene ontology (GO), protein-protein interaction analysis, and gene-pathway network analysis of the above genes were performed to identify a common target pool, including common genes, common hub genes, and common pathways, and to explore the specific pathogenesis of the two diseases, respectively. Drugs that target the common pathways/genes were identified through the Comparative Toxicogenomics Database (CTD), DrugBank database, and Drug-Gene Interaction (DGI) Database. Common hub genes were compared with the target genes of drugs approved for treating MS/AITD and drugs under investigation identified by DrugBank and ClinicalTrials, respectively. We identified a pool of shared targets containing genes and pathways, including 46 common genetic susceptibility pathways and 9 common differentially expressed pathways, including JAK-STAT signaling pathway, Th17 cell differentiation, Th1 and Th2 cell differentiation, PD-L1 expression and PD-1 checkpoint pathway in cancer, etc. In addition, a total of 29 hub genes, including TYK2, JAK1, STAT3, IL2RA, HLA-DRB1, and TLR3, were identified. Drugs approved for treating MS or AITD, such as methylprednisolone, cyclophosphamide, glatiramer, natalizumab, and methimazole, can target the shared genes and pathways, among which methylprednisolone and cyclophosphamide have been shown to be beneficial for the treatment of the two diseases, indicating that these drugs have the potential to become a priority in the treatment of comorbidities. Moreover, drugs targeting multiple common genes and pathways, including tacrolimus, deucravacitinib, and nivolumab, were identified as potential drugs for the treatment of MS, AITD, and their comorbidities. We observed that T-cell activation-related genes and pathways play a major role in the pathogenesis of both MS and AITD, which may be the molecular basis of the comorbidity. Moreover, we identified a variety of drugs which may be used as priority or potential treatments for comorbidities.

Abnormal Lymphatic Sphingosine-1-Phosphate Signaling Aggravates Lymphatic Dysfunction and Tissue Inflammation.

Lymphedema is a global health problem with no effective drug treatment. Enhanced T-cell immunity and abnormal lymphatic endothelial cell (LEC) signaling are promising therapeutic targets for this condition. Sphingosine-1-phosphate (S1P) mediates a key signaling pathway required for normal LEC function, and altered S1P signaling in LECs could lead to lymphatic disease and pathogenic T-cell activation. Characterizing this biology is relevant for developing much needed therapies. Human and mouse lymphedema was studied. Lymphedema was induced in mice by surgically ligating the tail lymphatics. Lymphedematous dermal tissue was assessed for S1P signaling. To verify the role of altered S1P signaling effects in lymphatic cells, LEC-specific S1pr1-deficient (S1pr1LECKO) mice were generated. Disease progression was quantified by tail-volumetric and -histopathologic measurements over time. LECs from mice and humans, with S1P signaling inhibition, were then cocultured with CD4 T cells, followed by an analysis of CD4 T-cell activation and pathway signaling. Last, animals were treated with a monoclonal antibody specific to P-selectin to assess its efficacy in reducing lymphedema and T-cell activation. Human and experimental lymphedema tissues exhibited decreased LEC S1P signaling through S1P receptor 1 (S1PR1). LEC S1pr1 loss-of-function exacerbated lymphatic vascular insufficiency, tail swelling, and increased CD4 T-cell infiltration in mouse lymphedema. LECs, isolated from S1pr1LECKO mice and cocultured with CD4 T cells, resulted in augmented lymphocyte differentiation. Inhibiting S1PR1 signaling in human dermal LECs promoted T-helper type 1 and 2 (Th1 and Th2) cell differentiation through direct cell contact with lymphocytes. Human dermal LECs with dampened S1P signaling exhibited enhanced P-selectin, an important cell adhesion molecule expressed on activated vascular cells. In vitro, P-selectin blockade reduced the activation and differentiation of Th cells cocultured with shS1PR1-treated human dermal LECs. P-selectin-directed antibody treatment improved tail swelling and reduced Th1/Th2 immune responses in mouse lymphedema. This study suggests that reduction of the LEC S1P signaling aggravates lymphedema by enhancing LEC adhesion and amplifying pathogenic CD4 T-cell responses. P-selectin inhibitors are suggested as a possible treatment for this pervasive condition.

Gene Expression Profiling and Signaling Pathway Analysis of Tolerant T cells Circulating in Adult Patients after PTCy Haplotransplantation

Abstract The mechanisms for the signaling pathways of T cell-dependent tolerance after haploidentical hematopoietic stem cell transplantation (haplo-HSCT), are incompletely understood. We tested 8 patients (Pt) with leukemia or myelodysplastic syndromes (median age:51-yrs old) following IRB approved consenting. All received Flu+TBI (+/− Cy) pre-conditioning and haplo-HSCT with post-transplant Cytoxan (PTCy) and FK506 as GvHD prophylaxis. The circulating T cells were hyporeactive in mixed lymphocyte reaction (MLR) assay to recipient DC in Pts at 1yr (or 2yrs in 1 Pt) post-haplo-HSCT (~6m post-ISD withdrawal), while these T cells responded vigorously to 3rd party APC. Neither depletion of Tregs, nor blockade of IL10R or PD1 could reverse T-cell hyporeactivity. Low-dose IL2 triggered mild anti-host responses. Cytokine profiling further confirmed the MLR responses. ImmunoSEQ™ revealed the disappearance of alloreactive T-cell clones from peripheral pool with time, even though a few alloreactive T clones remained. Low dose IL2 had minimal impact to reverse the diminishment. Unlike graft T-cell responses against host DC, tolerant T cells in circulation against hDC showed relative downregulation of allograft rejection, T-helper response, co-stimulation, and cell proliferation pathway, that was paired with upregulation of co-inhibition, PD1-PDL1, Ferroptosis and CREB pathways. In summary, hyporeactivity of donor-derived circulating T cells to recipient DC were evident. We found Treg, Tr1 activity to be insignificant in maintaining long-term tolerance. T-cell clonal deletion appears to be the dominant mechanism, along with systemically altered signaling pathways in tolerant T-cells.

Transcriptomics unravels molecular changes associated with cilia and COVID-19 in chronic rhinosinusitis with nasal polyps

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a common upper respiratory tract complication where the pathogenesis is largely unknown. Herein, we investigated the transcriptome profile in nasal mucosa biopsies of CRSwNP patients and healthy individuals. We further integrated the transcriptomics data with genes located in chromosomal regions containing genome-wide significant gene variants for COVID-19. Among the most significantly upregulated genes in polyp mucosa were CCL18, CLEC4G, CCL13 and SLC9A3. Pathways involving “Ciliated epithelial cells” were the most differentially expressed molecular pathways when polyp mucosa and non-polyp mucosa from the same patient was compared. Natural killer T-cell (NKT) and viral pathways were the most statistically significant pathways in the mucosa of CRSwNP patients compared with those of healthy control individuals. Upregulated genes in polyp mucosa, located within the genome-wide associated regions of COVID-19, included LZTFL1, CCR9, SLC6A20, IFNAR1, IFNAR2 and IL10RB. Interestingly, the second most over-expressed gene in our study, CLEC4G, has been shown to bind directly to SARS-CoV-2 spike's N-terminal domain and mediate its entry and infection. Our results on altered expression of genes related to cilia and viruses point to the de-regulation of viral defenses in CRSwNP patients, and may give clues to future intervention strategies.

A phase I/II study of the CXCR2 inhibitor, AZD5069, in combination with durvalumab, in patients (pts) with advanced hepatocellular carcinoma (HCC).

TPS631 Background: HCC is increasing rapidly in incidence worldwide driven by a rise in chronic liver disease including non-alcoholic steato-hepatitis (NASH). Most pts are not suitable for curative or loco-regional treatments and may be candidates for systemic therapies. Immune checkpoint inhibitors combined with VEGF inhibition is a standard of care in HCC. However, a meta-analysis of 3 phase III randomised trials of PD-1 or PDL-1 inhibitors (n > 1,600 pts) with HCC suggests that pts with NASH-related HCC treated with PD-1/PDL-1 inhibitors had reduced overall survival compared with other aetiologies. Neutrophils expressing the chemokine receptor CXCR2, crucial to neutrophil recruitment in acute-injury, are highly represented in NASH-HCC. In NASH-HCC murine models, lacking response to immune-checkpoint inhibitors, AZD5069 (CXCR2 inhibitor) in combination with anti-PDL-1 suppressed tumor burden and extended survival, accompanied by an increase in tumor-associated neutrophils which switched from a pro-tumor to anti-tumor progenitor-like neutrophil phenotype. We propose that inhibition of CXCR2 may potentiate the efficacy of anti-PDL-1 inhibition in pts with HCC. Methods: In this multi-centre (n = 10) study, pts with biopsy-confirmed HCC, PS ECOG < 1, Child-Pugh A, < 1 prior systemic therapies, receive 1 of escalating doses of AZD5069 (bid, po daily) with Durvalumab (1.5 gm iv on day 1) in 28-day cycles for up to 2 years to determine the recommended phase II dose using a Keyboard design, followed by an additional cohort of pts to determine the anti-tumor efficacy of this combination using a Simon’s two-stage design (min 18, max 35 pts; target objective response rate > 30%; unacceptable response rate < 10%). Dose limiting toxicities (DLTs) are assessed during cycle 1. Disease assessments are performed 8-weekly (12-weekly after 1 year). The 1st dose cohort has been completed with no DLTs. The 2nd dose cohort opened to recruitment in September 2022. Exploratory studies (blood; pre- & on-treatment tumor and non-malignant liver biopsies) include biomarkers of CXCR2 inhibition (blood); proof-of-mechanism (tumor: expression of CXCR2, PD-L1, PD-1, CD8, CD4, CD66b, CD69); proof-of-mechanism (blood: ctDNA); drug-induced changes of mRNA expression, CXCR2 ligands & signalling pathway genes, T-cell and myeloid cell pathways, neutrophil-associated genes; predictive biomarkers (blood and tumour) include biomarkers of the CXCR2/PD-L1 immune axis; aberrant CXCR2 signalling pathways; proliferation biomarkers and CD10 (neutrophils), CD68 (macrophages), CD103 (T-regs); tumour mutational status. This study is funded by a grant from Cancer Research UK (A29287) and is co-sponsored by University of Glasgow and NHS Greater Glasgow & Clyde. Study sites are supported by the Experimental Cancer Medicine Centre Network. AZD5069 and Durvalumab are provided by Astra Zeneca. Clinical trial information: 2020-003346-36 .

The IAP antagonist birinapant enhances chimeric antigen receptor T cell therapy for glioblastoma by overcoming antigen heterogeneity

Antigen heterogeneity that results in tumor antigenic escape is one of the major obstacles to successful chimeric antigen receptor (CAR) Tcell therapies in solid tumors including glioblastoma multiforme (GBM). To address this issue and improve the efficacy of CAR Tcell therapy for GBM, we developed an approach that combines CAR Tcells with inhibitor of apoptosis protein (IAP) antagonists, a new class of small molecules that mediate the degradation of IAPs, to treat GBM. Here, we demonstrated that the IAP antagonist birinapant could sensitize GBM cell lines and patient-derived primary GBM organoids to apoptosis induced by CAR Tcell-derived cytokines, such as tumor necrosis factor. Therefore, birinapant could enhance CAR Tcell-mediated bystander death of antigen-negative GBM cells, thus preventing tumor antigenic escape in antigen-heterogeneous tumor models invitro and invivo. In addition, birinapant could promote the activation of NF-κB signaling pathways in antigen-stimulated CAR Tcells, and with a birinapant-resistant tumor model we showed that birinapant had no deleterious effect on CAR Tcell functions invitro and invivo. Overall, we demonstrated the potential of combining the IAP antagonist birinapant with CAR Tcells as a novel and feasible approach to overcoming tumor antigen heterogeneity and enhancing CAR Tcell therapy forGBM.

Gene Expression Profiling and Signaling Pathway Analysis of Tolerant T Cells Circulating in Adult Patients after Ptcy Haplotransplantation

The mechanisms of T cell-dependent tolerance, especially its signaling pathways after haploidentical hematopoietic stem cell transplantation (haplo-HSCT), are incompletely understood. Both central and peripheral regulation mechanisms are suspected. Subjects and Methods: We tested 8 patients with leukemia (5) or myelodysplastic syndromes (MDS, 3) (age 35-73-years old, median: 51-years old) following IRB approved consenting. All received Flu+TBI (+/- Cy) pre-conditioning and haplo-HSCT with post-transplant Cytoxan (PTCy) and FK506 as GvHD prophylaxis. Chimerism at tolerance studies was donor dominant (97-100%, mean: 99% for both CD33+ myeloid cells and CD3+T cells). Immunosuppression drug (ISD) was withdrawn ~6 months before the time of the in vitro tolerance study described below. We described previously Treg depletion from purified T cells with IL2-immunotoxin (Denileukin Diftitox, Seragen Inc™) to test the involvement of these cells in long-term tolerance. The contribution of Tr1 or anergy was examined by either blocking IL10R or adding low dose IL2, respectively. T-cell exhaustion was tested by blocking the PD1 pathway with anti-PD1 MoAb (OPDIVO, Bristol Myers Squibb™). Purified T cell responses to recipient dendritic cells (DC) were measured in mixed lymphocyte reaction (MLR) assay, and further tested by quantifying cytokine secretion in MLR culture supernatants using Bio-Plex immunoassays (Bio-Rad Lab™). To test for clonal deletion, alloreactive T-cell clones with frequency >0.1% identified in vitro from the graft pre-infusion were tracked by monitoring their T-cell receptor CDR3 sequence serially, in an ImmunoSEQ™ assay (Adaptive Biotech™). Gene expression profiling and signaling pathway analysis on alloreactive or tolerant T-cells were performed by collecting T cells from in vitro MLR reactions, purifying total RNAs from these cells, running RNA-sequencing and analyzing with software of CLC Genomic Workbench®v21, GSEA®v4.1.0 and IPA®. Results: We have previously shown that circulating T cells were hyporeactive to recipient DC in patients at 1yr (or 2yrs in 1 patient) post-haplo-HSCT [6 months, (or 2-5 months in 3 patients) post-ISD withdrawal], while these T cells responded vigorously to 3rd party APC, As positive control, host DC triggered robust 3rd party T-cell response. Neither depletion of Tregs, nor blockade of IL10R or PD1 could reverse T-cell hypo-reactivity. Low-dose IL2 triggered mild anti-host proliferation responses. Similar scenarios were detected in graft T cells against self-DC, (negative control in autologous set up), while vigorous responses were shown in the graft T cells against host-DC pre-haplo-HSCT. Cytokine profiling further confirmed the MLR responses. ImmunoSEQ™ revealed disappearance of alloreactive T-cell clones from peripheral blood with time. Low dose IL2 had minimal impact to reverse hyporeactivity. While most alloreactive T clones were undetectable, a few remained in the peripheral pool in tolerant patients tested (Nee, T et al, Transplantation and Cellular Therapy, 2022;28(3):S44-S45). Unlike graft T-cell responses against host DC, tolerant T-cells in circulation (including remaining alloreactive T cells) against hDC showed distinct patterns of gene expressions, with relative downregulation of allograft rejection (GSEA-Hallmark) (Fig 1A, B, C) and T-helper response, co-stimulation, cell proliferation pathway, that was paired with upregulation of co-inhibition, PD1-PDL1, Ferroptosis, PTEN and CREB pathways (Fig1D). The profiles suggested systemic alterations in the signaling pathways on tolerant T cells post-haplo-HSCT. Conclusion: Hypo-reactivity of donor-derived circulating T cells to recipient DC and graft T cells to graft DC (negative control) were evident. We found Treg, Tr1 activity or T-cell exhaustion to be insignificant as their blockade or removal did not lead to 'flare’ in circulating tolerant T-cell reactivity against patient DC. T-cell clonal deletion appears to be the dominant mechanism explaining the in vitro hyporeactive proliferation, cytokine secretion, and alloreactive TCR clonotype attrition, along with systemically altered signaling pathways in tolerant T-cells including the alloreactive T cells remained. Anergy may play a minor supportive role. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Qingyi decoction attenuates intestinal epithelial cell injury via the calcineurin/nuclear factor of activated T-cells pathway.

BACKGROUNDRecent studies have demonstrated that dysfunction of the intestinal barrier is a significant contributing factor to the development of severe acute pancreatitis (SAP). A stable intestinal mucosa barrier functions as a major anatomic and functional barrier, owing to the balance between intestinal epithelial cell (IEC) proliferation and apoptosis. There is some evidence that calcium overload may trigger IEC apoptosis and that calcineurin (CaN)/nuclear factor of activated T-cells (NFAT) signaling might play an important role in calcium-mediated apoptosis.AIMTo investigate the potential mechanisms underlying the therapeutic effect of Qingyi decoction (QYD) in SAP.METHODSA rat model of SAP was created via retrograde infusion of sodium deoxycholate. Serum levels of amylase, tumor necrosis factor (TNF-α), interleukin (IL)-6, D-lactic acid, and diamine oxidase (DAO); histological changes; and apoptosis of IECs were examined in rats with or without QYD treatment. The expression of the two subunits of CaN and NFAT in intestinal tissue was measured via quantitative real-time polymerase chain reaction and western blotting. For in vitro studies, Caco-2 cells were treated with lipopolysaccharide (LPS) and QYD serum, and then cell viability and intracellular calcium levels were detected.RESULTSRetrograde infusion of sodium deoxycholate increased the severity of pancreatic and intestinal pathology and the levels of serum amylase, TNF-α, and IL-6. Both the indicators of intestinal mucosa damage (D-lactic acid and DAO) and the levels of IEC apoptosis were elevated in the SAP group. QYD treatment reduced the serum levels of amylase, TNF-α, IL-6, D-lactic acid, and DAO and attenuated the histological findings. IEC apoptosis associated with SAP was ameliorated under QYD treatment. In addition, the protein expression levels of the two subunits of CaN were remarkably elevated in the SAP group, and the NFATc3 gene was significantly upregulated at both the transcript and protein levels in the SAP group compared with the control group. QYD significantly restrained CaN and NFATc3 gene expression in the intestine, which was upregulated in the SAP group. Furthermore, QYD serum significantly decreased the LPS-induced elevation in intracellular free Ca2+ levels and inhibited cell death.CONCLUSIONQYD can exert protective effects against intestinal mucosa damage caused by SAP and the protective effects are mediated, at least partially, by restraining IEC apoptosis via the CaN/NFATc3 pathway.

Abstract 3911: Proteomic and transcriptomic subtypes of KRASG12C mutant lung cancer

Abstract Sotorasib (AMG510) is the first KRASG12C covalent inhibitor (KRASG12Ci) approved by the FDA in lung cancers harboring oncogenic KRASG12C mutation. However, it remains unclear why some patients show upfront resistance to the KRASG12Ci, and the mechanisms of adaptive resistance. To enhance the efficacy of the KRASG12Ci, several combination regimens are in clinical trials which includes Sotorasib combination with MEK, PD1, SHP2, pan-HER, PD-L1, and EGFR inhibitors. However, it remains unclear how to precisely identify an appropriate cohort for analysis and predict tumor-centric combination strategies that would be effective in the clinic. We hypothesized that subsets of KRASG12C lung cancer could be identified using transcriptomics and proteomics. To test our hypothesis, we performed hierarchical clustering of the microarray dataset on 87 NSCLC KRASG12C tumors and identified three novel transcriptomic subtypes - Subtype 1 had higher expression of extracellular matrix (ECM) genes, which are related to cellular adhesion, and lower expression of EMT genes. Subtype 2 had low ECM expression, high expression in EMT and cell cycle pathway genes suggesting that this is a more invasive subtype. Subtype 2 had lowest expression of small molecule metabolism, suggesting it may be more responsive to KRASG12Ci. Subtype 3 had the lowest expression in interferon gamma/cytotoxic T-cell, T-cell, and B-cell pathways. Subtype 3 had the highest expression of small molecule metabolism, suggesting that it could be more resistant to KRASG12Ci. Having observed these unique clusters, we next hypothesized that mass spectrometry-based expression and phosphoproteomic analysis (pY and pS/T/Y) will further refine these clusters and help identifying new subsets. Our previous work by Stewart et al., demonstrated the importance of proteomics integrated with genomic and transcriptomic analyses to define molecular subtypes in a cohort of 108 squamous cell lung cancer patients. We believe that a similar integrated proteogenomic study is important in context of KRASG12C tumors to understand the signaling diversity and phenotypically categorized them to optimize patient enrichment schemes for effective combination therapy. For that purpose, we performed an integrative proteogenomic analysis of 75 KRASG12C tumors (55 lung, 12 large bowel adenocarcinomas and 8 PDXs) with full clinical follow up and pathology data. We are presently classifying the 75 KRASG12C tumors into phosphoproteomic subtypes. We also carried out targeted exome sequencing for cancer-specific genes. Next, we plan to leverage existing gene expression microarray data and add exome sequencing proteomics, and phosphoproteomics for a comprehensive, protein-centric characterization on these tumors. Importantly, the inclusion of 8 PDXs being co-processed and co-analyzed along with the tumors will enable rapid follow-up experiments to validate the findings into preclinical therapy trials. Citation Format: Hitendra Singh Solanki, Paul A. Stewart, Eric A. Welsh, Victoria Izumi, Bin Fang, Sean J. Yoder, John M. Koomen, Eric B. Haura. Proteomic and transcriptomic subtypes of KRASG12C mutant lung cancer [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 3911.

Science, Medicine, and the Anesthesiologist

Science, Medicine, and the Anesthesiologist

STAT6 Blockade Abrogates Aspergillus-Induced Eosinophilic Chronic Rhinosinusitis and Asthma, A Model of Unified Airway Disease.

Unified airway disease, including concurrent asthma and chronic rhinosinusitis (CRS), is a common, but poorly understood disorder with no curative treatment options. To establish a murine model of chronic unified eosinophilic airway inflammation, mice were challenged with Aspergillus niger, and sinonasal mucosa and lung tissue were evaluated by immunohistochemistry, flow cytometry, and gene expression. Inhalation of A niger conidia resulted in a Th2-biased lung and sinus inflammation that typifies allergic asthma and CRS. Gene network and pathway analysis correlated with human disease with upregulation of not only the JAK-STAT and helper T-cell pathways, but also less expected pathways governing the spliceosome, osteoclast differentiation, and coagulation pathways. Utilizing a specific inhibitor and gene-deficient mice, we demonstrate that STAT6 is required for mycosis-induced sinus inflammation. These findings confirm the relevance of this new model and portend future studies that further extend our understanding of the immunopathologic basis of airway mycosis and unified airway disease.

Yunnan Baiyao Ameliorates Rheumatoid Arthritis in Rats by Shifting the Th17/Treg Cell Balance and Preventing Osteoclast Differentiation.

Yunnan Baiyao (YNB) is a traditional Chinese medicine that possesses anti-inflammatory effects. Previously, we have demonstrated the effects of YNB in rheumatoid arthritis (RA) animal models; however, the underlying mechanisms are unclear. In the present study, we aimed to investigate the effects of YNB on the T-helper (Th)17/T-regulatory (Treg) cell balance in a collagen-induced arthritis rat model orally administrated YNB or methotrexate, a widely used therapeutic agent for treating RA. Our results showed that YNB treatment significantly decreased the voix pedis thickness and joint functionality scores and alleviated joint histopathology in these rats. These YNB-induced effects were achieved by decreasing the number of Th17 cells and increasing that of Treg cells in the spleen. Moreover, the interleukin- (IL-) 17 level considerably decreased in the serum of YNB-treated rats, whereas the IL-10 level significantly increased. Furthermore, YNB could inhibit RANKL-induced osteoclast formation by regulating the tumor necrosis factor receptor-associated factor 6/NF-κB/nuclear factor of the activated T-cell pathway. In summary, our study shows that YNB exhibits antiarthritic activity by decreasing the ratio of Th17/Treg cells, regulating the cytokine balance, and inhibiting osteoclast activation, providing an experimental basis that supports the use of this traditional Chinese medicine for the clinical treatment of RA.