Surface Protein Expression Research Articles

CITEViz: interactively classify cell populations in CITE-Seq via a flow cytometry-like gating workflow using R-Shiny

BackgroundThe rapid advancement of new genomic sequencing technology has enabled the development of multi-omic single-cell sequencing assays. These assays profile multiple modalities in the same cell and can often yield new insights not revealed with a single modality. For example, Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq) simultaneously profiles the RNA transcriptome and the surface protein expression. The surface protein markers in CITE-Seq can be used to identify cell populations similar to the iterative filtration process in flow cytometry, also called “gating”, and is an essential step for downstream analyses and data interpretation. While several packages allow users to interactively gate cells, they often do not process multi-omic sequencing datasets and may require writing redundant code to specify gate boundaries. To streamline the gating process, we developed CITEViz which allows users to interactively gate cells in Seurat-processed CITE-Seq data. CITEViz can also visualize basic quality control (QC) metrics allowing for a rapid and holistic evaluation of CITE-Seq data.ResultsWe applied CITEViz to a peripheral blood mononuclear cell CITE-Seq dataset and gated for several major blood cell populations (CD14 monocytes, CD4 T cells, CD8 T cells, NK cells, B cells, and platelets) using canonical surface protein markers. The visualization features of CITEViz were used to investigate cellular heterogeneity in CD14 and CD16-expressing monocytes and to detect differential numbers of detected antibodies per patient donor. These results highlight the utility of CITEViz to enable the robust classification of single cell populations.ConclusionsCITEViz is an R-Shiny app that standardizes the gating workflow in CITE-Seq data for efficient classification of cell populations. Its secondary function is to generate basic feature plots and QC figures specific to multi-omic data. The user interface and internal workflow of CITEViz uniquely work together to produce an organized workflow and sensible data structures for easy data retrieval. This package leverages the strengths of biologists and computational scientists to assess and analyze multi-omic single-cell datasets. In conclusion, CITEViz streamlines the flow cytometry gating workflow in CITE-Seq data to help facilitate novel hypothesis generation.

A high-content screen of FDA approved drugs to enhance CAR T cell function: ingenol-3-angelate improves B7-H3-CAR T cell activity by upregulating B7-H3 on the target cell surface via PKCα activation

BackgroundCAR T cell therapy is a promising approach to improve outcomes and decrease toxicities for patients with cancer. While extraordinary success has been achieved using CAR T cells to treat patients with CD19-positive malignancies, multiple obstacles have so far limited the benefit of CAR T cell therapy for patients with solid tumors. Novel manufacturing and engineering approaches show great promise to enhance CAR T cell function against solid tumors. However, similar to single agent chemotherapy approaches, CAR T cell monotherapy may be unable to achieve high cure rates for patients with difficult to treat solid tumors. Thus, combinatorial drug plus CAR T cell approaches are likely required to achieve widespread clinical success.MethodsWe developed a novel, confocal microscopy based, high-content screen to evaluate 1114 FDA approved drugs for the potential to increase expression of the solid tumor antigen B7-H3 on the surface of osteosarcoma cells. Western blot, RT-qPCR, siRNA knockdown and flow cytometry assays were used to validate screening results and identify mechanisms of drug-induced B7-H3 upregulation. Cytokine and cytotoxicity assays were used to determine if drug pre-treatment enhanced B7-H3-CAR T cell effector function.ResultsFifty-five drugs were identified to increase B7-H3 expression on the surface of LM7 osteosarcoma cells using a novel high-content, high-throughput screen. One drug, ingenol-3-angelate (I3A), increased B7-H3 expression by up to 100%, and was evaluated in downstream experiments. Validation assays confirmed I3A increased B7-H3 expression in a biphasic dose response and cell dependent fashion. Mechanistic studies demonstrated that I3A increased B7-H3 (CD276) mRNA, total protein, and cell surface expression via protein kinase C alpha activation. Functionally, I3A induced B7-H3 expression enhanced B7-H3-CAR T cell function in cytokine production and cytotoxicity assays.ConclusionsThis study demonstrates a novel high-content and high-throughput screen can identify drugs to enhance CAR T cell activity. This and other high-content technologies will pave the way to develop clinical trials implementing rational drug plus CAR T cell combinatorial therapies. Importantly, the technique could also be repurposed for an array of basic and translational research applications where drugs are needed to modulate cell surface protein expression.

Abstract 6325: Lineage tracing defines responding CAR T cells in patients with B cell malignancies

Abstract Autologous T cells engineered to express a chimeric antigen receptor (CAR) have transformed the standard of care for patients with B cell malignancies, but >50% of patients progress following therapy. Here, we sought to understand key T cell intrinsic factors impacting efficacy: CAR T cell expansion, persistence, and homing to the tumor. Using an endogenous T cell receptor (TCR) sequence as a ‘barcode’, we followed individual T cell clonotypes at the single-cell level from pre-manufacture apheresis and infusion products to tumor-involved lymph nodes and blood at peak and late expansion in 22 adult patients with relapsed or refractory large B cell lymphoma (LBCL) or acute lymphoblastic leukemia (ALL) treated with axicabtagene ciloleucel, an FDA-approved CD19-CAR T cell therapy, or bispecific CD19/CD22-CAR T cells on an investigator-initiated trial (NCT03233854). The resulting CAR T cell atlas comprises matched transcriptome (scRNA-seq) and surface protein expression (CITE-seq) for 846,344 cells from 97 samples, with 251,175 unique TCR clonotypes identified, including 17,479 clonotypes that could be traced across ≥2 timepoints in CAR+ cells, enabling lineage tracing at scale for the first time. We found that T cells present in apheresis that preferentially expand during manufacturing primarily comprise T central memory (Tcm) cells and are enriched for predicted specificity to common viruses, including influenza, but also include a subset of T regulatory (Treg) cells. In infusion products, CAR T conventional (non-Treg) cells with superior abilities to home to the tumor, expand, and persist post-infusion share features of activated T effector memory (Tem) phenotype enriched for expression of BATF3 and high TCF4 transcription factor activity. Baseline apheresis and infusion products were enriched for cells classified as Tregs in patients with poor response. This observation aligned with our recent work linking post-infusion CAR Tregs to progression following CD19-CAR T cell therapy. We therefore performed lineage tracing and methylation analysis of the Treg-specific demethylated region (TSDR) in the FOXP3 gene and found that post-infusion CAR Tregs are primarily derived from pre-existing Tregs. Of interest, flow cytometry analysis of baseline blood from a cohort of 53 patients treated with CD19-CAR for LBCL showed that Tregs are elevated in baseline blood relative to healthy donors. Removing Tregs from healthy donor T cells prior to manufacturing enhanced CAR T cell expansion and anti-tumor activity against JeKo-1 human lymphoma cell line, whereas adding 5% Tregs to mimic patient T cells rendered the resulting CAR T cells less active. These analyses pinpoint the identities of apheresis T cells and infusion CAR T cells with properties impacting efficacy, and also identify pre-existing Tregs as limiting efficacy of CAR T cell immunotherapies. Citation Format: Zinaida Good, Mark P. Hamilton, Jay Y. Spiegel, Moksha H. Desai, Zachary J. Ehlinger, Patrick J. Quinn, Yiyun Chen, Snehit Prabhu, Shin-Heng Chiou, Sreevidya Kurra, Eric Yang, Michael G. Ozawa, Matthew J. Frank, Lori Muffly, Gursharan K. Claire, Sushma Bharadwaj, Saurabh Dahiya, Katherine A. Kong, Mark M. Davis, Sylvia K. Plevritis, Elena Sotillo, Bita Sahaf, David B. Miklos, Crystal L. Mackall. Lineage tracing defines responding CAR T cells in patients with B cell malignancies [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 6325.

Abstract 5458: Plasticity of CAF-like states in Ewing sarcoma is mediated by TGF-beta

Abstract Transcriptional plasticity of cancer cells promotes tumor progression and treatment resistance via generation of phenotypically distinct cell states. We previously showed that Ewing sarcoma (EwS) cells exhibit inter- and intra-tumoral transcriptional heterogeneity and that they undergo state changes in response to cues from the tumor microenvironment (TME). More recently, we identified phenotypically distinct EwS tumor cell subpopulations in vivo that deposit pro-tumorigenic extracellular matrix (ECM) proteins, adopting similar functions to cancer-associated fibroblasts (CAFs). Here, we sought to identify the upstream regulators of these CAF-like EwS cells with the goal of defining molecular mechanisms that control EwS plasticity and creation of ECM-remodeling states. Integration of single cell proteogenomic sequencing (CITE-seq) with immunofluorescence and digital spatial profiling of cell line xenografts and patient tumors validated the cell-surface protein CD73 (NT5E) as a biomarker of ECM-secreting EwS cell subpopulations in vitro and in vivo. The transcriptomes of CD73+ CAF-like cells were compared to isogenic CD73- cells (n=9 cell lines) and highly significant enrichment of gene ontology terms related to TGF-beta signaling (p.adj = <4.4x10-14) was noted. EwS cells were previously reported to be non-responsive to TGF-beta due to profound transcriptional repression of the TGF-beta type 2 receptor (TGFBR2) by EWS::FLI1. In support of this, TGFBR2 expression was low in bulk measurements of EwS cells. However, TGFBR2 mRNA and surface protein expression were significantly increased in CD73+ subpopulations. Exposure of EwS cells to TGF-beta ligands (TGFB1 or TGFB2) strongly induced the CAF-like gene signature, including ECM genes BGN, COL1A1, SPARC, and TNC (p. adj < 8.5x10-5), and this was augmented in CD73+ cell populations. Transduction of EwS cells with dominant negative-TGFBR2 inhibited both basal and TGF-beta-induced expression of CAF-like signature genes and blocked SMAD2 phosphorylation. To test the functional significance of the TGF-beta response, unsorted EwS cells were plated in 2D or as tumor spheroids in collagen I-rich 3D cultures and exposed to TGFB1 or SB-505124, a TGF-beta inhibitor. TGFB1 treatment had no effect on cell viability or proliferation in 2D but increased invasion into collagen in 3D. Inhibition of TGF-beta reduced CAF-like gene expression in 2D cultures and diminished both viability and invasion in 3D. Finally, our studies revealed that NT5E (CD73) is a TGF-beta induced target in EwS cells and that CD73+ cells upregulate expression of TGFB2. Thus, an autocrine signaling circuit is created in which TGF-beta activity in CAF-like tumor cell subpopulations is sustained by CD73+ tumor cell-derived ligand. Together these studies demonstrate that TGF-beta is a key determinant of cell plasticity and ECM remodeling in EwS. Citation Format: Emma Wrenn, April A. Apfelbaum, Katherine Braun, Erin R. Rudzinski, Xuemei Deng, Aya Miyaki, Trisha Lipson, Nicolas M. Garcia, Virginia J. Hoglund, Elizabeth R. Lawlor. Plasticity of CAF-like states in Ewing sarcoma is mediated by TGF-beta [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 5458.

Abstract 335: BD®OMICS-Guard Sample Preservation Buffer preserves mRNA and cell surface epitopes for single-cell sequencing applications

Abstract Multiomics has emerged as a powerful tool to derive insights on the transcriptomic and proteomic data of human cancer samples and preclinical models of blood and tumor malignancies. Due to the sensitivity of sequencing techniques, sample quality and stability can potentially interfere with results. BD® OMICS-Guard Sample Preservation Buffer (SPB), a novel preservation solution, allows cells and bulk tissues to be stored for up to 72h at 4 °C, while reliably capturing the true biology of cells. This reagent gently preserves mRNA and protein integrity without the traditional cross-linking and harsh fixatives, increasing flexibility and expanding options for study designs. Here, we showcase the preservation properties ofthis reagent for multiomic readouts in human cells and mouse tissue across three different timepoints.BD® OMICS-Guard SPB’s preservation of mRNA from human PBMC and bulk mouse spleen tissues wasevidenced by WTA for differentially expressed genes in samples preserved for 24, 48 and 72h comparedto fresh/unpreserved controls. Protein epitope preservation was demonstrated via CITE-Seq assay withthe BD® AbSeq Human Immune Discovery Panel (human PBMC) or a 30-plex panel of anti-mouse BD®AbSeq Antibody-Conjugated Oligonucleotides (mouse splenocytes) and confirmed with multicolor flowcytometry. For both sample types, high correlation R2 values (>0.87) were calculated for the preservation time points versus fresh controls. Concomitant flow cytometry analysis of major cell types and surface protein expression was consistent with sequencing data for all samples. Specificity and sensitivity of major cell type markers and cell surface proteins across cell types and time is visualized in the BD®AbSeq Antibody-generated heatmap for each sample. Additionally, BD® OMICS-Guard SPB maintains mRNA integrity in targeted gene assays and VDJ full-length assays, suggesting robust preservation of select transcripts readily interrogated in single-cell profiling. Batch effect across time and technical replicates for both sample types was limited with the BD Rhapsody™ HT Xpress System and delineated by donor/sample specific tSNEs across time. We demonstrate that the BD® OMICS-Guard Sample Preservation Buffer effectively preserves both mRNA and surface proteins for single-cell experiments. BD® OMICS-Guard SPB allows the convenient preservation of samples such as tumor biopsies to be collected and stored for up to 72h at 4 °C while maintaining mRNA and protein integrity, allowing the flexibility for precious samples to be potentially shipped to a central sequencing site to ensure standardized sequencing workflows. For Research Use Only. Not for use in diagnostic or therapeutic procedures.BD and BD Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2023 BD. All rights reserved. Citation Format: Samuel X. Shi, Cynthia Sakofsky, Zhiqi Zhang, Samatha Vadrevu, Xiaoshan Shi, Hye-Won Song, Aruna Ayer. BD®OMICS-Guard Sample Preservation Buffer preserves mRNA and cell surface epitopes for single-cell sequencing applications [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 335.

Abstract 5864: YAP1 defines a highly plastic, persister cell population in small cell lung cancer (SCLC) following relapse

Abstract SCLC is an aggressive, neuroendocrine malignancy notable for rapid, albeit transient, responses to therapy. We have previously shown that transcriptional heterogeneity among treatment-naïve SCLC tumors underlies four distinct transcriptional subtypes, each with distinct clinical vulnerabilities. Though previously hypothesized to delineate a distinct subtype, expression of YAP1 is largely absent from treatment-naïve SCLC. Less clearly defined is the transcriptional landscape of relapsed SCLC, the evolution of which may underlie SCLC’s capacity for insurmountable resistance. To better define the biology of relapsed SCLC, we performed mass cytometry analysis of patient circulating tumor cells (CTCs), immunohistochemistry for ASCL1, NEUROD1, POU2F3, MYC and YAP1, and single-cell (sc)RNAseq of core needle biopsies from SCLC patients and preclinical models following resistance to standard-of-care therapies. In contrast to treatment-naïve SCLC, YAP1 mRNA and protein are detectable in patient CTC populations and tumors from relapsed patients and PDXs, albeit at levels lower than subtype-defining transcription factors (e.g. ASCL1, NEUROD1, or POU2F3). Notably, however, scRNAseq reveals these YAP1 expressing cells to be mutually exclusive with ASCL1 and NEUROD1, though not necessarily POU2F3. YAP1-expressing cells possess unique mesenchymal, inflamed features and exhibit high levels of transcriptional plasticity. Consistent with the upregulation of YAP1 following chemotherapy, YAP1-positive cell populations express senescence associated secretory phenotype genes and cancer stem cell genes, suggesting that these populations represent drug tolerant persister cells that may serve as a source for renewal of classical neuroendocrine populations within the tumor. Targeting these resistant populations may be critical to re-establish response in relapsed SCLC, and these analyses identified high expression of several surface proteins that are currently under investigation as targets for antibody-drug conjugates in SCLC clinical trials, including B7H3, TROP2 and HER2 antibody drug conjugates. These data suggest that the acquisition of YAP1 expression delineates a particularly recalcitrant tumor cell population observed almost exclusively in relapsed SCLC that is capable of driving tumor persistence and treatment resistance. Clinically, these data underscore the importance of constraining transcriptional plasticity in the frontline management of SCLC, but also of developing therapies aimed specifically at emergent populations capable of driving resistance, as with the YAP1-high cells identified here. The emergence of YAP1 following resistance in SCLC and the observation of YAP1 as a common feature of other high-grade neuroendocrine carcinomas exclusive of SCLC, likely explains the initial mischaracterization of YAP1 as a subtype-defining feature of SCLC. Citation Format: C. Allison Stewart, Sarah M. Groves, Runsheng Wang, Kavya Ramkumar, Yuanxin Xi, Lixia Diao, Alejandra G. Serrano, Azusa Tanimoto, Ashley M. Victorian, Sayantan Bhattacharyya, Alexa J. Halliday, Neda Kalhor, Luisa Maren Soto, Pedro Filipe Da Rocha, Natalie Vokes, Loukia G. Karacosta, Jing Wang, John V. Heymach, Vito Quaranta, Bonnie Glisson, Lauren Averett Byers, Carl M. Gay. YAP1 defines a highly plastic, persister cell population in small cell lung cancer (SCLC) following relapse [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 5864.

Abstract 1127: Single cell network-based analyses reveal dose-dependent increase in myeloid suppressive cell sub-populations in response to radiation therapy

Abstract Background: More than half of all cancer patients undergo radiation therapy (RT) with curative or palliative intent. With the widespread use of immunotherapy to treat cancer, immune effects of radiation are an important consideration in design of sequential and combination therapies. While radiation has been found to deplete lymphocytes, less is known about its effects on the myeloid compartment, which includes both immune activating and suppressive populations. We hypothesized that a network-based analysis of single cell proteogenomic data generated from tumor-infiltrating immune cells can identify druggable proteins in pathways driving unwanted immunosuppressive changes with radiation, particularly among myeloid cells. Methods: Proteogenomic (CITE-Seq) analysis was conducted on CD45+ immune cells from unirradiated and irradiated orthotopic 4T1 murine mammary tumors three- and ten-days after tumor irradiation (8 Gy x 1 or 8 Gy x 3). Based on expression of select surface proteins, analysis of the CITE-Seq data resulted in eight distinct immune clusters, including three myeloid clusters — monocytes, macrophages, and granulocytes. Using VIPER (virtual inference of protein activity by enriched regulon), an algorithm that infers protein activities from weighted expression on each protein’s downstream targets, we identified distinct functional subpopulations within these eight clusters. Further profiling subclusters enriched by RT using the OncoTarget algorithm, we identified druggable proteins with significant inferred activity as potential targets of therapeutic interest. Results: As expected, lymphocytes were persistently depleted after RT on both radiotherapy schedules. However, three distinct subclusters of myeloid cells with immune-suppressive phenotype became enriched following tumor irradiation, with greater enrichment at the lower radiation dose of 8 Gy x 1: TAM2, PMN1, and PMN4. Surface protein expression enabled us to define the immunophenotype and isolate these three cell clusters from murine 4T1 tumors as well as human renal cell carcinoma tumors for further functional analyses. OncoTarget identified a number of druggable targets, including PARP1 for the PMN1 subcluster. Conclusion: Suppressive myeloid subpopulations induced by radiation were identified among the immune cells. Combining radiation therapy with inhibitors of these cell populations may provide therapeutic benefit over radiation therapy alone or in combination with checkpoint inhibitor immunotherapies. Future work will experimentally determine if inhibition of druggable protein targets in these myeloid subpopulations improves the overall efficacy of RT. Citation Format: Aparna Krishnan, Shruti Bansal, Samanta Sarti, Michael D. Kissner, Charles Karan, Andrea Califano, Aleksandar Z. Obradovic, Catherine S. Spina. Single cell network-based analyses reveal dose-dependent increase in myeloid suppressive cell sub-populations in response to radiation therapy [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 1127.

Abstract 1900: TORL-3-600, a novel antibody drug conjugate directed against cadherin 17 (CDH17), has preclinical efficacy in colorectal, gastric, and pancreatic cancer

Abstract Cadherin 17 (CDH17) is a cell-to-cell adhesion protein that is a member of the cadherin superfamily. In normal tissues, expression of this single pass transmembrane protein is restricted to the lateral surfaces of intestinal and pancreatic ductal epithelial cells. However, in cancer, CDH17 is frequently overexpressed in tumors of the colon, stomach, and pancreas. The selective expression of this cell surface protein in these hard-to-treat cancer makes it an attractive target for the development of antibody-based therapeutics. Here, we describe the preclinical development of TORL-3-600, a novel CDH17-targeting antibody drug conjugate (ADC). CDH17 specific monoclonal antibodies were generated using traditional hybridoma technology and splenocytes isolated from mice immunized with cocktails of NIH3T3 cells overexpressing full length hCHD17 plus purified mammalian expressed hCDH17 extracellular domain protein (aa 23-787). Selective mAb binding was confirmed by flow cytometry and mAb internalization rate was assessed by immunofluorescence (IF). TORL-3-600 was generated from a fully humanized CDH17 mAb by MMAE conjugation with a cleavable linker. Cell membrane staining of CDH17 in patient tumor microarrays (TMAs), cell line (CDX) and patient derived xenografts (PDX) was evaluated by IHC assay. Selective binding of the TORL-3-600 to CDH17 was confirmed in human cancer cell lines and cells engineered to overexpress CDH17. Binding of TORL-3-600 to cell surface CDH17 induced Internalization and translocation to the lysosome of the protein-ADC complex for release of the MMAE payload. Treatment with TORL-3-600 induced significant regressions and tumor growth inhibition (TGI) in four CDH17-positive (CDH17+) human colorectal cancer (CRC) CDXs (103.6 - 140.4% TGI) and three CDH17+ CRC PDXs (63.3 - 102.2% TGI). Responses in these models were sustained for up to nine-weeks following cessation of treatment. Sustained inhibition of xenograft tumor progression was also observed in a cell line model of CDH17+ pancreatic cancer (85.8% TGI). In contrast, greatly reduced responses (37.3 - 58.2% TGI) to TORL-3-600 were observed in the CDH17- human colon cancer CDX and PDX models. Each of the doses tested in this study were well tolerated in mice with no dose-limiting toxicities observed. Analyses of large human patient TMAs by CDH17 IHC assay demonstrated detectable expression of CDH17 in 90.1% (173/193) of CRC, 51.8% (86/166) of gastric and 20.4% (69/331) of pancreatic cancers. These numbers suggest that an ADC directed against CDH17 could provide benefit to a significant number of patients diagnosed with these cancers. These data support the clinical development of TORL-3-600 for the treatment of CDH17+ cancers. TORL-3-600 has completed IND enabling toxicity studies with acceptable PK and toxicity profiles and is now in phase 1 clinical testing (NCT05948826). Citation Format: Neil A. O'Brien, Martina SJ McDermott, Jun Zhang, Ming Lu, Ke Wei Gong, Benjamin Hoffstrom, Wei Ping Jia, Tong Luo, Athena M. Madrid, Min Liang, John A. Glaspy, Dennis J. Slamon. TORL-3-600, a novel antibody drug conjugate directed against cadherin 17 (CDH17), has preclinical efficacy in colorectal, gastric, and pancreatic 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 1900.

Abstract 4682: Membrane protein expression and activity following inhibition of B-cell receptor-initiated signaling by WBMP-4, a therapeutic antibody for leukemia/lymphoma

Abstract The B-cell Receptor (BCR), comprising membrane Ig (mIg) and CD79a/b, controls normal signaling in B-cells, but dysregulation of this signaling is the dominant driver of leukemia/lymphoma. The class-specific mIg subunit of the BCR is a rational drug target for B-cell malignancies. It plays a critical role in the functioning of these cells, and allows for restricted targeting of only B-cells expressing an Ig class, offering a more tumor-specific approach than current pan-B-cell treatments. We developed a first-in-class antibody, WBMP-4, to the mIgM-BCR, which inhibits cell growth and induces apoptosis via widespread down-regulation of tyrosine kinase activity. Given the potent inhibition of intracellular signaling induced upon WBMP-4 binding to the mIgM-BCR, we expect that translation of membrane proteins will be impacted. Here we investigate the expression and phosphorylation profile of cell surface proteins following treatment of malignant B-cells with WBMP-4. Using western blot and flow cytometry, we measure levels of CD79a/b, CD19, CD20, CD21, CD22, CD32a, CD81, and CXCR4 at 6, 24, and 48 hours after low (5ug/mL) and high (20ug/mL) dose WBMP-4 treatment, as compared to control, untreated Burkitt lymphoma (CA46) cells. We analyze phosphorylation levels of those surface proteins that serve as kinases in this system (CD79a/b, CD19, CXCR4, etc.). Using these same methods, we also examine the expression and phosphorylation of key B-cell transcription factors (e.g. NF-kB, c-MYC, STAT5, PAX5, EBF1, FOXO, and BCL-2). Preliminary analyses show a complete absence of CD79a protein (western blot), and decreased phospho-kinase activity of CD79b (Pamgene phosphokinase array) following treatment of Burkitt lymphoma cells with WBMP-4, as compared to untreated cells. While the effect of an antibody binding to mIg may be most pronounced for CD79a/b relative to other proteins, as mIg and CD79a/b form a complex, we expect that the absence of CD79a/b is a result of WBMP-4-induced mIgM inhibition modulating downstream signaling and transcription factor activity. By examining cell surface markers, we may gain insight into how WBMP-4 impacts a cell’s interaction with the tumor microenvironment, and this information is valuable for diagnostic and patient monitoring purposes, and to determine drug combination strategies. This work will be considered in the context of our ongoing research on WBMP-4’s inhibition of intracellular signaling pathways, and the resulting biologic effects, and will contribute to the continued development of WBMP-4 by providing an understanding of the effects achieved (cell growth inhibition, cell differentiation, apoptosis) at different dosages and if these effects are maintained over time. As a novel, efficacious, and low toxicity treatment strategy for B-cell leukemia and lymphoma, WBMP-4 has significant clinical potential. Citation Format: Rachel S. Welt, David Kostyal, Virginia Raymond, Jose M. Lobo, Sydney Welt. Membrane protein expression and activity following inhibition of B-cell receptor-initiated signaling by WBMP-4, a therapeutic antibody for leukemia/lymphoma [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 4682.

Abstract 1090: GPC2 directed CAR T cells are efficacious against fusion-positive and fusion-negative rhabdomyosarcomas

Abstract Background: Rhabdomyosarcoma (RMS) is an aggressive pediatric solid tumor with poor survival and new targeted therapies are needed. Aims: Using an integrative proteogenomic quantitative approach, we sought to identify candidate immunotherapeutic targets then validate top targets in RMS preclinical models. Methods: We first performed plasma membrane enrichment followed by mass spectrometry (MS) to define the surfaceome of 7 fusion-positive (FP) and 14 fusion-negative (FN) RMS patient-derived xenograft models. Surface proteins were scored by IMMUNOTAR, our custom pipeline which extracts and integrates quantitative features from diverse proteogenomic and annotation datasets to provide a score for each protein, using mean-average-precision for score optimization. Cell surface expression of IMMUNOTAR-prioritized proteins was validated using flow cytometry, focusing on targets with immunotherapies in development. CAR T cells targeting the lead protein were engineered and studied in RMS preclinical models. Results: A total of 847 and 882 high confidence cell surface proteins were detected in FP and FN RMS surfaceome, respectively, with 94% of proteins overlapping the phenotypes. IMMUNOTAR ranked anaplastic lymphoma kinase (ALK), a putative immunotherapeutic target in FP RMS, as the top FP target. Glypican 2 (GPC2), a differentially expressed cell surface proteoglycan identified in neuroblastoma and other neural-derived tumors, targetable with CAR T cells and antibody-drug conjugates [Bosse et al. Cancer Cell 2017], ranked as the top protein in FN and 5th protein in FP RMS. Overall, GPC2 was abundantly expressed in 16 of 21 RMS models profiled. Flow cytometry revealed expression of GPC2 comparable to neuroblastoma in 5 of 8 RMS cells lines. Additionally, both FP and FN GPC2+ RMS cell lines (FN: RH36, SMS-CTR; FP: RH41, RH30) activated GPC2 CAR T cells with either a CD28 or a 4-1BB costimulatory domain, inducing release of IFN-gamma and IL-2 upon co-incubation across different E:T ratios as quantified by ELISA. Further, GPC2 CAR T cells induced potent RMS cell cytotoxicity compared to control CD19 CAR T cells with near 100% specific cytotoxicity at 1:1 E:T ratio. In a pilot study, GPC2 CAR T cells with 4-1BB costimulatory domains induced significant tumor regression (p=0.03) and prolonged progression-free survival (p=0.01) compared to CD19 CAR T cells in an established murine RH41 flank xenograft model. Conclusion: In summary, we probed the RMS surfaceome using MS and identified candidate proteins amenable for immunotherapeutic targeting using our IMMUNOTAR algorithm. We validated GPC2 as a candidate target in FP and FN RMS and are corroborating our findings in additional pre-clinical RMS models. Demonstration of GPC2 CAR efficacy in these models could lead to inclusion of RMS in our ongoing phase 1 clinical trial (NCT05650749) in a planned expansion phase. Citation Format: Rawan Shraim, Patrick M. Schuerch, Anna Maria Giudice, Karina L. Conkrite, Amber K. Weiner, Alexander B. Radaoui, Brian Mooney, Raushan T. Kurmasheva, Peter J. Houghton, Gregg B. Morin, Yael B. Mosse, Poul H. Sorensen, Benjamin A. Garcia, Ahmet Sacan, John M. Maris, Kristopher R. Bosse, Sharon J. Diskin. GPC2 directed CAR T cells are efficacious against fusion-positive and fusion-negative rhabdomyosarcomas [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 1090.

Abstract 1854: Plasma proteomic biomarkers identify non-responders and reveal biological insights about the tumor microenvironment in melanoma patients after PD1 blockade

Abstract Most patients treated with immune checkpoint blockade (ICB) do not have durable treatment responses, stressing a critical need to identify early non-invasive biomarkers of response. Circulating biomarkers provide easy access for serial monitoring and can provide insight on the mechanisms of response to ICB. We performed plasma proteomics (~3000 proteins) on 250 metastatic melanoma patients before and on ICB treatment and performed analysis to identify response- and time point-associated peripheral protein biomarkers. We next generated patient-matched peripheral blood lymphocyte (PBL) surface proteomic data and tumor sample bulk transcriptomic data to build a multimodal dataset. We leveraged these data to train machine learning models for unsupervised analysis and to classify treatment responses and learned features predictive of survival and patient endotypes. Using univariate linear models to predict protein abundance based on time point, response and the interaction term for time point and response, we identified 343 time point-associated proteins, 141 response-associated proteins, and one interaction-associated protein. We further investigated response-related proteins by fitting multivariate logistic regression and cox proportional hazards regression models to further investigate the ability of these proteins to predict response, progression-free survival (PFS), and overall survival (OS) and identify those proteins that are most important in predicting response. We calculated covarying modules of proteins that were found to be either response-associated or time point-associated to discover underlying biological networks at different treatment time points and disease response states. Additionally, we leveraged these modules to endotype patients and identified trends in protein modules across patient subsets, including modules related to biological processes such as immune infiltration, apoptosis, metaplasia and cell adhesion. To understand the contribution of different compartments on plasma proteomic abundance, we studied correlations of plasma protein abundance with PBL surface protein abundance and tumor bulk mRNA-sequencing expression. Finally, we integrated our data modalities and fit various statistical models to evaluate the relative importance of plasma protein biomarkers in predicting response compared to PBL surface proteins and tumor bulk mRNA-sequencing expression. Together, these data represent one of the deepest peripheral biomarker studies using paired samples in melanoma patients treated with anti-PD1 therapy. Citation Format: Samuel J. Wright, Deepika Yeramosu, Milan Parikh, Marijana Rucevic, Ngan Nguyen, Russell W. Jenkins, Keith T. Flaherty, Nir Hacohen, Genevieve M. Boland, Arnav Mehta. Plasma proteomic biomarkers identify non-responders and reveal biological insights about the tumor microenvironment in melanoma patients after PD1 blockade [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 1854.

Abstract 3696: Liquid biopsy biomarker analysis during treatment with 177Lu-PSMA-617 in castrate resistant prostate cancer

Abstract Background: Prostate-Specific Membrane Antigen (PSMA) is a highly expressed prostate cancer (PCa) cell surface protein regulated by androgen receptor signaling and the target of the recently approved radioligand therapy 177Lu-PSMA-617. While some patients have dramatic responses to this therapy, rates of upfront or acquired resistance are high even in patients with PSMA-PET-avid disease, and better biomarkers of 177Lu PSMA-617 response and resistance are needed to select patients more effectively for this therapy. To address this need, we evaluated the relationship between longitudinal circulating tumor cell (CTC) androgen signaling and PSMA expression to 177Lu-PSMA-617 response or resistance in patients with metastatic prostate cancer. We leveraged a CTC androgen signaling signature that we have previously shown is prognostic for patients with metastatic prostate cancer treated with AR signaling inhibitors. Methods: Longitudinal blood samples were collected from 20 patients for circulating tumor cell (CTC) analysis at C1D1, C2D1, C4D1, and C6D1. CTCs were isolated with antibodies to EpCAM followed by parallel enumeration, analysis of cell surface expression of PSMA and RNA extraction for AR or neuroendocrine marker transcriptional profiling. Results: CTC samples were assessed for PSMA cell surface protein expression prior to treatment and on treatment. FOLH1 (PSMA) gene expression was detected from CTCs at baseline that persisted through treatment. Gene expression representative of AR pathway activation was present in 70% of the cohort at baseline and 50% of patients at C2D1. Concordant with these observations, no significant decrease in CTC number or PSMA protein expression was seen from baseline to C2D1. However, increased CTC AR pathway activation at C2D1 was associated with increased mean serum PSA (155 vs 21.93 ng/ml) and was associated with decreased duration of response. Conclusions: Improved tools to predict who will respond to 177Lu-PSMA-617 and detect early treatment resistance and disease progression are needed. Here, we show that persistent CTC burden, CTC AR signaling, and CTC PSMA expression are common early in treatment with 177Lu-PSMA-617 and may be associated with lower likelihood of clinical response. Further ongoing studies are needed to elucidate the relationship between these liquid biopsy results and clinical outcomes. Citation Format: Jamie M. Sperger, Marina N. Sharifi, Luke Nunamaker, Viridiana Carreno, Alex H. Chang, Shannon R. Reese, Charlotte Linebarger, Amy K. Taylor, Grace C. Bliitzer, John Floberg, Shuang G. Zhao, Joshua M. Lang. Liquid biopsy biomarker analysis during treatment with 177Lu-PSMA-617 in castrate resistant prostate 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 3696.

Abstract 6929: Inter- and intratumoral PIK3CA subclonal diversity in breast cancer contextualized by single-cell multiomics

Abstract Rare clonotypes within pre-cancerous tissues can drive progression to cancer. However, the evolution of rare clonotypes in tumors or normal tissue cannot be defined in the absence of single-cell resolution. At this single-cell level, multiomic interrogation across the Central Dogma of Biology provides enhanced power to reconstruct such evolutionary trajectories, defining the mutational profile, cell identify, and receptor expression within each subpopulation. Leveraging a multiomic approach, we aimed to define how different mutations in the same oncogenic driver observed in the same tumor resection associate with copy number variation (CNV) across the genome. We analyzed individual ductal carcinoma in situ/invasive ductal carcinoma cells using a unified whole-genome and full-transcript RNAseq workflow (ResolveOME™, BioSkryb Genomics) coupled with panel-level extracellular protein information through oligo-conjugated antibodies (BioLegend). We sequenced the exomes and transcriptomes of ResolveOME-amplified single cells from mastectomy samples from twelve patients. At the single nucleotide variant (SNV) level, we identified an allelic series of PIK3CA oncogenic driver mutations in the same tumor resection. A single amino acid, in-frame deletion of E109 dominated the sample, followed by H1047R and K111E in decreasing subclonal abundance. A fourth mutation, E345T, not present in the first sample, was detected as the sole PIK3CA variant in the second tumor sample. Intriguingly, each respective PIK3CA mutation class was associated with a distinct copy number alteration profile revealed by low-coverage whole-genome sequencing: Cells harboring the predominant ΔE109 mutation displayed chromosome 8p,16p, and 17 loss while the less abundant H1047R mutation was in single cells harboring 1q gain, 4q loss, and 22 loss in addition to the 8p and 16q loss present in the ΔE109 cells. PIK3CA K111E had a quiescent, 2n copy number profile. The transcriptomic arm of ResolveOME, containing an oligo-conjugated antibody readout of surface protein expression, jointly confirmed the epithelial identity for the cells harboring the oncogenic PIK3CA mutations. A subpopulation of cells harboring prototypical breast cancer CNV were typed as non-epithelial with increased stemness characteristics, indicative of the ability to resolve phenotypic cellular states. These results suggest a tight interrelationship between CNV and SNV influencing the relative rate of clonal expansion. They also provide the opportunity to explore CNV:SNV signature association with loci exclusive of PIK3CA, and to exploit the power of multiomic integration for lineage reconstruction and for defining common oncogenic signatures of the evolving tumors. Citation Format: Jon Zawistowski, Isai Salas-Gonzalez, Tia Tate, Tatiana Morozova, Katherine Kennedy, Durga Arvapalli, Jamie Remington, Jeffrey Marks, E. Shelley Hwang, Gary Harton, Victor Weigman, Jay A. West. Inter- and intratumoral PIK3CA subclonal diversity in breast cancer contextualized by single-cell multiomics [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 6929.

Abstract 2721: FF01: a novel humanized heavy chain antibody targeting endogenous retroviruses with anti-lymphoma activity

Abstract Background: Human endogenous retroviruses (HERVs) are transposable elements (TE) derived from retrovirus infections that occurred millions of years ago. Despite constituting 8% of our genome, their biological role still needs to be explored. HERV-K (HML-2), a subgroup of HERVs, represents the most recent retroviruses integrated into the human germline DNA. Members of the HML-2 subgroup retain complete ORF with coding potential and have garnered the most attention in disease-related studies. HERVs exhibit physiological expression in embryonic stem cells but are heavily methylated and remain almost silent in normal adult tissues. Although the mechanisms underlying HERV-K contribution to tumor cell growth and survival remain poorly understood, aberrant expression of HERVs has been demonstrated in various human tumor tissues and lymphoma. These findings led us to explore HERV-K as a potential therapeutic target in B cell lymphomas .Methods: RNA data were analyzed with Telescope. Validations were performed by qPCR, Western blot, flow cytometry, and ELISA. Env structure was obtained with AlphaFold and peptides were purchased from GeneScript. Antibody-dependent cellular cytotoxicity (ADCC) was performed with the ADCC Reporter Bioassay (Promega). Internalization was assessed using the Incucyte Fabfluor-pH Antibody labeling reagents. Results: We assessed the TE expression in RNA-seq datasets of diffuse large B cell lymphoma (DLBCL) and B cells from healthy individuals. An MDS plot showed that TEs could separate neoplastic from healthy samples, demonstrating that tumoral and normal specimens have distinct TE and HERV expression profiles. Focusing on tumor samples, DLBCLs were divided into four clusters with different prognoses. Similar results were obtained in a 47 B-cell lymphoma cell line panel. Focusing on the HML-2 family in lymphoma patients, we identified many of them upregulated in tumors. We validated RNA and protein expression of the HML-2 envelope (env) in seventeen lymphoma cell lines, and in two of them, we also proved the protein surface expression. Env-specific siRNA reduced env expression determined a decrease in cell proliferation, suggesting a contribution to lymphoma cell growth. In silico examination of the env identified four linear regions with potential for antibody binding, and peptides covering them were synthesized. We screened 150 lymphoma patients' serum samples for IgG antibodies binding to the peptides, and all peptides showed positive hits. We used a phage display screen to obtain one nanobody, FF01, binding one peptide. FF01 induced ADCC in the cell line with high surface env expression and not in a negative one. FF01 was internalized in an expressing cell, proving the principle for using it as a component of an antibody-drug conjugate. Conclusions: Our data demonstrate that HERVs are in DLBCL patients and propose FF01 as a new candidate to target HERV Env+ lymphomas. Citation Format: Filippo Spriano, Luciano Cascione, Jacopo Sgrignani, Nikolai Bendik, Sara Napoli, Giulio Sartori, Eleonora Cannas, Tao Gongo, Alberto J. Arribas, Davide Rossi, Davide F. Robbiani, Andrea Cavalli, Francesco Bertoni. FF01: a novel humanized heavy chain antibody targeting endogenous retroviruses with anti-lymphoma activity [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 2721.

A Yeast Modular Cloning (MoClo) Toolkit Expansion for Optimization of Heterologous Protein Secretion and Surface Display in Saccharomyces cerevisiae.

Saccharomyces cerevisiae is an attractive host for the expression of secreted proteins in a biotechnology context. Unfortunately, many heterologous proteins fail to enter, or efficiently progress through, the secretory pathway, resulting in poor yields. Similarly, yeast surface display has become a widely used technique in protein engineering but achieving sufficient levels of surface expression of recombinant proteins is often challenging. Signal peptides (SPs) and translational fusion partners (TFPs) can be used to direct heterologous proteins through the yeast secretory pathway, however, selection of the optimal secretion promoting sequence is largely a process of trial and error. The yeast modular cloning (MoClo) toolkit utilizes type IIS restriction enzymes to facilitate an efficient assembly of expression vectors from standardized parts. We have expanded this toolkit to enable the efficient incorporation of a panel of 16 well-characterized SPs and TFPs and five surface display anchor proteins into S. cerevisiae expression cassettes. The secretion promoting signals are validated by using five different proteins of interest. Comparison of intracellular and secreted protein levels reveals the optimal secretion promoting sequence for each individual protein. Large, protein of interest-specific variations in secretion efficiency are observed. SP sequences are also used with the five surface display anchors, and the combination of SP and anchor protein proves critical for efficient surface display. These observations highlight the value of the described panel of MoClo compatible parts to allow facile screening of SPs and TFPs and anchor proteins for optimal secretion and/or surface display of a given protein of interest in S. cerevisiae.

HPV16 E7 modulates the cell surface expression of MET and CD109 via the AP2 complex

Multiple cellular pathways are affected by HPV E6 and E7 oncoproteins, including endocytic and cellular trafficking. HPV-16 E7 can target the adaptor protein (AP) complex, which contains proteins important during endocytosis transport. To further investigate the role of HPV E7 during this process, we analysed the expression of cell surface proteins in NIKS cells expressing HPV-16 E7. We show that different cell surface proteins are regulated by HPV-16 E7 via interaction with AP2. We observed that the expression of MET and CD109 membrane protein seems to be upregulated in cells expressing E7. Moreover, the interaction of MET and CD109 with AP2 proteins is disrupted by HPV-16 E7. In addition, in the absence of HPV-16 E7, there is a downregulation of the cell membrane expression of MET and CD109 in HPV-positive cell lines. These results expand our knowledge of the functions of E7 and open new potential cellular pathways affected by this oncoprotein.

Decoding of the surfaceome and endocytome in primary glioblastoma cells identifies potential target antigens in the hypoxic tumor niche

Immunotherapies with antibody–drug-conjugates (ADC) and CAR-T cells, targeted at tumor surface antigens (surfaceome), currently revolutionize clinical oncology. However, target identification warrants a better understanding of the surfaceome and how it is modulated by the tumor microenvironment. Here, we decode the surfaceome and endocytome and its remodeling by hypoxic stress in glioblastoma (GBM), the most common and aggressive brain tumor in adults. We employed a comprehensive approach for global and dynamic profiling of the surfaceome and endocytosed (endocytome) proteins and their regulation by hypoxia in patient-derived GBM cultures. We found a heterogeneous surface-endocytome profile and a divergent response to hypoxia across GBM cultures. We provide a quantitative ranking of more than 600 surface resident and endocytosed proteins, and their regulation by hypoxia, serving as a resource to the cancer research community. As proof-of-concept, the established target antigen CD44 was identified as a commonly and abundantly expressed surface protein with high endocytic activity. Among hypoxia induced proteins, we reveal CXADR, CD47, CD81, BSG, and FXYD6 as potential targets of the stressed GBM niche. We could validate these findings by immunofluorescence analyses in patient tumors and by increased expression in the hypoxic core of GBM spheroids. Selected candidates were finally confronted by treatment studies, showing their high capacity for internalization and ADC delivery. Importantly, we highlight the limited correlation between transcriptomics and proteomics, emphasizing the critical role of membrane protein enrichment strategies and quantitative mass spectrometry. Our findings provide a comprehensive understanding of the surface-endocytome and its remodeling by hypoxia in GBM as a resource for exploration of targets for immunotherapeutic approaches in GBM.

Molecular mechanisms of selenite reduction by Lactiplantibacillus plantarum BSe: An integrated genomic and transcriptomic analysis

The reduction of selenite [Se(Ⅳ)] by microorganisms is a green and efficient detoxification strategy. We found that Se(Ⅳ) inhibited exopolysaccharide and protein secretion by Lactiplantibacillus plantarum BSe and compromised cell integrity. In this study, L. plantarum BSe reduced Se(Ⅳ) by increasing related enzyme activity and electron transfer. Genomic analysis demonstrated that L. plantarum BSe should be able to reduce Se(Ⅳ). Further transcriptome analysis showed that L. plantarum BSe enhanced its tolerance to Se(Ⅳ) by upregulating the expression of surface proteins and transporters, thus reducing the extracellular Se(Ⅳ) concentration through related enzymatic reactions and siderophore-mediated pathways. Lactiplantibacillus plantarum BSe was able to regulate the expression of related genes involved in quorum sensing and a two-component system and then select appropriate strategies for Se(Ⅳ) transformation in response to varying environmental Se(Ⅳ) concentrations. In addition, azo reductase was linked to the reduction of Se(Ⅳ) for the first time. The present study established a multipath model for the reduction of Se(Ⅳ) by L. plantarum, providing new insights into the biological reduction of Se(Ⅳ) and the biogeochemical cycle of selenium.

CD39 delineates chimeric antigen receptor regulatory T cell subsets with distinct cytotoxic & regulatory functions against human islets.

Human regulatory T cells (Treg) suppress other immune cells. Their dysfunction contributes to the pathophysiology of autoimmune diseases, including type 1 diabetes (T1D). Infusion of Tregs is being clinically evaluated as a novel way to prevent or treat T1D. Genetic modification of Tregs, most notably through the introduction of a chimeric antigen receptor (CAR) targeting Tregs to pancreatic islets, may improve their efficacy. We evaluated CAR targeting of human Tregs to monocytes, a human β cell line and human islet β cells in vitro. Targeting of HLA-A2-CAR (A2-CAR) bulk Tregs to HLA-A2+ cells resulted in dichotomous cytotoxic killing of human monocytes and islet β cells. In exploring subsets and mechanisms that may explain this pattern, we found that CD39 expression segregated CAR Treg cytotoxicity. CAR Tregs from individuals with more CD39low/- Tregs and from individuals with genetic polymorphism associated with lower CD39 expression (rs10748643) had more cytotoxicity. Isolated CD39- CAR Tregs had elevated granzyme B expression and cytotoxicity compared to the CD39+ CAR Treg subset. Genetic overexpression of CD39 in CD39low CAR Tregs reduced their cytotoxicity. Importantly, β cells upregulated protein surface expression of PD-L1 and PD-L2 in response to A2-CAR Tregs. Blockade of PD-L1/PD-L2 increased β cell death in A2-CAR Treg co-cultures suggesting that the PD-1/PD-L1 pathway is important in protecting islet β cells in the setting of CAR immunotherapy. In summary, introduction of CAR can enhance biological differences in subsets of Tregs. CD39+ Tregs represent a safer choice for CAR Treg therapies targeting tissues for tolerance induction.

Fecal Supernatants from Patients with Crohn's Disease Induce Inflammatory Alterations in M2 Macrophages and Fibroblasts.

Intestinal macrophages and fibroblasts act as microenvironmental sentinels mediating inflammation and disease progression in Crohn's disease (CD). We aimed to establish the effects of fecal supernatants (FSs) from patients with CD on macrophage and fibroblast phenotype and function. FS were obtained by ultracentrifugation, and the metabolites were analyzed. Monocyte-derived M2 macrophages and fibroblasts were conditioned with FS, and secreted proteins, surface proteins and gene expression were analyzed. M2 macrophage efferocytosis was evaluated. Patients with CD (n = 15) had a skewed fecal metabolite profile compared to healthy subjects (HS, n = 10). FS from CD patients (CD-FS) induced an anti-inflammatory response in M2 macrophages with higher expression of IL-10, IL1RA and CD206 as compared to healthy FS (HS-FS) while the efferocytotic capacity was unaltered. CD-FS did not affect extracellular matrix production from fibroblasts, but increased expression of the pro-inflammatory proteins IL-6 and MCP-1. Conditioned media from M2 macrophages treated with CD-FS modulated gene expression in fibroblasts for TGFβ superfamily members and reduced IL-4 expression compared to HS-FS. We show that M2 macrophages and fibroblasts react abnormally to the fecal microenvironment of CD patients, resulting in altered protein expression related to inflammation but not fibrosis. This implies that the gut microbiota and its metabolites have an important role in the generation and/or perpetuation of inflammation in CD.