Targeting metabolic dysregulation of T cells in kidney cancer.

2020 ◽  
Vol 38 (6_suppl) ◽  
pp. 722-722
Author(s):  
Katy Beckermann ◽  
Kirsten Young ◽  
Rachel Hongo ◽  
Xiang Ye ◽  
Diana Contreras ◽  
...  
Keyword(s):  
T Cells ◽  
Glucose Metabolism ◽  
Single Cell ◽  
Aerobic Glycolysis ◽  
Antigen Receptor ◽  
Effector T Cells ◽  
Mitochondrial Morphology ◽  
Metabolic Markers ◽  
Metabolic Defects

722 Background: Cancer cells can inhibit effector T cells through both immunomodulatory receptors and alteration of the tumor microenvironment. Rather than efficient use of aerobic glycolysis by activated effector T cells, Renal Cell Carcinoma (RCC) infiltrating T cells (TIL) fail to increase glucose metabolism, and instead display increased reactive oxygen species (ROS) and mitochondrial dysfunction. CD8 RCC TIL also have notable differences in mitochondrial morphology compared to healthy control CD8 T cells and were punctate and dispersed rather than fused in networks. Here we test if RCC TIL can be re-activated and identify metabolic requirements for inflammatory TIL function. Methods: De-identified samples from tumor or adjacent normal tissue donations from patients with RCC were collected under an approved IRB protocol and processed into single cell suspensions of tumor and associated cells by mechanical dissociation to test TIL activation and metabolic requirements upon in vitro re-stimulation. Results: RNA-seq data suggested that CD8 from TIL rely on distinct metabolic pathways compared to control. While control T cells increased effector cytokine production and glycolysis with antigen receptor alone and further augmented this pathway with co-stimulation, single cell RNAseq showed that CD8 RCC TIL required co-stimulation for this transition. Co-stimulation can promote T cell glycolysis and we found antigen receptor stimulation with CD28-mediated co-stimulation increased function of CD8 RCC TIL as indicated by increased surface markers of activation and IFNγproduction. This was accompanied by rescue of metabolic markers, including increased mitochondrial mass and markers of electron transport. Improved functional capacity was dependent upon glycolysis because inhibition with 2-deoxyglucose limited CD8 RCC TIL activation following CD28 co-stimulation. Conclusions: Bypassing metabolic defects restore markers of TIL activation and effector function.These datademonstrate that CD8 RCC TIL can be functionally restored but that this requires the ability to increase glucose metabolism. These findings may allow for combined therapies to improve response rates of checkpoint inhibition in this disease.

scholarly journals T lymphocytes coexpressing CCR4 and a chimeric antigen receptor targeting CD30 have improved homing and antitumor activity in a Hodgkin tumor model

Blood ◽  
2009 ◽  
Vol 113 (25) ◽  
pp. 6392-6402 ◽  
Author(s):  
Antonio Di Stasi ◽  
Biagio De Angelis ◽  
Cliona M. Rooney ◽  
Lan Zhang ◽  
Aruna Mahendravada ◽  
...  
Keyword(s):  
T Cells ◽  
T Lymphocytes ◽  
Chimeric Antigen Receptor ◽  
Chemokine Receptors ◽  
Antigen Receptor ◽  
Tumor Model ◽  
Effector T Cells ◽  
Th2 Cells ◽  
Tumor Control

Abstract For the adoptive transfer of tumor-directed T lymphocytes to prove effective, there will probably need to be a match between the chemokines the tumor produces and the chemokine receptors the effector T cells express. The Reed-Stemberg cells of Hodgkin lymphoma (HL) predominantly produce thymus- and activation-regulated chemokine/CC chemokine ligand 17 (TARC/CCL17) and macrophage-derived chemokine (MDC/CCL22), which preferentially attract type 2 T helper (Th2) cells and regulatory T cells (Tregs) that express the TARC/MDC-specific chemokine receptor CCR4, thus generating an immunosuppressed tumor environment. By contrast, effector CD8+ T cells lack CCR4, are nonresponsive to these chemokines and are rarely detected at the tumor site. We now show that forced expression of CCR4 by effector T cells enhances their migration to HL cells. Furthermore, T lymphocytes expressing both CCR4 and a chimeric antigen receptor directed to the HL associated antigen CD30 sustain their cytotoxic function and cytokine secretion in vitro, and produce enhanced tumor control when infused intravenously in mice engrafted with human HL. This approach may be of value in patients affected by HL.

scholarly journals Single-cell RNA sequencing reveals ex vivo signatures of SARS-CoV-2-reactive T cells through ‘reverse phenotyping’

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
David S. Fischer ◽  
Meshal Ansari ◽  
Karolin I. Wagner ◽  
Sebastian Jarosch ◽  
Yiqi Huang ◽  
...  
Keyword(s):  
T Cells ◽  
Single Cell ◽  
Rna Sequencing ◽  
Ex Vivo ◽  
Severe Disease ◽  
Human Leukocyte ◽  
Cell Receptor ◽  
Single Cell Rna Sequencing

AbstractThe in vivo phenotypic profile of T cells reactive to severe acute respiratory syndrome (SARS)-CoV-2 antigens remains poorly understood. Conventional methods to detect antigen-reactive T cells require in vitro antigenic re-stimulation or highly individualized peptide-human leukocyte antigen (pHLA) multimers. Here, we use single-cell RNA sequencing to identify and profile SARS-CoV-2-reactive T cells from Coronavirus Disease 2019 (COVID-19) patients. To do so, we induce transcriptional shifts by antigenic stimulation in vitro and take advantage of natural T cell receptor (TCR) sequences of clonally expanded T cells as barcodes for ‘reverse phenotyping’. This allows identification of SARS-CoV-2-reactive TCRs and reveals phenotypic effects introduced by antigen-specific stimulation. We characterize transcriptional signatures of currently and previously activated SARS-CoV-2-reactive T cells, and show correspondence with phenotypes of T cells from the respiratory tract of patients with severe disease in the presence or absence of virus in independent cohorts. Reverse phenotyping is a powerful tool to provide an integrated insight into cellular states of SARS-CoV-2-reactive T cells across tissues and activation states.

scholarly journals Single-cell characterization of a model of poly I:C-stimulated peripheral blood mononuclear cells in severe asthma

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Ailu Chen ◽  
Maria P. Diaz-Soto ◽  
Miguel F. Sanmamed ◽  
Taylor Adams ◽  
Jonas C. Schupp ◽  
...  
Keyword(s):  
T Cells ◽  
Single Cell ◽  
Severe Asthma ◽  
Mononuclear Cells ◽  
Effector T Cells ◽  
Poly I:C ◽  
Peripheral Blood Mononuclear ◽  
Main Cell ◽  
Blood Mononuclear Cells ◽  
Quantitative Changes

Abstract Background Asthma has been associated with impaired interferon response. Multiple cell types have been implicated in such response impairment and may be responsible for asthma immunopathology. However, existing models to study the immune response in asthma are limited by bulk profiling of cells. Our objective was to Characterize a model of peripheral blood mononuclear cells (PBMCs) of patients with severe asthma (SA) and its response to the TLR3 agonist Poly I:C using two single-cell methods. Methods Two complementary single-cell methods, DropSeq for single-cell RNA sequencing (scRNA-Seq) and mass cytometry (CyTOF), were used to profile PBMCs of SA patients and healthy controls (HC). Poly I:C-stimulated and unstimulated cells were analyzed in this study. Results PBMCs (n = 9414) from five SA (n = 6099) and three HC (n = 3315) were profiled using scRNA-Seq. Six main cell subsets, namely CD4 + T cells, CD8 + T cells, natural killer (NK) cells, B cells, dendritic cells (DCs), and monocytes, were identified. CD4 + T cells were the main cell type in SA and demonstrated a pro-inflammatory profile characterized by increased JAK1 expression. Following Poly I:C stimulation, PBMCs from SA had a robust induction of interferon pathways compared with HC. CyTOF profiling of Poly I:C stimulated and unstimulated PBMCs (n = 160,000) from the same individuals (SA = 5; HC = 3) demonstrated higher CD8 + and CD8 + effector T cells in SA at baseline, followed by a decrease of CD8 + effector T cells after poly I:C stimulation. Conclusions Single-cell profiling of an in vitro model using PBMCs in patients with SA identified activation of pro-inflammatory pathways at baseline and strong response to Poly I:C, as well as quantitative changes in CD8 + effector cells. Thus, transcriptomic and cell quantitative changes are associated with immune cell heterogeneity in this model to evaluate interferon responses in severe asthma.

scholarly journals Corrigendum: FB5P-seq: FACS-Based 5-Prime End Single-Cell RNA-seq for Integrative Analysis of Transcriptome and Antigen Receptor Repertoire in B and T Cells

2020 ◽  
Vol 11 ◽  
Author(s):  
Noudjoud Attaf ◽  
Iñaki Cervera-Marzal ◽  
Chuang Dong ◽  
Laurine Gil ◽  
Amédée Renand ◽  
...  
Keyword(s):  
T Cells ◽  
Single Cell ◽  
Antigen Receptor ◽  
Integrative Analysis ◽  
Rna Seq ◽  
Receptor Repertoire ◽  
Prime End

scholarly journals In Vitro Pre-Clinical Validation of Suicide Gene Modified Anti-CD33 Redirected Chimeric Antigen Receptor T-Cells for Acute Myeloid Leukemia

PLoS ONE ◽  
2016 ◽  
Vol 11 (12) ◽  
pp. e0166891 ◽  
Author(s):  
Kentaro Minagawa ◽  
Muhammad O. Jamil ◽  
Mustafa AL-Obaidi ◽  
Larisa Pereboeva ◽  
Donna Salzman ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Myeloid Leukemia ◽  
Chimeric Antigen Receptor ◽  
Myeloid Leukemia ◽  
Antigen Receptor ◽  
Suicide Gene ◽  
Clinical Validation ◽  
Acute Myeloid

scholarly journals Single Cell Transcriptome Analysis Reveals Disease-Defining T Cell Subsets in the Tumor Microenvironment of Classic Hodgkin Lymphoma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 547-547
Author(s):  
Tomohiro Aoki ◽  
Lauren C. Chong ◽  
Katsuyoshi Takata ◽  
Katy Milne ◽  
Monirath Hav ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Research Funding ◽  
Cell Clusters ◽  
Mhc Ii ◽  
Spatial Profiling ◽  
Classic Hodgkin Lymphoma ◽  
Cellular Components

INTRODUCTION: Classic Hodgkin lymphoma (cHL) is uniquely characterized by an extensively dominant microenvironment composed primarily of different types of non-cancerous immune cells with a rare population (~1%) of tumor cells. Detailed characterization of these cellular components and their spatial relationship is crucial to understand crosstalk and therapeutic targeting in the cellular ecosystem of the tumor microenvironment (TME). METHODS: In this study, we performed high dimensional and spatial profiling of immune cells in the TME of cHL. Single cell RNA sequencing (scRNA-seq) was performed with the 10x Genomics platform on cell suspensions collected from lymph nodes of 22 cHL patients, including 12 of nodular sclerosis subtype, 9 of mixed cellularity subtype and 1 of lymphocyte-rich subtype, with 5 reactive lymph nodes (RLNs) serving as normal controls. Illumina sequencing (HiSeq 2500) was performed to yield single-cell expression profiles for 127,786 cells. We also performed multicolor IHC and imaging mass cytometry (IMC) on TMA slides from the same patients. RESULTS: Unsupervised clustering using PhenoGraph identified 22 cell clusters including 12 T cell clusters, 7 B cell clusters and 1 macrophage cluster. While most immune cell populations were common between cHL and RLN, we observed an enrichment of cells from cHL in all 3 regulatory T cell (Treg) clusters. The most cHL-enriched cluster was characterized by high expression of LAG3, in addition to common Treg markers such as IL2RA (CD25) and TNFRSF18 (GITR), but lacked expression of FOXP3, consistent with a type 1 regulatory (Tr1) T cell population. LAG3+ T cells in cHL had high expression of immune-suppressive cytokines IL-10 and TGF-b . In vitro exposure of T cells to cHL cell line supernatant induced significantly higher levels of LAG3 in naïve T cells compared to co-culture with other lymphoma cell line supernatant or medium only. CD4+ LAG3+ T cells isolated by FACS also suppressed the proliferation of responder CD4+ T cells when co-cultured in vitro. Additionally, Luminex analysis revealed that cHL cell lines secrete substantial amounts of cytokines and chemokines that can promote Tr1 cell differentiation (e.g. IL-6). Our scRNA-seq analysis revealed that LAG3 expression was significantly higher in cHL cases with loss of major histocompatibility class II (MHC-II) expression on HRS cells as compared to MHC-II positive cases (P = 0.019), but was not correlated with EBV status or histological subtype. Strikingly, LAG3 was identified as the most up-regulated gene in cells from MHC-II negative cases compared to MHC-II positive cases. Topological analysis using multicolor IHC and IMC revealed that in MHC-II negative cases, HRS cells were surrounded by LAG3+ T cells. In these cases, the density of LAG3+ T cells in HRS cell-rich regions was significantly increased, and the average distance between an HRS cell and its closest LAG3+ T cell neighbor was significantly shorter. These associations were confirmed in an independent cohort of 166 cHL patients. Finally, we observed a trend towards an inferior disease-specific survival (DSS; P = 0.072) and overall survival (OS; P = 0.12) in cases with an increased number of LAG3+ T cells. A high proportion of LAG3+ T cells (> 20%) was identified as an independent prognostic factor for DSS by multivariate Cox regression. CONCLUSIONS: Our results reveal a diverse TME composition with inflammatory and immunosuppressive cellular components that are linked to MHC class II expression status on HRS cells (Figure). Unprecedented transcriptional and spatial profiling at the single cell level has established the pathogenic importance of HRS cell-induced CD4+ LAG3+ T cells as a mediator of immunosuppression in cHL, with potential implications for novel therapeutic approaches. Figure Disclosures Savage: Seattle Genetics, Inc.: Consultancy, Honoraria, Research Funding; BMS, Merck, Novartis, Verastem, Abbvie, Servier, and Seattle Genetics: Consultancy, Honoraria. Scott:Roche/Genentech: Research Funding; Celgene: Consultancy; Janssen: Consultancy, Research Funding; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoSting [Institution], Research Funding. Steidl:Bristol-Myers Squibb: Research Funding; Nanostring: Patents & Royalties: Filed patent on behalf of BC Cancer; Roche: Consultancy; Seattle Genetics: Consultancy; Bayer: Consultancy; Juno Therapeutics: Consultancy; Tioma: Research Funding.

scholarly journals Evaluation of chimeric antigen receptor T cell therapy in non-human primates infected with SHIV or SIV

PLoS ONE ◽  
2021 ◽  
Vol 16 (3) ◽  
pp. e0248973
Author(s):  
Nami Iwamoto ◽  
Bhavik Patel ◽  
Kaimei Song ◽  
Rosemarie Mason ◽  
Sara Bolivar-Wagers ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Chimeric Antigen Receptor ◽  
Viral Suppression ◽  
Antigen Receptor ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T

Achieving a functional cure is an important goal in the development of HIV therapy. Eliciting HIV-specific cellular immune responses has not been sufficient to achieve durable removal of HIV-infected cells due to the restriction on effective immune responses by mutation and establishment of latent reservoirs. Chimeric antigen receptor (CAR) T cells are an avenue to potentially develop more potent redirected cellular responses against infected T cells. We developed and tested a range of HIV- and SIV-specific chimeric antigen receptor (CAR) T cell reagents based on Env-binding proteins. In general, SHIV/SIV CAR T cells showed potent viral suppression in vitro, and adding additional CAR molecules in the same transduction resulted in more potent viral suppression than single CAR transduction. Importantly, the primary determinant of virus suppression potency by CAR was the accessibility to the Env epitope, and not the neutralization potency of the binding moiety. However, upon transduction of autologous T cells followed by infusion in vivo, none of these CAR T cells impacted either acquisition as a test of prevention, or viremia as a test of treatment. Our study illustrates limitations of the CAR T cells as possible antiviral therapeutics.

scholarly journals Sunitinib Exerts In Vitro Immunomodulatory Activity on Sarcomas via Dendritic Cells and Synergizes With PD-1 Blockade

2021 ◽  
Vol 12 ◽  
Author(s):  
Darina Ocadlikova ◽  
Mariangela Lecciso ◽  
Javier Martin Broto ◽  
Katia Scotlandi ◽  
Michele Cavo ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Cell Lines ◽  
Effector T Cells ◽  
Sarcoma Cell ◽  
Ifn Γ ◽  
Sarcoma Cells

BackgroundHigh-grade sarcomas are a heterogeneous group of aggressive tumors arising in bone and soft tissues. After relapse, treatment options are limited. The multi-targeted receptor tyrosine kinase inhibitors (TKIs) sunitinib and inhibitor of PD-1 (anti-PD-1) nivolumab have shown antitumor activity in selected subtypes. In this study, we examine the role of TKIs and PD-1 based therapy in in vitro cocultures of sarcoma.MethodsThe human osteosarcoma (SaOS-2) and synovial sarcoma (SYO-1) cell lines were treated with sunitinib. After cell death and proliferation assessment, expression of PD-L1 was analyzed by flow cytometry. Sunitinib-treated sarcoma cells were cocultured with dendritic cells (DCs), and the phenotype of mature DCs was determined by flow cytometry. Mature DCs were cultured with autologous T cells. PD-1 expression on T cells, their proliferation, T regulatory cell (Tregs) induction and IFN-γ production, before and after nivolumab exposure, were analyzed.ResultsAlong with its anti-proliferative and direct pro-apoptotic effect on sarcoma cell lines, sunitinib prompted PD-L1 upregulation on sarcoma cells. Interestingly, sunitinib-treated sarcoma cells drive DCs to full maturation and increase their capacity to induce sarcoma-reactive T cells to produce IFN-γ. Conversely, no effect on T cell proliferation and T cell subpopulation composition was observed. Moreover, both bone and synovial sarcoma cell lines induced Tregs through DCs but sunitinib treatment completely abrogated Treg induction. Finally, sarcoma cell lines induced PD-1 upregulation on both effector T cells and Tregs when loaded into DCs, providing a rationale for using PD-1 blockade. Indeed, PD-1 blockade by nivolumab synergized with sunitinib in inducing IFN-γ-producing effector T cells.ConclusionsTaken together, our in vitro data indicate that the treatment of sarcoma cells with sunitinib can exert significant changes on immune cell subsets toward immune activation, leading to DC-based cross-priming of IFN-γ-producing effector T cells and reduced Treg induction. PD-1 blockade with nivolumab has a synergistic effect with sunitinib, supporting the use of TKI and anti-PD-1 approach in sarcomas, and perhaps in other cancers. DC-targeted drugs, including toll-like receptor 3 inhibitors and CD47 inhibitors, are under development and our preclinical model might help to better design their clinical application.

scholarly journals Precision engineering of an anti-HLA-A2 chimeric antigen receptor in regulatory T cells for transplant immune tolerance

2021 ◽  
Author(s):  
Yannick D. Muller ◽  
Leonardo M.R. Ferreira ◽  
Emilie Ronin ◽  
Patrick Ho ◽  
Vinh Nguyen ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Chimeric Antigen Receptor ◽  
Mononuclear Cells ◽  
Antigen Receptor ◽  
Single Chain ◽  
Transplant Tolerance ◽  
Peripheral Blood Mononuclear

Infusion of regulatory T cells (Tregs) engineered with a chimeric antigen receptor (CAR) targeting donor-derived human leukocyte antigen (HLA) is a promising strategy to promote transplant tolerance. Here, we describe an anti-HLA-A2 CAR (A2-CAR) generated by grafting the complementarity-determining regions (CDRs) of a human monoclonal anti-HLA-A2 antibody into the framework regions of the Herceptin 4D5 single-chain variable fragment and fusing it with a CD28-zeta signaling domain. The CDR-grafted A2-CAR maintained the specificity of the original antibody. We then generated HLA-A2 mono-specific human CAR Tregs either by deleting the endogenous T-cell receptor (TCR) via CRISPR/Cas9 and introducing the A2-CAR using lentiviral transduction or by directly integrating the CAR construct into the TCR alpha constant locus using homology-directed repair. These A2-CAR+TCRdeficient human Tregs maintained both Treg phenotype and function in vitro. Moreover, they selectively accumulated in HLA-A2-expressing islets transplanted from either HLA-A2 transgenic mice or deceased human donors. A2-CAR+TCRdeficient Tregs did not impair the function of these HLA-A2+ islets, whereas similarly engineered A2-CAR+TCRdeficientCD4+ conventional T cells rejected the islets in less than 2 weeks. A2-CAR+TCRdeficient Tregs delayed graft-versus-host disease only in the presence of HLA-A2, expressed either by co-transferred peripheral blood mononuclear cells or by the recipient mice. Altogether, we demonstrate that genome-engineered mono-antigen-specific A2-CAR Tregs localize to HLA-A2-expressing grafts and exhibit antigen-dependent in vivo suppression, independent of TCR expression. These approaches may be applied towards developing precision Treg cell therapies for transplant tolerance.

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