scholarly journals 662 Dissecting the CD226 immune axis in the tumor microenvironment using CyTOF-based high-dimensional immunophenotyping

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A690-A690
Author(s):  
Katie Vowell ◽  
Michael Conner ◽  
Florence Perrin ◽  
Paul Bojczuk ◽  
Kenneth Hance ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Nk Cell ◽  
Expression Profiles ◽  
Large Body ◽  
Treg Cells ◽  
High Dimensional ◽  
Entire Axis ◽  
Immune Receptors ◽  
Study Protocols ◽  
Analytical Tools

BackgroundIn recent years, a regulatory network involving nectin/nectin-like immune receptors has emerged as a potential point of manipulation for cancer immunotherapy. Central to this axis, CD226 (DNAM-1) is a T and NK cell co-stimulatory receptor that competes for ligand (CD155 and CD112) binding with multiple inhibitory receptors (TIGIT, CD96, and PVRIG [CD112R]). Despite a large body of literature for TIGIT, detailed cellular characterization of the entire axis is still lacking. Therefore, we used mass cytometry (CyTOF) to systematically evaluate expression of the CD226 axis in tumors from a range of indications.MethodsTo thoroughly characterize the CD226 axis in the tumor microenvironment, we immunophenotyped approximately 100 tumor samples derived from a variety of cancer types using a bespoke 46-parameter CyTOF panel. Human biological samples were sourced ethically and their research use was in accord with the terms of the informed consents under an IRB/EC approved protocol. Using a suite of high-dimensional analytical tools, including FlowSOM, UMAP, and tSNE, we revealed distinct expression profiles for each receptor; a finding that was previously obscured due to a lack of sufficient resolution.ResultsWe observed a notable divergence in expression profiles between the CD226 axis members across tumor indications. For example, TIGIT expression was found to be highest on activated CD4+ regulatory T (Treg) cells, where its expression correlated strongly with ICOS, FoxP3, CD25, and CCR8. By contrast, CD96 and PVRIG exhibited broad expression across intratumoral T and NK cell populations. Other receptors (e.g., CD226) demonstrated variegated expression profiles across T and NK cell subsets. Finally, despite relatively consistent expression profiles of certain CD226 axis (i.e., TIGIT on Treg cells) across tumors, we also found several cell subsets/clusters unique to specific indications.ConclusionsUsing high-parameter CyTOF analysis, we were able to thoroughly characterize the CD226 axis (CD226, TIGIT, CD96, PVRIG) and related immune receptors across a range of tumor indications. These analyses revealed divergent expression profiles for each CD226 axis member, suggesting distinct/contextual biological role(s) for each receptor. However, future studies will need to dissect the importance of the distinct cellular representation for each CD226 axis member.Ethics ApprovalAll samples were purchased from Discovery Life Sciences (DLS). DLS represents and warrants that it has ownership of all Products available for sale and has properly obtained, where required under HHS/OHRP 45 CFR 46.102 (d) (f), IRB approval (or appropriate research approval for institutions outside the U.S.) for study protocols and informed consent documents for all human subject derived biological materials.

scholarly journals 492 Integration of high dimensional datasets in an immunocompetent mammary mouse model reveals pathways of tolerance and resistance to immune checkpoint blockade

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A528-A528
Author(s):  
Lin Ma ◽  
Jian-Hua Mao ◽  
Mary Helen Barcellos-Hoff ◽  
Jade Moore
Keyword(s):  
Tumor Microenvironment ◽  
Immune Cells ◽  
Transforming Growth Factor ◽  
Immune Cell ◽  
Systemic Disease ◽  
Ct Imaging ◽  
High Dimensional ◽  
Cytokine Signaling ◽  
Immune Infiltrate ◽  
Pet Ct

BackgroundCheckpoint inhibitors can induce robust and durable responses in a subset of patients. Extending this benefit to more patients could be facilitated by better understanding of how interacts with immune cells with the tumor microenvironment, which is a critical barrier to control both local and systemic disease. The composition and pattern of the immune infiltrate associates with the likelihood of response to immunotherapy. Inflamed tumors that exhibit a brisk immune cell infiltrate are responsive, while those in which immune cells are completely or partially excluded are not. Transforming growth factor β (TGFβ) is immunosuppressive and associated with the immune excluded phenotype.MethodsUsing an immune competent mammary tumor derived transplant (mTDT) model recently developed in our lab, exhibits inflamed, excluded or deserts immune infiltrate phenotypes based on localization of CD8 lymphocytes. Using whole transcriptome deep sequencing, cytof, and PET-CT imaging, we evaluated the tumor, microenvironment, and immune pathway activation among immune infiltrate phenotypes.ResultsThree distinct inflamed tumors phenotypes were identified: ‘classically’ inflamed characterized by pathway evidence of increased CD8+ T cells and decreased PD-L1 expression, inflamed tumors with pathways indicative of neovascularization and STAT3 signaling and reduced T cell mobilization, and an inflamed tumor with increased immunosuppressive myeloid phenotypes. Excluded tumors were characterized by TGFβ gene expression and pro-inflammatory cytokine signaling (e.g. TNFα, IL1β), associated with decreased leukocytes homing and increased immune cell death of cells. We visualized and quantified TGFβ activity using PET-CT imaging of 89Zr-fresolimumab, a TGFβ neutralizing antibody. TGFβ activity was significantly increased in excluded tumors compared to inflamed or desert tumors, which was supported by quantitative pathology (Perkin Elmer) of its canonical signaling target, phosphorylated SMAD2 (pSMAD2). pSMAD2 was positively correlated with PD-L1 expression in the stroma of excluded tumors. In contrast, in inflamed tumors, TGFβ activity positively correlated with increased F4/80 positive macrophages and negatively correlated with expression of PD-L1. CyTOF analysis of tumor and spleen immune phenotypes revealed increased trafficking of myeloid cells in mice bearing inflamed tumors compared to excluded and deserts.ConclusionsThe immunocompetent mTDT provides a model that bridges the gap between the immune landscape and tumor microenvironment. Integration of these high-dimensional data with further studies of response to immunotherapies will help to identify tumor features that favor response to treatment or the means to convert those that are unresponsive.

253 Anti-TIGIT antibodies require enhanced FcγR co-engagement for optimal T and NK cell-dependent anti-tumor immunity

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A275-A275
Author(s):  
Rebecca Ward ◽  
Elena Paltrinieri ◽  
Marilyn Marques ◽  
Priyadarshini Iyer ◽  
Sylvia Dietrich ◽  
...  
Keyword(s):  
T Cell ◽  
Tumor Microenvironment ◽  
Tumor Immunity ◽  
Cell Activation ◽  
Nk Cell ◽  
Tumor Control ◽  
Tumor Bearing ◽  
Anti Tumor Activity ◽  
Ct26 Tumor ◽  
Dependent Mechanism

BackgroundT-cell immunoreceptor with Ig and ITIM domains (TIGIT) is an important negative regulator of the immune response to cancer that contributes to resistance/relapse to anti-PD-1 therapy.1 In clinical trials, anti-human (h) TIGIT antibodies have shown promising activity in combination with anti-PD-1/PD-L1 antibodies for the treatment of various solid tumors.2 However, the optimal format for anti-TIGIT antibodies remains controversial. Here we describe a novel Fcγ receptor (FcγR)-dependent mechanism of action that is critical for enhancing T and NK cell anti-tumor immunity, and, further informs on the optimal design of anti-TIGIT antibodies.MethodsWe investigated a panel of Fc-silent, Fc-competent, and Fc-engineered anti-mouse (m) TIGIT antibody variants in syngeneic murine CT26 tumor-bearing or B16F10 pseudo-metastases models. To further elucidate the relative contribution of T and NK cells in controlling tumor growth, we assessed the activity of Fc-engineered anti-TIGIT antibodies in NK cell-depleted or T cell-deficient (Nu-Foxn1nu) CT26 tumor-bearing mice. Immune-related pharmacodynamic changes in the tumor microenvironment were assessed by flow cytometry. We further validated these findings in primary human T and NK cell activation assays using Fc-engineered anti-human TIGIT antibodies.ResultsThe Fc-engineered anti-mTIGIT antibody, which demonstrates enhanced binding to mouse FcγRIV, was the only variant to deliver single agent anti-tumor activity. The Fc-enhanced variant outperformed the Fc-competent variant while the Fc-inert variant had no anti-tumor activity. Tumor control by anti-mTIGIT antibodies was not dependent on Treg depletion, but rather on increased frequency of CD8+ T cells and activated NK cells (Ki67, IFNγ, CD107a and TRAIL) in the tumor microenvironment. Concordant with observations in the mouse, Fc-engineered anti-hTIGIT antibodies with improved binding to FcγRIIIA demonstrate superior T and NK cell activation in PBMC-based assays compared to a standard hIgG1 variant. Notably, superior activity of the Fc-engineered anti-hTIGIT antibody was observed from PBMC donors that express either high or low affinity FcγRIIIA. Blockade of FcγRIIIA or depletion of CD14+ and CD56+ cells reduced the functional activity of the Fc-enhanced anti-TIGIT antibody, confirming the requirement for FcγR co-engagement to maximize T cell responses.ConclusionsOur data demonstrate the importance of FcγR co-engagement by anti-TIGIT antibodies to promote immune activation and tumor control. First generation anti-TIGIT antibodies are not optimally designed to co-engage all FcγRIIIA variants. However, Fc-enhanced anti-TIGIT antibodies unlock a novel FcγR-dependent mechanism of action to enhance T and NK cell-dependent anti-tumor immunity and further improve therapeutic outcomes.ReferencesJohnston RJ, et al., The immunoreceptor TIGIT regulates antitumor and antiviral CD8(+) T cell effector function. Cancer Cell 2014; 26:923–37.Rodriguez-Abreu D, et al., Primary analysis of a randomized, double-blind, phase II study of the anti-TIGIT antibody tiragolumab (tira) plus atezolizumab (atezo) versus placebo plus atezo as first-line (1L) treatment in patients with PD-L1-selected NSCLC (CITYSCAPE). Journal of Clinical Oncology 2020; 38:15_suppl, 9503–9503.

scholarly journals FOXP3 expression accurately defines the population of intratumoral regulatory T cells that selectively accumulate in metastatic melanoma lesions

Blood ◽  
2008 ◽  
Vol 112 (13) ◽  
pp. 4953-4960 ◽  
Author(s):  
Mojgan Ahmadzadeh ◽  
Aloisio Felipe-Silva ◽  
Bianca Heemskerk ◽  
Daniel J. Powell ◽  
John R. Wunderlich ◽  
...  
Keyword(s):  
T Cells ◽  
Tumor Microenvironment ◽  
Metastatic Melanoma ◽  
Treg Cell ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Production Capacity ◽  
Biological Marker ◽  
Foxp3 Expression ◽  
Treg Cells

Abstract Regulatory T (Treg) cells are often found in human tumors; however, their functional characteristics have been difficult to evaluate due to low cell numbers and the inability to adequately distinguish between activated and Treg cell populations. Using a novel approach, we examined the intracellular cytokine production capacity of tumor-infiltrating T cells in the single-cell suspensions of enzymatically digested tumors to differentiate Treg cells from effector T cells. Similar to Treg cells in the peripheral blood of healthy individuals, tumor-infiltrating FOXP3+CD4 T cells, unlike FOXP3− T cells, were unable to produce IL-2 and IFN-γ upon ex vivo stimulation, indicating that FOXP3 expression is a valid biological marker for human Treg cells even in the tumor microenvironment. Accordingly, we enumerated FOXP3+CD4 Treg cells in intratumoral and peritumoral sections of metastatic melanoma tumors and found a significant increase in proportion of FOXP3+CD4 Treg cells in the intratumoral compared with peritumoral areas. Moreover, their frequencies were 3- to 5-fold higher in tumors than in peripheral blood from the same patients or healthy donors, respectively. These findings demonstrate that the tumor-infiltrating CD4 Treg cell population is accurately depicted by FOXP3 expression, they selectively accumulate in tumors, and their frequency in peripheral blood does not properly reflect tumor microenvironment.

scholarly journals In Vitro Evaluation of CD276-CAR NK-92 Functionality, Migration and Invasion Potential in the Presence of Immune Inhibitory Factors of the Tumor Microenvironment

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1020
Author(s):  
Stefan Grote ◽  
Guillermo Ureña-Bailén ◽  
Kenneth Chun-Ho Chan ◽  
Caroline Baden ◽  
Markus Mezger ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Solid Tumors ◽  
Immune Cells ◽  
Immune Cell ◽  
Nk Cell ◽  
Migration And Invasion ◽  
Cellular Immunotherapy ◽  
Knock Out ◽  
Immunosuppressive Tumor Microenvironment

Background: Melanoma is the most lethal of all skin-related cancers with incidences continuously rising. Novel therapeutic approaches are urgently needed, especially for the treatment of metastasizing or therapy-resistant melanoma. CAR-modified immune cells have shown excellent results in treating hematological malignancies and might represent a new treatment strategy for refractory melanoma. However, solid tumors pose some obstacles for cellular immunotherapy, including the identification of tumor-specific target antigens, insufficient homing and infiltration of immune cells as well as immune cell dysfunction in the immunosuppressive tumor microenvironment (TME). Methods: In order to investigate whether CAR NK cell-based immunotherapy can overcome the obstacles posed by the TME in melanoma, we generated CAR NK-92 cells targeting CD276 (B7-H3) which is abundantly expressed in solid tumors, including melanoma, and tested their effectivity in vitro in the presence of low pH, hypoxia and other known factors of the TME influencing anti-tumor responses. Moreover, the CRISPR/Cas9-induced disruption of the inhibitory receptor NKG2A was assessed for its potential enhancement of NK-92-mediated anti-tumor activity. Results: CD276-CAR NK-92 cells induced specific cytolysis of melanoma cell lines while being able to overcome a variety of the immunosuppressive effects normally exerted by the TME. NKG2A knock-out did not further improve CAR NK-92 cell-mediated cytotoxicity. Conclusions: The strong cytotoxic effect of a CD276-specific CAR in combination with an “off-the-shelf” NK-92 cell line not being impaired by some of the most prominent negative factors of the TME make CD276-CAR NK-92 cells a promising cellular product for the treatment of melanoma and beyond.

Comprehensive gene expression profiles of NK cell neoplasms identify vorinostat as an effective drug candidate

2013 ◽  
Vol 333 (1) ◽  
pp. 47-55 ◽  
Author(s):  
Kennosuke Karube ◽  
Shinobu Tsuzuki ◽  
Noriaki Yoshida ◽  
Kotaro Arita ◽  
Harumi Kato ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nk Cell ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Effective Drug ◽  
Drug Candidate

517 Regulatory T cell functional identity is sustained by a glucose:lactate axis that is exploited in the tumor microenvironment

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A553-A553
Author(s):  
McLane Watson ◽  
Paolo Vignali ◽  
Steven Mullet ◽  
Abigail Overacre-Delgoffe ◽  
Ronal Peralta ◽  
...  
Keyword(s):  
T Cells ◽  
Tumor Microenvironment ◽  
Treg Cell ◽  
High Glucose ◽  
Cell Function ◽  
Treg Cells ◽  
Effector T Cells ◽  
Flux Analysis ◽  
Major Barrier ◽  
Suppressive Function

BackgroundRegulatory T (Treg) cells are vital for preventing autoimmunity but are a major barrier to robust cancer immunity as the tumor microenvironment (TME) recruits and promotes their function. The deregulated cellular metabolism of tumor cells leads to a metabolite-depleted, hypoxic, and acidic TME. While the TME impairs the effector function of highly glycolytic tumor infiltrating CD8 T cells, Treg cell suppressive function is maintained. Further, studies of in vitro induced and ex vivo Treg cells reveal a distinct metabolic profile compared to effector T cells. Thus, it may be that the altered metabolic landscape of the TME and the increased activity of intratumoral Treg cells are linked.MethodsFlow cytometry, isotopic flux analysis, Foxp3 driven Cre-lox, glucose tracers, Seahorse extracellular flux analysis, RNA sequencing.ResultsHere we show Treg cells display heterogeneity in terms of their glucose metabolism and can engage an alternative metabolic pathway to maintain their high suppressive function and proliferation within the TME and other tissues. Tissue derived Treg cells (both at the steady state and under inflammatory conditions) show broad heterogeneity in their ability to take up glucose. However, glucose uptake correlates with poorer suppressive function and long-term functional stability, and culture of Treg cells in high glucose conditions decreased suppressive function. Treg cells under low glucose conditions upregulate genes associated with the uptake and metabolism of the glycolytic end-product lactic acid. Treg cells withstand high lactate conditions, and lactate treatment prevents the destabilizing effects of high glucose culture. Treg cells utilize lactate within the TCA cycle and generate phosphoenolpyruvate (PEP), a critical intermediate that can fuel intratumoral Treg cell proliferation in vivo. Using mice with a Treg cell-restricted deletion of lactate transporter Slc16a1 (MCT1) we show MCT1 is dispensable for peripheral Treg cell function but required intratumorally, resulting in slowed tumor growth and prolonged survival.ConclusionsThese data support a model in which Treg cells are metabolically flexible such that they can utilize ‘alternative’ metabolites present in the TME to maintain their suppressive identity. Further, our studies support the notion that tumors avoid immune destruction not only by depriving effector T cells of essential nutrients, but also by metabolically supporting regulatory T cells.

PATH-29. GLIOBLASTOMA TUMOR CLASSIFICATION BASED ON SPATIAL ANALYSIS OF ONCOGENIC AMPLIFICATIONS AND PROTEIN EXPRESSION

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi121-vi121
Author(s):  
Kacper Walentynowicz ◽  
Dalit Engelhardt ◽  
Shreya Yadav ◽  
Ugoma Onubogu ◽  
Roberto Salatino ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Protein Expression ◽  
Single Cell ◽  
Expression Profiles ◽  
Protein Profiling ◽  
Intratumor Heterogeneity ◽  
Neuronal Markers ◽  
Associated Proteins ◽  
Dual Amplification ◽  
Protein Expression Profiles

Abstract Heterogeneity of glioblastoma (GBM) has been extensively studied in recent years with identification of oncogenic drivers of GBM cellular subtypes. However, little is known about how these cells interact with each other or with the surrounding tumor microenvironment (TME). We employed spatial protein profiling targeting immune and neuronal markers (79 proteins) coupled with single-cell spatial maps of fluorescence in situ hybridization (FISH) for EGFR, CDK4, and PDGFRA on human GBM tissue sections. Several cores from 20 GBM samples were collected to create a tissue microarray, and 96 regions of interests were profiled with 37,844 nuclei for oncogenic amplification screen. Spatial protein profiling identified strong correlation of certain immune markers, TAU-associated proteins, and oligodendrocyte-enriched protein groups and overall high intratumor heterogeneity of TME. Our single-cell quantification of FISH signals showed differences among tumors based on the prevalence of dual amplification of EGFR and CDK4 within a cell relative to single oncogene amplified cells. High relative frequency of dual amplification was associated with increased expression of immune-related markers and decreased expression of EGFR protein. Moreover, this protein expression signature was associated with survival in another GBM dataset. Here, we present spatial genetic analysis at the single cell level coupled with protein expression profiles associated with tumor microenvironment. Our results suggest that assessment of genetic heterogeneity in GBM could potentially drive improved patient stratification and treatment.

scholarly journals Bcl11b prevents fatal autoimmunity by promoting Treg cell program and constraining innate lineages in Treg cells

2019 ◽  
Vol 5 (8) ◽  
pp. eaaw0480 ◽  
Author(s):  
Theodore T. Drashansky ◽  
Eric Helm ◽  
Zhiguang Huo ◽  
Nina Curkovic ◽  
Preet Kumar ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Steady State ◽  
Treg Cell ◽  
Nk Cell ◽  
Treg Cells ◽  
Chromatin Accessibility ◽  
Peripheral Tolerance ◽  
Identity Control ◽  
And Function ◽  
Human And Mouse

Regulatory T (Treg) cells are essential for peripheral tolerance and rely on the transcription factor (TF) Foxp3 for their generation and function. Several other TFs are critical for the Treg cell program. We found that mice deficient in Bcl11b TF solely in Treg cells developed fatal autoimmunity, and Bcl11b-deficient Treg cells had severely altered function. Bcl11b KO Treg cells showed decreased functional marker levels in homeostatic conditions, inflammation, and tumors. Bcl11b controlled expression of essential Treg program genes at steady state and in inflammation. Bcl11b bound to genomic regulatory regions of Treg program genes in both human and mouse Treg cells, overlapping with Foxp3 binding; these genes showed altered chromatin accessibility in the absence of Bcl11b. Additionally, Bcl11b restrained myeloid and NK cell programs in Treg cells. Our study provides new mechanistic insights on the Treg cell program and identity control, with major implications for therapies in autoimmunity and cancer.

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