scholarly journals High-dimensional cytometric analysis of colorectal cancer reveals novel mediators of antitumour immunity

Gut ◽  
2019 ◽  
Vol 69 (4) ◽  
pp. 691-703 ◽  
Author(s):  
Natasja L de Vries ◽  
Vincent van Unen ◽  
Marieke E Ijsselsteijn ◽  
Tamim Abdelaal ◽  
Ruud van der Breggen ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
T Cells ◽  
Lymph Nodes ◽  
Single Cell ◽  
Immune Cell ◽  
Γδ T Cells ◽  
High Dimensional ◽  
Hla Dr ◽  
Healthy Mucosa ◽  
Immune Contexture

ObjectiveA comprehensive understanding of anticancer immune responses is paramount for the optimal application and development of cancer immunotherapies. We unravelled local and systemic immune profiles in patients with colorectal cancer (CRC) by high-dimensional analysis to provide an unbiased characterisation of the immune contexture of CRC.DesignThirty-six immune cell markers were simultaneously assessed at the single-cell level by mass cytometry in 35 CRC tissues, 26 tumour-associated lymph nodes, 17 colorectal healthy mucosa and 19 peripheral blood samples from 31 patients with CRC. Additionally, functional, transcriptional and spatial analyses of tumour-infiltrating lymphocytes were performed by flow cytometry, single-cell RNA-sequencing and multispectral immunofluorescence.ResultsWe discovered that a previously unappreciated innate lymphocyte population (Lin–CD7+CD127–CD56+CD45RO+) was enriched in CRC tissues and displayed cytotoxic activity. This subset demonstrated a tissue-resident (CD103+CD69+) phenotype and was most abundant in immunogenic mismatch repair (MMR)-deficient CRCs. Their presence in tumours was correlated with the infiltration of tumour-resident cytotoxic, helper and γδ T cells with highly similar activated (HLA-DR+CD38+PD-1+) phenotypes. Remarkably, activated γδ T cells were almost exclusively found in MMR-deficient cancers. Non-activated counterparts of tumour-resident cytotoxic and γδ T cells were present in CRC and healthy mucosa tissues, but not in lymph nodes, with the exception of tumour-positive lymph nodes.ConclusionThis work provides a blueprint for the understanding of the heterogeneous and intricate immune landscape of CRC, including the identification of previously unappreciated immune cell subsets. The concomitant presence of tumour-resident innate and adaptive immune cell populations suggests a multitargeted exploitation of their antitumour properties in a therapeutic setting.

IMMU-43. IMMUNE CONTEXTURE OF ISOCITRATE DEHYDROGENASE STRATIFIED HUMAN GLIOMAS REVEALED BY SINGLE-CELL TRANSCRIPTOMICS AND ACCESSIBLE CHROMATIN

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi102-vi102
Author(s):  
Pravesh Gupta ◽  
Minghao Dang ◽  
Dapeng Hao Hao ◽  
Krishna Bojja ◽  
Tuan M Tran ◽  
...  
Keyword(s):  
T Cells ◽  
Antigen Presentation ◽  
Single Cell ◽  
Immune Cells ◽  
Isocitrate Dehydrogenase ◽  
Zinc Finger Protein ◽  
Immune Cell ◽  
Human Gliomas ◽  
Immune Contexture ◽  
Accessible Chromatin

Abstract The immune cell composition of isocitrate dehydrogenase wild type (IDH-wt) glioma patients significantly differs compared to IDH-mutant (IDH-mut) yet a detailed and unbiased understanding of their transcriptomic and epigenetic landscapes remains elusive. To this end, we performed single-cell RNA-sequencing (scRNA-seq) and single-cell Assay for Transposase-Accessible Chromatin using sequencing (sc-ATAC-seq) on ~100,000 tumor-associated immune cells from seventeen IDH mutation classified primary and recurrent human gliomas and non-glioma brains (NGBs). Our analyses revealed sixty-two transcriptionally distinct myeloid and lymphoid cell states within and across glioma subtypes and we noted microglial attrition with increasing disease severity concomitant with invading monocyte-derived cells (MDCs) and lymphocytes. Specifically, certain microglial and monocyte-derived subpopulations were associated with antigen presentation gene modules, akin to cross-presenting dendritic cells. As tissue macrophages exhibit multifaceted polarization in response to microenvironmental cues, we clarify the existence of microglia/macrophage functional states beyond M1/M2 paradigms exemplified by the presence of palmitic-, oleic- acid, and glucocorticoid responsive polarized states. We identified cytotoxic T cells with poly-functional cytolytic states mostly in recurrent IDH-wt gliomas. Furthermore, ligand-receptor interactome analyses showed a preponderance of antigen presentation/phagocytosis over the checkpoint axis in IDH-wt compared to IDH-mut gliomas. Additionally, our sc-ATAC-seq analyses revealed differences in regulatory networks in NGBs, IDH-mut, and IDH-wt glioma-associated immune cells. In particular, we noted reduced microglial usage of an iron recycling SPIC transcription factor and Interferon Regulatory Factors (IRFs); IRF1 and IRF2 in IDH-wt relative to IDH-mut and NGBs. Unique features such as amplification of 11-Zinc Finger Protein accessibility were restricted to MDCs. Finally, sc-ATAC-seq profiles of CD8+ exhausted T cells from IDH-wt showed strong enhancer accessibility on CTLA-4, Layilin, and TIM-3 but no enrichment on PD1 was seen. In summary, our study provides unprecedented granular detail of transcriptionally and epigenomically defined glioma-specific immune contexture that can be exploited for immunotherapy applications.

scholarly journals 661 Five immunotypic signatures identified in human glioblastoma correlate with tumor contact with the lateral ventricle, immune suppression, and patient outcome

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A698-A698
Author(s):  
Todd Bartkowiak ◽  
Sierra Barone ◽  
Madeline Hayes ◽  
Allison Greenplate ◽  
Justine Sinnaeve ◽  
...  
Keyword(s):  
Machine Learning ◽  
Patient Outcome ◽  
T Cells ◽  
Single Cell ◽  
Lateral Ventricle ◽  
Immune Cell ◽  
Cell Mass ◽  
Γδ T Cells ◽  
Survival Outcomes ◽  
Mass Cytometry

BackgroundGlioblastomas make up more than 60% of adult primary brain tumors and carry a median survival of less than 15 months despite aggressive therapy. Immunotherapy, now standard of care for many peripheral solid tumors, offers an appealing alternative platform that may improve survival outcomes for patients with glioblastoma; however, predictive features that could inform responsiveness to different immunotherapeutic modalities remains to be elucidated. Recent studies have demonstrated that patients whose tumors show radiographic contact with the lateral ventricle have diminished survival outcomes compared to patients whose tumors do not contact the lateral ventricle. While greater immune infiltrate correlates with more favorable outcomes and more effectual responses to immunotherapy, the anti-tumor immune response in the ventricle is unknown. We hypothesized that ventricle contact may provide a uniquely immunosuppressive microenvironment within the brain that promotes tumor growth by suppressing anti-tumor immunity, that may be overcome with appropriate targeting strategies.MethodsPrimary glioblastoma tumors obtained in accordance with the Declaration of Helsinki and with institutional IRB approval (#131870) were disaggregated into single-cell suspensions. Radiographic contact with the LV was identified by MRI imaging and confirmed by a trained neurosurgeon. Multi-dimensional single-cell mass cytometry (CyTOF) then measured >30 immune parameters in thirteen immune subpopulations infiltrating human glioblastomas, including T cells, natural killer cells, B cells, microglia, peripheral macrophages, and myeloid-derived suppressors cells (MDSC). Computational machine-learning pipelines including Citrus, t-SNE, FlowSOM, and MEM identified key differences in the abundance and phenotypes of immune infiltrates.ResultsOn the basis of glioblastoma contact with the ventricle, we computationally identified consequential distinctions in the abundance of T cell, macrophage, and microglia subsets constituting five immunotype signatures among glioblastoma patients. Immunotypes associated with CD69+CD32+CD44+ peripheral macrophages and PD-1+TIGIT+ CD8 T cells correlated with ventricle contact, whereas immunotypes associated with enriched γδ T cells, B, NK cell, and tissue-resident microglial cells correlated with tumors distal to the ventricle. Further, immune infiltration in the tumor microenvironment correlated with patient outcome, with higher lymphocyte infiltrates correlating with more favorable outcomes, and immune exhaustion correlating with less favorable outcomes.ConclusionsSingle-cell mass cytometry in conjunction with the machine learning tools identified key differences in immune cell abundance between lateral ventricle contacting and non-contacting glioblastomas. These results provide key insights into the immune microenvironment of glioblastomas and elucidate several clinically actionable immunotherapeutic targets that may be used to optimize treatment strategies for glioblastomas based on ventricle contact status.Ethics ApprovalThis study was approved by Vanderbilt University’s Institutional Ethics Board, approval number 131870

scholarly journals The ratio of exhausted to resident infiltrating lymphocytes is prognostic for colorectal cancer patient outcome

2020 ◽  
Author(s):  
Momeneh Foroutan ◽  
Ramyar Molania ◽  
Aline Pfefferle ◽  
Corina Behrenbruch ◽  
Axel Kallies ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
T Cells ◽  
Nk Cells ◽  
Single Cell ◽  
Immune Cells ◽  
Immune Cell ◽  
Colorectal Cancer Patient ◽  
Colon Adenocarcinoma ◽  
Rna Seq ◽  
Infiltrating Lymphocytes

AbstractImmunotherapy success in colorectal cancer (CRC) is mainly limited to patients whose tumours exhibit high microsatellite instability (MSI). However, there is variability in treatment outcomes within this group, which is in part driven by the frequency and characteristics of tumour infiltrating immune cells. Indeed, the presence of specific infiltrating immune cell subsets has been shown to correlate with immunotherapy responses and is in many cases prognostic of treatment outcome. Tumour-infiltrating lymphocytes (TILs) can undergo distinct differentiation programs such as acquire features of tissue-residency or exhaustion, a process during which T cells upregulate inhibitory receptors such as PD-1 and loose functionality. While residency and exhaustion programs of CD8 T cells are relatively well-studied, these programs have only recently been appreciated in CD4 T cells and remain largely unknown in tumour-infiltrating natural killer (NK) cells. In this study, we use single cell RNA-seq data to identify signatures of residency and exhaustion in CRC infiltrating lymphocytes, including CD8, CD4 and NK cells. We then test these signatures in independent single cell data from tumour and normal tissue infiltrating immune cells. Further, we use versions of these signatures adapted for bulk RNA-seq data to identify a list of tumour intrinsic mutations associated with residency and exhaustion from TCGA data. Finally, using two independent transcriptomic data sets from patients with colon adenocarcinoma, we show that combinations of these signatures, in particular NK signatures, as well as tumour-associated signatures, such as TGF-β signalling, are associated with distinct survival outcomes in colorectal cancer patients.

Abstract 439: High Dimensional Single-Cell Immune Contexture of Human Atherosclerotic Plaques and Blood

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Adeeb Rahman ◽  
Aleksey Chudnovskiy ◽  
El-ad David Amir ◽  
Seunghee Kim-Schulze ◽  
Jennifer R Li ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Cd8 T Cells ◽  
Immune Cells ◽  
High Dimensional ◽  
Specific Cell ◽  
Single Cell Level ◽  
Cell Level ◽  
Immune Contexture

Atherosclerosis is a disease characterized by immune infiltration of the arterial wall in response to tissue damage and systemic inflammation. In the era of precision medicine, is essential to gain insights on immune contexture of atherosclerotic tissue taking into account disease-specific cell variation in patients. We applied high-dimensional technologies for the analysis of multiple parameters at the single-cell level in clinical samples of patients undergoing carotid endatherectomy (CEA, n=15). Using time-of-flight mass-cytometry (CyTOF), we simultaneously analyzed 32 parameters at the single-cell level in peripheral blood mononuclear cells (PBMCs) and atherosclerotic-tissue associated immune cells of the same patient. Using viSNE, we mapped single-cell heterogeneity into two dimensions to discriminate PBMCs and tissue-associated CD45+ immune cells. Next, we employed Phenograph to cluster cells into phenotypically related populations, which were annotated based on canonical marker expression patterns. We identified several major immune subsets including two subsets of macrophages (CD163 low and CD163 high ), monocytes, dendritic cells (DCs), B and T cells. The most prevalent CD45+ cells identified in atherosclerotic tissue were CD4 + (25.8%) and CD8 + (25.2%) T cells, macrophages (12.8%), monocytes (7.7%) and B (2.1%) cells. Using a regression analysis similar to that employed by CITRUS, we determined that macrophages and a subset of CD8 T cells characterized by low expression of CD127 were selectively enriched in tissue vs. blood. Multiplexed immunohistochemistry confirmed that T cells comprised a major portion of the CD45+ cells in atherosclerotic tissue, even more abundant than macrophages. This study of deep phenotyping across-atherosclerotic tissue and blood demonstrate a significant T cell tissue infiltration of a specific subset of CD8 T cells. This suggests that adaptive T cell immunity plays a critical role in advanced atherosclerosis. The extension of this systems biology analysis pipeline to larger datasets can improve our understanding of the core mechanisms of chronic inflammation in atherosclerosis.

scholarly journals TAMI-01. NEOADJUVANT PD-1 ANTIBODY BLOCKADE REMODELS THE IMMUNE MICROENVIRONMENT OF METASTATIC BRAIN TUMORS

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii212-ii213
Author(s):  
Lu Sun ◽  
Jenny Kienzler ◽  
Alexander Lee ◽  
Frances Chow ◽  
Carolina Chavez ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Single Cell ◽  
Immune Cell ◽  
Cytotoxic T Cells ◽  
Lymphoid Cells ◽  
New Combination ◽  
Inflammatory Microenvironment ◽  
Hla Dr ◽  
Line Standard

Abstract Brain metastases (BM) commonly arise in patients with melanoma, lung, and breast cancer. Currently, there are limited options for GBM and BM patients who have failed the first-line standard treatment, underscoring the importance of developing new therapeutic strategies. Last year, we and other groups evaluated the neoadjuvant timing of anti-PD-1 checkpoint blockade therapy in recurrent GBM (rGBM) patients, which resulted in a modest survival benefit. In light of the known effectiveness of anti-PD-1 as a systemic therapy to control melanoma and non-small cell lung cancer BM, we set out to study the anti-tumor immune response of BM patients to anti-PD-1 in the neoadjuvant setting. We posited that neoadjuvant anti-PD-1 in patients with BM would result in a stronger antitumoral immune response, which could be quantified at the single cell level. To test this, we made use of contemporary single cell techniques, including multiplex immunofluorescence, time-of-flight mass cytometry (CyTOF) and single-cell RNA sequencing (scRNAseq), to characterize the intratumoral immune cell populations and their transcriptomic profiles. We found that neoadjuvant anti-PD-1 significantly increased the number of tumor infiltrating T lymphocytes in BM compared to rGBM (2.5 fold in BM, p= 0.02 vs. 1.4 fold in rGBM, p= 0.19). Multiplex immunofluorescence analysis of T cells in BM samples revealed a change from T cell exclusion to a diffusely infiltrating phenotype after anti-PD-1 treatment. Importantly, BM showed a higher fraction of effector/cytotoxic T cells compared to rGBM (7.3% vs. 0.9% of lymphoid cells, p= 0.005) and anti-PD-1 further enhanced this population. In the myeloid compartment of BM, neoadjuvant anti-PD-1 increased the frequency of HLA-DR+CD206- M1-like macrophages, implicating a pro-inflammatory microenvironment. In summary, our study delineated the immune cell subtypes altered by neoadjuvant anti-PD-1 and offers insights into new combination therapies that can help understand the clinical efficacy of immunotherapy for BM and GBM patients.

scholarly journals Mixed-effects association of single cells identifies an expanded effector CD4+T cell subset in rheumatoid arthritis

2018 ◽  
Vol 10 (463) ◽  
pp. eaaq0305 ◽  
Author(s):  
Chamith Y. Fonseka ◽  
Deepak A. Rao ◽  
Nikola C. Teslovich ◽  
Ilya Korsunsky ◽  
Susan K. Hannes ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
T Cells ◽  
Single Cell ◽  
Single Cells ◽  
Mixed Effects ◽  
Interindividual Variation ◽  
Effector Memory ◽  
High Dimensional ◽  
Mass Cytometry ◽  
Hla Dr

High-dimensional single-cell analyses have improved the ability to resolve complex mixtures of cells from human disease samples; however, identifying disease-associated cell types or cell states in patient samples remains challenging because of technical and interindividual variation. Here, we present mixed-effects modeling of associations of single cells (MASC), a reverse single-cell association strategy for testing whether case-control status influences the membership of single cells in any of multiple cellular subsets while accounting for technical confounders and biological variation. Applying MASC to mass cytometry analyses of CD4+T cells from the blood of rheumatoid arthritis (RA) patients and controls revealed a significantly expanded population of CD4+T cells, identified as CD27−HLA-DR+effector memory cells, in RA patients (odds ratio, 1.7;P= 1.1 × 10−3). The frequency of CD27−HLA-DR+cells was similarly elevated in blood samples from a second RA patient cohort, and CD27−HLA-DR+cell frequency decreased in RA patients who responded to immunosuppressive therapy. Mass cytometry and flow cytometry analyses indicated that CD27−HLA-DR+cells were associated with RA (meta-analysisP= 2.3 × 10−4). Compared to peripheral blood, synovial fluid and synovial tissue samples from RA patients contained about fivefold higher frequencies of CD27−HLA-DR+cells, which comprised ~10% of synovial CD4+T cells. CD27−HLA-DR+cells expressed a distinctive effector memory transcriptomic program with T helper 1 (TH1)– and cytotoxicity-associated features and produced abundant interferon-γ (IFN-γ) and granzyme A protein upon stimulation. We propose that MASC is a broadly applicable method to identify disease-associated cell populations in high-dimensional single-cell data.

scholarly journals Mixed Effects Association of Single Cells Identifies an Expanded Th1-Skewed Cytotoxic Effector CD4+ T Cell Subset in Rheumatoid Arthritis

2017 ◽  
Author(s):  
Chamith Y. Fonseka ◽  
Deepak A. Rao ◽  
Nikola C. Teslovich ◽  
Susan K. Hannes ◽  
Kamil Slowikowsi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Cd4 T Cells ◽  
Single Cells ◽  
Cell Subset ◽  
Mixed Effects ◽  
Cd4 T Cell ◽  
High Dimensional ◽  
Hla Dr

AbstractHigh dimensional single-cell analyses have dramatically improved the ability to resolve complex mixtures of cells from human disease samples; however, identifying disease-associated cell types or cell states in patient samples remains challenging due to technical and inter-individual variation. Here we present Mixed effects modeling of Associations of Single Cells (MASC), a novel reverse single cell association strategy for testing whether case-control status influences the membership of single cells in any of multiple cellular subsets while accounting for technical confounds and biological variation. Applying MASC to mass cytometry analyses of CD4+ T cells from blood of rheumatoid arthritis (RA) patients and controls revealed a significantly expanded population of CD4+ T cells, identified as CD27- HLA-DR+ effector memory cells, in RA patients (OR = 1.7; p = 1.1 × 10−3). The frequency of CD27- HLA-DR+ cells was similarly elevated in blood samples from a second RA patient cohort, and CD27- HLA-DR+ cell frequency decreased in RA patients who respond to immunosuppressive therapy. Compared to peripheral blood, synovial fluid and synovial tissue samples from RA patients contained ∼5-fold higher frequencies of CD27- HLA-DR+ cells, which comprised ∼10% of synovial CD4+ T cells. We find that CD27- HLA-DR+ cells are abundant producers of IFN-γ and also express perforin and granzyme A at elevated levels. Thus MASC identified the expansion of a unique Th1 skewed effector T cell population with cytotoxic capacity in RA. We propose that MASC is a broadly applicable method to identify disease-associated cell populations in high-dimensional single cell data.One Sentence SummaryMixed-effects regression of single cells identifies a cytotoxic Th1-like CD4+ T cell subset while accounting for inter-individual and technical variation.

773 Adoptive Cell Therapy Response in Melanoma is Mediated by Stem-like CD8 T cells

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A822-A822
Author(s):  
Sri Krishna ◽  
Frank Lowery ◽  
Amy Copeland ◽  
Stephanie Goff ◽  
Grégoire Altan-Bonnet ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Single Cell ◽  
Cd8 T Cells ◽  
Tumor Infiltrating Lymphocytes ◽  
High Dimensional ◽  
Tumor Control ◽  
Intrinsic Factors ◽  
Infiltrating Lymphocytes

BackgroundAdoptive T cell therapy (ACT) utilizing ex vivo-expanded autologous tumor infiltrating lymphocytes (TILs) can result in complete regression of human cancers.1 Successful immunotherapy is influenced by several tumor-intrinsic factors.2 3 Recently, T cell-intrinsic factors have been associated with immunotherapy response in murine and human studies.4 5 Analyses of tumor-reactive TILs have concluded that anti-tumor neoantigen-specific TILs are enriched in subsets defined by the expression of PD-1 or CD39.6 7 Thus, there is a lack of consensus regarding the tumor-reactive TIL subset that is directly responsible for successful immunotherapies such as ICB and ACT. In this study, we attempted to define the fitness landscape of TIL-enriched infusion products to specifically understand its phenotypic impact on human immunotherapy responses.MethodsWe compared the phenotypic differences that could distinguish bulk ACT infusion products (I.P.) administered to patients who had complete response to therapy (complete responders, CRs, N = 24) from those whose disease progressed following ACT (non-responders, NRs, N = 30) by high dimensional single cell protein and RNA analysis of the I.P. We further analyzed the phenotypic states of anti-tumor neoantigen specific TILs from patient I.P (N = 26) by flow cytometry and single cell transcriptomics.ResultsWe identified two CD8+ TIL populations associated with clinical outcomes: a memory-progenitor CD39-negative stem-like TIL (CD39-CD69-) in the I.P. associated with complete cancer regression (overall survival, P < 0.0001, HR = 0.217, 95% CI 0.101 to 0.463) and TIL persistence, and a terminally differentiated CD39-positive TIL (CD39+CD69+) population associated with poor TIL persistence post-treatment. Although the majority (>65%) of neoantigen-reactive TILs in both responders and non-responders to ACT were found in the differentiated CD39+ state, CR infusion products also contained a pool of CD39- stem-like neoantigen-specific TILs (median = 8.8%) that was lacking in NR infusion products (median = 23.6%, P = 1.86 x 10-5). Tumor-reactive stem-like T cells were capable of self-renewal, expansion, and persistence, and mediated superior anti-tumor response in vivo.ConclusionsOur results support the hypothesis that responders to ACT received infusion products containing a pool of stem-like neoantigen-specific TILs that are able to undergo prolific expansion, give rise to differentiated subsets, and mediate long-term tumor control and T cell persistence, in line with recent murine ICB studies mediated by TCF+ progenitor T cells.4 5 Our data also suggest that TIL subsets mediating ACT-response (stem-like CD39-) might be distinct from TIL subsets enriched for anti-tumor-reactivity (terminally differentiated CD39+) in human TIL.6 7AcknowledgementsWe thank Don White for curating the melanoma patient cohort, and J. Panopoulos (Flowjo) for helpful discussions on high-dimensional analysis, and NCI Surgery Branch members for helpful insights and suggestions. S. Krishna acknowledges funding support from NCI Director’s Innovation Award from the National Cancer Institute.Trial RegistrationNAEthics ApprovalThe study was approved by NCI’s IRB ethics board.ReferencesGoff SL, et al. Randomized, prospective evaluation comparing intensity of lymphodepletion before adoptive transfer of tumor-infiltrating lymphocytes for patients with metastatic melanoma. J Clin Oncol 2016;34:2389–2397.Snyder A, et al. Genetic basis for clinical response to CTLA-4 blockade in melanoma. N Engl J Med 2014;371:2189–2199.McGranahan N, et al. Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade. Science 2016;351:1463–1469.Sade-Feldman M, et al. Defining T cell states associated with response to checkpoint immunotherapy in melanoma. Cell 2019;176:404.Miller BC, et al. Subsets of exhausted CD8 T cells differentially mediate tumor control and respond to checkpoint blockade. Nat. Immunol 2019;20:326–336.Simoni Y, et al. Bystander CD8 T cells are abundant and phenotypically distinct in human tumour infiltrates. Nature 2018;557:575–579.Gros A, et al. PD-1 identifies the patient-specific CD8+ tumor-reactive repertoire infiltrating human tumors. J Clin Invest 2014;124:2246–2259.

540 Transcriptionally defined immune landscape in human gliomas

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A576-A576
Author(s):  
Pravesh Gupta ◽  
Minghao Dang ◽  
Krishna Bojja ◽  
Huma Shehwana ◽  
Tuan Tran ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cell ◽  
Single Cell ◽  
Rna Sequencing ◽  
Immune Cell ◽  
Myeloid Cell ◽  
Wild Type ◽  
Cell Clusters ◽  
Md Anderson ◽  
Antigen Presenting

BackgroundBrain immunity is largely myeloid cell dominated rather than lymphoid cells in healthy and diseased state including malignancies of glial origins called as gliomas. Despite this skewed myeloid centric immune contexture, immune checkpoint and T cell based therapeutic modalities are generalizably pursued in gliomas ignoring the following facts i) T cells are sparse in tumor brain ii) glioma patients are lymphopenic iii) gliomas harbor abundant and highly complex myeloid cell repertoire. We recognized these paradoxes pertaining to fundamental understanding of constituent immune cells and their functional states in the tumor immune microenvironment (TIME) of gliomas, which remains elusive beyond a priori cell types and/or states.MethodsTo dissect the TIME in gliomas, we performed single-cell RNA-sequencing on ~123,000 tumor-derived sorted CD45+ leukocytes from fifteen genomically classified patients comprising IDH-mutant primary (IMP; n=4), IDH-mutant recurrent (IMR; n=4), IDH-wild type primary (IWP; n=3), or IDH-wild type recurrent (IWR; n=4) gliomas (hereafter referred as glioma subtypes) and two non-glioma brains (NGBs) as controls.ResultsUnsupervised clustering analyses delineated predominant 34-myeloid cell clusters (~75%) over 28-lymphoid cell clusters (~25%) reflecting enormous heterogeneity within and across glioma subtypes. The glioma immune diversity spanned functionally imprinted phagocytic, antigen-presenting, hypoxia, angiogenesis and, tumoricidal myeloid to classical cytotoxic lymphoid subpopulations. Specifically, IDH-mutant gliomas were predominantly enriched for brain-resident microglial subpopulations in contrast to enriched bone barrow-derived infiltrates in IDH-wild type especially in a recurrent setting. Microglia attrition in IWP and IWR gliomas were concomitant with invading monocyte-derived cells with semblance to dendritic cell and macrophage like transcriptomic features. Additionally, microglial functional diversification was noted with disease severity and mostly converged to inflammatory states in IWR gliomas. Beyond dendritic cells, multiple antigen-presenting cellular states expanded with glioma severity especially in IWP and IWR gliomas. Furthermore, we noted differential microglia and dendritic cell inherent antigen presentation axis viz, osteopontin, and classical HLAs in IDH subtypes and, glioma-wide non-PD1 checkpoints associations in T cells like Galectin9 and Tim-3. As a general utility, our immune cell deconvolution approach with single-cell-matched bulk RNA sequencing data faithfully resolved 58-cell states which provides glioma specific immune reference for digital cytometry application to genomics datasets.ConclusionsAltogether, we identified prognosticator immune cell-signatures from TCGA cohorts as one of many potential immune responsiveness applications of the curated signatures for basic and translational immune-genomics efforts. Thus, we not only provide an unprecedented insight of glioma TIME but also present an immune data resource that can be exploited for immunotherapy applications.Ethics ApprovalThe brain tumor/tissue samples were collected as per MD Anderson internal review board (IRB)-approved protocol numbers LAB03-0687 and, LAB04-0001. One non-tumor brain tissue sample was collected from patient undergoing neurosurgery for epilepsy as per Baylor College of Medicine IRB-approved protocol number H-13798. All experiments were compliant with the review board of MD Anderson Cancer Center, USA.ConsentWritten informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal

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