Single-Cell mRNA Sequencing of Murine and Human Alopecia Areata Identifies Immune Cell Profiles Predictive of Human Disease State

2020 ◽  
Vol 154 (Supplement_1) ◽  
pp. S5-S5
Author(s):  
Nicholas Borcherding ◽  
Sydney Crotts ◽  
Luana Ortolan ◽  
Nicholas Bormann ◽  
Ali Jabbari
Keyword(s):  
T Cells ◽  
Single Cell ◽  
Hair Follicle ◽  
Cd8 T Cells ◽  
Alopecia Areata ◽  
Predictive Models ◽  
Expression Profiles ◽  
Mrna Sequencing ◽  
Single Cell Sequencing ◽  
Autoimmune Attack

Abstract Alopecia areata (AA) is one of the most common autoimmune conditions, presenting initially with loss of hair without overt skin changes. The unremarkable appearance of the skin surface contrasts with the complex immune activity occurring at the hair follicle. AA pathogenesis is due to the loss of immune privilege of the hair follicle leading to autoimmune attack. Although the literature has focused on CD8+ T cells, vital roles for CD4+ T cells and antigen-presenting cells have been suggested. Here, we use single-cell mRNA sequencing to reveal distinct expression profiles of immune cells in AA. We found clonal expansions of both CD4+ and CD8+ T cells, with shared clonotypes across varied transcriptional states. Demonstrating distinct gene and clonotypic variations, AA murine data were used to generate highly predictive models of human AA disease. In order to corroborate the results, single-cell sequencing of T cells in human AA recapitulated the clonotypic findings and the gene expression of the predictive models. Taken together, this work demonstrates the unique transcriptomic environment of AA skin, not just limited to CD8+ T cells. This work also represents the first single-cell sequencing for the autoimmune condition AA.

Abstract 4943: Leveraging single-cell sequencing to discover novel exhaustion markers of CD8 T cells

2019 ◽  
Author(s):  
Xiaoshan Shi ◽  
Savita Jayaram ◽  
Kayla Lee ◽  
Keshav Bhojak ◽  
Vasumathi Kode ◽  
...  
Keyword(s):  
T Cells ◽  
Single Cell ◽  
Cd8 T Cells ◽  
Single Cell Sequencing

scholarly journals Single-Cell Analysis of Antigen-Specific CD8+ T-Cell Transcripts Reveals Profiles Specific to mRNA or Adjuvanted Protein Vaccines

2021 ◽  
Vol 12 ◽  
Author(s):  
Trine Sundebo Meldgaard ◽  
Fabiola Blengio ◽  
Denise Maffione ◽  
Chiara Sammicheli ◽  
Simona Tavarini ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Cd8 T Cells ◽  
Single Cell Analysis ◽  
Expression Profiles ◽  
Cd8 T Cell ◽  
Cellular Heterogeneity ◽  
Cell Analysis

CD8+ T cells play a key role in mediating protective immunity after immune challenges such as infection or vaccination. Several subsets of differentiated CD8+ T cells have been identified, however, a deeper understanding of the molecular mechanism that underlies T-cell differentiation is lacking. Conventional approaches to the study of immune responses are typically limited to the analysis of bulk groups of cells that mask the cells’ heterogeneity (RNA-seq, microarray) and to the assessment of a relatively limited number of biomarkers that can be evaluated simultaneously at the population level (flow and mass cytometry). Single-cell analysis, on the other hand, represents a possible alternative that enables a deeper characterization of the underlying cellular heterogeneity. In this study, a murine model was used to characterize immunodominant hemagglutinin (HA533-541)-specific CD8+ T-cell responses to nucleic- and protein-based influenza vaccine candidates, using single-cell sorting followed by transcriptomic analysis. Investigation of single-cell gene expression profiles enabled the discovery of unique subsets of CD8+ T cells that co-expressed cytotoxic genes after vaccination. Moreover, this method enabled the characterization of antigen specific CD8+ T cells that were previously undetected. Single-cell transcriptome profiling has the potential to allow for qualitative discrimination of cells, which could lead to novel insights on biological pathways involved in cellular responses. This approach could be further validated and allow for more informed decision making in preclinical and clinical settings.

scholarly journals Deciphering the Prognostic Implications of the Components and Signatures in the Immune Microenvironment of Pancreatic Ductal Adenocarcinoma

2021 ◽  
Vol 12 ◽  
Author(s):  
Rong Tang ◽  
Xiaomeng Liu ◽  
Chen Liang ◽  
Jie Hua ◽  
Jin Xu ◽  
...  
Keyword(s):  
T Cells ◽  
Tumor Microenvironment ◽  
Single Cell ◽  
Pancreatic Ductal Adenocarcinoma ◽  
Cd8 T Cells ◽  
Nkt Cells ◽  
Ductal Adenocarcinoma ◽  
Hematopoietic Stem ◽  
Single Cell Sequencing ◽  
Prognostic Implications

Background: The treatment modalities for pancreatic ductal adenocarcinoma (PDAC) are limited and unsatisfactory. Although many novel drugs targeting the tumor microenvironment, such as immune checkpoint inhibitors, have shown promising efficacy for some tumors, few of them significantly prolong the survival of patients with PDAC due to insufficient knowledge on the tumor microenvironment.Methods: A single-cell RNA sequencing (scRNA-seq) dataset and seven PDAC cohorts with complete clinical and bulk sequencing data were collected for bioinformatics analysis. The relative proportions of each cell type were estimated using the gene set variation analysis (GSVA) algorithm based on the signatures identified by scRNA-seq or previous literature.Results: A meta-analysis of 883 PDAC patients showed that neutrophils are associated with worse overall survival (OS) for PDAC, while CD8+ T cells, CD4+ T cells, and B cells are related to prolonged OS for PDAC, with marginal statistical significance. Seventeen cell categories were identified by clustering analysis based on single-cell sequencing. Among them, CD8+ T cells and NKT cells were universally exhausted by expressing exhaustion-associated molecular markers. Interestingly, signatures of CD8+ T cells and NKT cells predicted prolonged OS for PDAC only in the presence of “targets” for pyroptosis and ferroptosis induction. Moreover, a specific state of T cells with overexpression of ribosome-related proteins was associated with a good prognosis. In addition, the hematopoietic stem cell (HSC)-like signature predicted prolonged OS in PDAC. Weighted gene co-expression network analysis identified 5 hub genes whose downregulation may mediate the observed survival benefits of the HSC-like signature. Moreover, trajectory analysis revealed that myeloid cells evolutionarily consisted of 7 states, and antigen-presenting molecules and complement-associated genes were lost along the pseudotime flow. Consensus clustering based on the differentially expressed genes between two states harboring the longest pseudotime span identified two PDAC groups with prognostic differences, and more infiltrated immune cells and activated immune signatures may account for the survival benefits.Conclusion: This study systematically investigated the prognostic implications of the components of the PDAC tumor microenvironment by integrating single-cell sequencing and bulk sequencing, and future studies are expected to develop novel targeted agents for PDAC treatment.

scholarly journals Aberrant peripheral immune responses in acute Kawasaki disease with single-cell sequencing

2020 ◽  
Author(s):  
Zhen Wang ◽  
Lijian Xie ◽  
Sirui Song ◽  
Liqin Chen ◽  
Guang Li ◽  
...  
Keyword(s):  
T Cells ◽  
Kawasaki Disease ◽  
Immune Responses ◽  
Single Cell ◽  
Cd8 T Cells ◽  
Developed Countries ◽  
Interferon Response ◽  
Effector Memory ◽  
Intravenous Immunoglobulin Therapy ◽  
Single Cell Sequencing

AbstractKawasaki disease (KD) is the most common cause of acquired heart disease in children in developed countries. Although diverse immune aberrance was reported, a global understanding of immune responses underlying acute KD was lacking. Based on single-cell sequencing, we profiled peripheral blood mononuclear cells from patients with acute KD before and after intravenous immunoglobulin therapy and from healthy controls. Most differentially expressed genes were derived from monocytes, with upregulation of immunoglobulin receptors, complement and receptors and downregulation of MHC class II receptors before therapy. The percentage of B cells was significantly increased before therapy and rapidly returned to normal after therapy. There was also an increased abundance of B-cell receptors with IGHA and IGHG after therapy, accompanied by massive oligoclonal expansion. The percentage of CD8 T cells was remarkably decreased during acute KD, especially the subset of effector memory CD8 T cells. All lymphocyte compartments were characterized by underexpressed interferon response pathways before therapy. The identification of unique innate and adaptive immune responses suggests potential mechanisms underlying pathogenesis and progression of KD.

Abstract 4943: Leveraging single-cell sequencing to discover novel exhaustion markers of CD8 T cells

2019 ◽  
Author(s):  
Xiaoshan Shi ◽  
Savita Jayaram ◽  
Kayla Lee ◽  
Keshav Bhojak ◽  
Vasumathi Kode ◽  
...  
Keyword(s):  
T Cells ◽  
Single Cell ◽  
Cd8 T Cells ◽  
Single Cell Sequencing

scholarly journals Single-cell sequencing reveals a clonal expansion of pro-inflammatory synovial CD8 T cells expressing tissue homing receptors in psoriatic arthritis

2019 ◽  
Author(s):  
Frank Penkava ◽  
Martin Del Castillo Velasco-Herrera ◽  
Matthew D Young ◽  
Nicole Yager ◽  
Alicia Lledo Lara ◽  
...  
Keyword(s):  
T Cells ◽  
Psoriatic Arthritis ◽  
T Cell ◽  
Single Cell ◽  
Cd8 T Cells ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Developmental State ◽  
Cell Receptor ◽  
Memory Cd8 T Cells

AbstractPsoriatic arthritis (PsA) is a debilitating immune-mediated inflammatory arthritis of unknown pathogenesis commonly affecting patients with skin psoriasis. We used three complementary single cell approaches to study leukocytes from PsA joints. Mass cytometry (CyTOF) demonstrated marked (>3 fold) expansion of memory CD8 T cells in the joints compared to matched blood. Further exploration of the memory CD8 compartment using both droplet and plate based single cell RNA sequencing of paired alpha and beta chain T cell receptor sequences identified pronounced CD8 T cell clonal expansions within the joints, strongly suggesting antigen driven expansion. These clonotypes exhibited distinct gene expression profiles including cycling, activation, tissue homing and tissue residency markers. Pseudotime analysis of these clonal CD8 populations identified trajectories in which tissue residency can represent an intermediate developmental state giving rise to activated, cycling and exhausted CD8 populations. Comparing T-cell clonality across patients further revealed specificity convergence of clones against a putative common antigen. We identify chemokine receptor CXCR3 as upregulated in expanded synovial clones, and elevation of two CXCR3 ligands, CXCL9 and CXCL10, in PsA synovial fluid.

scholarly journals Identification and Characterization of Antigen-Specific CD8+ T Cells Using Surface-Trapped TNF-α and Single-Cell Sequencing

2021 ◽  
pp. ji2100535
Author(s):  
Shaheed Abdulhaqq ◽  
Abigail B. Ventura ◽  
Jason S. Reed ◽  
Arman A. Bashirova ◽  
Katherine B. Bateman ◽  
...  
Keyword(s):  
T Cells ◽  
Single Cell ◽  
Cd8 T Cells ◽  
Single Cell Sequencing ◽  
Tnf Α ◽  
Identification And Characterization

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.

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