scholarly journals Single-cell differential splicing analysis reveals high heterogeneity of liver tumor-infiltrating T cells

2020 ◽  
Author(s):  
Shang Liu ◽  
Biaofeng Zhou ◽  
Liang Wu ◽  
Yan Sun ◽  
Jie Chen ◽  
...  
Keyword(s):  
T Cells ◽  
Alternative Splicing ◽  
Single Cell ◽  
Tumor Infiltrating Lymphocytes ◽  
Differential Splicing ◽  
Cell Level ◽  
Comprehensive Investigation ◽  
Cancer Initiation ◽  
Cell Groups ◽  
Infiltrating Lymphocytes

AbstractRecent advances in single-cell RNA sequencing (scRNA-seq), enriched the knowledge of the heterogeneity of the tumor-infiltrating lymphocytes (TIL) for understanding the mechanisms of cancer initiation and progression. However, alternative splicing (AS), as one of the important regulatory factors of heterogeneity, has been poorly investigated. Here, we proposed a computational tool, DESJ-detection, which could fast and accurately detect the differentially expressed splicing junction (DESJ) between cell groups at single-cell level. We analyzed 5,063 T cells of hepatocellular carcinoma (HCC) and identified 1,176 DESJs across 11 T cell subtypes. Cell subtypes with a similar function clustered closer rather than the lineage at the AS level. Meanwhile, we identified two novel cell states, pre-exhaustion and pre-activation with the marker isoform CD103-201 and ARHGAP15-205. In summary, we presented a comprehensive investigation of alternative splicing differences, which provided novel insights for heterogeneity of T cells and can be applied in other full-length scRNA-seq datasets.

scholarly journals Single-cell Differential Splicing Analysis Reveals High Heterogeneity of Liver Tumor-infiltrating T Cells

2020 ◽  
Author(s):  
Shang Liu ◽  
Biaofeng Zhou ◽  
Liang Wu ◽  
Yan Sun ◽  
Jie Chen ◽  
...  
Keyword(s):  
T Cells ◽  
Alternative Splicing ◽  
T Cell ◽  
Single Cell ◽  
Regulatory Factor ◽  
Differential Splicing ◽  
Cell Level ◽  
Comprehensive Investigation ◽  
Cancer Initiation ◽  
Cell Groups

Abstract Recent advances in single-cell RNA sequencing (scRNA-seq) have improved our understanding of the association between tumor-infiltrating lymphocyte (TILs) heterogeneity and cancer initiation and progression. However, studies investigating alternative splicing (AS) as an important regulatory factor of heterogeneity remain limited. Here, we developed a new computational tool, DESJ-detection, which accurately detects differentially expressed splicing junctions (DESJs) between cell groups at the single-cell level. We analyzed 5,063 T cells of hepatocellular carcinoma (HCC) and identified 1,176 DESJs across 11 T cell subtypes. Interestingly, DESJs were enriched in UTRs, and have putative effects on heterogeneity. Cell subtypes with a similar function closely clustered together at the AS level. Meanwhile, we identified two novel cell states, pre-exhaustion and pre-activation with the isoform markers CD103-201 and ARHGAP15-205. In summary, we present a comprehensive investigation of alternative splicing differences, which provided novel insights into T cell heterogeneity and can be applied to other full-length scRNA-seq datasets.

scholarly journals Single-cell differential splicing analysis reveals high heterogeneity of liver tumor-infiltrating T cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shang Liu ◽  
Biaofeng Zhou ◽  
Liang Wu ◽  
Yan Sun ◽  
Jie Chen ◽  
...  
Keyword(s):  
T Cells ◽  
Alternative Splicing ◽  
T Cell ◽  
Single Cell ◽  
Regulatory Factor ◽  
Differential Splicing ◽  
Cell Level ◽  
Comprehensive Investigation ◽  
Cancer Initiation ◽  
Cell Groups

AbstractRecent advances in single-cell RNA sequencing (scRNA-seq) have improved our understanding of the association between tumor-infiltrating lymphocyte (TILs) heterogeneity and cancer initiation and progression. However, studies investigating alternative splicing (AS) as an important regulatory factor of heterogeneity remain limited. Here, we developed a new computational tool, DESJ-detection, which accurately detects differentially expressed splicing junctions (DESJs) between cell groups at the single-cell level. We analyzed 5063 T cells of hepatocellular carcinoma (HCC) and identified 1176 DESJs across 11 T cell subtypes. Interestingly, DESJs were enriched in UTRs, and have putative effects on heterogeneity. Cell subtypes with a similar function closely clustered together at the AS level. Meanwhile, we identified a novel cell state, pre-activation with the isoform markers ARHGAP15-205. In summary, we present a comprehensive investigation of alternative splicing differences, which provided novel insights into T cell heterogeneity and can be applied to other full-length scRNA-seq datasets.

scholarly journals IMMU-02. NEOANTIGENS ARISING FROM ALTERNATIVE SPLICING EVENTS MAY BE TARGETED BY TUMOR INFILTRATING LYMPHOCYTES IN GLIOBLASTOMAS

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi118-vi119
Author(s):  
Alexander Lee ◽  
Yang Pan ◽  
Aaron Mochizuki ◽  
Mildred Galvez ◽  
Frances Chow ◽  
...  
Keyword(s):  
T Cells ◽  
Alternative Splicing ◽  
Single Cell ◽  
Rna Splicing ◽  
Ex Vivo ◽  
Tumor Infiltrating Lymphocytes ◽  
Sequencing Data ◽  
Synonymous Mutations ◽  
Alternative Splicing Events ◽  
Infiltrating Lymphocytes

Abstract INTRODUCTION Alternative splicing, the cellular process that converts premature mRNA to mature mRNA and allows for single genes to produce multiple protein products, is frequently dysregulated in many cancers, including glioblastoma. However, along with non-synonymous mutations in the DNA, altered splicing mechanisms in cancers may produce novel antigens (so-called neoantigens) that distinguish cancer cells from healthy cells and can thus be targeted by the immune system. METHODS We developed a new computation pipeline (IRIS – Isoform peptides from RNA splicing for Immunotherapy targets Screening) that took bulk RNA-sequencing data from 23 glioblastoma patient tumor samples and predicted neoantigens that may arise from alternative splicing events. We prioritized predicted neoantigens that arose in HLA*A02:01 and HLA*A03:01 patients and selected 8 potential neoantigens to generate peptide:MHC Class 1 dextramers. We tested PBMCs and/or ex vivo expanded tumor infiltrating lymphocytes (TIL) from 6 of our glioblastoma patients against these dextramers, sorted for any neoantigen-reactive T cells, and performed single-cell RNAsequencing on the sorted population to determine the TCR sequence. RESULTS Among the 8 predicted neoantigens tested, 7 of the neoantigens were recognized by at least 1 patient’s T cells. 1 HLA*A03:01 epitope was recognized in 3 of the 4 HLA*A03:01 patients tested and this epitope was highly positive in an expanded TIL population, representing 1.7% of all CD3+ CD8+ cells. When we sorted for those neoantigen reactive T cells from the expanded TIL population and performed single-cell RNAsequencing, we found 325 unique T cell clonotypes, but the top 10 clonotypes represented 83.6% of all TCR clonotypes. The most frequent TCR clonotype represented 39.1% of the repertoire and suggests that clonal expansion of a select few TCR clones occurred within the tumor. CONCLUSIONS In total, our data indicates that neoantigens arising from alternative splicing events may represent a potential target for immunotherapy in glioblastoma.

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.

scholarly journals 64 A cloning and expression system of the neoantigen-specific TCRs from tumor-infiltrating lymphocytes by single-cell sequencing of paired TCRα and TCRβ chains

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A71-A71
Author(s):  
Yukari Kobayashi ◽  
Koji Nagaoka ◽  
Kaori Kubo ◽  
Toshikazu Nishie ◽  
Sachiko Okamoto ◽  
...  
Keyword(s):  
Lung Cancer ◽  
T Cells ◽  
Single Cell ◽  
Expression System ◽  
Tumor Infiltrating Lymphocytes ◽  
Cloning And Expression ◽  
Class I ◽  
Autologous Tumor ◽  
Single Cell Sequencing ◽  
Infiltrating Lymphocytes

BackgroundT-cells that target tumor neoantigens arising from cancer mutations are the primary mediators of cancer immunotherapies. Identifying neoantigens and T-cells that recognize them is essential for T-cell-based immunotherapy. However, neoantigen-reactive Tumor-infiltrating lymphocytes (TILs) are highly differentiated or exhausted with a limited proliferative capacity; it is challenging to expand them for a sufficient number to probe their specificity. Therefore, we developed a novel cloning and expression system to examine TCRs discovered by single-cell sequencing of TILs for their neoantigen-specificity.MethodsTILs of lung cancer and sarcoma were analyzed. Surgically removed tumors were divided into several pieces. They were enzymatically digested to prepare fresh tumor digest (FTD) and cryopreserved. They were used to generate TIL cultures and perform WES and RNA-Seq to identify tumor-specific mutations. MHCflurry was used to predict the binding affinity of potential epitopes arising from these mutations to HLA class I. Peptides that were predicted to bind to patients‘ own MHC class I molecules strongly were then synthesized. Single TILs isolated with the ICELL8® cx system (Takara Bio) were dispensed into a nanowell TCR chip containing preprinted barcodes. Barcoded cDNAs were PCR-amplified in-chip, pooled off-chip, and used as a template in the TCR-specific PCR or for the whole transcriptome library generation of 5’ ends of all transcripts. Based on single-cell transcriptome data and TCR profiles of TILs, we predict and prioritize neoantigen-specific TCRs and cloned them into siTCR® retrovirus vectors. These TCRs were transduced into SUP-T1-based reporter cells in which ZsGreen fluorescent protein expression is controlled by AP-1 and NFAT binding sites. TCR-expressing reporter cells were cocultured with patient autologous APCs pulsed with a pool of candidate neoantigen peptides. ZsGreen expression indicates that TCRs match their cognate neoantigens.ResultsIn a lung cancer patient, we set up 18 TIL cultures and obtained 12 TILs. TILs were cocultured with FTD; IFN-γ production was measured by ELISA to evaluate their reactivity to the autologous tumor. NGS identified 197 somatic mutations, 4 fusion genes, and 8 highly expressed cancer-testis antigens. Among them, 339 candidate peptides were synthesized and screened. In addition, we cloned 3 pairs of TCRαβ chains from most expanded TIL cultures and 4 TCRs from ex vivo TILs with exhausted phenotype. Two reporter cells that express TCRs from exhausted TILs responded to the same neoantigen peptide.ConclusionsGenerating TCR expressing cell lines facilitated the identifying neoantigens and their cognate TCR sequences from patients.Ethics ApprovalG3545

scholarly journals Tumor microenvironment in triple-negative breast cancer: the correlation of tumor-associated macrophages and tumor-infiltrating lymphocytes

2021 ◽  
Author(s):  
H. Kuroda ◽  
T. Jamiyan ◽  
R. Yamaguchi ◽  
A. Kakumoto ◽  
A. Abe ◽  
...  
Keyword(s):  
Breast Cancer ◽  
T Cells ◽  
Tumor Microenvironment ◽  
Triple Negative Breast Cancer ◽  
Triple Negative ◽  
Cytotoxic T Cells ◽  
Tumor Infiltrating Lymphocytes ◽  
Tumor Associated Macrophages ◽  
Free Survival ◽  
Infiltrating Lymphocytes

Abstract Purpose Immune cells such as cytotoxic T cells, helper T cells, B cells or tumor-associated macrophages (TAMs) contribute to the anti-tumor response or pro-tumorigenic effect in triple negative breast cancer (TNBC). The interrelation of TAMs, T and B tumor-infiltrating lymphocytes (TILs) in TNBC has not been fully elucidated. Methods We evaluated the association of tumor-associated macrophages, T and B TILs in TNBC. Results TNBCs with a high CD68+, CD163+ TAMs and low CD4+, CD8+, CD20+ TILs had a significantly shorter relapse-free survival (RFS) and overall survival (OS) than those with low CD68+, CD163+ TAMs and high CD4+, CD8+, CD20+ TILs. TNBCs with high CD68+ TAMs/low CD8+ TILs showed a significantly shorter RFS and OS and a significantly poorer prognosis than those with high CD68+ TAMs/high CD8+ TILs, low CD68+ TAMs/high CD8+ TILs, and low CD68+/low CD8+. TNBCs with high CD163+ TAMs/low CD8+, low CD20 + TILs showed a significantly shorter RFS and OS and a significantly poorer prognosis than those with high CD163+ TAMs/high CD8+ TILs and high CD163+ TAMs /high CD20+ TILs. Conclusions Our study suggests that TAMs further create an optimal tumor microenvironment (TME) for growth and invasion of cancer cells when evasion of immunoreactions due to T and B TILs occurs. In TNBCs, all these events combine to affect prognosis. The process of TME is highly complex in TNBCs and for an improved understanding, larger validation studies are necessary to confirm these findings.

Detection of intracellular expression and secretion of interferon-γ at the single-cell level after activation of human T cells with tetanus toxoidin vitro

1990 ◽  
Vol 20 (5) ◽  
pp. 1085-1089 ◽  
Author(s):  
Lalitha Kabilan ◽  
Gudrun Andersson ◽  
Francesco Lolli ◽  
Hans-peter Ekre ◽  
Tomas Olsson ◽  
...  
Keyword(s):  
T Cells ◽  
Single Cell ◽  
Interferon Γ ◽  
Single Cell Level ◽  
Cell Level ◽  
Human T Cells ◽  
Intracellular Expression

scholarly journals Tumor infiltrating lymphocytes in seminoma lesions comprise clonally expanded cytotoxic T cells

2006 ◽  
Vol 119 (4) ◽  
pp. 831-838 ◽  
Author(s):  
Sine Reker Hadrup ◽  
Otto Brændstrup ◽  
Grete Krag Jacobsen ◽  
Svend Mortensen ◽  
Lars Østergaard Pedersen ◽  
...  
Keyword(s):  
T Cells ◽  
Cytotoxic T Cells ◽  
Tumor Infiltrating Lymphocytes ◽  
Infiltrating Lymphocytes

scholarly journals Nomograms to Predict the Density of Tumor-Infiltrating Lymphocytes in Patients With High-Grade Serous Ovarian Cancer

2021 ◽  
Vol 11 ◽  
Author(s):  
Danian Dai ◽  
Lili Liu ◽  
He Huang ◽  
Shangqiu Chen ◽  
Bo Chen ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
T Cells ◽  
Menopausal Status ◽  
Tumor Infiltrating Lymphocytes ◽  
Roc Curves ◽  
Clinicopathological Characteristics ◽  
High Grade ◽  
Serous Ovarian Cancer ◽  
Ki 67 ◽  
Infiltrating Lymphocytes

BackgroundTumor-infiltrating lymphocytes (TILs) have important roles in predicting tumor therapeutic responses and progression, however, the method of evaluating TILs is complicated. We attempted to explore the association of TILs with clinicopathological characteristics and blood indicators, and to develop nomograms to predict the density of TILs in patients with high-grade serous ovarian cancer (HGSOC).MethodsThe clinical profiles of 197 consecutive postoperative HGSOC patients were retrospectively analyzed. Tumor tissues and matched normal fallopian tubes were immunostained for CD3+, CD8+, and CD4+ T cells on corresponding tissue microarrays and the numbers of TILs were counted using the NIH ImageJ software. The patients were classified into low- or high-density groups for each marker (CD3, CD4, CD8). The associations of the investigated TILs to clinicopathological characteristics and blood indicators were assessed and the related predictors for densities of TILs were used to develop nomograms; which were then further evaluated using the C-index, receiver operating characteristic (ROC) curves and calibration plots.ResultsMenopausal status, estrogen receptor (ER), Ki-67 index, white blood cell (WBC), platelets (PLT), lactate dehydrogenase (LDH), and carbohydrate antigen 153 (CA153) had significant association with densities of tumor-infiltrating CD3+, CD8+, or CD4+ T cells. The calibration curves of the CD3+ (C-index = 0.748), CD8+ (C-index = 0.683) and CD4+ TILs nomogram (C-index = 0.759) demonstrated excellent agreement between predictions and actual observations. ROC curves of internal validation indicated good discrimination for the CD8+ TILs nomogram [area under the curve (AUC) = 0.659, 95% CI 0.582–0.736] and encouraging performance for the CD3+ (AUC= 0.708, 95% CI 0.636–0.781) and CD4+ TILs nomogram (AUC = 0.730, 95% CI 0.659–0.801).ConclusionMenopausal status, ER, Ki-67 index, WBC, PLT, LDH, and CA153 could reflect the densities of T cells in the tumor microenvironment. Novel nomograms are conducive to monitor the immune status of patients with HGSOC and help doctors to formulate the appropriate treatment strategies.

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