scholarly journals Erratum: Corrigendum: High-throughput epitope discovery reveals frequent recognition of neo-antigens by CD4+ T cells in human melanoma

2016 ◽  
Vol 22 (10) ◽  
pp. 1192-1192 ◽  
Author(s):  
Carsten Linnemann ◽  
Marit M van Buuren ◽  
Laura Bies ◽  
Els M E Verdegaal ◽  
Remko Schotte ◽  
...  
Keyword(s):  
T Cells ◽  
High Throughput ◽  
Cd4 T Cells ◽  
Human Melanoma ◽  
Epitope Discovery

High-throughput epitope discovery reveals frequent recognition of neo-antigens by CD4+ T cells in human melanoma

2014 ◽  
Vol 21 (1) ◽  
pp. 81-85 ◽  
Author(s):  
Carsten Linnemann ◽  
Marit M van Buuren ◽  
Laura Bies ◽  
Els M E Verdegaal ◽  
Remko Schotte ◽  
...  
Keyword(s):  
T Cells ◽  
High Throughput ◽  
Cd4 T Cells ◽  
Human Melanoma ◽  
Epitope Discovery

Functional Characterization of CD4+ T-Cells in Aplastic Anemia (AA)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1340-1340 ◽  
Author(s):  
Shahram Y Kordasti ◽  
Judith C. W. Marsh ◽  
Sufyan Al-Khan ◽  
Jie Jiang ◽  
Alexander E Smith ◽  
...  
Keyword(s):  
T Cells ◽  
Treatment Group ◽  
High Throughput ◽  
Cd4 T Cells ◽  
Th17 Cells ◽  
Absolute Number ◽  
Th2 Cells ◽  
Th1 Cells ◽  
Ifn Γ ◽  
Pre Treatment

Abstract Abstract 1340 We have examined the role of CD4+ T-cells in the pathogenesis of AA in 63 patients, 48 of whom were analyzed at diagnosis and 15 following immunosuppressive therapy (IST). Absolute numbers of CD4+ regulatory T cells (Tregs, defined as CD3+CD4+CD25highCD27+Foxp3+) were lower in pre-treatment AA patients compared to 10 healthy donors (HDs) (5.5 × 106 v 1.4 × 107)(p=0.01). In patients with severe (SAA) and very severe AA (VSAA), the absolute number and frequency of Tregs were lower than non-severe AA (NSAA) (4.4 × 106/L v 1 × 107/L)(p=0.01) and HDs (4.4 × 106/L v 3 × 107/L) (p<0.001). Absolute numbers of Th1 and Th2 cells in all pre-treatment patients were higher compared to HDs (6.4 × 107/L v 1.8 × 107/L)(p=0.03) for Th1 and (2.6 × 107/L v 2.4 × 106/L)(p=0.006) Th2 cells. Although mean percentages of AA Th17 cells were higher than in HDs (1.5% v 0.15%)(p=0.04), differences in absolute numbers were not significant. Absolute numbers of Th2 and Th17 cells were increased in SAA (1.3 × 107/L v 7.4 × 106/L for Th2)(p=0.01) compared to NSAA (5.7 × 106/L v 2.15 × 106/L for Th17)(p=0.02). Ratios of Th1/Tregs (p=0.003), Th2/Tregs (p=0.02), and Th17/Tregs (p=0.001) were higher in SAA and VSAA compared to NSAA. Percentage of both activated (CD4+CD45RA−CD25highFoxp3high) and resting (CD4+CD45RA+ CD25highFoxp3low) Tregs was decreased in AA patients, compared to HDs (p=0.004 and p=0.01), whereas cytokine secreting Tregs (CD4+CD45RA−CD25high Foxp3low) were increased in AA (p<0.003). Sorted Tregs from AA patients did not suppress cytokine secretion by autologous or HD T effectors (Te) cells in 1:1 co-cultures, whereas IL-2 and IFN-γ secretion by AA Te (CD4+CD25lowCD127high) was suppressible by allogeneic Tregs from HDs, confirming Tregs dysfunction. AA Tregs did not inhibit either CD154 or CD69 expression on Te cells. Tregs from AA patients secreted significantly more IFN-γ, TNF-α and IL-17 (p=0.02, p=0.02 and p=0.01, respectively) after 4 hours stimulation with PMA/Ionomycine compared to HDs. Expression levels of FoxP3, ROR□c and T-bet in AA Tregs was normal. IFN-γ secreting cells (Th1) were enriched using enrichment kit then further enriched by FACS sorting. CDR3 region products of TCR Vβ-chain were amplified using Vβ specific forward and Cβ reverse primers. CDR3 PCR products from AA patients and HDs were subjected 454 sequencing (Roche GS FLX titanium). Sequencing was performed to yield an average ‘depth’ in excess of 1000 clonally reads (1000x) for each sample specific CDR3 PCR amp icon. Reads were processed using Roche Amp icon Variant Analyzer software (AVA). Diversity of TCR receptors (measured by spectratyping and confirmed by high throughput deep sequencing) in AA Th1 cells was lower than HDs (p=0.037), as shown by the percentage and number of consensus clusters in total sequence reads. Interestingly, percentages of the most dominant CDR3 clones, revealed by high throughput sequencing, were higher in AA compared to HDs, regardless of spectratyping pattern. Global gene expression of Tregs was compared in 3 pre-IST AA patients and 5 HDs. A unique gene signature consisting of 86 genes that were significant was identified. There were 8 down regulated genes (fold change) in the pre-treatment group; PIN4 (−4.1), OR2T12 (−3.3), AMAC1 (−2.73), PERP (−2.69), UTS2 (−2.27), RNF139 (−2.13), COMMD9 (−2.09) and LOC100128356 (−2.01). The top 10 of 78 up-regulated genes in the pre-treatment group were HBB (19.5), PSME2 (13.8), CSDA (13.07), FAM127A (7.78), EXOSC1 (7.73), BPGM (7.43), CYSLTR1 (7.17), CHPT1 (6.96) and PLAC8 (6.71). qPCR analysis for CSDA, HBB, PSMiE2, PERP, PIN4, and UTS2 confirmed a similar trend to the microarray results. Interestingly absolute number of Tregs, and Th2/Treg ratio were higher in 10 IST responsive patients compared to 5 non-responsive patients (p=0.005 and 0.02, respectively). We show that expansion of Th1, Th2, Th17, and decreased/skewed Tregs immunophenotype and function are a consistent and defining feature of SAA and VSAA. Clonal expansion of Th1 cells is likely to be antigen driven and the presence of dysfunctional Tregs aggravates this autoimmune response. Increases of Tregs, and Th2/Treg ratios following IST predicts a favourable response to this treatment. Disclosures: No relevant conflicts of interest to declare.

scholarly journals High-Throughput Sequencing of Islet-Infiltrating Memory CD4+ T Cells Reveals a Similar Pattern of TCR Vβ Usage in Prediabetic and Diabetic NOD Mice

PLoS ONE ◽  
2013 ◽  
Vol 8 (10) ◽  
pp. e76546 ◽  
Author(s):  
Idania Marrero ◽  
David E. Hamm ◽  
Joanna D. Davies
Keyword(s):  
T Cells ◽  
High Throughput ◽  
Cd4 T Cells ◽  
Similar Pattern ◽  
High Throughput Sequencing ◽  
Nod Mice ◽  
Memory Cd4 T Cells

scholarly journals Comprehensive Analysis of the Immune Infiltrates and Aberrant Pathways Activation in Atherosclerotic Plaque

2021 ◽  
Vol 7 ◽  
Author(s):  
Hukui Han ◽  
Rongsheng Du ◽  
Panke Cheng ◽  
Jiancheng Zhang ◽  
Yang Chen ◽  
...  
Keyword(s):  
T Cells ◽  
High Throughput ◽  
Cd4 T Cells ◽  
High Throughput Sequencing ◽  
Close Association ◽  
Γδ T Cells ◽  
Cerebrovascular Diseases ◽  
Cytolytic Activity ◽  
Immune Microenvironment ◽  
Sequencing Technologies

Atherosclerosis is the pathological basis of many cardiovascular and cerebrovascular diseases. The development of gene chip and high-throughput sequencing technologies revealed that the immune microenvironment of coronary artery disease (CAD) in high-risk populations played an important role in the formation and development of atherosclerotic plaques. Three gene expression datasets related to CAD were assessed using high-throughput profiling. CIBERSORT analysis revealed significant differences in five types of immune cells: activated dendritic cells (DCs), T follicular helper cells (Tfhs), resting CD4+ T cells, regulatory T cells (Tregs), and γδ T cells. Immune transcriptome analysis indicated higher levels of inflammatory markers (cytolytic activity, antigen presentation, chemokines, and cytokines) in the cases than in the controls. The level of activated DCs and the lipid clearance signaling score were negatively correlated. We observed a positive correlation between the fraction of Tfhs and lipid biosynthesis. Resting CD4+ T cells and the activity of pathways related to ossification in bone remodeling and glutathione synthesis showed a negative correlation. Gamma delta T cells negatively correlated with IL-23 signaling activity. GSEA revealed a close association with the inflammatory immune microenvironment. The present study revealed that CAD patients may have an inflammatory immune microenvironment and provides a timely update on anti-inflammatory therapies under current investigation.

scholarly journals 661 Neoantigen-specific CD4+ T cells in human melanoma have diverse differentiation states and correlate with CD8+ T cell, macrophage, and B cell function

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A689-A689
Author(s):  
Naina Singhi ◽  
Carolyn Shasha ◽  
Sylvia Lee ◽  
Julia Szeto ◽  
Ata Moshiri ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
Cd8 T Cells ◽  
Immune Cells ◽  
Tumor Antigen ◽  
Cd4 T Cells ◽  
Viral Antigen ◽  
Tumor Antigens ◽  
Human Melanoma

BackgroundTumor-antigen specific CD4+ T cells are crucial for the efficacy of antibodies that block immune checkpoint proteins in mouse tumor models, but their activities in human tumor immunity are less clear. CD8+ T cells infiltrating human tumors, including those specific for tumor antigens, have been studied using single cell profiling techniques and exist in a variety of dysfunctional states. The transcriptional states of tumor-specific CD4+ T cells present in tumors and their potential contributions to the tumor microenvironment are less well understood.MethodsWe used targeted single cell RNA sequencing and matching of T cell receptor (TCR) sequences to identify phenotypic signatures that discriminated tumor antigen- and viral antigen-specific CD4+ T cells infiltrating human melanoma tumors in four patients. The presence of CD4+ T cells with these signatures was correlated with the number and phenotype of other immune cells in the tumor microenvironment in an extended cohort of 20 patients.ResultsWe identified 259 CD4+ T cells representing 40 different TCR clonotypes specific for 13 neoantigens and 108 cells representing 14 TCR clonotypes specific for self-antigens in four melanoma patients. High expression of CXCL13 defined conventional CD4+ T cells that recognize tumor associated neoantigens and self-antigens from bystander and viral antigen-specific CD4+ T cells. Tumor-reactive CD4+ T cells could be subdivided into clusters expressing memory and T follicular helper markers, and those expressing cytolytic markers and IFN-g. In an extended cohort of 20 patients with melanoma, the frequency of CXCL13+ CD4+ T cells in the tumor microenvironment correlated with the presence and proliferation of CD8+ T cells, the presence and maturation of B cells, the activation of interferon responsive genes in tumor associated macrophages, and patient survival. CD4+ T cells with similar transcriptional signatures were identified in data sets from breast and non-small cell lung cancer, suggesting these markers may enrich for tumor-reactive CD4+ T cells in many cancers.ConclusionsThese results identify a subset of tumor infiltrating conventional CD4+ T cells in melanoma that are enriched for reactivity to tumor antigens and exist in multiple phenotypic states. Correlations of the presence of these cells with the frequency and phenotype of other immune cells suggest roles for these tumor antigen-specific CD4+ T cells in providing CD8+ T cell help, driving recruitment and maturation of B cells, and activating macrophages. Isolating such cells based on their unique phenotype and utilizing them for adoptive therapy could alter the tumor microenvironment for therapeutic benefit.Ethics ApprovalAll Patient samples in this study were obtained from patients who signed informed consent in a study approved by the institutional review board of the Fred Hutchinson Cancer Research Center (protocol #2643).

Abstract 193: HDL-Small RNA Intercellular Communication in Systemic Lupus Erythematosus

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Danielle L Michell ◽  
Jared L Moore ◽  
Michelle J Ormseth ◽  
Stuart R Landstreet ◽  
Shilin Zhao ◽  
...  
Keyword(s):  
Systemic Lupus Erythematosus ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
High Throughput ◽  
Intercellular Communication ◽  
Lupus Erythematosus ◽  
Cd4 T Cells ◽  
High Density Lipoproteins ◽  
Systemic Lupus

Extracellular small non-coding RNAs (sRNAs) are a new class of disease biomarkers and are transferred between cells by high-density lipoproteins (HDL) in a novel form of intercellular communication. In chronic inflammatory states and auto-immunity, HDL can become dysfunctional, likely through alterations in its diverse cargo, including changes to sRNA signatures. We have previously found that HDL-microRNAs (miRNA) are altered in systemic lupus erythematosus (SLE); however, miRNAs are just one type of sRNAs. As such we hypothesized that changes to HDL-sRNA cargo and cell-to-cell communication in SLE extend beyond miRNAs. To test this hypothesis, HDL was isolated from SLE and control (n=6-8) subjects by density-gradient ultracentrifugation followed by size-exclusion chromatography. High-throughput sRNA sequencing of HDL demonstrated that tRNA-derived sRNAs (tDRs) were the most abundant class of sRNAs on HDL and were significantly altered in SLE subjects compared to controls (26 up, 10 down). In addition, circulating levels of angiogenin, an RNaseIII enzyme capable of cleaving parent tRNAs into tDRs, was also significantly ( P <0.05) increased in SLE plasma. To determine if tDRs are altered in CD4+ T cells in SLE subjects, real-time PCR was used to quantify candidate tDRs, and we found that tDR-GlyGCC levels were significantly increased 4.2-fold in SLE ( P <0.01). Most importantly, we found that T cells exported tDR-GlyGCC to HDL. To determine if T cell exported tDR-GlyGCC is transferred to other cells by HDL, ex vivo studies were completed using Trans-PhotoActivatable-Ribonucleoside-CrossLinking-ImmunoPrecipitation high-throughput Sequencing (Trans-PAR-CLIPseq) to trace sRNAs from human CD4+ T cells to HDL and then to recipient CD14+ monocytes and CD19+ B-cells. Using this approach, we found a cassette of T cell originating sRNAs, including tDR-GlyGCC, transferred by HDL to recipient B cells and monocytes and loaded onto RNA-Induced Silencing Complexes (RISC) targeting genes associated with inflammation. Here, we demonstrate that HDL facilitates the intercellular transfer of sRNAs between immune cells and these sRNAs are altered in SLE. This altered communication may contribute to T cell imbalance and B cell activation observed in SLE.

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