TCR sequencing paired with massively parallel 3′ RNA-seq reveals clonotypic T cell signatures

Tu, Ang A.; Gierahn, Todd M.; Monian, Brinda; Morgan, Duncan M.; Mehta, Naveen K.; Ruiter, Bert; Shreffler, Wayne G.; Shalek, Alex K.; Love, J. Christopher

doi:10.1038/s41590-019-0544-5

Cited by 102 publications

(89 citation statements)

References 57 publications

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“…Commercial reverse-emulsion droplet-based methods, like 10x, overcome these inefficiencies and provide streamlined workflows, but add substantial cost, both with respect to consumables and instrumentation, and constrain where and how samples can be run. Although the state-of-the-art continues to evolve rapidly, Seq-Well S 3 provides a competitive alternative that is uniquely suited for clinical studies because of its efficiency, simplicity, compatibility with fragile cells, limited peripherals, flexible stopping points (post-reverse transcription), ability to be parallelized (up to 20 samples in a one-day experiment), high degree of technical reproducibility, and open molecular biology (which enables targeted enrichment of molecules of interest) ( Tu et al., 2019 ; van Galen et al., 2019 ).…”

Section: Discussionmentioning

confidence: 99%

Second-Strand Synthesis-Based Massively Parallel scRNA-Seq Reveals Cellular States and Molecular Features of Human Inflammatory Skin Pathologies

et al. 2020

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Summary High-throughput single-cell RNA-sequencing (scRNA-seq) methodologies enable characterization of complex biological samples by increasing the number of cells that can be profiled contemporaneously. Nevertheless, these approaches recover less information per cell than low-throughput strategies. To accurately report the expression of key phenotypic features of cells, scRNA-seq platforms are needed that are both high fidelity and high throughput. To address this need, we created Seq-Well S 3 (“Second-Strand Synthesis”), a massively parallel scRNA-seq protocol that uses a randomly primed second-strand synthesis to recover complementary DNA (cDNA) molecules that were successfully reverse transcribed but to which a second oligonucleotide handle, necessary for subsequent whole transcriptome amplification, was not appended due to inefficient template switching. Seq-Well S 3 increased the efficiency of transcript capture and gene detection compared with that of previous iterations by up to 10- and 5-fold, respectively. We used Seq-Well S 3 to chart the transcriptional landscape of five human inflammatory skin diseases, thus providing a resource for the further study of human skin inflammation.

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Section: Discussionmentioning

confidence: 99%

Second-Strand Synthesis-Based Massively Parallel scRNA-Seq Reveals Cellular States and Molecular Features of Human Inflammatory Skin Pathologies

et al. 2020

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“…This analysis platform enables the assessment of gene expression in each individual cell which is superior to bulk analysis that often overlooks shifts in transcriptomic patterns by averaging the mRNA expression data [150]. In the field of allergy, scRNA-seq has already propelled knowledge of B cell class switching [151], B cell memory [46•], airway T cell metabolism [152], transcriptional differences between asthmatics with and without allergy [153], IgE responses [154], T cell clonotypes [155], and functional heterogeneity within allergen-specific T cell subset [156].…”

Section: Addressing the Unmet Needs And The Current Challenges In Biomentioning

confidence: 99%

Mechanisms of Allergen Immunotherapy in Allergic Rhinitis

Drazdauskaité

Layhadi

Shamji

2020

Curr Allergy Asthma Rep

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Purpose of Review Allergic rhinitis (AR) is a chronic inflammatory immunoglobulin (Ig) E-mediated disease of the nasal mucosa that can be triggered by the inhalation of seasonal or perennial allergens. Typical symptoms include sneezing, rhinorrhea, nasal itching, nasal congestion and symptoms of allergic conjunctivitis. AR affects a quarter of the population in the United States of America and Europe. Recent Findings AR has been shown to reduce work productivity in 36–59% of the patients with 20% reporting deteriorated job attendance. Moreover, 42% of children with AR report reduced at-school productivity and lower grades. Most importantly, AR impacts the patient’s quality of life, due to sleep deprivation. However, a proportion of patients fails to respond to conventional medication and opts for the allergen immunotherapy (AIT), which currently is the only disease-modifying therapeutic option. AIT can be administered by either subcutaneous (SCIT) or sublingual (SLIT) route. Both routes of administration are safe, effective, and can lead to tolerance lasting years after treatment cessation. Both innate and adaptive immune responses that contribute to allergic inflammation are suppressed by AIT. Innate responses are ameliorated by reducing local mast cell, basophil, eosinophil, and circulating group 2 innate lymphoid cell frequencies which is accompanied by decreased basophil sensitivity. Induction of allergen-specific blocking antibodies, immunosuppressive cytokines, and regulatory T and B cell phenotypes are key pro-tolerogenic adaptive immune responses. Conclusion A comprehensive understanding of these mechanisms is necessary for optimal selection of AIT-responsive patients and monitoring treatment efficacy. Moreover, it could inspire novel and more efficient AIT approaches.

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“…Given the strong association between the abundance of specific T cell phenotypes and control of Mtb, we wondered whether these T cells might target common antigens (i.e., share common T cell receptors; TCRs). To investigate whether there was clonal enrichment among T cells, we reconstructed CDR3 sequences from granuloma T cells by performing targeted pulldowns of αβ TCR sequences from the granuloma whole transcriptome amplification libraries to generate secondary sequencing libraries (STAR* Methods) (Tu et al, 2019). Initially, we examined the extent of TCR-α and TCR-β recovery and enrichment (i.e.…”

Section: Tcr Repertoires Of Tb Lung Granulomasmentioning

confidence: 99%

Multimodal profiling of lung granulomas reveals cellular correlates of tuberculosis control

Gideon

Behar

Flynn

et al. 2020

Preprint

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In humans and nonhuman primates, Mycobacterium tuberculosis lung infection yields a complex multicellular structure: the tuberculosis granuloma. All granulomas are not equivalent, however, even within the same host: in some, local immune activity promotes bacterial clearance, while in others, it allows persistence or outgrowth. Here, we used single-cell RNA-sequencing to define holistically cellular responses associated with control in cynomolgus macaques. Granulomas that facilitated bacterial killing contained significantly higher proportions of CD4+ and CD8+ T cells expressing hybrid Type1-Type17 immune responses or stem-like features and CD8-enriched T cells with specific cytotoxic functions; failure to control correlated with mast cell, plasma cell and fibroblast abundance. Co-registering these data with serial PET-CT imaging suggests that a degree of early immune control can be achieved through cytotoxic activity, but that more robust restriction only arises after the priming of specific adaptive immune responses, defining new targets for vaccination and treatment.

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TCR sequencing paired with massively parallel 3′ RNA-seq reveals clonotypic T cell signatures

Cited by 102 publications

References 57 publications

Second-Strand Synthesis-Based Massively Parallel scRNA-Seq Reveals Cellular States and Molecular Features of Human Inflammatory Skin Pathologies

Second-Strand Synthesis-Based Massively Parallel scRNA-Seq Reveals Cellular States and Molecular Features of Human Inflammatory Skin Pathologies

Mechanisms of Allergen Immunotherapy in Allergic Rhinitis

Multimodal profiling of lung granulomas reveals cellular correlates of tuberculosis control

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