Analysis of TCR β CDR3 sequencing data for tracking anti-tumor immunity

2019 ◽  
pp. 443-464
Author(s):  
Jiajia Zhang ◽  
Zhicheng Ji ◽  
Kellie N. Smith
Keyword(s):  
Tumor Immunity ◽  
Sequencing Data ◽  
Cdr3 Sequencing

IMMU-28. DECONVOLUTION OF SPATIALLY RESOLVED T CELL RECEPTOR PROFILING (SPTCR-seq) UNCOVERED REGIONAL ANTI-TUMOR IMMUNITY IN GLIOBLASTOMA

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi98-vi98
Author(s):  
Jasim Kada Benotmane ◽  
Jan Kückelhaus ◽  
Kevin Joseph ◽  
Jürgen Beck ◽  
Oliver Schnell ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
Tumor Immunity ◽  
Clonal Evolution ◽  
Cell Receptor ◽  
Sequencing Data ◽  
Spatially Resolved ◽  
Long Read ◽  
T Cell Clonality ◽  
Cell Clonality

Abstract The diversity to T cell responses and clonality in spatially heterogeneous glioblastoma is of paramount importance to explore underlying mechanisms of anti-tumor immunity. Spatial transcriptomics, a novel technology to map the transcriptional architecture, is technically limited to discover T cell receptor (TCR) sequences as the 3' approach lacks sufficient coverage. Here, we established SPTCR-seq, a method to capture TCR sequences followed by long-read sequencing to enable full-length TCR reconstruction. We performed 10X Visium spatial transcriptomics on 9 primary and recurrent glioblastoma with both 3’-sequencing and SPTCR-seq. For SPTCR-seq, we target enriched T cell receptor sequences by capturing by hybridization followed by Oxford-Nanopore long-read sequencing. The on-target rate was above 80% for captured TCR genes and spatial barcode was successfully aligned in more than 60%. IgBlast and MixCR were used to reconstruct the TCR and map T cell clonality. Within our recent developed spatial transcriptomic analysis framework (SPATA2), we build a novel toolbox, SPATA-Immunology, which enables integration of stRNA-sequencing data and spatially resolved TCR sequencing. Our data showed that clonal evolution of T cells is limited to regional areas underpinned by significant spatial autocorrelation coefficient (0.6-0.95, padj< 0.001). In the surrounding tumor cell spots, the recently described transcriptional program “reactive immune” (RI), was significantly enriched. Using spotlight, a computational approach to project scRNA-sequencing into the spatial space, we found a local enrichment of CD163 positive macrophages exclusively in areas of large T cell clonality. Imaging mass cytometry of a consecutive section confirmed the spatial confluence of T-cell infiltration and CD163-positive macrophages. Through DeepTCR we uncovered potential epitopes which correlate with T cell clonality and might help to discover novel targets for CART therapy. Spatial profiling of TCR sequences through SPTCR-seq is a powerful tool to investigate anti-tumor immunity in glioblastoma and allows to discover general and personalized targets for immunotherapy.

scholarly journals Tissue-resident memory CD8+ T cells amplify anti-tumor immunity by triggering antigen spreading through dendritic cells

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Evelyn Menares ◽  
Felipe Gálvez-Cancino ◽  
Pablo Cáceres-Morgado ◽  
Ehsan Ghorani ◽  
Ernesto López ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
T Cell ◽  
Tumor Immunity ◽  
T Cell Responses ◽  
Gene Signature ◽  
Tumor Rejection ◽  
Sequencing Data ◽  
Memory Cd8 T Cells ◽  
Cell Responses ◽  
And Migration

Abstract Tissue-resident memory CD8+ T (Trm) cells mediate potent local innate and adaptive immune responses and play a central role against solid tumors. However, whether Trm cells cross-talk with dendritic cells (DCs) to support anti-tumor immunity remains unclear. Here we show that antigen-specific activation of skin Trm cells leads to maturation and migration to draining lymph nodes of cross-presenting dermal DCs. Tumor rejection mediated by Trm cells triggers the spread of cytotoxic CD8+ T cell responses against tumor-derived neo- and self-antigens via dermal DCs. These responses suppress the growth of intradermal tumors and disseminated melanoma lacking the Trm cell-targeted epitope. Moreover, analysis of RNA sequencing data from human melanoma tumors reveals that enrichment of a Trm cell gene signature associates with DC activation and improved survival. This work unveils the ability of Trm cells to amplify the breath of cytotoxic CD8+ T cell responses through DCs, thereby strengthening anti-tumor immunity.

Escape Mechanisms in Tumor Immunity: An Update

2002 ◽  
Vol 21 (4) ◽  
pp. 54 ◽  
Author(s):  
Ludmila Muller ◽  
Rolf Kiessling ◽  
Robert C. Rees ◽  
Graham Pawelec
Keyword(s):  
Tumor Immunity

Prime-editors (nickases), hRad51–Cas9 nickase fusions and dCas9 have the same problem as conventional CRISPR-Cas9 of plasmid/Cas9 integration after making a double stranded break

2019 ◽  
Author(s):  
Sandeep Chakraborty
Keyword(s):  
Cellular Response ◽  
Sequencing Data ◽  
Dna Breaks ◽  
Precise Integration ◽  
Guide Rna ◽  
Double Strand Dna Breaks ◽  
Human Therapy ◽  
P53 Activation ◽  
Programmable Nucleases

‘Prime-editing’ proposes to replace traditional programmable nucleases (CRISPR-Cas9) using a catalytically impaired Cas9 (dCas9) connected to a engineered reverse transcriptase, and a guide RNA encoding both the target site and the desired change. With just a ‘nick’ on one strand, it is hypothe- sized, the negative, uncontrollable effects arising from double-strand DNA breaks (DSBs) - translocations, complex proteins, integrations and p53 activation - will be eliminated. However, sequencing data pro- vided (Accid:PRJNA565979) reveal plasmid integration, indicating that DSBs occur. Also, looking at only 16 off-targets is inadequate to assert that Prime-editing is more precise. Integration of plasmid occurs in all three versions (PE1/2/3). Interestingly, dCas9 which is known to be toxic in E. coli and yeast, is shown to have residual endonuclease activity. This also affects studies that use dCas9, like base- editors and de/methylations systems. Previous work using hRad51–Cas9 nickases also show significant integration in on-targets, as well as off-target integration [1]. Thus, we show that cellular response to nicking involves DSBs, and subsequent plasmid/Cas9 integration. This is an unacceptable outcome for any in vivo application in human therapy.

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