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Profiling tissue-resident T cell repertoires by RNA sequencing.

Brown SD, Raeburn LA, Holt RA - Genome Med (2015)

Bottom Line: Deep sequencing of recombined T cell receptor (TCR) genes and transcripts has provided a view of T cell repertoire diversity at an unprecedented resolution.Beyond profiling peripheral blood, analysis of tissue-resident T cells provides further insight into immune-related diseases.We describe the extraction of TCR sequence information directly from RNA-sequencing data from 6738 tumor and 604 control tissues, with a typical yield of 1 TCR per 10 million reads.

View Article: PubMed Central - PubMed

Affiliation: Canada's Michael Smith Genome Sciences Centre, BC Cancer Agency, Vancouver, British Columbia, V5Z 1L3, Canada.

ABSTRACT
Deep sequencing of recombined T cell receptor (TCR) genes and transcripts has provided a view of T cell repertoire diversity at an unprecedented resolution. Beyond profiling peripheral blood, analysis of tissue-resident T cells provides further insight into immune-related diseases. We describe the extraction of TCR sequence information directly from RNA-sequencing data from 6738 tumor and 604 control tissues, with a typical yield of 1 TCR per 10 million reads. This method circumvents the need for PCR amplification of the TCR template and provides TCR information in the context of global gene expression, allowing integrated analysis of extensive RNA-sequencing data resources.

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The number of reads containing CDR3 sequences varies across tumor sites. ACC adrenocortical carcinoma, BLCA bladder urothelial carcinoma, BRCA breast invasive carcinoma, CESC cervical squamous cell carcinoma and endocervical adenocarcinoma, CRAD colon and rectum adenocarcinoma, ESCA esophageal carcinoma, GBM glioblastoma multiforme, HNSC head and neck squamous cell carcinoma, KICH kidney chromophobe, KIRC kidney renal clear cell carcinoma, KIRP kidney renal papillary cell carcinoma, LGG brain lower grade glioma, LIHC liver hepatocellular carcinoma, LUAD lung adenocarcinoma, LUSC lung squamous cell carcinoma, OV ovarian serous cystadenocarcinoma, PAAD pancreatic adenocarcinoma, PCPG pheochromocytoma and paraganglioma, PRAD prostate adenocarcinoma, SKCM skin cutaneous melanoma, STAD stomach adenocarcinoma, TCR T cell receptor, THCA thyroid carcinoma, UCEC uterine corpus endometrial carcinoma, UCS uterine carcinosarcoma
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Fig2: The number of reads containing CDR3 sequences varies across tumor sites. ACC adrenocortical carcinoma, BLCA bladder urothelial carcinoma, BRCA breast invasive carcinoma, CESC cervical squamous cell carcinoma and endocervical adenocarcinoma, CRAD colon and rectum adenocarcinoma, ESCA esophageal carcinoma, GBM glioblastoma multiforme, HNSC head and neck squamous cell carcinoma, KICH kidney chromophobe, KIRC kidney renal clear cell carcinoma, KIRP kidney renal papillary cell carcinoma, LGG brain lower grade glioma, LIHC liver hepatocellular carcinoma, LUAD lung adenocarcinoma, LUSC lung squamous cell carcinoma, OV ovarian serous cystadenocarcinoma, PAAD pancreatic adenocarcinoma, PCPG pheochromocytoma and paraganglioma, PRAD prostate adenocarcinoma, SKCM skin cutaneous melanoma, STAD stomach adenocarcinoma, TCR T cell receptor, THCA thyroid carcinoma, UCEC uterine corpus endometrial carcinoma, UCS uterine carcinosarcoma

Mentions: We extracted TCR alpha and beta chain CDR3 sequences from all available RNA-seq datasets from the TCGA project. This included 7342 total datasets derived from 6738 solid tumor and 604 matched normal tissues, from 24 different tumor sites. In tumors, the yield per individual ranged from 0 to 702 (median of 9) reads containing a full CDR3 sequence (Fig. 2), and this translated to a range of 0 to 538 (median of 7) distinct CDR3 amino acid sequences per individual. Kidney renal clear cell carcinoma (KIRC) produced the greatest yield of CDR3s, whereas brain lower grade glioma produced the lowest. As expected, there was a strong correlation between number of distinct CDR3 amino acid sequences and CD3 expression (Additional file 1: Figure S4). Comparing the gene expression of HLA Class I and Class II genes with the number of distinct CDR3 amino acid sequences per individual, we observed a positive correlation, with markedly stronger correlations seen for Class II genes (Additional file 1: Figure S5; P = 9.3 × 10−10, paired t test). This is consistent with recent reports highlighting the immunoreactivity of T cells with specificity for MHC Class II presented tumor antigens [28–30]. Next, we evaluated the differential abundance of CDR3s between tumors and matched normal control tissues for all individuals where the RNA-seq data were available for both (n = 462; Fig. 3 and Additional file 1: Table S5). Of 6611 total alpha chains in this set, 3560 (53.8 %) were unique to tumor samples and 2826 (42.7 %) were unique to matched control samples. Likewise, of the 7664 beta chains, 4279 (55.8 %) were unique to tumor and 3277 (42.8 %) were unique to matched control samples. A total of 225 unique CDR3α and 108 unique CDR3β sequences were present in both tumor and control tissues. Thus, while there is evidence for a larger and more diverse T cell infiltrate in tumor compared to control tissues (P < 2.2 × 10−16, chi-squared test), these results suggest that a large proportion of tumor-associated T cells are bystanders, not readily distinguishable from the normal population of tissue resident T cells. A single individual with KIRC was a notable outlier in this analysis. The tumor sample from this individual yielded the three most abundant tumor-specific CDR3αs and the two most abundant tumor-specific CDR3βs in the entire cohort, suggesting the possibility of an acute anti-tumor T cell response in this individual.Fig. 2


Profiling tissue-resident T cell repertoires by RNA sequencing.

Brown SD, Raeburn LA, Holt RA - Genome Med (2015)

The number of reads containing CDR3 sequences varies across tumor sites. ACC adrenocortical carcinoma, BLCA bladder urothelial carcinoma, BRCA breast invasive carcinoma, CESC cervical squamous cell carcinoma and endocervical adenocarcinoma, CRAD colon and rectum adenocarcinoma, ESCA esophageal carcinoma, GBM glioblastoma multiforme, HNSC head and neck squamous cell carcinoma, KICH kidney chromophobe, KIRC kidney renal clear cell carcinoma, KIRP kidney renal papillary cell carcinoma, LGG brain lower grade glioma, LIHC liver hepatocellular carcinoma, LUAD lung adenocarcinoma, LUSC lung squamous cell carcinoma, OV ovarian serous cystadenocarcinoma, PAAD pancreatic adenocarcinoma, PCPG pheochromocytoma and paraganglioma, PRAD prostate adenocarcinoma, SKCM skin cutaneous melanoma, STAD stomach adenocarcinoma, TCR T cell receptor, THCA thyroid carcinoma, UCEC uterine corpus endometrial carcinoma, UCS uterine carcinosarcoma
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4666197&req=5

Fig2: The number of reads containing CDR3 sequences varies across tumor sites. ACC adrenocortical carcinoma, BLCA bladder urothelial carcinoma, BRCA breast invasive carcinoma, CESC cervical squamous cell carcinoma and endocervical adenocarcinoma, CRAD colon and rectum adenocarcinoma, ESCA esophageal carcinoma, GBM glioblastoma multiforme, HNSC head and neck squamous cell carcinoma, KICH kidney chromophobe, KIRC kidney renal clear cell carcinoma, KIRP kidney renal papillary cell carcinoma, LGG brain lower grade glioma, LIHC liver hepatocellular carcinoma, LUAD lung adenocarcinoma, LUSC lung squamous cell carcinoma, OV ovarian serous cystadenocarcinoma, PAAD pancreatic adenocarcinoma, PCPG pheochromocytoma and paraganglioma, PRAD prostate adenocarcinoma, SKCM skin cutaneous melanoma, STAD stomach adenocarcinoma, TCR T cell receptor, THCA thyroid carcinoma, UCEC uterine corpus endometrial carcinoma, UCS uterine carcinosarcoma
Mentions: We extracted TCR alpha and beta chain CDR3 sequences from all available RNA-seq datasets from the TCGA project. This included 7342 total datasets derived from 6738 solid tumor and 604 matched normal tissues, from 24 different tumor sites. In tumors, the yield per individual ranged from 0 to 702 (median of 9) reads containing a full CDR3 sequence (Fig. 2), and this translated to a range of 0 to 538 (median of 7) distinct CDR3 amino acid sequences per individual. Kidney renal clear cell carcinoma (KIRC) produced the greatest yield of CDR3s, whereas brain lower grade glioma produced the lowest. As expected, there was a strong correlation between number of distinct CDR3 amino acid sequences and CD3 expression (Additional file 1: Figure S4). Comparing the gene expression of HLA Class I and Class II genes with the number of distinct CDR3 amino acid sequences per individual, we observed a positive correlation, with markedly stronger correlations seen for Class II genes (Additional file 1: Figure S5; P = 9.3 × 10−10, paired t test). This is consistent with recent reports highlighting the immunoreactivity of T cells with specificity for MHC Class II presented tumor antigens [28–30]. Next, we evaluated the differential abundance of CDR3s between tumors and matched normal control tissues for all individuals where the RNA-seq data were available for both (n = 462; Fig. 3 and Additional file 1: Table S5). Of 6611 total alpha chains in this set, 3560 (53.8 %) were unique to tumor samples and 2826 (42.7 %) were unique to matched control samples. Likewise, of the 7664 beta chains, 4279 (55.8 %) were unique to tumor and 3277 (42.8 %) were unique to matched control samples. A total of 225 unique CDR3α and 108 unique CDR3β sequences were present in both tumor and control tissues. Thus, while there is evidence for a larger and more diverse T cell infiltrate in tumor compared to control tissues (P < 2.2 × 10−16, chi-squared test), these results suggest that a large proportion of tumor-associated T cells are bystanders, not readily distinguishable from the normal population of tissue resident T cells. A single individual with KIRC was a notable outlier in this analysis. The tumor sample from this individual yielded the three most abundant tumor-specific CDR3αs and the two most abundant tumor-specific CDR3βs in the entire cohort, suggesting the possibility of an acute anti-tumor T cell response in this individual.Fig. 2

Bottom Line: Deep sequencing of recombined T cell receptor (TCR) genes and transcripts has provided a view of T cell repertoire diversity at an unprecedented resolution.Beyond profiling peripheral blood, analysis of tissue-resident T cells provides further insight into immune-related diseases.We describe the extraction of TCR sequence information directly from RNA-sequencing data from 6738 tumor and 604 control tissues, with a typical yield of 1 TCR per 10 million reads.

View Article: PubMed Central - PubMed

Affiliation: Canada's Michael Smith Genome Sciences Centre, BC Cancer Agency, Vancouver, British Columbia, V5Z 1L3, Canada.

ABSTRACT
Deep sequencing of recombined T cell receptor (TCR) genes and transcripts has provided a view of T cell repertoire diversity at an unprecedented resolution. Beyond profiling peripheral blood, analysis of tissue-resident T cells provides further insight into immune-related diseases. We describe the extraction of TCR sequence information directly from RNA-sequencing data from 6738 tumor and 604 control tissues, with a typical yield of 1 TCR per 10 million reads. This method circumvents the need for PCR amplification of the TCR template and provides TCR information in the context of global gene expression, allowing integrated analysis of extensive RNA-sequencing data resources.

Show MeSH
Related in: MedlinePlus