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High throughput sequencing reveals a complex pattern of dynamic interrelationships among human T cell subsets

Proc Natl Acad Sci U S A. 2010 January 26; 107(4): 1518–1523.




Developing T cells face a series of cell fate choices in the thymus and in the periphery. The role of the individual T cell receptor (TCR) in determining decisions of cell fate remains unresolved. The stochastic/selection model postulates that the initial fate of the cell is independent of TCR specificity, with survival dependent on additional TCR/coreceptor “rescue” signals. The “instructive” model holds that cell fate is initiated by the interaction of the TCR with a cognate peptide-MHC complex. T cells are then segregated on the basis of TCR specificity with the aid of critical coreceptors and signal modulators [Chan S, Correia-Neves M, Benoist C, Mathis (1998) Immunol Rev 165: 195–207]. The former would predict a random representation of individual TCR across divergent T cell lineages whereas the latter would predict minimal overlap between divergent T cell subsets. To address this issue, we have used high-throughput sequencing to evaluate the TCR distribution among key T cell developmental and effector subsets from a single donor. We found numerous examples of individual subsets sharing identical TCR sequence, supporting a model of a stochastic process of cell fate determination coupled with dynamic patterns of clonal expansion of T cells bearing the same TCR sequence among both CD4+ and CD8+ populations.


Comprehensive and semi-quantitative TCR repertoire analysis with a novel multiplex PCR method and 454 sequencing 




CDR3 sequences, composed by V(D)J combination, form the center of the antigen binding site where they often play a critical role in defining the affinity and specificity of the receptor for individual peptide-MHC complexes of both the TCRα and TCRβ chains. The goal of our study was to produce comprehensive, unrestricted profiles of TCR diversity among key developmental and effector subsets of T cells isolated from the blood of a single, healthy donor at sequence-level resolution using a novel multiplex PCR method combined with Roche 454 Life Sciences high-throughput sequencing technology.  From the sequence reads, about 1.48 million CDR3 intervals were identified, totaling 169,977 and 113,290 unique CDR3 intervals for TCRα and for TCRβ, respectively.  Our data also show numerous examples of identical CDR3 sequences shared by different T subsets.  Using the compound Poisson process model, we estimated that the diversity of the TCRα and β chains expressed by our donor is around 0.47 x 10⁶ and 0.35 x 10⁶ unique CDR3 nucleotide sequences, respectively.  Our comprehensive data demonstrates that our new method has overcome past challenges in studying the T cell immune repertoire and is highly sensitive, repeatable, and semi-quantitative.  This approach provides a useful tool for assessing immune competence, tracking T cell expansion kinetics, assessing vaccine efficiency, and detecting antigen-specific T cell clones in patients with infection or cancer. 

Immunorepertoire Analysis by Multiplex PCR Amplification and High-throughput Sequencing




An immunorepertoire is the sum of functionally diverse B and T cells in one’s circulation at any given moment. Three key factors shape the personal immunorepertoire:  HLA type, antigen exposure history, and immunoregulation. Two major challenges have made studying the immunorepertoire difficult:  the enormous diversity of V(D)Js, and the scarcity of each V(D)J in a sample. To overcome these challenges, a powerful multiplex PCR method has been developed to amplify inclusively and unbiased, all expressed V(D)Js in a sample. The amplicon mixture was sequenced directly with Roche 454 platform. In one run, for example, more than 174,495 and 176,095 unique V(D)Js were obtained from B and T cells, respectively. High resolution HLA typing was also carried our with the same sequencing run. This novel technology can be used to uncover possible disease mechanisms associated with abnormal immuoreperotoires, to evaluate vaccine efficiency, to identify new biomarkers, and to develop new therapeutics. An online database and unique analytical tools have been developed for data sharing, deposition, and mining.

Immune Repertoire High-throughput Sequencing Analysis Web Service




The advent of the next-generation sequencing technologies enables determining antigen receptor sequences of millions of lymphocytes in a high-throughput fashion and in-depth studying the immune repertoire of a particular sample. However, the huge amount of sequencing data poses big challenges to immunologists including data storage, data analysis and data presentation. To address these challenges, we developed the IRSA web service to help researchers cross the bridge. The IRSA web service includes storing and managing sequencing data, removing sequencing artifacts, mapping reference sequences, identifying CDR3 junctions and generating various distribution plots such as domain usage, nucleotide nibbling and addition at the junction sites, CDR3 length. The backend of the IRSA web service is  several high-end computers including a computer cluster of more than 100 CPU cores, a powerful database server and a web server. Through the web portal, researchers can work on their data interactively without the need of high-end computers. Most of plots through web portal are of publication quality. Various summary data tables can be downloaded from the web pages. The web service was designed from the researchers’ point of view. Best of all, the web service is free-of-charge.

Development of an alternative method for the identification and production of antigen-specific monoclonal antibodies




A critical step in the production of monoclonal antibodies (Abs) is the initial identification of the antigen-specific Abs, which is usually performed by multiple rounds of panning in both hybridoma and phage display. We have developed an alternative method that allows for the rapid and direct identification of antigen-specific Abs from peripheral blood via high-throughput immune repertoire sequencing and LC MS/MS peptide matching. Our B-cell repertoire technology combines novel amplicon rescued multiplex PCR (arm-PCR, patent pending) with high-throughput gene sequencing to access the sequence of a broad spectrum of heavy and light chain V-regions. As a proof of concept, we have sequenced the immune repertoire of 2 healthy individuals at various time points after administration of the 2009-2010 seasonal influenza vaccine. Antigen-specific Abs were purified directly from immune peripheral blood serum and identified using LC MS/MS peptide sequencing, exploiting the B-cell repertoire gene sequencing results as a database for identification. During our study, several unique peptides were successfully matched for each individual’s response to both Flu A strains in the vaccine. Future work includes the development of a method to rapidly clone and express these identified Abs in a human in vitro glycoexpression system. The recombinant Abs will then be tested for their ability to bind flu hemagglutinin, demonstrating the utility of our technology towards the production of mAbs.


Data Analysis Pipeline For Immune Repertoire Sequencing Data




Immune repertoire diversity is a fundamental determinant of the competence of the immune system. The loss of diversity of an immune repertoire has been linked to agin and implicated in various disease states. Previous methods extrapolate the full diversity of the human immune repertoire from only a small fraction of VJ combinations, which in turn was chosen at random, thus making it difficult to determine whether the actual diversity or the extent of clonal amplification of the repertoire could have been quantified. Next generation sequencing technology, in combination with robust multiplex PCR technique, is becoming a powerful took for profiling immune repertoire diversity. However, errors accumulated during PCR and sequencing make it hard to measure diversity accurately. Here we describe a newly developed SMART filtering method, which include sequencing error filter, mosaic sequence filter, amplification error filter, reference sequence filter, and frequency threshold filter, to remove both sequencing errors and PCR errors, as demonstrated with control constructs sequencing. In addition, the large volume of sequencing data limit accessibility to this technique to regular bench-work researchers. We outline our data analysis pipeline and web interface for immune repertoire sequencing projects.

R10K:  an international collaborative project for biomarker discovery




The R10K project is an international collaborative effort to sequence the immune repertoire (T and B cells) from 10,000 samples that cover 100 diseases. We have developed a novel multiplex PCR method, which can amplify targets from a complex genetic material, inclusively and semi-quantitatively. This easy-to-use technology has been successfully applied to amplify immune repertoire libraries from peripheral blood samples or other tissue samples. The natural duty of the adaptive immune system is to detect any internal and external threats and mount specific and measured reactions accordingly. Our pilot studies have demonstrated that immune repertoire sequencing can be used to measure this specific repertoire sequencing can be used to measure this specific response and identify disease-related T cell CDR3s. Therefore, analyzing the immune repertoire of several disease states with high throughput sequencing may lead to the discovery of disease-specific biomarkers for diagnosis, prognosis, and treatment evaluations. The R10K project is organized and supported by the HudsonAlpha Institute for Biotechnology, a non-profit research institute specialized in genomic research. A Scientific Advisory Board has been established to hep identify projects from online submitted proposals. The R10K data will be made public 6 months after it has been generated.

Identification and temporal monitoring of breast cancer-associated T cell receptors with high throughput sequencing




Tumor-specific antigens may trigger an immune response that leads to T lymphocytes infiltrating the tumor tissue. We have developed a method to study the immune repertoire of a sample by utilizing a patented multiplex PCR amplification strategy, arm-PCR (Patent No. 7,999,092), coupled with high throughput sequencing (Wang et al., PNAS 2010). Using this method, we sequenced CDR3 fragments amplified from cancer tissue and normal tissue surrounding the cancer sites, which were surgically removed from a patient during operation. The patient’s sorted peripheral blood was also examined at three months, six months, and one year after surgery. Dominant T cell clones with specific CDR3 sequences were identified from the breast cancer tissue. Some of these same clones were found expressed at high levels in the nearby normal tissue and peripheral CD8+ cells. After the treatment, dynamic changes in these cancer-associated clones were apparent, demonstrating the capability of the current technology to identify specific T cells associated with the patient’s cancer tissue. These specific T cells can serve as a personalized biomarkers for prognosis, treatment evaluations, and early detection for recurrence. They can also be used to develop personalized treatment strategies. Currently, this study has been extended to examine additional breast cancer patients.

Evaluation of three next-generation sequencing platforms for immune repertoire sequencing




The advent of next generation sequencing (NGS) techniques, combined with a semi-quantitative multiplex PCR method, enables us to comprehensively profile the lymphocyte receptor diversity (immune repertoire) of the entire collection of B or T cells from a particular sample. Different NGS platforms vary in read length, throughput, sequencing error rate and error pattern, hands-on time, turnaround time and price. Three popular NGS techniques (Illumina HiSeq, 454 FLX, and Ion Torrent) were employed to sequence amplicons from T cell receptor clones generated with arm-PCR in order to evaluate the usage of those techniques on immune repertoire sequencing. The error pattern and rate of the three techniques in the context of the T cell receptor sequences were profiled. Based on the differences of the three techniques, we discussed the usage of each NGS platform in the context of immune repertoire sequencing.