Estimating T-cell repertoire diversity: limitations of classical estimators and a new approach

Laydon, Daniel J; Bangham, Charles R. M.; Asquith, Becca

doi:10.1098/rstb.2014.0291

Cited by 176 publications

(177 citation statements)

References 97 publications

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“…We performed sequencing on CD4 + and CD8 + T cells separately, allowing for key comparisons between the 2 distinct repertoires. Consistent with prior studies (20,22), the alloreactive populations were not only composed of large numbers of unique clones ( Figure 1A), but were also qualitatively diverse in terms of CDR3 amino acid length and Vβ-and Jβ-gene usage. Since the structure of the TCR is determined in part by the number of amino acids that gives rise to the CDR3 region, we compared the distribution of CDR3 amino acid lengths between the unstimulated and alloreactive populations and found no significant difference (Figure 2, A and B, and Supplemental Figure 3).…”

Section: Identification and Qualitative Characterization Of Human Allsupporting

confidence: 74%

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Quantifying size and diversity of the human T cell alloresponse

DeWolf

Grinshpun²,

Savage

et al. 2018

JCI Insight

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Section: Identification and Qualitative Characterization Of Human Allsupporting

confidence: 74%

“…Based on approaches developed in ecology to study population diversity, different strategies have been proposed to address the unseen species question (14,22). Here we present a new computational approach to model T cell clonal frequency distribution.…”

Section: Introductionmentioning

confidence: 99%

Quantifying size and diversity of the human T cell alloresponse

DeWolf

Grinshpun²,

Savage

et al. 2018

JCI Insight

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“…Further variability and antigen recognition capacity is introduced by nucleotide insertion (NI) in the recombined TCR a and b VDJ sequences. This generates a vast T-cell repertoire, yielding in excess of a trillion potential TCRab combinations capable of reacting to non-self (and self) peptides [3]. Since the advent of next generation sequencing techniques, the TCR repertoire, as estimated by TCR b clonal frequency measurement has revealed that the T-cell repertoire in healthy individuals is complex with thousands of clones in each individual spanning a spectrum of high and low frequencies [4,5].…”

Section: Introductionmentioning

confidence: 99%

On the organization of human T-cell receptor loci: log-periodic distribution of T-cell receptor gene segments

Toor

Rahmani

et al. 2016

J. R. Soc. Interface.

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We dedicate this work to Mr Omair Ahmed Toor and other people with Down's Syndrome and patients with congenital neurological disorders from around the world, whose constant struggle to overcome the challenges of everyday life and better themselves are an inspiration to all.On the organization of human T-cell receptor loci: log-periodic distribution of T-cell receptor gene segments School of Engineering, Virginia Commonwealth University, Richmond, VA, USAThe human T-cell repertoire is complex and is generated by the rearrangement of variable (V), diversity (D) and joining (J) segments on the T-cell receptor (TCR) loci. The T-cell repertoire demonstrates self-similarity in terms clonal frequencies when defined by V, D and J gene segment usage; therefore to determine whether the structural ordering of these gene segments on the TCR loci contributes to the observed clonal frequencies, the TCR loci were examined for self-similarity and periodicity in terms of gene segment organization. Logarithmic transformation of numeric sequence order demonstrated that the V and J gene segments for both T-cell receptor a (TRA) and b (TRB) loci are arranged in a self-similar manner when the spacing between the adjacent segments was considered as a function of the size of the neighbouring gene segment, with an average fractal dimension of approximately 1.5. Accounting for the gene segments occurring on helical DNA molecules with a logarithmic distribution, sine and cosine functions of the log-transformed angular coordinates of the start and stop nucleotides of successive TCR gene segments showed an ordered progression from the 5 0 to the 3 0 end of the locus, supporting a log-periodic organization. T-cell clonal frequency estimates, based on V and J segment usage, from normal stem cell donors were plotted against the V and J segment on TRB locus and demonstrated a periodic distribution. We hypothesize that this quasi-periodic variation in genesegment representation in the T-cell clonal repertoire may be influenced by the location of the gene segments on the periodic-logarithmically scaled TCR loci. Interactions between the two strands of DNA in the double helix may influence the probability of gene segment usage by means of either constructive or destructive interference resulting from the superposition of the two helices.

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“…These results are consistent with the findings reported previously by other groups. 32,33 Spectratyping assays have been widely used in clinical settings for more than a decade to detect the TCR clonality of patients. This method separates V/D/J DNA clones based on the DNA amplicon size in the TCR repertoire.…”

Section: Variability Across Different Biological Replicate Librariesmentioning

confidence: 99%

Methodologic Considerations in the Application of Next-Generation Sequencing of Human TRB Repertoires for Clinical Use

You

Zheng

et al. 2017

The Journal of Molecular Diagnostics

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Next-generation sequencing (NGS) of immune receptors has become a standard tool to assess minimal residual disease (MRD) in patients treated for lymphoid malignancy, and it is being used to study the T-cell repertoire in many clinical settings. To better understanding the potential clinical utility and limitations of this application outside of MRD, we developed a BIOMED-2 primerebased NGS method and characterized its performance in controls and patients with graft-versus-host disease (GVHD) after allogeneic hematopoietic transplant. For controls and patients with GVHD, replicate sequencing of the same T-cell receptor b (TRB) libraries was highly reproducible. Higher variability was observed in sequencing of different TRB libraries made from the same DNA stock. Variability was increased in patients with GVHD compared with controls; patients with GVHD also had lower diversity than controls. In the T-cell repertoire of a healthy person, approximately 99.6% of the CDR3 clones were in low abundance, with frequency <10À3 . A single library could identify >93% of the clones with frequency !10 À3 in the repertoire. Sequencing in duplicate increased the average detection rate to >97%. This work demonstrates that NGS reliably and robustly characterizes TRB populations in healthy individuals and patients with GVHD with frequency !10 À3 and provides a methodologic framework for applying NGS immune repertoire methods to clinical testing applications beyond MRD. (J Mol Diagn 2017, 19: 72e83; http://dx.doi.org/10.1016/j.jmoldx.2016 Study and analysis of T-cell receptor (TCR) repertoires are essential for better understanding the development and reconstitution of immune systems, for providing insights into the pathogenesis of immune disorders, and for diagnosing and assessing therapeutic interventions for diseases driven by T cells. Next-generation sequencing (NGS) technology has become a standard method for assessing minimal residual disease (MRD) in patients with lymphoid malignancies. From a technical standpoint, MRD is a relatively simple application of NGS technology, essentially looking for known malignant or related somatically mutated clones; by combining replicate libraries, a sensitivity of 1 in 10 À5 has been demonstrated for MRD. Beyond detecting MRD, NGS is also an innovative and promising approach to assess clonal populations of T cells in healthy people during immune system development, patients with suspected malignancy, response to targeted therapies, immune reconstitution in patients undergoing hematopoietic cell transplant, and autoimmune diseases. 7,9e25 However, assessment of the T-cell repertoire in individuals is a far more complex task than assessment of MRD. In consideration of extending NGS methods beyond MRD applications in clinical laboratories, we examined the opportunity and technical limitations of the widely used BIOMED-2 primer sets and the MiSeq system (Illumina Inc., San Diego, CA) as an NGS application to assess the TCR repertoire. We evaluated replicate testing of individual libraries (th...

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Estimating T-cell repertoire diversity: limitations of classical estimators and a new approach

Cited by 176 publications

References 97 publications

Quantifying size and diversity of the human T cell alloresponse

Quantifying size and diversity of the human T cell alloresponse

On the organization of human T-cell receptor loci: log-periodic distribution of T-cell receptor gene segments

Methodologic Considerations in the Application of Next-Generation Sequencing of Human TRB Repertoires for Clinical Use

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