Qualifying high-throughput immune repertoire sequencing

Niklas, Norbert; Pröll, Johannes; Weinberger, Johannes; Zopf, Agnes; Wiesinger, Karin; Krismer, Konstantin; Bettelheim, Peter; Gabriel, Christian

doi:10.1016/j.cellimm.2014.02.001

Cited by 12 publications

(12 citation statements)

References 28 publications

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“…The results did not fully reflect the CDR3␦ repertoire. Future studies will require immune repertoire sequencing techniques, based on specific PCR amplification and high-throughput sequencing (22,23), for a comprehensive and systematic analysis.…”

Section: Discussionmentioning

confidence: 99%

Chaperonin-containing T-complex Protein 1 Subunit ζ Serves as an Autoantigen Recognized by Human Vδ2 γδ T Cells in Autoimmune Diseases

Chen

You

Wang

et al. 2016

Journal of Biological Chemistry

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Human γδ T cells recognize conserved endogenous and stress-induced antigens typically associated with autoimmune diseases. However, the role of γδ T cells in autoimmune diseases is not clear. Few autoimmune disease-related antigens recognized by T cell receptor (TCR) γδ have been defined. In this study, we compared Vδ2 TCR complementarity-determining region 3 (CDR3) between systemic lupus erythematosus (SLE) patients and healthy donors. Results show that CDR3 length distribution differed significantly and displayed oligoclonal characteristics in SLE patients when compared with healthy donors. We found no difference in the frequency of Jδ gene fragment usage between these two groups. According to the dominant CDR3δ sequences in SLE patients, synthesized SL2 peptides specifically bound to human renal proximal tubular epithelial cell line HK-2; SL2-Vm, a mutant V sequence of SL2, did not bind. We identified the putative protein ligand chaperonin-containing T-complex protein 1 subunit ζ (CCT6A) using SL2 as a probe in HK-2 cell protein extracts by affinity chromatography and liquid chromatography-electrospray ionization-tandem mass spectrometry analysis. We found CCT6A expression on the surface of HK-2 cells. Cytotoxicity of only Vδ2 γδ T cells to HK-2 cells was blocked by anti-CCT6A antibody. Finally, we note that CCT6A concentration was significantly increased in plasma of SLE and rheumatoid arthritis patients. These data suggest that CCT6A is a novel autoantigen recognized by Vδ2 γδ T cells, which deepens our understanding of mechanisms in autoimmune diseases.

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Section: Discussionmentioning

confidence: 99%

Chaperonin-containing T-complex Protein 1 Subunit ζ Serves as an Autoantigen Recognized by Human Vδ2 γδ T Cells in Autoimmune Diseases

Chen

You

Wang

et al. 2016

Journal of Biological Chemistry

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“…Others studies have used alternative statistics, including mean clone size, the number of unique IgHV-D-J allele combinations, and genetic distances between IgV segment sequences and their respective germline homologues (e.g. [ 7 ]). Both sets of approaches have shown promise in studying B-cell cancers, which result from significant clonal expansions of usually one BCR lineage [ 20 ], but it is unknown if a similar approach will be informative when studying immune responses to HIV infection.…”

Section: Introductionmentioning

confidence: 99%

Dynamics of immunoglobulin sequence diversity in HIV-1 infected individuals

Hoehn

Gall

Bashford-Rogers

et al. 2015

Phil. Trans. R. Soc. B

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Advances in immunoglobulin (Ig) sequencing technology are leading to new perspectives on immune system dynamics. Much research in this nascent field has focused on resolving immune responses to viral infection. However, the dynamics of B-cell diversity in early HIV infection, and in response to anti-retroviral therapy, are still poorly understood. Here, we investigate these dynamics through bulk Ig sequencing of samples collected over 2 years from a group of eight HIV-1 infected patients, five of whom received anti-retroviral therapy during the first half of the study period. We applied previously published methods for visualizing and quantifying B-cell sequence diversity, including the Gini index, and compared their efficacy to alternative measures. While we found significantly greater clonal structure in HIV-infected patients versus healthy controls, within HIV patients, we observed no significant relationships between statistics of B-cell clonal expansion and clinical variables such as viral load and CD4+ count. Although there are many potential explanations for this, we suggest that important factors include poor sampling resolution and complex B-cell dynamics that are difficult to summarize using simple summary statistics. Importantly, we find a significant association between observed Gini indices and sequencing read depth, and we conclude that more robust analytical methods and a closer integration of experimental and theoretical work is needed to further our understanding of B-cell repertoire diversity during viral infection.

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“…(2) Immunosequencing, of which there are many types [reviewed in Ref. ( 8 , 12 , 19 , 25 – 27 )], is carried out on the B-cell sample. Here, we carried out bulk sequencing of the murine IGH BCR locus, focusing on the CDR3 region.…”

Section: Methodsmentioning

confidence: 99%

“…Given the stochastic nature of individual germline repertoires and antigen-specific selective pressures, B-cell repertoires can vary widely depending on the subject, antigen or vaccine conditions, and time (e.g., time postexposure) ( 17 , 18 ). Despite the fact that various statistical approaches have been applied to analyze B-cell repertoire data, a consensus on the best method remains elusive ( 2 , 8 , 12 , 19 – 27 ).…”

Section: Introductionmentioning

confidence: 99%

Quantitative Analysis of Repertoire-Scale Immunoglobulin Properties in Vaccine-Induced B-Cell Responses

et al. 2017

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Recent advances in the next-generation sequencing of B-cell receptors (BCRs) enable the characterization of humoral responses at a repertoire-wide scale and provide the capability for identifying unique features of immune repertoires in response to disease, vaccination, or infection. Immunosequencing now readily generates 103–105 sequences per sample; however, statistical analysis of these repertoires is challenging because of the high genetic diversity of BCRs and the elaborate clonal relationships among them. To date, most immunosequencing analyses have focused on reporting qualitative trends in immunoglobulin (Ig) properties, such as usage or somatic hypermutation (SHM) percentage of the Ig heavy chain variable (IGHV) gene segment family, and on reducing complex Ig property distributions to simple summary statistics. However, because Ig properties are typically not normally distributed, any approach that fails to assess the distribution as a whole may be inadequate in (1) properly assessing the statistical significance of repertoire differences, (2) identifying how two repertoires differ, and (3) determining appropriate confidence intervals for assessing the size of the differences and their potential biological relevance. To address these issues, we have developed a technique that uses Wilcox’ robust statistics toolbox to identify statistically significant vaccine-specific differences between Ig repertoire properties. The advantage of this technique is that it can determine not only whether but also where the distributions differ, even when the Ig repertoire properties are non-normally distributed. We used this technique to characterize murine germinal center (GC) B-cell repertoires in response to a complex Ebola virus-like particle (eVLP) vaccine candidate with known protective efficacy. The eVLP-mediated GC B-cell responses were highly diverse, consisting of thousands of clonotypes. Despite this staggering diversity, we identified statistically significant differences between non-immunized, vaccine only, and vaccine-plus-adjuvant groups in terms of Ig properties, including IGHV-family usage, SHM percentage, and characteristics of the BCR complementarity-determining region. Most notably, our analyses identified a robust eVLP-specific feature—enhanced IGHV8-family usage in B-cell repertoires. These findings demonstrate the utility of our technique in identifying statistically significant BCR repertoire differences following vaccination. More generally, our approach is potentially applicable to a wide range of studies in infection, vaccination, auto-immunity, and cancer.

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Qualifying high-throughput immune repertoire sequencing

Cited by 12 publications

References 28 publications

Chaperonin-containing T-complex Protein 1 Subunit ζ Serves as an Autoantigen Recognized by Human Vδ2 γδ T Cells in Autoimmune Diseases

Chaperonin-containing T-complex Protein 1 Subunit ζ Serves as an Autoantigen Recognized by Human Vδ2 γδ T Cells in Autoimmune Diseases

Dynamics of immunoglobulin sequence diversity in HIV-1 infected individuals

Quantitative Analysis of Repertoire-Scale Immunoglobulin Properties in Vaccine-Induced B-Cell Responses

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