TCR repertoire profiling revealed antigen-driven CD8+ T cell clonal groups shared in synovial fluid of patients with spondyloarthritis.

Spondyloarthritis (SpA) comprises a number of inflammatory rheumatic diseases with overlapping clinical manifestations. Strong association with several HLA-I alleles and T cell infiltration into an inflamed joint suggest involvement of T cells in SpA pathogenesis. In this study, we performed high-throughput T cell repertoire profiling of synovial fluid (SF) and peripheral blood (PB) samples collected from a large cohort of SpA patients. We showed that synovial fluid is enriched with expanded T cell clones that are shared between patients with similar HLA genotypes and persist during recurrent synovitis. Using an algorithm for identification of TCRs involved in immune response we discovered several antigen-driven CD8+ clonal groups associated with risk HLA-B*27 or HLA-B*38 alleles. We further show that these clonal groups were enriched in SF and had higher frequency in PB of SpA patients vs healthy donors, implying their relevance to SpA pathogenesis. Several of the groups were shared among patients with different SpAs that suggests a common immunopathological mechanism of the diseases. In summary, our results provide evidence for the role of specific CD8+ T cell clones in pathogenesis of SpA.

Surface NKG2C Identifies Differentiated αßT-Cell Clones Expanded in Peripheral Blood.

T cells that express CD56 in peripheral blood of healthy humans represent a heterogeneous and poorly studied subset. In this work, we analyzed this subset for NKG2C expression. In both CD56+ and CD56- subsets most of the NKG2C+ T cells had a phenotype of highly differentiated CD8+ TEMRA cells. The CD56+NKG2C+ T cells also expressed a number of NK cell receptors, such as NKG2D, CD16, KIR2DL2/DL3, and maturation marker CD57 more often than the CD56-NKG2C+CD3+ cells. TCR ß-chain repertoire of the CD3+CD56+NKG2C+ cell fraction was limited by the prevalence of one or several clonotypes which can be found within the most abundant clonotypes in total or CD8+ T cell fraction TCRß repertoire. Thus, NKG2C expression in highly differentiated CD56+ T cells was associated with the most expanded αß T cell clones. NKG2C+ T cells produced almost no IFN-γ in response to stimulation with HCMV pp65-derived peptides. This may be partially due to the high content of CD45RA+CD57+ cells in the fraction. CD3+NKG2C+ cells showed signs of activation, and the frequency of this T-cell subset in HCMV-positive individuals was positively correlated with the frequency of NKG2C+ NK cells that may imply a coordinated in a certain extent development of the NKG2C+ T and NK cell subsets under HCMV infection.

Persisting fetal clonotypes influence the structure and overlap of adult human T cell receptor repertoires.

The diversity of T-cell receptors recognizing foreign pathogens is generated through a highly stochastic recombination process, making the independent production of the same sequence rare. Yet unrelated individuals do share receptors, which together constitute a "public" repertoire of abundant clonotypes. The TCR repertoire is initially formed prenatally, when the enzyme inserting random nucleotides is downregulated, producing a limited diversity subset. By statistically analyzing deep sequencing T-cell repertoire data from twins, unrelated individuals of various ages, and cord blood, we show that T-cell clones generated before birth persist and maintain high abundances in adult organisms for decades, slowly decaying with age. Our results suggest that large, low-diversity public clones are created during pre-natal life, and survive over long periods, providing the basis of the public repertoire.

tcR: an R package for T cell receptor repertoire advanced data analysis.

BACKGROUND: The Immunoglobulins (IG) and the T cell receptors (TR) play the key role in antigen recognition during the adaptive immune response. Recent progress in next-generation sequencing technologies has provided an opportunity for the deep T cell receptor repertoire profiling. However, a specialised software is required for the rational analysis of massive data generated by next-generation sequencing. RESULTS: Here we introduce tcR, a new R package, representing a platform for the advanced analysis of T cell receptor repertoires, which includes diversity measures, shared T cell receptor sequences identification, gene usage statistics computation and other widely used methods. The tool has proven its utility in recent research studies. CONCLUSIONS: tcR is an R package for the advanced analysis of T cell receptor repertoires after primary TR sequences extraction from raw sequencing reads. The stable version can be directly installed from The Comprehensive R Archive Network ( http://cran.r-project.org/mirrors.html ). The source code and development version are available at tcR GitHub ( http://imminfo.github.io/tcr/ ) along with the full documentation and typical usage examples.

Next generation sequencing for TCR repertoire profiling: platform-specific features and correction algorithms.

The TCR repertoire is a mirror of the human immune system that reflects processes caused by infections, cancer, autoimmunity, and aging. Next generation sequencing (NGS) is becoming a powerful tool for deep TCR profiling; yet, questions abound regarding the methodological approaches for sample preparation and correct data interpretation. Accumulated PCR and sequencing errors along with library preparation bottlenecks and uneven PCR efficiencies lead to information loss, biased quantification, and generation of huge artificial TCR diversity. Here, we compare Illumina, 454, and Ion Torrent platforms for individual TCR profiling, evaluate the rate and character of errors, and propose advanced platform-specific algorithms to correct massive sequencing data. These developments are applicable to a wide variety of next generation sequencing applications. We demonstrate that advanced correction allows the removal of the majority of artificial TCR diversity with concomitant rescue of most of the sequencing information. Thus, this correction enhances the accuracy of clonotype identification and quantification as well as overall TCR diversity measurements.

A new set of markers for human identification based on 32 polymorphic Alu insertions.

A number of genetic systems for human genetic identification based on short tandem repeats or single nucleotide polymorphisms are widely used for crime detection, kinship studies and in analysis of victims of mass disasters. Here, we have developed a new set of 32 molecular genetic markers for human genetic identification based on polymorphic retroelement insertions. Allele frequencies were determined in a group of 90 unrelated individuals from four genetically distant populations of the Russian Federation. The mean match probability and probability of paternal exclusion, calculated based on population data, were 5.53 x 10(-14) and 99.784%, respectively. The developed system is cheap and easy to use as compared to all previously published methods. The application of fluorescence-based methods for allele discrimination allows to use the human genetic identification set in automatic and high-throughput formats.