Quantitative analysis of T cell receptor diversity in clinical samples of human peripheral blood

Abstract

The analysis of T cell receptor diversity provides a clinically relevant and sensitive marker of repertoire loss, gain, or skewing. Spectratyping is a broadly utilized technique to measure global TCR diversity by the analysis of the lengths of CDR3 fragments in each Vβ family. However the common use of large numbers of T cells to obtain a global view of TCR Vβ CDR3 diversity has restricted spectratyping analyses when limited T-cell numbers are available in clinical setting, such as following transplant regimens. We here demonstrate that one hundred thousand T cells are sufficient to obtain a robust, highly reproducible measure of the global TCR Vβ repertoire diversity among twenty Vβ families in human peripheral blood. We also show that use of lower cell number results not in a dwindling of observed diversity but rather in non- reproducible patterns in replicate spectratypes. Finally, we report here a simple to use but sensitive method to quantify repertoire divergence in patient samples by comparison to a standard repertoire profile we generated from fifteen normal donors. We provide examples using this method to statistically evaluate the changes in the global TCR Vβ repertoire diversity that may take place during T subset immune reconstitution after hematopoietic stem cell transplantation or after immune modulating therapies.

Methods for diversity and overlap analysis in T-cell receptor populations

“…By enumeration of all such possible recombinations alone, one concludes that there are 10 18 distinct TCR chains in humans (Janeway, 2005) and 10 15 in mice (Davis and Bjorkman, 1988). The experimentally observed numbers of different recombinations seem to confirm this order of magnitude (Arstila et al, 1999; Memon et al, 2012). In the presence of such a large number of different antigen receptor chain types, the statistical analysis of the samples obtained from different TCR populations presents a formidable challenge, due to the unavoidable issue of chain types under-sampling, even with the use of modern high-hroughput methods of TCR data collection (Salameire et al, 2009; Van Den Berg et al, 2011).…”

Age-Related Decrease in TCR Repertoire Diversity Measured with Deep and Normalized Sequence Profiling

“…7). As such, extrapolating spectratype data to whole repertoires, although a powerful and widely used approach (27,53,54), can lead to inaccurate estimations. In contrast, the direct identification of hundreds of thousands or millions of unique TCR b clonotypes using deep normalized profiling followed by strict correction of PCR and sequencing errors provides full information on clonal composition and relative sample diversity.…”

Quantitative assessment of T cell repertoire recovery after hematopoietic stem cell transplantation

“…One strategy aims to identify the presence of different TCR families, by using flow cytometry or PCR to determine the usage of different TCR variable (V) genes 13,14 . A second strategy, called CDR3 size spectratyping, aims to determine polyclonality of the repertoire, by using fluorescent primers to measure length variation of the CDR3 region within each TCR V family 15,16 . Spectratyping in particular has been useful to document substantial abnormalities in T-cell repertoire composition after allo-HSCT 17–19 .…”