TCR-repertoire analysis by NGS in a murine infection model. Mus musculus breed:C57BL/6

Infections represent challenges for the immune system and affect immunological imprints within the T-cell-receptor (TCR)-repertoire of an individual. The complexity and dynamics of the TCR-repertoire remain largely unknown as the dimension cannot be addressed by conventional sequence-analysis. Next-generation-sequencing (NGS)-technology, in combination with advanced computational algorithms, may overcome the technical limitations of current methods and offers a new and powerful tool to investigate repertoire-dynamics. It might also reveal characteristic immune signatures, providing a basis for the development of predictive diagnostics and immunotherapeutic approaches for personalized medicine. Due to the huge amounts of TCR NGS-data, bioinformatic solutions for the analysis by defined algorithms are absolutely necessary. Therefore, an improved NGS data analysis platform is needed that offers better analysis capacity, data visualization and shorter analysis time.We established a novel bioinformatic solution - AptaIT´s COMmon PAtternS Software COMPAS - as a new method to analyze big data from NGS. Using 5´-RACE (rapid-amplification-of-cDNA-ends)-multiplexed PCR, combined with Illumina-NGS-technology, we monitored the entire TCR-β-repertoire during the course of an in vivo infection with murine γ-herpesvirus 68 (MHV-68), a well characterized mouse model for infections with the human γ-herpesviruses EBV and KSHV. The results determined via COMPAS were confirmed by additional analyses: i) Published CDR3-sequences could be detected and verified in silico after infection. ii) The ratio of two Vβ-subtypes detected via TCR-NGS-analysis in MHV-68-infected mice (the most frequent Vβ13 family and the infrequent Vβ2 family) was confirmed by flow cytometry. iii) The Vβ13 family was selected and tested for its specific cytolytic ca-pacity. Vβ13+ T-cells derived from MHV-68-infected mice specifically lysed target-cells, while Vβ13-depleted T cells showed a decreased reactivity.In sum, we present an innovative and fast technical approach - COMPAS - to profile the TCR-repertoire.