NGS data analysis algorithm for evaluating repertoire of T-cell receptors involved in the antitumor immune response

Abstract

Introduction. The repertoire of T- and B cellular receptors is represented by a large number of sequences. Therefore, the characterization of the immune receptor repertoire is a technically difficult task. The most advanced method of analyzing the immune receptor repertoires is NGS sequencing. To reduce the risk of error when working with sequences, computational biology methods are used, which simplify the processing of large amounts of data.

Aim of study - development and testing of NGS data analysis algorithm for comparative evaluation of the T-cell receptor repertoire in syngeneic and allogeneic responses to the tumor.

Material and methods. As a part of this work we have created a chain of program scenarios that allow to increase the efficiency of analyzing the repertoire of T-cell receptors. The developed scenarios allow us to automatically start processing of «raw» data in MIGEC software, merge sequences within groups, and analyze clonal composition of T-cell populations taking into account the specificity of the immune response. The conditions and parameters of the analysis have been tested in a biological experiment in comparison with the antitumor response in groups of mice immunized by EL-4 line tumor cells.

Tumor cells of EL-4 line were grafted into C57BL/6 mice to evaluate the syngeneic response, and tumor cells of B10.D2 (R101) line were grafted into B10.D2 (R101) mice to evaluate the allogeneic response. To exclude allospecific component in allogeneic response to the tumor, an additional group of animals of B10.D2 (R101) line was immunized with normal thymocytes of C57BL/6 mice.

Results. When comparing the repertoire of T-cell receptors in the examined groups, it was found that the antitumor T-cell response is characterized by a decrease in diversity and an increase in the clonality of the immune response to the tumor, most pronounced in the allogeneic group of B10.D2 (R101) mice. The analysis of antitumor clones in the syngeneic and allogeneic models showed reliable differences in qualitative composition of T-cell repertoire. Using the developed program scenarios, the clonal composition of T-cell populations was analyzed, taking into account the allospecific component of the immune response, and specific clones providing antitumor response in the syngeneic and allogeneic models were identified.

Conclusion. As a result of the research, an algorithm for analyzing the data of NGS-sequencing processed in MIGEC software was created. The conditions and parameters of the analysis were tested in a biological experiment in comparison with the antitumor response in groups of mice immunized by EL-4 line tumor cells. T-cell receptor sequencing of α- and β-chains in immunized mice and control animals were performed. A comparative analysis of repertoire of T-cell receptors in mice in a syngeneic and allogeneic model in the development of antitumor immune response was carried out. It was found that the syngeneic and allogeneic antitumor response, as well as allogeneic response to intact tissue, differ from each other. The structure of the immune response in the allogeneic model seems to contain tumor-specific clones that are absent in the syngeneic model.

Keywords:T-cell receptor; NGS; evaluation of TCR repertoire; antitumor immune response

For citation: Bulusheva I.A., Kozlov I.B., Mitin A.N., Korostin D.O., Kofiadi I.A. NGS data analysis algorithm for evaluating repertoire of T-cell receptors involved in the antitumor immune response. Immunologiya. 2020; 41 (5): 400-10. DOI: https://doi.org/10.33029/0206-4952-2020-41-5-400-410 (in Russian)

Funding. The investigation of the syngeneic and allogeneic response to the tumor was carried out with financial support from the Perspective Research Foundation. Research to develop an algorithm for data analysis was carried out with financial support from RFBR under scientific project No. 19-33-90076.

Conflict of interests. The authors declare no conflict of interests.

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