Characterization of the T-cell Repertoire after Autologous HSCT in Patients with Ankylosing Spondylitis

Abstract

Autologous hematopoietic stem cell transplantation (HSCT), a safer type of HSCT than allogeneic HSCT, is a promising therapy for patients with severe autoimmune diseases (ADs). Despite the long history of medical practice, structural changes in the adaptive immune system as a result of autologous HSCT in patients with various types of ADs remain poorly understood. In this study, we used high-throughput sequencing to investigate the structural changes in the peripheral blood T-cell repertoire in adult patients with ankylosing spondylitis (AS) during two years after autologous HSCT. The implementation of unique molecular identifiers allowed us to substantially reduce the impact of the biases occurring during the preparation of libraries, to carry out a comparative analysis of the various properties of the T-cell repertoire between different time points, and to track the dynamics of both distinct T-cell clonotypes and T-cell subpopulations. In the first year of the reconstitution, clonal diversity of the T-cell repertoire remained lower than the initial one in both patients. During the second year after HSCT, clonal diversity continued to increase and reached a normal value in one of the patients. The increase in the diversity was associated with the emergence of a large number of low-frequency clonotypes, which were not identified before HSCT. Efficiency of clonotypes detection after HSCT was dependent on their abundance in the initial repertoire. Almost all of the 100 most abundant clonotypes observed before HSCT were detected 2 years after transplantation and remained highly abundant irrespective of their CD4+ or CD8+ phenotype. A total of up to 25% of peripheral blood T cells 2 years after HSCT were represented by clonotypes from the initial repertoire.

Keywords: NGS; TCR repertoire; ankylosing spondylitis; autologous HSCT.

Fig. 1

Dynamics of T-cell diversity during 2 years after HSCT. Estimation of the lower bound of clonal diversity using the Chao1 index [16]. Blue dots represent healthy donors (n=6, age 22–34 years; the data were reported by Britanova et al. 2014 [21]); orange dots – patients with AS (n=5, age 22–34; the data were reported by Komech et al. 2018 [22]). The dashed vertical line shows the time point of HSCT. The 95% confidence interval for each value is marked with an error bar.

Fig. 2

Clonal frequency distribution in the initial repertoire according to the detection of clonotypes in samples after HSCT. White box plots show the frequency distribution of clonotypes found in all the analyzed samples, including the time point before HSCT; blue box plots show the frequency distribution of clonotypes present at point 0 but not found in at least one sample after HSCT (points 4, 12 or 24). *The p-value < 2.2×10^-16 (Mann–Whitney U-test).

Fig. 3

Clonal dynamics of the initial T-cell repertoire. (A) Reproducibility of clonal frequency in two parallel blood samples collected from one donor. Each dot represents a TCRβ clonotype. Black dots represent the clonotypes that do not reproduce in replica. (B) The dynamics of the top 100 clonotypes from the initial repertoire. CD4+ clonotypes are shown in red; CD8+ clonotypes, in blue. The black horizontal line represents the lower bound of clonal frequency for the top 100 clonotypes at point 24.

Fig. 4

The structure of the clonal repertoire two years after HSCT. (A) Proportion of clonotypes belonging to different frequency groups with respect to the total number of clonotypes in the sample: high-abundance (0.1–10%), medium-abundance (0.01–0.1%), and low-abundance ( < 0.01%). Structures of T-cell repertoires are given for two patients with AS before and 2 years after HSCT (points 0 and 24) and for one representative healthy donor. (B) All clones at point 24 are divided into groups according to the time point when they were detected for the first time. Y-axis: the cumulative proportion of clonotypes from each group with respect to all cells in a sample 2 years after HSCT.

Fig. 5

Degree of clonal repertoire renewal after HSCT. (A) Correlation between the normalized number of the clonotypes shared between point 0 and points 4 (p4), 12 (p12), 24 (in two replicas: p24_R1 and p24_R2) and the diversity of a reference sample. Points p24_R1R2 represent the comparison of replicas of point 24. (B) Composition of the 100 most abundant clonotypes in the repertoire of point 24. CD8+ cytotoxic clonotypes are shown in blue and light blue; CD4+ T-helper clonotypes are shown in red and orange.