EHA Library - The official digital education library of European Hematology Association (EHA)

Chronic lymphocytic leukemia (CLL) is characterized by a remarkably restricted immunoglobulin (IG) gene repertoire, mainly attributable to the existence of subsets of patients with stereotyped B-cell receptor (BcR) IGs strongly implying clonal selection by a restricted set of antigens. Our preliminary high-throughput, next-generation sequencing studies of the T-cell receptor beta chain (TRB) gene repertoire in CLL cases from various subsets indicated repertoire skewing, pointing to antigenic selection of the T cells as well, which is relevant in view of the bidirectional CLL-T cell interactions.

We performed in-depth profiling of the TRB gene repertoire in CLL subset #4 which became our focus for the following reasons: (i) it is the most indolent subgroup of CLL patients identified thus far; (ii) previous studies have strongly supported ongoing antigenic stimulation attested by the intraclonal diversification of the clonotypic IG.

We studied 11 untreated CLL subset #4 cases and a healthy control. RNA was isolated from peripheral blood mononuclear cells (n=12) or purified CD4⁺ and CD8⁺ T cells (n=2 CLL cases). Two patients were studied overtime. TRBV-TRBD-TRBJ gene rearrangements were amplified on cDNA according to the BIOMED2 protocol and subjected to paired-end NGS (MiSeq Illumina Platform). The experimental design allowed sequencing of the complementarity determining region 3 (CDR3) twice/read, so as to increase the accuracy of results. Computational processing of raw data was performed using a purpose-built algorithm and a bioinformatics platform was developed for IMGT/HighV-QUEST metadata clustering and analysis.

Overall, 12,261,280 TRBV-TRBD-TRBJ reads were produced (median 397,035 reads/sample, median Q-score 38.4). Poor quality, incomplete, out-of-frame and unproductive rearrangements were filtered out. For repertoire analyses, clonotypes (i.e. TRB rearrangements with identical TRBV gene usage and amino acid CDR3 sequence) rather than single rearrangement reads were considered (median 77020 distinct clonotypes/sample, 54733 singletons versus 22287 expanded). Among the 53 functional TRBV genes identified, 5 predominated: TRBV12-3/12-4 (9.9%), TRBV29-1 (8.6%), TRBV19 (7.5%), TRBV5-1 (5.5%), and TRBV6-5 (4.9%), collectively accounting for 36.4% of the TRBV repertoire. Comparison of the TRBV gene repertoire of CD8⁺ vs CD4⁺ cells showed that TRBV19 was overrepresented in the CD4+ compartment (9.4% versus 6.9%, p<0.001). The TRB repertoire was significantly more oligoclonal in CLL compared to the healthy control (median frequency of the predominant clonotype: 3.6% versus 0.47%, respectively, p<0.001), and this skewing stemmed mainly from the CD8⁺ rather than the CD4⁺ compartment (median frequency of the predominant clonotype 10.7% versus 1.0%, respectively, p<0.001). Cluster analysis of all CLL cases identified 37303 different clonotypes (excluding singletons) shared by different patients and not present in the healthy control. The longitudinal analysis of 2 cases showed contrasting results, with 14.6% of all expanded clonotypes persisting over time in one case, but only 0.06% in the other. In the former case, 5 of the persisting clonotypes ranked among the 10 most expanded within the patient's T cell repertoire, whereas in the latter case only minor clonotypes persisted.

Our study provides large-scale evidence of TR repertoire skewing and oligoclonality in CLL subset #4, strongly supporting antigenic selection. The nature of selecting antigens remains to be elucidated.