High Throughput Immunoprofiling of Low Count B Lymphocytosis Reveals a Distinct T Cell Receptor Repertoire from Chronic Lymphocytic Leukemia

Mounting evidence supports the notion that T cells are crucial for the survival and expansion of the malignant clone in chronic lymphocytic leukemia (CLL). Recently, NGS immunoprofiling of the T cell receptor (TR) gene repertoire in CLL revealed "disease-specific" TR clonotypes shared by different patients that persisted and further expanded over time, suggesting that antigen drive likely underlies T-cell expansions in CLL. CLL-like Monoclonal B-cell Lymphocytosis (MBL) is characterized by the presence of CLL-like clonal B cells in lower numbers compared to CLL with no other evidence of disease. MBL is distinguished into high and low count (HC-MBL and LC-MBL, respectively). HC-MBL may evolve to CLL requiring treatment and is considered a pre-leukemic condition, while LC-MBL has an unclear, if any, link to CLL, posing a particular conundrum, especially considering that despite sharing CLL-associated genomic aberrations it displays a distinct immunoglobulin gene repertoire. The precise mechanisms underlying MBL onset and/or progression into overt CLL remain unknown. Arguably, these could entail the acquisition of particular genetic lesions and/or extrinsic signals delivered to the clonal B cells, including those emanating from T cells. Considering the above, here we aimed at characterizing T cells in the early steps of CLL ontogeny by detailed immunoprofiling of the TR gene repertoire in LC-MBL, a thus far unexplored area.

The study group comprised 43 individuals with CLL-like (CD5⁺) LC-MBL and 7 individuals with non CLL-like LC-MBL (atypical CLL-like and CD5^- LC-MBL). The presence of a LC-MBL population was confirmed by a standardized flow cytometry protocol. TR beta chain (TRB) gene rearrangements were RT-PCR amplified with the use of the BIOMED 2 protocol. Sequencing libraries were prepared with the TruSeq DNA LT Kit and run on the MiSeq Sequencer using the MiSeq Reagent Kit v3 (500 cycles). Raw NGS reads were subjected to data filtering based on read length and quality. Filtered-in sequences were submitted to IMGT/HighV-QUEST and an in-house bioinformatics pipeline for clonotype computation and repertoire characterization.

Overall, 9,385,319 productive filtered-in TRBV-TRBD-TRBJ rearrangement sequences were evaluated corresponding to an average of 32,205 different (unique) clonotypes/sample (clonotype: same TRBV gene and CDR3 amino acid sequence). Skewing of the TRBV repertoire was identified in both LC-MBL categories. In specific, the 4/5 most frequently utilized TRBV genes were identical between the 2 categories (TRBV29-1, TRBV12-3, TRBV19, TRBV6-5) and cumulatively accounted for a virtually identical part of the respective repertoire (28.7% versus 28.6%). Comparison to our published data from CLL revealed similarities regarding TRBV gene usage, particularly for CLL-like LC-MBL. Oligoclonality was evident in both MBL categories with the 10 most frequent clonotypes accounting for 21.3% of the total repertoire in CLL-like LC-MBL and 20.3% in non CLL-like LC-MBL, again not differing significantly from CLL (26.3%). All the above 3 entities were clearly more oligoclonal in comparison to 2 healthy control samples from aged-matched used as a reference. Cluster analysis among the 10 most frequent clonotypes of all LC-MBL and CLL samples revealed infrequent matches between: (i) different CLL-like LC-MBL samples (24/430, 0.055%); (ii) CLL-like and non CLL-like LC-MBL (8/500, 0.016%); and, (iii) CLL-like LC-MBL and CLL (2/680, 0.002%).

Altogether, these findings indicate that, similar to CLL, antigen drive likely underlies T-cell expansions also in LC-MBL. However, despite similar TRBV gene usage and degree of oligoclonality, scant shared clonotypes were identified, thus indicating that the antigenic stimuli and/or immune processes shaping the TR profiles in LC-MBL and CLL are distinct.