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. 2020 Mar 10;26(1):25.
doi: 10.1186/s10020-020-00151-9.

Tracing CLL-biased stereotyped immunoglobulin gene rearrangements in normal B cell subsets using a high-throughput immunogenetic approach

Monica Colombo  1 , Davide Bagnara  2 , Daniele Reverberi  3 , Serena Matis  3 , Martina Cardillo  3 , Rosanna Massara  3 , Luca Mastracci  4   5 , Jean Louis Ravetti  4 , Luca Agnelli  6 , Antonino Neri  6 , Michela Mazzocco  7 , Margherita Squillario  8 , Andrea Nicola Mazzarello  9 , Giovanna Cutrona  3 , Andreas Agathangelidis  10 , Kostas Stamatopoulos  10 , Manlio Ferrarini  2 , Franco Fais  3   2
Affiliations

Tracing CLL-biased stereotyped immunoglobulin gene rearrangements in normal B cell subsets using a high-throughput immunogenetic approach

Monica Colombo et al. Mol Med. .

Abstract

Background: B cell receptor Immunoglobulin (BcR IG) repertoire of Chronic Lymphocytic Leukemia (CLL) is characterized by the expression of quasi-identical BcR IG. These are observed in approximately 30% of patients, defined as stereotyped receptors and subdivided into subsets based on specific VH CDR3 aa motifs and phylogenetically related IGHV genes. Although relevant to CLL ontogeny, the distribution of CLL-biased stereotyped immunoglobulin rearrangements (CBS-IG) in normal B cells has not been so far specifically addressed using modern sequencing technologies. Here, we have investigated the presence of CBS-IG in splenic B cell subpopulations (s-BCS) and in CD5+ and CD5- B cells from the spleen and peripheral blood (PB).

Methods: Fractionation of splenic B cells into 9 different B cell subsets and that of spleen and PB into CD5+ and CD5- cells were carried out by FACS sorting. cDNA sequences of BcR IG gene rearrangements were obtained by NGS. Identification of amino acidic motifs typical of CLL stereotyped subsets was carried out on IGHV1-carrying gene sequences and statistical evaluation has been subsequently performed to assess stereotypes distribution.

Results: CBS-IG represented the 0.26% average of IGHV1 genes expressing sequences, were detected in all of the BCS investigated. CBS-IG were more abundant in splenic and circulating CD5+ B (0.57%) cells compared to CD5- B cells (0.17%). In all instances, most CBS IG did not exhibit somatic hypermutation similar to CLL stereotyped receptors. However, compared to CLL, they exhibited a different CLL subset distribution and a broader utilization of the genes of the IGHV1 family.

Conclusions: CBS-IG receptors appear to represent a part of the "public" BcR repertoire in normal B cells. This repertoire is observed in all BCS excluding the hypothesis that CLL stereotyped BcR accumulate in a specific B cell subset, potentially capable of originating a leukemic clone. The different relative representation of CBS-IG in normal B cell subgroups suggests the requirement for additional selective processes before a full transformation into CLL is achieved.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Relative frequency of different stereotype subsets among the CBS-IG rearrangements of s-BCS. a Frequency of subsets used by CBS-IG rearrangements of B cells considered in bulk. b Relative frequency of the subsets found among the CBS-IG rearrangements expressed by the various s-BCS. Stereotype subsets are marked by different colours as indicated
Fig. 2
Fig. 2
Utilization of IGHV1 family genes by CBS-IG and by CLL stereotype rearrangements. Normal (s-BCS) CBS-IG rearrangements are classified as typical (grey) or non-typical (white) based upon whether the IGHV1 genes utilized were the same or different from those commonly used by CLL clones for a given rearrangement (black). The relative frequency of typical and non-typical CBS-IG in s-BCS (considered in bulk) for each stereotype subset is reported on the right of the figure. Numbers in boxes indicate the total number of sequences investigated for each subset
Fig. 3
Fig. 3
SHM in CBS-IG rearrangements of s-BCS. a. Frequency of mutated and unmutated rearrangements in CBS-IG and in non-stereotyped rearrangements (non CBS-IG) in the different s-BCS as indicated. b Frequency of mutated and unmutated CBS-IG rearrangements stratified according to CLL stereotype subsets. Numbers in the boxes indicate the total number of clones analyzed
Fig. 4
Fig. 4
a Box-plots of the frequency (%) of CSB-IG rearrangements in CD5+ and CD5- B cell fractions from three PB and from three spleen samples. Each dot represents a single individual. CD5+ B cells expressed higher number of CBS-IG rearrangements (p = 0.002 PB; p = 0.01 spleens) irrespective of their location, PB or spleen. b CLL stereotyped subset distribution in the CBS-IG rearrangements of CD5+ and CD5-B cells. CLL stereotype subsets are marked by different colours as indicated. c Frequency of mutated and unmutated rearrangements in CBS-IG and in non CBS-IG rearrangements in the CD5+ and CD5-B cells as indicated
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