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Potential Relevance of B-cell Maturation Pathways in Defining the Cell(s) of Origin for Chronic Lymphocytic Leukemia

  • Anita Ng
    Affiliations
    The Karches Center for Oncology Research, Institute for Molecular Medicine, The Feinstein Institutes for Medical Research, 350 Community Drive, Manhasset, NY 11030, USA

    Donald and Barbara Zucker School of Medicine at Hofstra/Northwell, 350 Community Drive, Manhasset, NY 11030, USA
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  • Nicholas Chiorazzi
    Correspondence
    Corresponding author.
    Affiliations
    The Karches Center for Oncology Research, Institute for Molecular Medicine, The Feinstein Institutes for Medical Research, 350 Community Drive, Manhasset, NY 11030, USA

    Donald and Barbara Zucker School of Medicine at Hofstra/Northwell, 350 Community Drive, Manhasset, NY 11030, USA
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Open AccessPublished:June 01, 2021DOI:https://doi.org/10.1016/j.hoc.2021.03.002

      Keywords

      Key points

      • Chronic lymphocytic leukemia (CLL) is a clonal B-cell disease with a distinct membrane phenotype.
      • Although both immunoglobulin heavy-chain variable region (IGHV) unmutated and IGHV mutated CLL clones show mature B-cell phenotypes, the difference in IGHV mutations has led to the hypotheses of multiple cells of origin that are separated by having traversed or not having traversed a germinal center, the landmark in classic B-cell development.
      • Recent immunogenetic and epigenetic studies have suggested that all CLL clones are most similar to antigen-experienced, memorylike normal B lymphocytes.
      • In normal B-cell biology, the memory stage of maturation can be reached by several pathways that differ in T-cell dependence, and the resultant memory cells can be subdivided into subtypes.
      • This article emphasizes the need to incorporate considerations of the type of B-cell maturation pathways, and possibly the memory B-cell subtypes to which they lead, when trying to define the CLL cell of origin.

      Introduction

      Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease characterized by the expansion of clonal cluster of differentiation (CD) 5+CD19+ B cells in the blood, bone marrow (BM), and lymphoid tissues.
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      Chronic lymphocytic leukaemia.
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      Chronic Lymphocytic leukemia.
      The understanding and hypotheses about the underlying molecular cause of CLL have evolved as technological advances were made. Historically, when analyzed by light microscopy, CLL cells were considered small, mature, resting B lymphocytes.
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      However, when flow cytometry became available, it was recognized that CLL cells resembled activated lymphocytes.
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      B-cell chronic lymphocytic leukemia cells express a surface membrane phenotype of activated, antigen-experienced B lymphocytes.
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      The history and future of the fluorescence activated cell sorter and flow cytometry: a view from Stanford.
      Similarly, with the availability of Sanger sequencing, the discovery of differences in immunoglobulin heavy-chain variable region (IGHV) mutations that divided patients with CLL into subsets
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      Chronic lymphocytic leukemia B cells express restricted sets of mutated and unmutated antigen receptors.
      with different patient outcomes
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      Unmutated Ig V(H) genes are associated with a more aggressive form of chronic lymphocytic leukemia.
      was made. This discovery provided a valuable prognostic indicator and led to 2 cells of origin (COOs) hypotheses.
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      Next, however, the capacity to measure gene expression by microarray analyses indicated relatively homogenous transcriptomes for unmutated CLL (U-CLL) and mutated CLL (M-CLL),
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      Gene expression profiling of B cell chronic lymphocytic leukemia reveals a homogeneous phenotype related to memory B cells.
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      Relation of gene expression phenotype to immunoglobulin mutation genotype in B cell chronic lymphocytic leukemia.
      giving rise to new hypotheses that attempted to reconcile the apparent homogeneity of the transcriptome with the clinical and immunologic heterogeneity. Subsequently, again based on DNA sequencing, subsets of patients with CLL expressing remarkably similar stereotyped B-cell receptors (BCRs) were identified.
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      Chronic lymphocytic leukemias utilizing the VH3-21 gene display highly restricted Vλ2-14 gene use and homologous CDR3s: implicating recognition of a common antigen epitope.
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      Remarkably similar antigen receptors among a subset of patients with chronic lymphocytic leukemia.
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      Multiple distinct sets of stereotyped antigen receptors indicate a role for antigen in promoting chronic lymphocytic leukemia.
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      Subsets with restricted immunoglobulin gene rearrangement features indicate a role for antigen selection in the development of chronic lymphocytic leukemia.
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      Chronic lymphocytic leukemia B cells of more than 1% of patients express virtually identical immunoglobulins.
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      Stereotyped patterns of somatic hypermutation in subsets of patients with chronic lymphocytic leukemia: implications for the role of antigen selection in leukemogenesis.
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      Stereotyped B-cell receptors in one-third of chronic lymphocytic leukemia: a molecular classification with implications for targeted therapies.
      This finding again suggested that more than 1 COO might exist. Most recently, massively parallel, deep sequencing of DNA and RNA has reshaped the hypotheses of CLL differentiation and clonal architectures.
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      Mutations driving CLL and their evolution in progression and relapse.
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      Epigenetic evolution and lineage histories of chronic lymphocytic leukaemia.
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      Tracing CLL-biased stereotyped immunoglobulin gene rearrangements in normal B cell subsets using a high-throughput immunogenetic approach.
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      SF3B1 and other novel cancer genes in chronic lymphocytic leukemia.
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      Exome sequencing identifies recurrent mutations of the splicing factor SF3B1 gene in chronic lymphocytic leukemia.
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      DNA methylation dynamics during B cell maturation underlie a continuum of disease phenotypes in chronic lymphocytic leukemia.
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      A B-cell epigenetic signature defines three biologic subgroups of chronic lymphocytic leukemia with clinical impact.
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      The proliferative history shapes the DNA methylome of B-cell tumors and predicts clinical outcome.
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      Methylome-based cell-of-origin modeling (Methyl-COOM) identifies aberrant expression of immune regulatory molecules in CLL.
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      Growth dynamics in naturally progressing chronic lymphocytic leukaemia.
      This article reviews the literature relating to this issue, focusing on recent findings, made through epigenetic analyses, that strongly support the disease developing from a normal B lymphocyte that is antigen (Ag) experienced and memory B-cell–like. It also relates the pathways whereby normal B lymphocytes mature after Ag challenge and proposes that this information is relevant when trying to define the COOs of this disease.

      Normal B-lymphocyte development and maturation

      Early B-cell Development

      B cells derive from hematopoietic stem cells (HSCs) in the fetal liver or BM, maturing by a stepwise series of maturational events (Fig. 1A). After lymphoid progenitor cells become committed to the B-cell lineage, defined by the expression of CD19, serial gene recombination events, involving IGHV, immunoglobulin heavy-chain diversity (IGHD), and immunoglobulin heavy-chain joining (IGHJ) genes, take place at the pro–B cell and pre–B-cell stages. Accompanying recombinations of immunoglobulin light-chain variable (IGLV) and IGLJ genes initiate at the immature B-cell stage. Immature B lymphocytes then can transition from the BM to the periphery as transitional B cells (TrBCs), which mature into naive B cells (NBCs). Collectively, these B-lymphocyte subsets express diverse, functional BCR repertoires.
      Figure thumbnail gr1
      Fig. 1Normal B-cell differentiation. B-cell maturation and BCR-mediated selection of B cells occurs in 2 phases that differ in the type of Ag encountered. The first phase begins in early development when foreign Ag is normally absent. The second phase is mediated by foreign and pathogenic Ag that mainly concerns B cells in the periphery, lymph nodes, and secondary lymphoid organs (SLOs). (A) Early B-cell development in the BM. In the BM, BCR-mediated signaling begins from the progenitor stage, being induced by dimerization of the pre-B BCR. Productive pairing of IGHV-D-J with surrogate light-chain (SLC) leads to survival and proliferation of the pre-B cells. Although SLC pairs nonrandomly with IGHV-D-J μ chains, because of the monomorphic nature of SLC and the autologous nature of Ag, the restricted repertoire is biased toward autoreactivity at this stage. When the translated IGκJ or IGλJ are paired with IGHV-D-J μ chain, immature B cells with autoreactive BCRs are selected against by central tolerance mechanisms: clonal deletion, receptor editing of κ/λ V genes, and receptor revision of IGHV. The selected cells exit to the periphery as transitional B cells (TrBCs) or newly formed B (NF-B) cells. (B) T cell–dependent maturation at extrafollicular sites (EF-TD). In the primary immune response, Ag-specific B cells and T cells interact and the activated B cells proliferate rapidly and differentiate into IGHV-unmutated MBCs, and short-lived PBs are generated in this pre-GC phase. (C) T cell–dependent B-cell maturation in lymphoid follicles of SLOs (GC-TD pathway). Naive B cells (NBCs) arrive at lymphoid follicles ∼5 days after TD-Ag engagement and most become FO B cells. After the formation of GCs, GCBs in dark zone downregulate BCRs, proliferate, and undergo somatic hypermutation (SHM) in an activation-induced cytidine deaminase (AID)–dependent manner, referred as the GC reaction. Subsequently, these cells migrate into the GC light zone and reexpress BCRs. In order to receive survival signals, centrocytes compete for the opportunity to bind the initiating Ag presented by follicular dendritic cells (FDCs). T-follicular helper cells (Tfh) then rescue B cells after binding major histocompatibility complex (MHC)–processed Ag. The affinity-matured B cells can become either memory B cells (MBCs) or age-associated B cells (ASCs) with higher-affinity BCRs that are often isotype switched. (D) Early development of murine B-1 cells in fetal liver. In the fetal stage, B-cell development takes place in the liver and precursors migrate to the BM after birth. Afterward, early B-1 cells differentiate by the series of recombination and rearrangement events similar to development of non–B-1 cells in BM. However, negative selection at the pre-B stage seems to be less stringent. Note that the origin of B-1 cells is still debated: do they arise from a genetically distinct progenitor cell and give rise exclusively to B-1 cells, or do they come from a common B-cell progenitor that can acquire B-1 or B-2 cell features and fates based on antigenic and microenvironmental drive? (E) T cell–independent (TI) maturation at extrafollicular sites (EF-TI pathway). Depending on the specific cell type, mature B cells can be activated in a TI manner outside of lymphoid follicles and mature to MBCs and ASCs. SHM and CSR occurring at these EF sites are mediated by AID and the result is termed the EF reaction. MBCs and plasma cells (PCs) generated in this manner differ from GC-experienced effector cells in terms of isotypes, SHM load, and BCR reactivity. Marginal zone B cells (MZBs) and murine B-1 cells are examples of B cells differentiating at an EF site. (B) T cell–dependent maturation at extrafollicular sites (EF-TD). FO, follicular; TD, T-cell dependent.
      To maintain a BCR repertoire of sufficient diversity while reducing the potential for autoimmunity, central (BM) and peripheral (secondary lymphoid organs) mechanisms to eliminate self-reactivity are necessary. Such mechanisms include anergy,
      • Cambier J.C.
      • Gauld S.B.
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      B-cell anergy: from transgenic models to naturally occurring anergic B cells?.
      clonal deletion,
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      • Brink R.
      • et al.
      Elimination from peripheral lymphoid tissues of self-reactive B lymphocytes recognizing membrane-bound antigens.
      IGLV receptor editing, and IGHV receptor revision.
      • Nemazee D.
      Receptor editing in lymphocyte development and central tolerance.
      The last 3 options seem restricted to the BM, whereas anergy seems most relevant in the periphery. Consequently, cells with autoreactive BCRs are greatly reduced as B cells mature, being ∼50% of immature B cells, 20% of NBCs, and 2% in immunoglobulin M+ (IgM+) memory B cells (MBCs).
      • Tsuiji M.
      • Yurasov S.
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      A checkpoint for autoreactivity in human IgM memory B cell development.
      ,
      • Tiller T.
      • Tsuiji M.
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      • et al.
      Autoreactivity in human IgG+ memory B cells.
      B-cell clones with polyreactive BCRs decrease from 7% in immature subsets to 4% in NBCs.
      • Tsuiji M.
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      • et al.
      A checkpoint for autoreactivity in human IgM memory B cell development.
      ,
      • Tiller T.
      • Tsuiji M.
      • Yurasov S.
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      Autoreactivity in human IgG+ memory B cells.
      In addition, IGHV gene usage, particularly the relative use of IGHV1 and IGHV3 family genes, differs between B-cell subsets, with IGHV3 family usage decreasing from the immature to naive developmental stages,
      • Martin V.
      • Wu Y.-C.
      • Kipling D.
      • et al.
      Age-related aspects of human IgM(+) B cell heterogeneity.
      the most significant decrease occurring between NBCs and isotype-switched MBCs.
      • Martin V.
      • Wu Y.-C.
      • Kipling D.
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      Age-related aspects of human IgM(+) B cell heterogeneity.
      • Wu Y.-C.
      • Kipling D.
      • Leong H.S.
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      High-throughput immunoglobulin repertoire analysis distinguishes between human IgM memory and switched memory B-cell populations.
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      • Townsend C.L.
      • et al.
      Transitional B cells in early human B cell development - time to revisit the Paradigm?.
      A significant change in the repertoire occurs between IgM and switched MBCs, suggesting that some IgM MBCs are subjected to different selective pressures, potentially by following a different developmental pathway than non-IgM MBCs.
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      • Kipling D.
      • Leong H.S.
      • et al.
      High-throughput immunoglobulin repertoire analysis distinguishes between human IgM memory and switched memory B-cell populations.

      B-cell Differentiation Pathways

      B lymphocytes can follow several differentiation pathways that differ in the anatomic locations at which they occur, types of initiating Ag, requirement for T-cell help, and the degree that they promote somatic hypermutation (SHM) and class switch recombination (CSR). However, it is important to remember that these dynamics are most often and most easily studied in rodent systems, so understanding of the human response is often derivative.
      The current understanding is that MBCs and antibody (Ab) secreting cell (ASCs) are generated via 3 maturation pathways: T cell–dependent (TD) activation, occurring outside of lymphoid follicles, at extrafollicular (EF) sites (EF-TD path); TD activation, occurring within lymphoid follicles (classic germinal center [GC] reaction; GC-TD path); and T cell–independent (TI) activation occurring at EF sites (EF-TI path).

      T cell–dependent maturation at extrafollicular sites response

      On the initial encounter with immunostimulatory Ags, NBCs migrate to the splenic red pulp, where T cells and interdigitating dendritic cells reside. This phase is the first part of the immune response that develops outside of lymphoid follicles (Fig. 1B). Although B-cell expansion is meager at this point, maturation occurs rapidly (∼3 days), generating short-lived (also ∼3 days) plasmablasts (PBs) along with IgG and IgM MBCs.
      • Takemori T.
      • Kaji T.
      • Takahashi Y.
      • et al.
      Generation of memory B cells inside and outside germinal centers.
      ,
      • Inamine A.
      • Takahashi Y.
      • Baba N.
      • et al.
      Two waves of memory B-cell generation in the primary immune response.
      The Abs secreted by PBs and found on the membranes of MBCs carry minimal IGHV mutations.
      • Takemori T.
      • Kaji T.
      • Takahashi Y.
      • et al.
      Generation of memory B cells inside and outside germinal centers.
      ,
      • Inamine A.
      • Takahashi Y.
      • Baba N.
      • et al.
      Two waves of memory B-cell generation in the primary immune response.

      Classic germinal center T cell–dependent response

      Concurrently, a portion of B cells primed during this EF phase migrate to the splenic white pulp, where nests of follicular dendritic cells (FDCs) provide the nidus for B-cell follicle development (Fig. 1C).
      • Zotos D.
      • Tarlinton D.M.
      Determining germinal centre B cell fate.
      B-cell follicle development occurs on influx of Ag-activated, IgMlowIgDhigh cells that divide considerably in situ, downregulate membrane BCRs, and upregulate the activation-induced cytidine deaminase (AID) enzyme.
      • Rajewsky K.
      Clonal selection and learning in the antibody system.
      These cells, now considered centroblasts, create the GC dark zone. Based on the actions of AID, the cells can undergo CSR and SHM of IGHV-IGHD-IGHJ (IGHV-D-J) and IGLV-IGLJ (IGLV-J) rearranged genes.
      • Casellas R.
      • Basu U.
      • Yewdell W.T.
      • et al.
      Mutations, kataegis and translocations in B cells: understanding AID promiscuous activity.
      B cells that have exited the cell cycle (centrocytes) congregate at a pole, constituting the light zone of the GC. Enormous death occurs there and only those cells that bind and internalize native FDC-expressed Ag and present them on the cell surface receive a final rescue signal from GC T cells (T-follicular helper cells [Tfh]), which bind major histocompatibility complex II (MHCII)–associated processed Ag on the B-cell surface. Competition for FDC-displayed Ag is a major mechanism whereby improved BCR affinity is achieved. This T cell–mediated, B-cell diversity–generating response is referred to as the classic GC reaction and leads to the development of MBCs and plasma cells (PCs). The specificity of the BCRs of the former and the secreted Abs from the latter reflects the Ag that initiated the immune response. Because of the enormous number of GC reactions that occur normally over time, the Ag-binding repertoire is diverse, as reflected best by isotope-switched Abs.
      • Shlomchik M.J.
      • Weisel F.
      Germinal center selection and the development of memory B and plasma cells.
      ,
      • Cyster J.G.
      • Allen C.D.C.
      B cell responses: cell interaction dynamics and decisions.
      Eventually, the GC reaction wanes (1 week to several weeks) and GCs involute; the rates at which these happen seem to be controlled by the type and availability of Ag, and the level and persistence of T-cell help.
      • MacLennan I.C.M.
      Germinal Centers.
      ,
      • Seifert M.
      • Küppers R.
      Human memory B cells.

      Extrafollicular T cell–independent responses

      B-cell differentiation can also occur without T-cell help, called the TI response. B cells undergoing TI responses often recognize repetitive structural epitopes that can be found on natural pathogens, such as polysaccharides, viral capsids, and nucleic acids, or autologous cellular debris.
      • Ramiscal R.R.
      • Vinuesa C.G.
      T-cell subsets in the germinal center.
      Furthermore, maturation driven by TI-Ags often involves more innatelike B cells that mature in an EF manner, such as marginal zone B cells (MZBs) and murine B-1 cells. MZBs primarily reside in the white pulp of the spleen, subepithelial areas of the tonsil, Peyer patches, and subcapsular regions of lymph nodes.
      • Cerutti A.
      • Cols M.
      • Puga I.
      Marginal zone B cells: virtues of innate-like antibody-producing lymphocytes.
      Their strategic locations and rapid innatelike Ab responses provide the host with a first line of defense. ASCs generated in a TI manner elaborate mostly IgMs of low affinity and polyreactivity, although, because AID can be upregulated in this setting, SHM and BCR affinity maturation can occur to a lesser degree.
      • Shlomchik M.J.
      • Weisel F.
      Germinal center selection and the development of memory B and plasma cells.
      In addition to MZBs, B cells of various types can upregulate AID and undergo CSR and SHM outside of GCs.
      • Weller S.
      • Braun M.C.
      • Tan B.K.
      • et al.
      Human blood IgM “memory” B cells are circulating splenic marginal zone B cells harboring a prediversified immunoglobulin repertoire.
      • Weller S.
      • Mamani-Matsuda M.
      • Picard C.
      • et al.
      Somatic diversification in the absence of antigen-driven responses is the hallmark of the IgM+ IgD+ CD27+ B cell repertoire in infants.
      • Scheeren F.A.
      • Nagasawa M.
      • Weijer K.
      • et al.
      T cell-independent development and induction of somatic hypermutation in human IgM+ IgD+ CD27+ B cells.
      Because these AID-mediated events occur outside of lymphoid follicles, the process is referred to as an EF reaction. The types of ancillary cells present at the sites where EF reactions take place are not equivalent to those where GC reactions occur, nor is their organization as defined as in GCs (Fig. 1E).
      • Ramiscal R.R.
      • Vinuesa C.G.
      T-cell subsets in the germinal center.
      ,
      • Nutt S.L.
      • Tarlinton D.M.
      Germinal center B and follicular helper T cells: siblings, cousins or just good friends?.
      Possibly because of these differences, the SHM load and fidelity of selection for the initiating Ag are not as rigid in an EF reaction as in a GC reaction.
      In this regard, patients with hyper-IgM syndrome, type 1, who genetically lack functional CD154 and hence cannot form GCs, generate IgM+IgD+CD27+ MBCs with IGHV-mutated Abs.
      • Kuraoka M.
      • Liao D.
      • Yang K.
      • et al.
      Activation-induced cytidine deaminase expression and activity in the absence of germinal centers: insights into hyper-IgM syndrome.
      ,
      • Weller S.
      • Faili A.
      • Garcia C.
      • et al.
      CD40-CD40L independent Ig gene hypermutation suggests a second B cell diversification pathway in humans.
      In addition, analyses of human fetuses revealed a small population of IgM+IgD+CD27+ cells with IGHV-mutated genes in liver and mesenteric lymph nodes.
      • Scheeren F.A.
      • Nagasawa M.
      • Weijer K.
      • et al.
      T cell-independent development and induction of somatic hypermutation in human IgM+ IgD+ CD27+ B cells.
      ,
      • Kuraoka M.
      • Liao D.
      • Yang K.
      • et al.
      Activation-induced cytidine deaminase expression and activity in the absence of germinal centers: insights into hyper-IgM syndrome.
      Hence, CSR and SHM do not occur exclusively in GCs, and these are 2 spontaneous examples of EF reactions culminating in CSR and SHM in the absence of GCs.
      In addition, AID can be expressed in immature B cells and TrBCs from fetal liver,
      • Kuraoka M.
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      • Yang K.
      • et al.
      Activation-induced cytidine deaminase expression and activity in the absence of germinal centers: insights into hyper-IgM syndrome.
      BM, and umbilical cord blood.
      • Martin V.G.
      • Wu Y.-C.B.
      • Townsend C.L.
      • et al.
      Transitional B cells in early human B cell development - time to revisit the Paradigm?.
      Moreover, within the phenotypic window of TrBCs, there is a newly characterized population of recent BM emigrants that are not merely precursors for NBCs, termed newly formed B (NF-B) cells.
      • Giltiay N.V.
      • Giordano D.
      • Clark E.A.
      The plasticity of newly formed B cells.
      Although they are immature, NF-B cells can undergo CSR, SHM, and become ASCs after BCR activation by self-Ag and signal via Toll-like receptor (TLR) 7.
      • Giltiay N.V.
      • Giordano D.
      • Clark E.A.
      The plasticity of newly formed B cells.
      ,
      • Capolunghi F.
      • Cascioli S.
      • Giorda E.
      • et al.
      CpG drives human transitional B cells to terminal differentiation and production of natural antibodies.
      In addition, AID can be upregulated in vitro by both TD and TI stimuli.
      • Cantaert T.
      • Schickel J.-N.
      • Bannock J.M.
      • et al.
      Activation-induced cytidine deaminase expression in human B cell precursors is essential for central B cell tolerance.
      For example, activating B cells via BCR and TLRs, or engaging BCRs with sufficient signaling strength, can drive maturation and proliferation without costimulation from Tfh.
      • Capolunghi F.
      • Cascioli S.
      • Giorda E.
      • et al.
      CpG drives human transitional B cells to terminal differentiation and production of natural antibodies.
      In addition to the development of IgM+IgD+CD27+ MBCs, murine B-1 cells travel an EF pathway that leads primarily to the maturation of IgM-producing B cells.
      • Sindhava V.J.
      • Bondada S.
      Multiple regulatory mechanisms control B-1 B cell activation.
      This murine B-lymphocyte compartment is divided into 2 categories: B-1 and B-2 cells. Although there is debate as to whether the 2 populations are genetically distinct
      • Kantor A.B.
      • Herzenberg L.A.
      Origin of murine B cell lineages.
      ,
      • Ghosn E.E.B.
      • Yang Y.
      Hematopoietic stem cell-independent B-1a lineage: HSC-independent hematopoiesis.
      or reflect differences based on Ag drive,
      • Casola S.
      • Otipoby K.L.
      • Alimzhanov M.
      • et al.
      B cell receptor signal strength determines B cell fate.
      ,
      • Wong J.B.
      • Hewitt S.L.
      • Heltemes-Harris L.M.
      • et al.
      B-1a cells acquire their unique characteristics by bypassing the pre-BCR selection stage.
      these 2 subsets follow distinct maturation pathways.
      B-2 cells are BM-derived, CD5 B lymphocytes that give rise to follicular (FO) B cells and MZBs. B-1 cells are long-lived B lymphocytes that develop in fetal liver and BM. B-1 cells can express CD5 (B-1a), or not (B-1b). In fetal liver, B-1a precursors can differentiate via an alternative pathway that bypasses the surrogate light-chain selection checkpoint,
      • Wong J.B.
      • Hewitt S.L.
      • Heltemes-Harris L.M.
      • et al.
      B-1a cells acquire their unique characteristics by bypassing the pre-BCR selection stage.
      generating polyreactive and autoreactive BCRs with a restricted repertoire (Fig. 1E). B-1 cells have self-renewing properties and perform more innatelike B-cell responses such as binding microbial and self-Ags,
      • Berland R.
      • Wortis H.H.
      Origins and functions of B-1 cells with notes on the role of CD5.
      • Baumgarth N.
      The double life of a B-1 cell: self-reactivity selects for protective effector functions.
      • Duan B.
      • Morel L.
      Role of B-1a cells in autoimmunity.
      the latter property helping to clear altered and dying cells. B-1 cells do not require the participation of T lymphocytes for activation and differentiation, and are found primarily in the peritoneal and pleural cavities.
      • Sindhava V.J.
      • Bondada S.
      Multiple regulatory mechanisms control B-1 B cell activation.
      Moreover, B-1 cells rarely switch to non-IgM isotypes, and their Ag-binding sites are not as specific as B-2 cells, because the immunoglobulin variable domains of B-1 cells are usually unmutated, with few N additions.
      • Kantor A.B.
      • Merrill C.E.
      • Herzenberg L.A.
      • et al.
      An unbiased analysis of V(H)-D-J(H) sequences from B-1a, B-1b, and conventional B cells.
      However, it seems that a subset of B-1 cells bearing surface membrane CD25 express AID, implying that they could undergo CSR and SHM.
      • Kaku H.
      • Holodick N.E.
      • Tumang J.R.
      • et al.
      CD25 + B-1a cells express Aicda.
      In addition, as B-1 cells age, the population seems to express BCRs with different structural features, including an increase of clones with more N additions.
      • Holodick N.E.
      • Repetny K.
      • Zhong X.
      • et al.
      Adult BM generates CD5+ B1 cells containing abundant N-region additions.
      In addition, there is a population of murine B cells that develop with age and have unique phenotypic, molecular, and functional properties; these are termed age-associated B cells (ABCs).
      • Rubtsova K.
      • Rubtsov A.V.
      • Cancro M.P.
      • et al.
      Age-associated B cells: a T-bet–dependent effector with roles in protective and pathogenic immunity.
      • Rubtsov A.V.
      • Rubtsova K.
      • Fischer A.
      • et al.
      Toll-like receptor 7 (TLR7)-driven accumulation of a novel CD11c+ B-cell population is important for the development of autoimmunity.
      • Cancro M.P.
      Age-Associated B Cells.
      Human ABCs bear a CD11c+CD86+CD5intCD21CD23CD43 surface phenotype
      • Rubtsova K.
      • Rubtsov A.V.
      • Cancro M.P.
      • et al.
      Age-associated B cells: a T-bet–dependent effector with roles in protective and pathogenic immunity.
      and are directed by a T-bet (T-box expressed in T cells) transcriptional program, unlike conventional B-cell subsets.
      • Rubtsova K.
      • Rubtsov A.V.
      • Cancro M.P.
      • et al.
      Age-associated B cells: a T-bet–dependent effector with roles in protective and pathogenic immunity.
      • Rubtsov A.V.
      • Rubtsova K.
      • Fischer A.
      • et al.
      Toll-like receptor 7 (TLR7)-driven accumulation of a novel CD11c+ B-cell population is important for the development of autoimmunity.
      • Cancro M.P.
      Age-Associated B Cells.
      Furthermore, ABCs carry IGHV-unmutated or IGHV-mutated BCRs showing an innatelike repertoire, akin to B-1 cells and MZBs. ABCs can differentiate via TD and TI pathways.
      • Zumaquero E.
      • Stone S.L.
      • Scharer C.D.
      • et al.
      IFNγ induces epigenetic programming of human T-bethi B cells and promotes TLR7/8 and IL-21 induced differentiation.
      • Di Niro R.
      • Lee S.-J.
      • Vander Heiden J.A.
      • et al.
      Salmonella infection drives promiscuous B cell activation followed by extrafollicular affinity maturation.
      • Naradikian M.S.
      • Myles A.
      • Beiting D.P.
      • et al.
      Cutting edge: IL-4, IL-21, and IFN-γ interact to govern T-bet and CD11c expression in TLR-activated B cells.

      Features of chronic lymphocytic leukemia cells to consider when assigning cell of origin

      Membrane Phenotype

      CLL has the characteristic immunophenotype of CD5+CD19+CD27+CD43+, with coexpression of IgM and IgD (IgG in 5%–10% of all cases).
      • Chiorazzi N.
      • Chen S.-S.
      • Rai K.R.
      Chronic Lymphocytic leukemia.
      ,
      • Geisler C.H.
      • Larsen J.K.
      • Hansen N.E.
      • et al.
      Prognostic importance of flow cytometric immunophenotyping of 540 consecutive patients with B-cell chronic lymphocytic leukemia.
      ,
      • Potter K.N.
      • Mockridge C.I.
      • Neville L.
      • et al.
      Structural and functional features of the B-cell receptor in IgG-positive chronic lymphocytic leukemia.
      Ever since the discovery of CD5 expression on CLL cells in the early 1980s,
      • Royston I.
      • Majda J.A.
      • Baird S.M.
      • et al.
      Human T cell antigens defined by monoclonal antibodies: the 65,000-dalton antigen of T cells (T65) is also found on chronic lymphocytic leukemia cells bearing surface immunoglobulin.
      ,
      • Wang C.Y.
      • Good R.A.
      • Ammirati P.
      • et al.
      Identification of a p69,71 complex expressed on human T cells sharing determinants with B-type chronic lymphatic leukemic cells.
      CD5 has been an essential membrane feature to assign the diagnosis of CLL (Table 1). Like murine B-1 cells, human CD5+ B lymphocytes constitute a major subset at birth and wane with age.
      • Caligaris-Cappio F.
      • Ghia P.
      The nature and origin of the B-chronic lymphocytic leukemia cell: a tentative model.
      ,
      • Hardy R.R.
      • Hayakawa K.
      • Shimizu M.
      • et al.
      Rheumatoid factor secretion from human Leu-1+ B cells.
      Moreover, the human CD5+ B-cell compartment comprises TrBCs, mature follicular mantle zone cells,
      • Caligaris-Cappio F.
      • Gobbi M.
      • Bofill M.
      • et al.
      Infrequent normal B lymphocytes express features of B-chronic lymphocytic leukemia.
      and ABCs, which predominantly produce IgM and carry polyreactive IGHV-unmutated BCRs.
      • Martin V.
      • Wu Y.-C.
      • Kipling D.
      • et al.
      Age-related aspects of human IgM(+) B cell heterogeneity.
      ,
      • Rubtsova K.
      • Rubtsov A.V.
      • Cancro M.P.
      • et al.
      Age-associated B cells: a T-bet–dependent effector with roles in protective and pathogenic immunity.
      • Rubtsov A.V.
      • Rubtsova K.
      • Fischer A.
      • et al.
      Toll-like receptor 7 (TLR7)-driven accumulation of a novel CD11c+ B-cell population is important for the development of autoimmunity.
      • Cancro M.P.
      Age-Associated B Cells.
      The equivalent of murine B-1 cells in humans is a matter of debate, although human B cells with a CD43+CD20+CD27+CD70 membrane phenotype and the capacity to spontaneously secrete Abs are a possibility.
      • Rothstein T.L.
      • Quach T.D.
      The human counterpart of mouse B-1 cells.
      Table 1Chronic lymphocytic leukemia–related characteristics in normal B-cell subsets
      CLL CharacteristicsTransitional B CellsNewly Formed B Cells
      • Giltiay N.V.
      • Giordano D.
      • Clark E.A.
      The plasticity of newly formed B cells.
      Naive B CellsMarginal Zone B CellsCD5+ B Cells (PB)
      • Seifert M.
      • Sellmann L.
      • Bloehdorn J.
      • et al.
      Cellular origin and pathophysiology of chronic lymphocytic leukemia.
      Age-Associated B Cells
      • Holodick N.E.
      • Repetny K.
      • Zhong X.
      • et al.
      Adult BM generates CD5+ B1 cells containing abundant N-region additions.
      • Rubtsova K.
      • Rubtsov A.V.
      • Cancro M.P.
      • et al.
      Age-associated B cells: a T-bet–dependent effector with roles in protective and pathogenic immunity.
      • Rubtsov A.V.
      • Rubtsova K.
      • Fischer A.
      • et al.
      Toll-like receptor 7 (TLR7)-driven accumulation of a novel CD11c+ B-cell population is important for the development of autoimmunity.
      MBCs
      Membrane phenotype

      (4) CD5+CD27+CD43+: sIgMhigh sIgDlow
      Partial (only CD5+)Partial (only CD5+)NoNo (CD5)Partial (both CLL types are CD27+ but only CD27 cells are IGHV unmutated)Partial (unknown for hCD43)No (only CD27+)
      • IGHV mutation statues
        • Unmutated or mutated
      Yes (on activation)Yes (on activation)Yes (on activation)YesYes (CD27 unmutated; CD27+ mutated)YesPartial (only mutated in post-GC MBCs)
      • BCR reactivity
        • Polyreactive
        • Autoreactive
      YesYesNoYesYesYesNo
      BCR restriction/stereotypyNoYesNoYesYesYesNo
      GC maturation pathwayPartial (AID+: SHM?)Partial (AID+: SHM?)YesYesYes (CD27+)YesYes
      EF maturation pathwayPartial (AID+: SHM?)YesYes (in TD response)YesYes (CD27+)YesYes (only in EF MBCs)
      Gene expression profile
      Comparative data unavailable.
      Comparative data unavailable.
      NoNoYes (CD27+: U-CLL) (CD27+: M-CLL)
      Comparative data unavailable.
      Yes
      Methylation landscape
      Comparative data unavailable.
      Comparative data unavailable.
      Yes (more U-CLL)Yes
      Comparative data unavailable.
      Comparative data unavailable.
      Yes
      a Comparative data unavailable.
      Consistent with the concept that CLL cells are Ag experienced is the uniform expression of CD27, a marker for activated or MBCs. Consistent with their maturational age, TrBCs and mature NBCs do not express CD27. In addition to MBCs, MZB cells display CD27 and share a IgMhighIgDlowCD27+ membrane phenotype with MBCs in circulation.
      • Weill J.-C.
      • Weller S.
      • Reynaud C.-A.
      Human marginal zone B cells.
      • Carsetti R.
      • Rosado M.M.
      • Wardmann H.
      Peripheral development of B cells in mouse and man.
      • Klein U.
      • Rajewsky K.
      • Küppers R.
      Human immunoglobulin (Ig)M+IgD+ peripheral blood B cells expressing the CD27 cell surface antigen carry somatically mutated variable region genes: CD27 as a general marker for somatically mutated (memory) B cells.
      In addition, CD43 is another pan T-cell marker expressed on most CLL cells that is frequently used in conjunction with CD5 to distinguish normal and neoplastic B lymphocytes.
      • Wikén M.
      • Björck P.
      • Axelsson B.
      • et al.
      Studies on the role of CD43 in human B-cell activation and differentiation.
      CD43 is expressed on most human B-cell progenitors in BM, 25% to 30% of human B cells in the periphery,
      • Wiken M.
      • Robertsson E.S.
      • Axelsson B.
      • et al.
      Characterization of a subpopulation of human peripheral lymphocytes-t expressing the large sialoglycoprotein, GP150.
      a portion of activated B cells in the tonsil,
      • Wikén M.
      • Björck P.
      • Axelsson B.
      • et al.
      Studies on the role of CD43 in human B-cell activation and differentiation.
      and on most terminally differentiated PCs.
      • Wikén M.
      • Björck P.
      • Axelsson B.
      • et al.
      Induction of CD43 expression during activation and terminal differentiation of human B cells.
      CD43 expression seems to correlate with activation and proliferation in in vivo and in vitro settings.
      • Wikén M.
      • Björck P.
      • Axelsson B.
      • et al.
      Studies on the role of CD43 in human B-cell activation and differentiation.
      ,
      • Wikén M.
      • Björck P.
      • Axelsson B.
      • et al.
      Induction of CD43 expression during activation and terminal differentiation of human B cells.
      ,
      • Björck P.
      • Axelsson B.
      • Paulie S.
      Expression of CD40 and CD43 during activation of human B lymphocytes.

      Presence of Immunoglobulin Heavy-Chain Variable Region Gene Mutations

      The difference in IGHV gene mutations among patients with CLL remains a key distinction. In particular, 2 key unresolved issues need to be taken into account.
      First is the lack of IGHV mutations in U-CLL, which is their main similarity to NBCs. However, U-CLL could have gone down a maturation pathway but been unable to perform SHM; alternatively, induced mutations could have been selected against. In this regard, ex vivo U-CLL clones can express AICDA messenger RNA
      • Palacios F.
      • Moreno P.
      • Morande P.
      • et al.
      High expression of AID and active class switch recombination might account for a more aggressive disease in unmutated CLL patients: link with an activated microenvironment in CLL disease.
      • Patten P.E.M.
      • Chu C.C.
      • Albesiano E.
      • et al.
      IGHV-unmutated and IGHV-mutated chronic lymphocytic leukemia cells produce activation-induced deaminase protein with a full range of biologic functions.
      • Oppezzo P.
      • Vuillier F.
      • Vasconcelos Y.
      • et al.
      Chronic lymphocytic leukemia B cells expressing AID display dissociation between class switch recombination and somatic hypermutation.
      and clonal IGHV-D-J transcripts of non-IgM isotypes.
      • Oppezzo P.
      • Magnac C.
      • Bianchi S.
      • et al.
      Do CLL B cells correspond to naive or memory B-lymphocytes? Evidence for an active Ig switch unrelated to phenotype expression and Ig mutational pattern in B-CLL cells.
      ,
      • Vardi A.
      • Agathangelidis A.
      • Sutton L.-A.
      • et al.
      IgG-switched CLL has a distinct immunogenetic signature from the common MD variant: ontogenetic implications.
      The latter is consistent with detecting in these cells circle transcripts containing DNA coding the switch region for IgM (Sμ) and those for the targeted isotype (γ, α).
      • Oppezzo P.
      • Vuillier F.
      • Vasconcelos Y.
      • et al.
      Chronic lymphocytic leukemia B cells expressing AID display dissociation between class switch recombination and somatic hypermutation.
      In addition, when provided with adequate costimulation, U-CLL clones can perform CSR
      • Patten P.E.M.
      • Chu C.C.
      • Albesiano E.
      • et al.
      IGHV-unmutated and IGHV-mutated chronic lymphocytic leukemia cells produce activation-induced deaminase protein with a full range of biologic functions.
      ,
      • Oppezzo P.
      • Vuillier F.
      • Vasconcelos Y.
      • et al.
      Chronic lymphocytic leukemia B cells expressing AID display dissociation between class switch recombination and somatic hypermutation.
      and SHM
      • Patten P.E.M.
      • Chu C.C.
      • Albesiano E.
      • et al.
      IGHV-unmutated and IGHV-mutated chronic lymphocytic leukemia cells produce activation-induced deaminase protein with a full range of biologic functions.
      in vitro and in vivo, suggesting that the environment within which maturation occurs determines the mutational landscape, not an inherent inability to do so.
      Second is the differentiation pathways that the precursors of M-CLL clones followed when IGHV mutations developed. Could a shared precursor follow the same pathways but not develop IGHV mutations?

      B-cell Receptor Polyreactivity and Autoreactivity

      CLL immunoglobulins, particularly those from U-CLL clones, are low affinity and polyreactive, characteristics that resemble natural Abs in the circulation of healthy individuals that recognize epitopes in commensal and pathogenic organisms,
      • Borche L.
      • Lim A.
      • Binet J.L.
      • et al.
      Evidence that chronic lymphocytic leukemia B lymphocytes are frequently committed to production of natural autoantibodies.
      as well as apoptotic cells,
      • Catera R.
      • Silverman G.J.
      • Hatzi K.
      • et al.
      Chronic lymphocytic leukemia cells recognize conserved epitopes associated with apoptosis and oxidation.
      cellular debris (including single-strand DNA and double-strand DNA), and catabolized proteins.
      • Catera R.
      • Silverman G.J.
      • Hatzi K.
      • et al.
      Chronic lymphocytic leukemia cells recognize conserved epitopes associated with apoptosis and oxidation.
      Besides the commonly identified self-Ag epitopes, such as cytoskeletal proteins and myosin heavy chain,
      • Lanemo Myhrinder A.
      • Hellqvist E.
      • Sidorova E.
      • et al.
      A new perspective: molecular motifs on oxidized LDL, apoptotic cells, and bacteria are targets for chronic lymphocytic leukemia antibodies.
      self-association of membrane IGs (homodimerization resulting from BCR-BCR interactions), which can lead to cell-autonomous signaling, is another example of self-reactivity.
      • Ohnishi K.
      • Melchers F.
      The nonimmunoglobulin portion of lambda5 mediates cell-autonomous pre-B cell receptor signaling.
      • Meixlsperger S.
      • Köhler F.
      • Wossning T.
      • et al.
      Conventional light chains inhibit the autonomous signaling capacity of the B cell receptor.
      • Minici C.
      • Gounari M.
      • Übelhart R.
      • et al.
      Distinct homotypic B-cell receptor interactions shape the outcome of chronic lymphocytic leukaemia.
      Notably, this type of BCR engagement occurs predominantly with polyreactive IGs.
      • Meixlsperger S.
      • Köhler F.
      • Wossning T.
      • et al.
      Conventional light chains inhibit the autonomous signaling capacity of the B cell receptor.
      In vivo, CLL cells differentiate poorly into ASCs, secreting low levels of Abs. However, when adequate T-cell help
      • Fluckiger A.C.
      • Rossi J.F.
      • Bussel A.
      • et al.
      Responsiveness of chronic lymphocytic leukemia B cells activated via surface Igs or CD40 to B-cell tropic factors.
      or CpG
      • Gutierrez Jr., A.
      • Arendt B.K.
      • Tschumper R.C.
      • et al.
      Differentiation of chronic lymphocytic leukemia B cells into immunoglobulin secreting cells decreases LEF-1 expression.
      • Hoogeboom R.
      • Reinten R.J.A.
      • Schot J.-J.
      • et al.
      In vitro induction of antibody secretion of primary B-cell chronic lymphocytic leukaemia cells.
      • Duckworth A.
      • Glenn M.
      • Slupsky J.R.
      • et al.
      Variable induction of PRDM1 and differentiation in chronic lymphocytic leukemia is associated with anergy.
      (TLR9 ligand) is provided in vitro, CLL cells proliferate and differentiate into ASCs.
      Compared with U-CLL, M-CLL Abs recognize a more restricted set of antigenic epitopes, and are generally less polyreactive. However in their germline configuration, M-CLL Abs often display similar reactivity against a broad range of (auto)Ags,
      • Moens L.
      • Tangye S.G.
      Cytokine-mediated regulation of plasma cell generation: IL-21 takes center stage.
      suggesting that most, if not all, CLL cells emanate from a polyreactive B-cell precursor.

      B-cell Receptor Repertoire Restriction

      CLL BCRs seem to differ from those of normal IGs in several ways.
      • Chiorazzi N.
      • Ferrarini M.
      B cell chronic lymphocytic leukemia: lessons learned from studies of the B cell antigen receptor.
      The most notable feature is the remarkable amino acid similarity structure that can be found in unrelated CLL cases. These stereotyped BCRs are often coded by identical IGHV gene segments.
      • Stamatopoulos K.
      • Belessi C.
      • Moreno C.
      • et al.
      Over 20% of patients with chronic lymphocytic leukemia carry stereotyped receptors: pathogenetic implications and clinical correlations.
      ,
      • Darzentas N.
      • Hadzidimitriou A.
      • Murray F.
      • et al.
      A different ontogenesis for chronic lymphocytic leukemia cases carrying stereotyped antigen receptors: molecular and computational evidence.
      At present, ∼34% of CLL cases present BCR clones with stereotypy, with a higher occurrence in U-CLL.
      • Stamatopoulos K.
      • Belessi C.
      • Moreno C.
      • et al.
      Over 20% of patients with chronic lymphocytic leukemia carry stereotyped receptors: pathogenetic implications and clinical correlations.
      ,
      • Agathangelidis A.
      • Darzentas N.
      • Hadzidimitriou A.
      • et al.
      Stereotyped B-cell receptors in one-third of chronic lymphocytic leukemia: a molecular classification with implications for targeted therapies.
      Although stereotyped BCRs are found most frequently and easily among IGHV-unmutated sequences, they exist in both U-CLL and M-CLL, suggesting the precursor cells likely derived from a pool with highly restricted BCR repertoire, brought about genetically or by selection.
      • Messmer B.T.
      • Albesiano E.
      • Efremov D.G.
      • et al.
      Multiple distinct sets of stereotyped antigen receptors indicate a role for antigen in promoting chronic lymphocytic leukemia.

      The Transcriptomes and Epigenomes of Chronic Lymphocytic Leukemia Cells

      In early microarray-based gene expression profiling studies, a similar profile was found for U-CLL and M-CLL, with a small number of differentially expressed genes, less than the differences between normal B-cell subsets.
      • Klein U.
      • Tu Y.
      • Stolovitzky G.A.
      • et al.
      Gene expression profiling of B cell chronic lymphocytic leukemia reveals a homogeneous phenotype related to memory B cells.
      ,
      • Rosenwald A.
      • Alizadeh A.A.
      • Widhopf G.
      • et al.
      Relation of gene expression phenotype to immunoglobulin mutation genotype in B cell chronic lymphocytic leukemia.
      A subsequent study revealed closer resemblances between U-CLL and M-CLL and normal CD5+ B cells that were either CD27 or CD27+.
      • Seifert M.
      • Sellmann L.
      • Bloehdorn J.
      • et al.
      Cellular origin and pathophysiology of chronic lymphocytic leukemia.
      However, there are a few differences between pre-GC NBCs and U-CLL, because the latter is CD27+sIgM sIgD and expresses AID (see Table 1).
      The cancer methylome reflects the normal cell type from which the tumor initiated or the maturation stage that the fully transformed and expanded neoplastic cell achieved.
      • Easwaran H.
      • Baylin S.B.
      Origin and mechanisms of DNA methylation dynamics in cancers.
      ,
      • Duran-Ferrer M.
      • Clot G.
      • Nadeu F.
      • et al.
      The proliferative history shapes the DNA methylome of B-cell tumors and predicts clinical outcome.
      Hence, comparative analyses of the methylome represent strategies to search for the COO in cancer.
      In CLL, major progress has been made using this approach. Characterizing the CLL epigenetic landscapes has provided 2 implications: identification of pathogenic aberrant methylation programming leading to transcription factor dysregulation in CLL progression, and finding similar methylation imprints between CLL and various normal B-cell subsets. M-CLL clones mostly resembled GC-experienced/MBCs and U-CLL pre-GC/naivelike B cells.
      • Queirós A.C.
      • Villamor N.
      • Clot G.
      • et al.
      A B-cell epigenetic signature defines three biologic subgroups of chronic lymphocytic leukemia with clinical impact.
      ,
      • Kulis M.
      • Merkel A.
      • Heath S.
      • et al.
      Whole-genome fingerprint of the DNA methylome during human B cell differentiation.
      ,
      • Kulis M.
      • Heath S.
      • Bibikova M.
      • et al.
      Epigenomic analysis detects widespread gene-body DNA hypomethylation in chronic lymphocytic leukemia.
      The naivelike subgroup includes mostly U-CLL, whereas the MBC-like subgroup contains mostly M-CLL.
      • Queirós A.C.
      • Villamor N.
      • Clot G.
      • et al.
      A B-cell epigenetic signature defines three biologic subgroups of chronic lymphocytic leukemia with clinical impact.
      ,
      • Kulis M.
      • Merkel A.
      • Heath S.
      • et al.
      Whole-genome fingerprint of the DNA methylome during human B cell differentiation.
      ,
      • Kulis M.
      • Heath S.
      • Bibikova M.
      • et al.
      Epigenomic analysis detects widespread gene-body DNA hypomethylation in chronic lymphocytic leukemia.
      Interestingly, 66% to 69% of the hypomethylation that occurs during normal B-cell differentiation is seen between NBCs and U-CLL.
      • Kulis M.
      • Heath S.
      • Bibikova M.
      • et al.
      Epigenomic analysis detects widespread gene-body DNA hypomethylation in chronic lymphocytic leukemia.
      Because normal B cells undergo extensive epigenetic programming during differentiation, several studies have taken the in silico approach to reconstruct developmental trajectories based on the inherent characteristic methylome of normal B-cell subsets, ranging from progenitors to PCs.
      • Gaiti F.
      • Chaligne R.
      • Gu H.
      • et al.
      Epigenetic evolution and lineage histories of chronic lymphocytic leukaemia.
      ,
      • Oakes C.C.
      • Seifert M.
      • Assenov Y.
      • et al.
      DNA methylation dynamics during B cell maturation underlie a continuum of disease phenotypes in chronic lymphocytic leukemia.
      ,
      • Wierzbinska J.A.
      • Toth R.
      • Ishaque N.
      • et al.
      Methylome-based cell-of-origin modeling (Methyl-COOM) identifies aberrant expression of immune regulatory molecules in CLL.
      ,
      • Kretzmer H.
      • Biran A.
      • Purroy N.
      • et al.
      Preneoplastic alterations define CLL DNA methylome and persist through disease progression and therapy.
      After mapping CLL samples onto the reconstructed trajectories, the inferred COOs were defined by the position of the closest normal B-cell node on the lineage tree.
      Using this approach, in 1 study each CLL clone seemed to derive from a continuum of maturation states along the normal B-cell pathway.
      • Oakes C.C.
      • Seifert M.
      • Assenov Y.
      • et al.
      DNA methylation dynamics during B cell maturation underlie a continuum of disease phenotypes in chronic lymphocytic leukemia.
      After mapping CLL samples onto the reconstructed trajectories, the closest normal B-cell subset node to U-CLL was IgM+IgD+CD27+ MBCs and IgM+CD21highCD27+ splenic marginal zone B cells (sMZBs), and for M-CLL was IgG+CD27+ MBCs. Subsequent epigenetic trajectory studies also reported that both U-CLL and M-CLL were developmentally closer to the branch of MBCs than to the branch of NBCs.
      • Wierzbinska J.A.
      • Toth R.
      • Ishaque N.
      • et al.
      Methylome-based cell-of-origin modeling (Methyl-COOM) identifies aberrant expression of immune regulatory molecules in CLL.
      ,
      • Kretzmer H.
      • Biran A.
      • Purroy N.
      • et al.
      Preneoplastic alterations define CLL DNA methylome and persist through disease progression and therapy.
      Moreover, the switch to aberrant methylation landscape was established before the preleukemic monoclonal B-cell lymphocytosis (MBL) stage.
      • Kretzmer H.
      • Biran A.
      • Purroy N.
      • et al.
      Preneoplastic alterations define CLL DNA methylome and persist through disease progression and therapy.
      Taken together, these studies suggest that the precursors for both U-CLL and M-CLL reached the Ag-experienced, memory stage of normal B-cell development. In order to deviate from normal development, B cells at this maturation stage must have acquired sufficient oncogenic hits to initiate final transformation.

      Genetic Abnormalities

      Similar to other neoplasias, CLL is genetically heterogeneous, often containing multiple subclones. Early fluorescence in situ hybridization studies revealed that the most frequent chromosomal abnormalities observed in CLL included del13q14, trisomy12, del11q, and del17p.
      • Bosch F.
      • Dalla-Favera R.
      Chronic lymphocytic leukaemia: from genetics to treatment.
      Since the advance in molecular techniques and next-generation sequencing, recurrent mutations involving discrete genes or genes in distinct pathways (NOTCH1 and nuclear factor kappa-B pathways, RNA processing, DNA damage response, and chromatin modifications) have been identified.
      • Bosch F.
      • Dalla-Favera R.
      Chronic lymphocytic leukaemia: from genetics to treatment.
      Based on clonal evolution analyses, for some clones, del13q, del11q, and trisomy12 correspond with earlier driving events, whereas subclonal drivers such as Tp53 are acquired later in the course of disease.
      • Landau D.A.
      • Tausch E.
      • Taylor-Weiner A.N.
      • et al.
      Mutations driving CLL and their evolution in progression and relapse.
      In addition, CLL-related genetic lesions have been detected in HSCs and their progeny
      • Damm F.
      • Mylonas E.
      • Cosson A.
      • et al.
      Acquired initiating mutations in early hematopoietic cells of CLL patients.
      ,
      • Marsilio S.
      • Khiabanian H.
      • Fabbri G.
      • et al.
      Somatic CLL mutations occur at multiple distinct hematopoietic maturation stages: documentation and cautionary note regarding cell fraction purity.
      and in B-progenitor cells.
      • Marsilio S.
      • Khiabanian H.
      • Fabbri G.
      • et al.
      Somatic CLL mutations occur at multiple distinct hematopoietic maturation stages: documentation and cautionary note regarding cell fraction purity.
      In addition, HSCs from patients with CLL engrafted into alymphoid mice can generate monoclonal or oligoclonal expansions of CD5+CD19+ B cells.
      • Kikushige Y.
      • Ishikawa F.
      • Miyamoto T.
      • et al.
      Self-renewing hematopoietic stem cell is the primary target in pathogenesis of human chronic lymphocytic leukemia.
      Although some of these carried stereotyped IGHV-D-J rearrangements, they were unrelated to the IGHV-D-J rearrangement in the same patient’s leukemic clone and did not carry the genetic changes found in the autologous CLL cell.
      • Damm F.
      • Mylonas E.
      • Cosson A.
      • et al.
      Acquired initiating mutations in early hematopoietic cells of CLL patients.

      Scenarios addressing the potential relevance of B-cell maturation pathways in defining the cells of origin in chronic lymphocytic leukemia

      Over the years, several investigators have proposed putative COOs based on the characteristics mentioned earlier.
      • Chiorazzi N.
      • Ferrarini M.
      B cell chronic lymphocytic leukemia: lessons learned from studies of the B cell antigen receptor.
      • Caligaris-Cappio F.
      • Ghia P.
      The nature and origin of the B-chronic lymphocytic leukemia cell: a tentative model.
      • Chiorazzi N.
      • Rai K.R.
      • Ferrarini M.
      Chronic lymphocytic leukemia.
      • Caligaris-Cappio F.
      • Ghia P.
      The normal counterpart to the chronic lymphocytic leukemia B cell.
      • Seifert M.
      • Sellmann L.
      • Bloehdorn J.
      • et al.
      Cellular origin and pathophysiology of chronic lymphocytic leukemia.
      • Chiorazzi N.
      • Ferrarini M.
      Cellular origin(s) of chronic lymphocytic leukemia: cautionary notes and additional considerations and possibilities.
      • Darwiche W.
      • Gubler B.
      • Marolleau J.-P.
      • et al.
      Chronic Lymphocytic Leukemia B-cell normal cellular counterpart: clues from a functional perspective.
      Although most of the previously considered cell types remain possibilities, and 2 additional types need to be taken into account (NF-B cells and ABCs) (see Table 1), the observations that the COO imprints of all CLL clones most resemble the MBC methylome are compelling in assigning memory B lymphocytes as the likely COO,
      • Gaiti F.
      • Chaligne R.
      • Gu H.
      • et al.
      Epigenetic evolution and lineage histories of chronic lymphocytic leukaemia.
      ,
      • Oakes C.C.
      • Seifert M.
      • Assenov Y.
      • et al.
      DNA methylation dynamics during B cell maturation underlie a continuum of disease phenotypes in chronic lymphocytic leukemia.
      ,
      • Queirós A.C.
      • Villamor N.
      • Clot G.
      • et al.
      A B-cell epigenetic signature defines three biologic subgroups of chronic lymphocytic leukemia with clinical impact.
      ,
      • Kulis M.
      • Merkel A.
      • Heath S.
      • et al.
      Whole-genome fingerprint of the DNA methylome during human B cell differentiation.
      ,
      • Wierzbinska J.A.
      • Toth R.
      • Ishaque N.
      • et al.
      Methylome-based cell-of-origin modeling (Methyl-COOM) identifies aberrant expression of immune regulatory molecules in CLL.
      ,
      • Kulis M.
      • Heath S.
      • Bibikova M.
      • et al.
      Epigenomic analysis detects widespread gene-body DNA hypomethylation in chronic lymphocytic leukemia.
      ,
      • Kretzmer H.
      • Biran A.
      • Purroy N.
      • et al.
      Preneoplastic alterations define CLL DNA methylome and persist through disease progression and therapy.
      although this is surprising in light of the imperfect correlations listed in Table 1.
      However, note that (1) the phenotypic definition of an MBC derives from studies of B cells stimulated T dependently and requires the expression of CD27. (2) Within the CD27+ memory compartment, several distinct MBC types have been defined (reviewed in Pauli and colleagues
      • Pauli N.T.
      • Henry Dunand C.J.
      • Wilson P.C.
      Exploiting human memory B cell heterogeneity for improved vaccine efficacy.
      ). (3) CD27 cells have also been ascribed to MBCs.
      • Fecteau J.F.
      • Côté G.
      • Néron S.
      A new memory CD27-IgG+ B cell population in peripheral blood expressing VH genes with low frequency of somatic mutation.
      ,
      • Wei C.
      • Anolik J.
      • Cappione A.
      • et al.
      A new population of cells lacking expression of CD27 represents a notable component of the B cell memory compartment in systemic lupus erythematosus.
      (4) As highlighted earlier, MBCs can be generated by several distinct maturational pathways that differ in T-cell involvement. (5) The methylomes of subtypes of MBCs have not been studied.
      Hence, several distinct cell types can arrive at a CD27+ phenotype, and these can follow distinct pathways to achieve this. In addition, although the resultant CD27+ MBCs might have similar methylomes, they might be heterogeneous based on other molecular features. If so, the differences could result from distinct microenvironmental inputs provided by the different maturation paths followed. The authors propose that this needs to be considered in assigning the COO in CLL.
      In our constructions about the COO in CLL, we are assuming that the features under consideration, especially the epigenetic imprints, are directly related to the leukemic cells expanded in patients; that is, that the final transformation event occurred in the clonally expanded cell.
      Generation of MBCs resembling CLL cells in the various B-cell maturation pathways is shown in Fig. 2.
      Figure thumbnail gr2
      Fig. 2B-cell maturation pathways that can give rise to the MBC-like cells that could be the COOs in CLL. (A) Classic GC-TD pathway. Within GCs, B cells proliferate massively and undergo CSR and SHM, processes that further diversity the BCR repertoire and produce ASCs and MBCs. BCRs on post-GC MBCs are typically mutated with higher-affinity IGs. (B) EF-TD pathway. MBCs can be generated early in a primary immune response via this pathway. The MBCs are IGHV unmutated but often, albeit not always, have undergone CSR to IgG. (C) EF-TI pathway. MBCs generated via this pathway are often IGHV-unmutated effector cells with restricted BCR repertoires biased toward autoreactive and polyreactive Abs. MZBs, ABCs (not shown), TrBCs, and NF-B cells are cell types that can be activated and can differentiate in this manner. However, SHM and CSR can also take place because of an EF reaction.

      Classic Germinal Center T Cell–dependent Pathway

      If U-CLL cells were generated from MBCs that followed this route, the activated precursor would not have reached/traversed a classic GC and consequently could not undergo a GC reaction (pre-GC); this is a temporally short maturation window. Alternatively, the precursor could have completed a GC reaction, accumulated replacement, nonsynonymous IGHV mutations, but had these negatively selected to retain an IGHV-unmutated, germline BCR amino acid structure. This alternative would be possible if there were a strong selection such as that required to maintain autonomous (or standard BCR) signaling, without which the mutant cells would die. In comparison, the M-CLL precursor would have completed the GC-TD reaction and developed IGHV mutations (post-GC B cells). For MBC-like cells generated via the GC-TD pathway, the most likely precursors, based on the parameters in Table 1, are activated NBCs, FOs, and some ABCs. This possibility takes into account the mixed IGHV mutation status in CLL and is supported by the detection of bcl6 mutations in patients with M-CLL, a GC-associated feature. However, B cells matured in this pathway have a diverse repertoire that is different from both CLL subtypes, and some do not express CD5.

      T Cell–dependent Maturation at Extrafollicular Sites Pathway in Early Extrafollicular Responses

      In this setting, U-CLL would come about by precursors being activated in a TD manner, at the initiation of an immune response, thereby generating MBCs with IGHV-unmutated BCRs; because this pathway often, but not necessarily, leads to IgG-producing cells,
      • Takemori T.
      • Kaji T.
      • Takahashi Y.
      • et al.
      Generation of memory B cells inside and outside germinal centers.
      this U-CLL could be of the type seen in stereotyped subset 8, which bears an unmutated, IgG BCR. Moreover, in mice, these MBCs can generate IGHV-mutated BCRs after Ag reexposure.
      • Kaji T.
      • Furukawa K.
      • Ishige A.
      • et al.
      Both mutated and unmutated memory B cells accumulate mutations in the course of the secondary response and develop a new antibody repertoire optimally adapted to the secondary stimulus.
      For MBCs generated via the EF-TD pathway, the most likely precursors are activated NBCs in the initial response and Ag-primed FOs and some ABCs in the secondary response.

      Extrafollicular T Cell–independent Pathway

      As mentioned earlier, both IGHV-unmutated and IGHV-mutated MBCs can be generated by this pathway and hence could give rise to U-CLL and M-CLL. MZBs and B-1 cells are well-characterized examples of cell types that can undergo TI activation outside of the lymphoid follicles. Thus, MBCs could come from MZBs and give rise to both U-CLL and M-CLL. CD5+ B cells, and the assumed human B-1 equivalent, could give rise to IGHV-unmutated MBCs, and hence U-CLL, and possibly mutated MBCs and M-CLL if the starting cells were CD5+CD27+. Moreover, ABCs share some unique CLL-like characteristics that are lacking in MZBs, such as CD5 expression, T-bet-driven transcription, and responsiveness to TLR7/9 signaling.
      • Rubtsova K.
      • Rubtsov A.V.
      • Cancro M.P.
      • et al.
      Age-associated B cells: a T-bet–dependent effector with roles in protective and pathogenic immunity.
      ,
      • Cancro M.P.
      Age-Associated B Cells.
      Based on the information discussed earlier, the authors suggest that EF pathways unify the cellular possibilities, making them the most parsimonious option. However, none of the scenarios can be discounted. It will be important going forward to analyze, epigenetically and transcriptomically, human B-cell subsets passing through the various differentiation paths and the subsets of MBCs that they produce.

      Final qualification

      The hypotheses for the COOs provided earlier assume that the cell types that developed the final transformation event correspond with those that are clonally expanded in a patient’s leukemic B cells. However, it is possible that the final transformation event occurred at a maturation stage earlier than that of the clinically dominant population. There are immature B cells (eg, TrBCs and NF-B cells) that can express AID and could give rise to IGHV-unmutated and IGHV-mutated BCRs. In this scenario, these precursor cell types could have received the final transformative event but still matured along the differentiation pathways mentioned, triggered by microenvironmental cues. Based on sharing Ag polyspecificity and BCR restrictions with CLL, NF-B cells are an attractive candidate in such a possibility.
      • Giltiay N.V.
      • Giordano D.
      • Clark E.A.
      The plasticity of newly formed B cells.

      Clinics care points

      • Defining the COO in cancer is important to understand how the neoplastic transformation occurred and how to best diagnose and treat the disease.
      • CLL, which is the most frequent leukemia among people of the United States and Europe, remains incurable, and the COO in the disease has been unclear.
      • Recent immunogenetic and epigenetic studies have suggested that the COO in CLL clones is a memorylike B lymphocyte.
      • However, previous studies have not taken into account how various B-cell maturation pathways could affect the transformation of a memorylike B cell.
      • Therefore, this article presents possibilities whereby the type of pathway leading to a memory state could influence the occurrence of malignancy.

      Acknowledgments

      The authors thank The Nash Family Foundation, the Karches Foundation, The Marks Foundation, and the Jean Walton Fund for Leukemia, Lymphoma and Myeloma Research for their support.

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