Background: B-cell receptor (BCR) signaling is crucial for normal B-cell development and adaptive immunity. In chronic lymphocytic leukemia (CLL), the malignant B cells display many features of normal mature B lymphocytes, including the expression of functional B-cell receptors (BCRs). Cross talk between CLL cells and the microenvironment in secondary lymphatic organs results in BCR signaling and BCR-driven proliferation of the CLL cells. This critical pathomechanism can be targeted by blocking BCR-related kinases (BTK, PI3K, spleen tyrosine kinase) using small-molecule inhibitors. Among these targets, Bruton tyrosine kinase (BTK) inhibitors have the highest therapeutic efficacy; they effectively block leukemia cell proliferation and generally induce durable remissions in CLL patients, even in patients with high-risk disease. By disrupting tissue homing receptor (i.e., chemokine receptor and adhesion molecule) signaling, these kinase inhibitors also mobilize CLL cells from the lymphatic tissues into the peripheral blood (PB), causing a transient redistribution lymphocytosis, thereby depriving CLL cells from nurturing factors within the tissue niches. Summary: The clinical success of the BTK inhibitors in CLL underscores the central importance of the BCR in CLL pathogenesis. Here, we review CLL pathogenesis with a focus on the role of the BCR and other microenvironment cues. Key Messages: (i) CLL cells rely on signals from their microenvironment for proliferation and survival. (ii) These signals are mediated by the BCR as well as chemokine and integrin receptors and their respective ligands. (iii) Targeting the CLL/microenvironment interaction with small-molecule inhibitors provides a highly effective treatment strategy, even in high-risk patients.

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of monoclonal mature B lymphocytes expressing CD5 and CD23 in the blood, bone marrow (BM), and secondary lymphatic organs (SLO, i.e., lymph nodes, spleen). CLL cells retain many functional properties of normal B cells, including key signaling pathways such as the B-cell receptor (BCR) and its downstream signaling cascade that normally organizes B-cell selection and proliferation as part of adaptive immune responses [1]. CLL cell survival and proliferation are dependent on signals from the microenvironment in SLO. Direct in vivo measurements demonstrated that the SLO are the primary site of CLL cell proliferation [2, 3]. When removed from the supportive microenvironment, CLL cells undergo spontaneous apoptosis ex vivo unless co-cultured with bone marrow stromal cells (BMSC) [4, 5] or monocyte-derived nurse-like cells (NLC) [6]. The clinical efficacy of kinase inhibitors that disrupt BCR signaling and other CLL cell interactions with the tissue microenvironment further corroborates the central importance of BCR signaling and the tissue microenvironment in CLL pathogenesis [7].

The entire CLL clone consists of a small fraction of proliferating CLL cells, which are primarily found in SLO, and a vast majority of resting B cells [8]. CLL cells from these compartments are distinct in their morphology, gene expression, and immunophenotype. The proliferative compartment in SLO forms characteristic “proliferation centers (PC),” also called “pseudofollicles” that are a typical finding in CLL histopathology. CLL cell proliferation can be quantified in patients by heavy (deuterated) water labeling, ranging between 0.1 to greater than 1% of the entire clone per day [9]. Small resting lymphocytes, the progeny of the proliferating compartment, accumulate in the blood and tissues (SLO, marrow) and traffic between these compartments, in an active process of migration that is organized by chemokine receptors and adhesion molecules, which are expressed on the CLL cell surface, and respective tissue ligands (Table 1).

Table 1.

Constituents of the CLL microenvironment

Receptors/ligandsFunctionPrognostic impact
BCR Main driver of CLL cell proliferation and survival; activation is mediated through exogenous (auto) antigens or autonomous BCR activation IGHV mutation status distinguishes indolent from more active disease courses [10
CXCR4 Surface receptor for CXCL12; mediates the migration and chemotaxis of CLL cells CXCR4 expression levels allow the distinction of quiescent CXCR4bright/CD5dim and proliferating CXCR4dim/CD5bright CLL cells [11
CXCR5 Surface receptor for CXCL13 expressed on CLL cells; mediates chemotaxis and actin polymerization 
CCL3/4 Secreted by CLL cells to attract T cells and monocytes to the microenvironment Serum CCL3 levels serve as a prognostic marker for time to treatment [12
VLA-4 Expressed on CLL cells; facilitates CLL cell adhesion through binding of fibronectin or VCAM-1 VLA-4 expression levels on CLL cells predict time to treatment and overall survival [13
Receptors/ligandsFunctionPrognostic impact
BCR Main driver of CLL cell proliferation and survival; activation is mediated through exogenous (auto) antigens or autonomous BCR activation IGHV mutation status distinguishes indolent from more active disease courses [10
CXCR4 Surface receptor for CXCL12; mediates the migration and chemotaxis of CLL cells CXCR4 expression levels allow the distinction of quiescent CXCR4bright/CD5dim and proliferating CXCR4dim/CD5bright CLL cells [11
CXCR5 Surface receptor for CXCL13 expressed on CLL cells; mediates chemotaxis and actin polymerization 
CCL3/4 Secreted by CLL cells to attract T cells and monocytes to the microenvironment Serum CCL3 levels serve as a prognostic marker for time to treatment [12
VLA-4 Expressed on CLL cells; facilitates CLL cell adhesion through binding of fibronectin or VCAM-1 VLA-4 expression levels on CLL cells predict time to treatment and overall survival [13

NLCs (CLL-Associated Macrophages)

The first evidence that myeloid cells are contributing to the pathogenesis of CLL came from the in vitro observation that blood monocytes, in the presence of CLL cells, spontaneously differentiate into large adherent feeder cells termed NLC which protect CLL cells from apoptosis [6, 14]. NLC secrete the chemokines CXCL12 and CXCL13, activating CLL cells via the corresponding chemokine receptors CXCR4 and CXCR5, respectively [6, 15], thereby contributing to the survival and tissue homing of CLL cells. In addition, NLC also express other CLL survival factors, such as B-cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) [16, 17]. Gene expression profiling of CLL cells revealed that NLC activate BCR and NF-κB signaling [18], recapitulating in vitro the gene expression signature of CLL cells isolated from CLL lymph nodes [2]. NLC express antigens such as vimentin and calreticulin that can directly crosslink the BCR and activate BCR signaling in CLL cells [19, 20]. They also carry high levels of CD31 and plexin-B1, which deliver survival and proliferation signals to CD38+/CD100+ CLL cells [21]. Co-culture with NLC induces BCR signaling-dependent transcription and secretion of the chemokines CCL3 and CCL4 by CLL cells that can attract CLL-supportive immune cells, especially T cells and monocytes, into the tissue microenvironment [18, 22]. In addition, NLC secrete other cytokines including IL-6, IL-8, and IL-10 that promote CLL cell proliferation and/or survival.

Based on gene expression profiles, surface markers, and cytokine expression, NLC resemble M2-skewed tumor-associated macrophages (TAM). NLC express high levels of the scavenger receptor CD163, a marker of M2 tissue macrophages, along with CD68. In the tissues (lymph nodes, BM), NLC form cell membrane extensions for intimate contact with CLL cells, which are disrupted by BTK inhibitor therapy with ibrutinib [23]. Increased numbers of CD163+ or CD68+ cells in LN are associated with active disease requiring treatment and shorter overall survival [24], and high density of CD163+ cells in BM correlated with disease persistence with ibrutinib therapy [25], corroborating the importance of NLC in CLL. Further evidence that NLC are the key players in the CLL microenvironment was generated in CLL mouse models. In the Eµ-TCL1 mouse model, monocytes and M2-skewed macrophages co-localize to sites where CLL cells accumulate, such as in the peritoneal cavity, spleen, and BM. Targeted deletion of macrophage migration inhibitory factor (MIF) leads to reduced numbers of macrophages in the spleen and BM and significantly delays the development of CLL in Eµ-TCL1 mice [26]. Accordingly, depletion of monocytes and macrophages significantly delayed the development of CLL and improved survival in several mouse models [27, 28]. Furthermore, monocytes and macrophages facilitate leukemia engraftment in xenotransplantation models of CLL [29, 30].

Mesenchymal Stromal Cells

Mesenchymal stromal cells (MSC) are well known for supporting the growth and maturation of normal hematopoietic progenitor cells (HPC). These cells are primarily found in the BM, where they are called BMSC, but also are a part of the stromal cell network in normal [31] and malignant SLO, for example, in CLL patients [32, 33]. The initial evidence that MSC are important for CLL cell survival came from studies that demonstrated the rescue of CLL cells from spontaneous and drug-induced apoptosis when co-cultured with BMSC [34]. The high affinity of CLL cells for BMSC is illustrated by pseudo-emperipolesis, the spontaneous migration of CLL cells beneath and underneath BMSC layers in vitro [35]. BMSC constitutively secrete stromal cell-derived factor-1 (SDF-1, now designated CXCL12), a tissue homing chemokine that attracts CLL cells via the corresponding chemokine receptor CXCR4, which CLL cells express highly on their cell surface [18] (Fig. 1). Besides attracting CLL cells into the tissues, CXCL12 also has modest pro-survival activity and is an important factor in BMSC-mediated protection of CLL cells from spontaneous or chemotherapy-induced apoptosis [6, 36]. Adhesion molecules are also highly important for interactions between CLL cells, MSC, and other cells in the tissue microenvironment. For example, VLA-4 integrins (CD49d/CD29) on CLL cells can bind to VCAM-1 or vimentin, both present in the tissue microenvironment and on MSC, and binding subsequently activates CLL cells and rescues them from apoptosis via Bcl-2 and Bcl-XL up-regulation. The importance of VLA-4 in CLL is further emphasized by its robust prognostic impact; high VLA-4 levels on CLL cells correlate with increased homing to the BM and inferior clinical outcome [37].

Fig. 1.

Composition of the CLL microenvironment. CLL cells rely on a multitude of signals from the tissue microenvironment in SLO to promote their survival and proliferation. These signals are received through CLL surface receptors that are engaged by corresponding microenvironmental ligands, including signals from the BCR, CD40, chemokine receptors (CXCR4, CXCR5), and adhesion molecules, such as integrins, and TNF-family receptors including BAFF-R, TACI, and BCMA. BTK inhibitors inhibit several of these signaling cascades and effectively disrupt the crosstalk between CLL cells and the microenvironment, thereby depriving them from essential survival factors, and disrupting tissue homing signals, resulting in mobilization of tissue CLL cells from SLO into the PB.

Fig. 1.

Composition of the CLL microenvironment. CLL cells rely on a multitude of signals from the tissue microenvironment in SLO to promote their survival and proliferation. These signals are received through CLL surface receptors that are engaged by corresponding microenvironmental ligands, including signals from the BCR, CD40, chemokine receptors (CXCR4, CXCR5), and adhesion molecules, such as integrins, and TNF-family receptors including BAFF-R, TACI, and BCMA. BTK inhibitors inhibit several of these signaling cascades and effectively disrupt the crosstalk between CLL cells and the microenvironment, thereby depriving them from essential survival factors, and disrupting tissue homing signals, resulting in mobilization of tissue CLL cells from SLO into the PB.

Close modal

Given the multitude of surface molecules and downstream signaling pathways activated by contact between CLL cells and MSC, it is no surprise that MSC induce substantial changes in CLL cell gene and protein expression. Co-culture with BMSC leads to substantial up-regulation of the T-cell leukemia/lymphoma 1 (TCL1) proto-oncogene in CLL cells [38]. MSC induce up-regulation of ZAP-70, CD38, and CD49d, which are markers associated with more aggressive clinical behavior. Contact with MSC also induces CLL cell resistance to chemotherapeutic agents, a mechanism to explain why residual disease after chemotherapy-based regimen typically is more predominant in the tissues than in the blood. CLL cells not only benefit from the contact with tissue MSC, they also, vice versa, activate and reshape MSC. Through the release of exosomes and via direct cell-cell contact, CLL cells can induce protein kinase C beta II (PKCβII) expression and nuclear factor kappa B (NF-κB) pathway activation in MSC, inducing an inflammatory cancer-associated fibroblast (CAF) phenotype. Activated stromal cells, in turn, secrete pro-tumorigenic cytokines, such as IL-1 and IL-15 which further propagate this symbiotic relation between CLL and MSC. In an elegant series of experiments, vom Stein et al. [39] recently demonstrated that the LYN kinase, which is overexpressed in lymph node stromal cells from CLL patients, induces the inflammatory CAF phenotype via modulation of cytokine secretion and extracellular matrix (ECM) components. In turn, such activated CAF effectively promoted CLL cell survival.

T Cells

During normal adaptive immune responses, T cells migrate to germinal centers (GCs) for providing help to antigen-selected B cells and constitute 5–20% of the GC. The GC response depends on CD4+ T cells and CD40, as well as the costimulatory molecules CD28 and ICOS, and the cytokines IL-4 and IL-21 [40]. GC B cells compete for contact/help by these T cells, resulting in the selection and expansion of B cells carrying B-cell receptors (BCRs) with high affinity to the antigen. The chemokines CCL3 and CCL4, secreted by B cells in response to BCR triggering [41], attract T cells and seem to play an important role in this selection process, a mechanism which also seems to play a role in CLL [18, 42].

Normal GC reactions are regulated by specialized CD4+ T helper cell subpopulations, especially T follicular helper (Tfh) cells, which provide the majority of T cell help to GC B cells by activating B cells via CD40 ligand and IL-21. Tfh cells are characterized by high expression of the CXCR5 chemokine receptor, also highly expressed on CLL cells [15, 43], which allows normal B and Tfh cells to home to the SLO. Tfh cells also express the inducible co stimulator molecule (ICOS), programmed cell death protein-1 (PD-1), transcriptional repressor B-cell lymphoma 6 (BCL6), and produce IL-21. Tfh maturation stages can be distinguished based on expression levels of CXCR5 and PD-1. For example, Tfh cells which exit GCs are less activated and become memory Tfh cells, with low or absent PD-1 expression. Another important T helper cell subset in SLO is the regulatory T (Treg) cell which regulates immune tolerance and homeostasis and express the transcription factor FOXP3.

In patients with CLL, peripheral blood T-cell numbers are increased and oligoclonal [44], and BM and LN are also enriched in T cells, predominantly with CD4+ helper cells, constituting a substantial portion of the lymphoid infiltrate. T cells localize both inside and outside of proliferation centers [45‒48]. When investigating T-cell subsets in CLL, several studies demonstrated increased frequencies of regulatory T cells (Tregs), interleukin 17-secreting T cells (Th17), and follicular T helper cells (Tfh) in PB [49]. T-cell populations in SLO from CLL patients remain incompletely defined. Generally, untreated CLL patients have increased numbers of CD4+ T cells in their PB, and CLL lymph nodes also are densely infiltrated by T helper cells [42, 50] which are in intimate contact with proliferating CLL cells [48] and express CD40L [47]. On the other hand, immunosurveillance by T cells is compromised in CLL, due to, among others, defective cytoskeletal signaling and immune synapse formation with CLL cells due to immunosuppressive signaling [51]. CLL T cells express high levels of exhaustion markers, including programmed cell death protein-1 (PD-1); accordingly, CLL cells express high levels of PD-L1. Additionally, CLL T cells demonstrate increased expression of the inhibitory receptor cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Besides the well-recognized functional deficiencies in the T-cell compartment in CLL patients, there is also growing evidence that specialized T helper cells, which are expanded in untreated patients, support the growth of the CLL clone in the SLO. The requirement for activated CD4+ T cells for survival and growth of CLL cells in patient-derived xenograft (PDX) models was among the first robust evidence for this model [29]. This observation was extended by demonstrating that in vitro pre-activation of CLL T cells with CD3/CD28 beads and IL-2 resulted in robust transient CLL cell expansion in vivo [52].

TCR sequencing in CLL patients documented a restricted TCR gene repertoire, with an oligoclonal T-cell expansion and shared T-cell receptor clonotypes among different patients, suggesting T-cell selection and expansion by restricted antigens, possibly on CLL cells. This is supported by experiments in which T cells isolated from CLL patients were stimulated with autologous CLL cells, resulting in a clonal T-cell expansion. Similarly, in the CLL Eμ-TCL1 mouse model, a restricted TCR repertoire was found [53]. Vice versa, when CLL patients receive effective therapy (with ibrutinib), the TCR repertoire diversifies, along with deletion of high-frequency clones and normalization of T-cell counts [54]. In addition, in a recent study in patients with stereotyped BCR immunoglobulins, TCR repertoire analyses demonstrated remarkable repertoire skewing and T-cell oligoclonality, further supporting the concept of co-evolution of CLL and supportive T-cell clones [44]. Most studies about T helper cells in CLL have been based on PB samples, even though, as discussed, their primary role appears to be in the SLO. One study recently reported, based on lymph node aspirates, about enrichment in Tfh and Treg in CLL lymph nodes, as compared to PB, and their depletion from the PB after treatment with obinutuzumab plus venetoclax or with ibrutinib plus venetoclax [55]. We recently reported that co-culture of CLL T cells in the NLC model resulted in the expansion of Tfh cells based on PD-1, BCL6, and ICOS expression [56]. Tregs, which promote immune tolerance, also expanded in NLC co-cultures. T-cell receptor gene repertoire analyses confirmed the clonal expansion of T helper cells, with an enrichment of TR clonotypes commonly expanded also in primary CLL samples. Multicolor confocal microscopy revealed that Tfh, but not Tregs co-localize with proliferating CLL cells in CLL lymph node sections [56].

Follicular Dendritic Cells

Follicular dendritic cells (FDC) are an important component of normal lymphoid follicles, where they present antigens to selected B cells. However, the role of FDC in CLL remains ill-defined, based on variable results when staining CLL tissues for FDC and detection of FDC only during early-stage CLL [57]. A FDC cell line protected CLL cells from apoptosis in vitro [58]. In the Eµ-TCL1 mouse model, CXCR5-expressing leukemia cells migrate towards CXCL13-secreting FDC to gain access to the growth-promoting follicle microenvironment [59], and, in turn, lymphotoxin alpha beta (LTαβ) secretion by CLL cells promoted CXCR13 production by FDC [59].

In the lymph node microenvironment, BCR signaling is activated in the CLL cells [2], resulting in leukemia cell proliferation [3]. Signaling downstream of the BCR activates upstream kinases (LYN, spleen tyrosine kinase [SYK], BTK, PI3K). Among these, BTK emerged as the most successful therapeutic target. Downstream signaling via ERK and NFκB results in transcriptional activation, for example, of the T-cell chemo-attractants CCL3 and CCL4, which are secreted at high concentrations by BCR-activated CLL [18] and function as plasma biomarkers when targeting BTK [60] or PI3K [61]. The evidence that CLL cell proliferation is BCR-driven is based on the following findings: first, the pseudo-follicular architecture [62] and engagement of proliferating CLL cells with activated T helper cells [47, 48] and specifically with Tfh [56] replicates the cellular architecture of normal B-cell follicles. Second, SLO are the primary site of CLL cell proliferation, as demonstrated directly in patients after labeling of CLL cells with deuterated (heavy) water [3]. Third, gene expression studies demonstrated that BCR and NF-κB signaling are exclusively activated in CLL cells isolated from SLO but not in blood or BM CLL cells [2]. Fourth, inhibition of BCR signaling with kinase inhibitors, such as ibrutinib, results in immediate and almost complete abrogation of CLL cell proliferation [63]. As with normal B cells, additional costimulatory signals are necessary for CLL cell expansion, which appear to derive from T cells and macrophages/NLC, as discussed earlier.

The mutational status of the Ig heavy chain variable (IGHV) genes is a powerful prognostic marker in CLL. IGHV mutation analysis distinguishes mutated CLL (M-CLL) with >2% deviation from the IGHV germline sequence, who generally have indolent disease, from patients with unmutated CLL (U-CLL) with ≤2% deviation [10, 64], who present with more active disease. These differences in clinical behavior have been linked to differences in BCR responsiveness. U-CLL clones respond to various (auto) antigens, triggering robust BCR signaling, whereas M-CLL display more selective binding to restricted antigens, and less active BCR signaling (Fig. 2a). Consequently, M-CLL clones remain stable or expand at a slower rate than clones from U-CLL patients. Interestingly, the negative prognostic impact of U-CLL is overcome when patients are treated with the new targeted agents. Long-term follow-up of CLL patients receiving ibrutinib frontline therapy demonstrated that the survival of U-CLL patients was excellent and not different from those with M-CLL [65].

Fig. 2.

CLL BCR subsets and BCR signaling inhibitors. a CLL patients can be distinguished based on the mutational status of IGHV gene sequences with the variable region of the BCR heavy chains (VH) and categorized into U-CLL or M-CLL, which differ in terms of activation properties and antigen affinity and selectivity, as highlighted in the figure. b Engagement of the BCR results in the activation of the receptor-associated kinases LYN and SYK which further propagate the signal via BTK, PI3K, and PLCg2. This ultimately results in the activation of ERK and NFkB and the modulation of their transcriptional targets. The BCR-associated kinases also serve as important hubs for signals emanating from receptors other than the BCR, such as integrin and chemokine receptors. SYK, PI3K, and BTK are targets for small-molecule kinase inhibitors, including the SYK inhibitor fostamatinib (not approved), the BTK kinase inhibitors ibrutinib, acalabrutinib, zanubrutinib, and pirtobrutinib, and the PI3K inhibitors idelalisib and duvelisib.

Fig. 2.

CLL BCR subsets and BCR signaling inhibitors. a CLL patients can be distinguished based on the mutational status of IGHV gene sequences with the variable region of the BCR heavy chains (VH) and categorized into U-CLL or M-CLL, which differ in terms of activation properties and antigen affinity and selectivity, as highlighted in the figure. b Engagement of the BCR results in the activation of the receptor-associated kinases LYN and SYK which further propagate the signal via BTK, PI3K, and PLCg2. This ultimately results in the activation of ERK and NFkB and the modulation of their transcriptional targets. The BCR-associated kinases also serve as important hubs for signals emanating from receptors other than the BCR, such as integrin and chemokine receptors. SYK, PI3K, and BTK are targets for small-molecule kinase inhibitors, including the SYK inhibitor fostamatinib (not approved), the BTK kinase inhibitors ibrutinib, acalabrutinib, zanubrutinib, and pirtobrutinib, and the PI3K inhibitors idelalisib and duvelisib.

Close modal

Originally, the presence or absence of hypermutations in IGHV sequences of CLL patients was interpreted as a sign of antigenic stimulation or lack thereof in M-CLL versus U-CLL, respectively. However, this initial view has been replaced by the model that all CLL cases, regardless of IGHV mutation status, encounter antigen(s) through their BCR, with differential signaling effects. This is supported by the CLL cell surface marker pattern that resembles antigen-experienced B cells [66], with characteristic up-regulation of CD27 and CD23, and down-modulation of CD79b and surface IG [66]. Low-surface IG levels on CLL cells can be recovered to normal, high levels when cultured in vitro, in the absence of antigenic stimulation [67]. Vice versa, sIg levels can be down-modulated by BCR activation in co-culture of CLL cells with NLC, and recover/up-regulated by removal from NLC [68]. BCR cross-linking induces potent pro-survival and proliferative signals in U-CLL cells, while M-CLL are less BCR-responsive [69, 70]. The evidence of antigenic stimulation in CLL, including U-CLL, ignited a quest for putative CLL antigens, particularly in patients with stereotyped BCR, because these clones are most probably selected by antigen. Structural studies demonstrated that, generally, IG from U-CLL clones are polyreactive, resembling natural antibodies [71, 72]; in contrast, the IG from M-CLL have higher specificity and more restricted binding, for example, to fungal antigens [73]. Regarding the putative antigens, of particular interest is CLL BCR binding of molecules on apoptotic bodies, such as non-muscle myosin heavy chain IIA [74] or oxidized LDL [72], a profile reminiscent of natural antibodies engaged in the removal of apoptotic cell debris. Foreign antigens can also be recognized in some instances, including bacterial polysaccharides [72] or fungal glucans [73], and strong associations with viral infections (CMV or EBV) [75] have also been demonstrated in distinct stereotyped subsets. Also relevant to mention, CLL IG may show a rheumatoid factor activity (i.e. auto-reactivity against the FC portion of IgG antibodies), a finding reported more than 50 years ago [76, 77], though more recently associated with distinct stereotyped BCR IG, likely in the context of HCV infection [78].

The concept of BCR activation through exogenous (auto) antigens in CLL has been challenged by an alternative model, activation through autonomous BCR signaling [79], which is due to interactions between two adjacent BCR molecules mediated by CDR3 recognition. This phenomenon appears to be CLL-specific and the internal epitopes involved in the BCR interaction are distinct for different IGs [80]. How this autonomous BCR stimulation plays a role in predisposing to CLL transformation or progression or integrates with classic antigenic stimulation in CLL remains to be determined. In one recently postulated scenario, classical antigen-induced BCR signaling is required for CLL cell growth and proliferation in SLO, whereas cell autonomous signals exclusively derived from the IgM-BCR ensure CLL cell survival outside of their supportive microenvironment [81]. Despite the central importance of the BCR signaling in CLL pathogenesis, it is surprising that the extensive leukemic genome sequencing efforts did not report about pathogenic variants in molecules of the upstream BCR signaling pathway that are targeted by kinase inhibitors in CLL (Fig. 2b). The notable exception are mutations detected in the BTK and PLCG2 genes identified in patients relapsing on therapy with covalent [82, 83] or non-covalent [84] BTK inhibitors.

Stereotyped BCRs in CLL

The discovery of virtually identical (stereotyped) BCR sequences in unrelated CLL patients solidified the concept of common antigens driving CLL development and progression. Approximately 30% of patients can be grouped into CLL subsets based on shared immunoglobulin sequence motifs [85], with distinct clinical behavior and functional differences in terms of BCR activation [86]. Subset #2, for instance, is associated with an aggressive clinical course and poor treatment response irrespective of the underlying IGHV mutational status, whereas subset #4 is characterized by a more indolent disease phenotype [87]. Stereotyped BCRs in CLL were first described in 2003, when cases with BCRs encoded by the IGHV3-21 gene were found to carry highly homologous or identical amino acid sequences within the variable heavy complementarity determining region 3 (VH CDR3) and exhibited highly restricted usage of the IGLV3-21 gene [88]. Soon thereafter, additional cases with stereotyped BCR IG were identified in M-CLL and U-CLL using other IGHV genes [71, 89‒91]. After these original publications, it was established that a large fraction of patients with CLL can be assigned to different subsets with distinct, stereotyped VH CDR3 sequences [85, 91‒94]: some of these subsets are more common and thus named “major” subsets [95]. Interestingly, only six IGHV genes (IGHV1-69, IGHV1-3, IGHV1-2, IGHV3-21, IGHV4-34, and IGHV4-39) account for approximately 80% of the major stereotyped subsets [93, 95]. Moreover, certain IGHV genes (e.g., IGHV3-21, IGHV1-69, IGHV1-2) are over-represented while others (e.g. IGHV3-7, IGHV3-23, IGHV3-30) are under-represented among the stereotyped fraction of CLL [93]. More recently, after sequencing approximately 30,000 patients with CLL, Agathangelidis et al. [95] reported that 41% of patients expressed stereotyped BCR IG. This is remarkable considering the extremely low probability of randomly finding identical VH CDR3 sequences in different B cells, which is in the range of 1:10−16 to 1:10−18 [96].

CXCR4 (CD184)

CXCR4 is expressed at high levels on the surface of PB CLL cells [35], and mediates CLL cell chemotaxis, migration across vascular endothelium, actin polymerization, and migration beneath and underneath CXCL12-secreting BMSC [35]. CXCL12 also has a pro-survival effect on CLL cells [6, 16, 97] which is not surprising, given that CXCL12 initially was characterized as pre-B-cell growth-stimulating factor (PBSF) [98]. CXCR4 surface expression is regulated by its ligand CXCL12 (previously called stromal cell-derived factor-1/SDF-1) via receptor endocytosis [35], with down-regulation of surface CXCR4 on tissue CLL cells by CXCL12 present at high levels in the tissues. This characteristic can be used to distinguish tissue (lymphatic tissue- and marrow-derived) from blood CLL cells, which express low or high CXCR4 levels, respectively [2, 35]. Proliferating, Ki-67+ CLL cells from marrow and lymphatic tissue displayed significantly lower levels of CXCR4 and CXCR5 than non-proliferating CLL cells [99]. In vivo deuterium (2H) labeling of CLL cells revealed that patients with higher CXCR4 expression on their CLL cells had delayed appearance of newly produced CD38+ cells in the blood and increased the risk for lymphoid organ infiltration and poor outcome [11]. These 2H studies also revealed intraclonal heterogeneity of CXCR4 expression, with an enrichment of CLL cells expressing lower CXCR4 surface levels in the CD38+/CD5bright fraction, along with increased 2H incorporation [11]. Co-culture with BMSC down-regulates CXCR4 on CLL cells due to the receptor internalization. Interestingly, this mechanism of CXCR4 receptor endocytosis is the basis for distinguishing recently divided from older quiescent CLL cells in the PB [11] and the tissue compartments [3]. The proliferative fraction contains CLL cells which recently exited the SLO into the PB with a characteristic surface signature of CXCR4dimCD5bright cells. Vice versa, CXCR4brightCD5dim cells are a quiescent CLL cell population destined for re-circulating from PB back into the tissue compartment. Heavy water labeling demonstrated significantly increased labeling of CXCR4dimCD5bright cells in patients [3].

B-cell antigen receptor (BCR) signaling results in down-modulation of CXCR4 [100, 101], along with enhanced chemotaxis towards CXCL12 and CXCL13, at least in our hands [100]. This may explain why ZAP-70+ CLL cells display increased chemotaxis and survival in response to CXCL12 when compared to ZAP-70-negative CLL cells [97], given that ZAP-70 expression is associated with a higher responsiveness to BCR stimulation [102]. CXCR4 signaling in CLL cells is pertussis toxin-sensitive and induces calcium mobilization, activation of PI3 kinases [35], p44/42 MAP kinases [6], and serine phosphorylation of signal transducer and activator of transcription 3 (STAT3) [36]. CXCR4 signaling can be inhibited by isoform-selective PI3 kinase inhibitors [103], including idelalisib, and inhibitors of SYK [100], and BTK [60], leading to impaired CLL cell migration.


CXCR5 (CD185) is the receptor for CXCL13, a chemokine that regulates lymphocyte homing and positioning within lymph follicles [104]. CXCR5 is expressed by mature B cells, a small subset of T cells, and skin-derived dendritic cells (reviewed in [105]). CXCR5 gene-deleted mice have defective primary follicles and GCs in the spleen and Payer’s patches and lack inguinal lymph nodes [106]. The ligand for CXCR5 is designated CXCL13 and is constitutively secreted by stromal cells in B-cell areas of lymphoid follicles [104, 107]. CXCR5 induces recruitment of circulating naïve B cell to follicles [104, 107] and is responsible for positioning within the GC [108‒111]. In addition, it has been suggested that the primordial function of CXCL13 may be the recruitment of primitive B cells to body cavities for T-independent responses [112]. CLL and MCL cells express high levels of CXCR5 [15, 113]. Stimulation of CLL cells with CXCL13 induces actin polymerization, CXCR5 endocytosis, chemotaxis [114], and prolonged activation of MAPK (ERK 1/2). In CLL, CXCR5 signals through Gi proteins, PI3 kinases, and p44/42 MAPK pathway [15]. CXCL13 mRNA and protein is expressed by NLC in vitro and in vivo [15]. These data suggest that CXCR5 plays a role in positioning and interactions between malignant B cells and CXCL13-secreting stromal cells, such as NLC/LAM in lymphoid tissues.

CCL3 and CCL4 are chemo-attractants for monocytes and lymphocytes [115]. CCL3 expression in normal B cells is induced by BCR triggering and CD40 ligand [41, 116, 117], and repressed by Bcl-6 [118]. Activated CLL cells express and secrete CCL3/4 [18, 22, 119] in response to BCR stimulation and in co-culture with NLC [18]. This BCR- and NLC-dependent induction of CCL3/4 is sensitive to inhibition of BCR signaling, using SYK, BTK, and PI3K inhibitors [18, 60, 100, 120]. CLL patients have elevated CCL3/4 plasma levels [18] and plasma levels of CCL3 were strongly associated with established prognostic markers and time to treatment. A multivariable analysis revealed that CCL3, advanced clinical stage, poor risk cytogenetics, and CD38 expression were independent prognostic markers in a cohort of 351 CLL patients [12]. The function of CCL3/4 in CLL remains poorly defined, but based upon the function of B-cell-derived CCL3/4 in normal immune responses, increased CCL3/4 secretion by CLL cells may induce trafficking and homing of accessory cells, particularly of T cells and monocytes to CLL cells in the tissue microenvironments [18, 121].

Integrins are a superfamily of heterodimeric glycoproteins, consisting of various α (1 through 11) and β (1 through 6) subunits, whose function is to mediate cell-cell and cell-matrix adhesion in various cell types. The term “integrin” was first proposed in 1986 to describe membrane complexes involved in the transmembrane association between fibronectin (FN) as part of the ECM and the actin cytoskeleton [122]. Integrins are categorized into subfamilies with members sharing a common β subunit pairing with a unique α subunit. β1 integrins are very late activation antigens (VLA) that have the same β1 subunit but various α chains (α 1 through 6) [123]. The α4β1 integrin VLA-4 (CD49d) is a receptor for FN and vascular cell adhesion molecule-1 (VCAM-1/CD106). VLA-4 is expressed on lymphocytes, monocytes, and most other hematopoietic cells (except for neutrophils); VLA-4 is involved in both cell-cell and cell-ECM adhesion and plays a role in lymphocyte trafficking and homing as part of immune surveillance [124], trafficking and homing of other hematopoietic cells, and inflammation. Integrins are highly versatile adhesion molecules; their adhesiveness can rapidly be regulated by the cells on which they are expressed, for example, by chemokine receptor activation [125]. VLA-4 mediates lymphocyte adhesion to the VCAM-1 (CD106), which is expressed on cytokine-activated endothelium. VCAM-1 mediates leukocyte-endothelial cell adhesion and may play a role in the development of artherosclerosis and rheumatoid arthritis. VLA-4 also binds FN, an ECM component expressed on MSC [126], by interacting with at least three FN sites: CS-1 and REDV in the IIICS region and H1 in the HepII region [127]. VLA-4 plays a particularly important role for interactions between normal and malignant hematopoietic cells and the marrow microenvironment. VLA-4 integrins cooperate with chemokine receptors in CLL cell adhesion to stromal cells [35, 128], they cooperate with CD38 [129], and their function can be inhibited by the BTK inhibitor ibrutinib [130]. Moreover, VLA-4 expression on CLL cells has a prognostic impact [13, 131], indicating the relevance of these interactions in vivo. Collectively, these studies indicate that VLA-4 integrins play a key role for adhesion of CLL and other leukemia cells to stromal cells and ECM, and provide a rationale to further explore and target this molecule in CLL.

Normal and malignant B cells contain cytoplasmic kinases that are essential for transmitting signals from surface receptors into the cell, thereby regulating B-cell survival and proliferation. These include SYK, Bruton’s tyrosine kinase (BTK) and PI3 kinase isoforms, especially the delta isoform (PI3Kδ). These kinases are closely related to BCR signaling, as well as the signaling of B-cell chemokine receptors and adhesion molecules, which regulate the trafficking and homing of B lymphocytes. The SYK inhibitor fostamatinib was the first targeted agent to be tested in a clinical trial in patients with different B-cell malignancies including CLL patients [132]. Shortly thereafter, other inhibitors that target BCR signaling, especially BTK became available and were found to be highly effective for treating CLL patients. Building on basic and translational research that emphasized the importance of the BCR [133] and the CLL microenvironment [134] in CLL disease biology, we now can look back at 2 decades of transformative research in CLL, resulting in the approval of drugs that are targeting these key disease pathways. Within a short time, the BTK inhibitors have overturned CLL treatment paradigms, replacing chemotherapy-based treatments for patients with CLL. The efficacy of these kinase inhibitors in CLL is related to a high dependency of CLL cells on external signals derived from the microenvironment, especially the BCR. These developments demonstrate that CLL basic and translational research has come full circle, from studying the CLL microenvironment and the role of the BCR in CLL pathogenesis to effective targeted therapies replacing chemo-(immuno) therapy.

CLL is a paradigmatic malignancy in which a delicate balance between cell-extrinsic triggers from the tissue microenvironment and cell-intrinsic aberrations regulates the growth of the malignant B cells, which in turn determines the clinical behavior of individual patients. Signals from the microenvironment of secondary lymphoid tissues result in downstream signaling that promotes CLL cell growth. Among the surface receptors initiating these signaling cascades, the BCR has been shown to be central for the growth of the malignant B cells. The BCR structure is finely tuned by somatic hypermutation and dictates the signaling response of the BCR in individual patients, which appears to be the basis for the heterogenous clinical behavior in patients with CLL. Moreover, the BCR is key to treating CLL, as shown by the impressive clinical results in patients treated with kinase inhibitors targeting BCR signaling, such as the BTK inhibitors, which have become the first choice of therapy for many patients with CLL. The BCR appears to be the central driver of CLL disease progression, which is supported by the markedly different clinical outcomes of patients with CLL displaying a similar background of genomic aberrations, but different IGHV mutation statuses (M-CLL vs. U-CLL), and the remarkable therapeutic efficacy of BTK inhibitors even in patients with major cell cycle de-regulation due to cell-intrinsic aberrations (e.g., TP53 or ATM defects), underscoring the critical dependence of CLL cells on the BCR and the tissue microenvironment.

We apologize that we were not able to discuss and cite a number of additional studies of other investigators that are related to CXCR4 in neoplastic diseases because of space limitation.

The authors have no conflict of interest related to this review article.

J.A.B. is supported by MD Anderson’s Moon Shot Program in CLL and in part by the MD Anderson Cancer Center Support Grant CA016672.

S.K. and J.A.B. wrote the manuscript and designed the figures together.

Burger JA, Wiestner A. Targeting B cell receptor signalling in cancer: preclinical and clinical advances. Nat Rev Cancer. 2018 Mar;18(3):148–67.
Herishanu Y, Perez-Galan P, Liu D, Biancotto A, Pittaluga S, Vire B, et al. The lymph node microenvironment promotes B-cell receptor signaling, NF-kappaB activation, and tumor proliferation in chronic lymphocytic leukemia. Blood. 2011 Jan 13;117(2):563–74.
Herndon TM, Chen SS, Saba NS, Valdez J, Emson C, Gatmaitan M, et al. Direct in vivo evidence for increased proliferation of CLL cells in lymph nodes compared to bone marrow and peripheral blood. Leukemia. 2017 Jun;31(6):1340–7.
Collins RJ, Verschuer LA, Harmon BV, Prentice RL, Pope JH, Kerr JF. Spontaneous programmed death (apoptosis) of B-chronic lymphocytic leukaemia cells following their culture in vitro. Br J Haematol. 1989;71(3):343–50.
Panayiotidis P, Jones D, Ganeshaguru K, Foroni L, Hoffbrand AV. Human bone marrow stromal cells prevent apoptosis and support the survival of chronic lymphocytic leukaemia cells in vitro. Br J Haematol. 1996;92(1):97–103.
Burger JA, Tsukada N, Burger M, Zvaifler NJ, Dell’Aquila M, Kipps TJ. Blood-derived nurse-like cells protect chronic lymphocytic leukemia B cells from spontaneous apoptosis through stromal cell-derived factor-1. Blood. 2000 Oct 15;96(8):2655–63.
Burger JA. Treatment of chronic lymphocytic leukemia. N Engl J Med Overseas Ed. 2020 Jul 30;383(5):460–73.
Caligaris-Cappio F, Ghia P. Novel insights in chronic lymphocytic leukemia: are we getting closer to understanding the pathogenesis of the disease?J Clin Oncol. 2008 Sep 20;26(27):4497–503.
Messmer BT, Messmer D, Allen SL, Kolitz JE, Kudalkar P, Cesar D, et al. In vivo measurements document the dynamic cellular kinetics of chronic lymphocytic leukemia B cells. J Clin Invest. 2005 Mar;115(3):755–64.
Hashimoto S, Dono M, Wakai M, Allen SL, Lichtman SM, Schulman P, et al. Somatic diversification and selection of immunoglobulin heavy and light chain variable region genes in IgG+ CD5+ chronic lymphocytic leukemia B cells. J Exp Med. 1995 Apr 1;181(4):1507–17.
Calissano C, Damle RN, Hayes G, Murphy EJ, Hellerstein MK, Moreno C, et al. In vivo intraclonal and interclonal kinetic heterogeneity in B-cell chronic lymphocytic leukemia. Blood. 2009 Nov 26;114(23):4832–42.
Sivina M, Hartmann E, Kipps TJ, Rassenti L, Krupnik D, Lerner S, et al. CCL3 (MIP-1α) plasma levels and the risk for disease progression in chronic lymphocytic leukemia. Blood. 2011 Feb 3;117(5):1662–9.
Gattei V, Bulian P, Del Principe MI, Zucchetto A, Maurillo L, Buccisano F, et al. Relevance of CD49d protein expression as overall survival and progressive disease prognosticator in chronic lymphocytic leukemia. Blood. 2008 Jan 15;111(2):865–73.
Tsukada N, Burger JA, Zvaifler NJ, Kipps TJ. Distinctive features of “nurselike” cells that differentiate in the context of chronic lymphocytic leukemia. Blood. 2002 Feb 1;99(3):1030–7.
Burkle A, Niedermeier M, Schmitt-Graff A, Wierda WG, Keating MJ, Burger JA. Overexpression of the CXCR5 chemokine receptor, and its ligand, CXCL13 in B-cell chronic lymphocytic leukemia. Blood. 2007 Nov 1;110(9):3316–25.
Nishio M, Endo T, Tsukada N, Ohata J, Kitada S, Reed JC, et al. Nurselike cells express BAFF and APRIL, which can promote survival of chronic lymphocytic leukemia cells via a paracrine pathway distinct from that of SDF-1α. Blood. 2005 Aug 1;106(3):1012–20.
Endo T, Nishio M, Enzler T, Cottam HB, Fukuda T, James DF, et al. BAFF and APRIL support chronic lymphocytic leukemia B-cell survival through activation of the canonical NF-kappaB pathway. Blood. 2007 Jan 15;109(2):703–10.
Burger JA, Quiroga MP, Hartmann E, Burkle A, Wierda WG, Keating MJ, et al. High-level expression of the T-cell chemokines CCL3 and CCL4 by chronic lymphocytic leukemia B cells in nurselike cell cocultures and after BCR stimulation. Blood. 2009 Mar 26;113(13):3050–8.
Binder M, Lechenne B, Ummanni R, Scharf C, Balabanov S, Trusch M, et al. Stereotypical chronic lymphocytic leukemia B-cell receptors recognize survival promoting antigens on stromal cells. PLoS One. 2010;5(12):e15992.
Hacken ET, Gounari M, Back JW, Shimanovskaya E, Scarfo L, Kim E, et al. Calreticulin as a novel B-cell receptor antigen in chronic lymphocytic leukemia. Haematologica. 2017 Oct;102(10):e394–6.
Deaglio S, Vaisitti T, Bergui L, Bonello L, Horenstein AL, Tamagnone L, et al. CD38 and CD100 lead a network of surface receptors relaying positive signals for B-CLL growth and survival. Blood. 2005 Apr 15;105(8):3042–50.
Zucchetto A, Benedetti D, Tripodo C, Bomben R, Dal Bo M, Marconi D, et al. CD38/CD31, the CCL3 and CCL4 chemokines, and CD49d/vascular cell adhesion molecule-1 are interchained by sequential events sustaining chronic lymphocytic leukemia cell survival. Cancer Res. 2009 May 1;69(9):4001–9.
Niemann CU, Herman SE, Maric I, Gomez-Rodriguez J, Biancotto A, Chang BY, et al. Disruption of in vivo chronic lymphocytic leukemia tumor-microenvironment interactions by ibrutinib--findings from an investigator-initiated phase II study. Clin Cancer Res. 2016 Apr 01;22(7):1572–82.
Boissard F, Laurent C, Ramsay AG, Quillet-Mary A, Fournie JJ, Poupot M, et al. Nurse-like cells impact on disease progression in chronic lymphocytic leukemia. Blood Cancer J. 2016 Jan 15;6(1):e381.
Strati P, Schlette EJ, Solis Soto LM, Duenas DE, Sivina M, Kim E, et al. Achieving complete remission in CLL patients treated with ibrutinib: clinical significance and predictive factors. Blood. 2020 Feb 13;135(7):510–3.
Reinart N, Nguyen PH, Boucas J, Rosen N, Kvasnicka HM, Heukamp L, et al. Delayed development of chronic lymphocytic leukemia in the absence of macrophage migration inhibitory factor. Blood. 2013 Jan 31;121(5):812–21.
Gatjen M, Brand F, Grau M, Gerlach K, Kettritz R, Westermann J, et al. Splenic marginal zone granulocytes acquire an accentuated neutrophil B-cell helper phenotype in chronic lymphocytic leukemia. Cancer Res. 2016 Sep 15;76(18):5253–65.
Martines C, Chakraborty S, Vujovikj M, Gobessi S, Vaisitti T, Deaglio S, et al. Macrophage- and BCR-derived but not TLR-derived signals support the growth of CLL and Richter syndrome murine models in vivo. Blood. 2022 Dec 1;140(22):2335–47.
Bagnara D, Kaufman MS, Calissano C, Marsilio S, Patten PE, Simone R, et al. A novel adoptive transfer model of chronic lymphocytic leukemia suggests a key role for T lymphocytes in the disease. Blood. 2011 May 19;117(20):5463–72.
Galletti G, Scielzo C, Barbaglio F, Rodriguez TV, Riba M, Lazarevic D, et al. Targeting macrophages sensitizes chronic lymphocytic leukemia to apoptosis and inhibits disease progression. Cell Rep. 2016 Feb 23;14(7):1748–60.
Rodda LB, Bannard O, Ludewig B, Nagasawa T, Cyster JG. Phenotypic and morphological properties of germinal center dark zone cxcl12-expressing reticular cells. J Immunol. 2015 Nov 15;195(10):4781–91.
Ruan J, Hyjek E, Kermani P, Christos PJ, Hooper AT, Coleman M, et al. Magnitude of stromal hemangiogenesis correlates with histologic subtype of non-Hodgkin’s lymphoma. Clin Cancer Res. 2006 Oct 1;12(19):5622–31.
Ding W, Nowakowski GS, Knox TR, Boysen JC, Maas ML, Schwager SM, et al. Bi-directional activation between mesenchymal stem cells and CLL B-cells: implication for CLL disease progression. Br J Haematol. 2009 Nov;147(4):471–83.
Kurtova AV, Balakrishnan K, Chen R, Ding W, Schnabl S, Quiroga MP, et al. Diverse marrow stromal cells protect CLL cells from spontaneous and drug-induced apoptosis: development of a reliable and reproducible system to assess stromal cell adhesion-mediated drug resistance. Blood. 2009 Nov 12;114(20):4441–50.
Burger JA, Burger M, Kipps TJ. Chronic lymphocytic leukemia B cells express functional CXCR4 chemokine receptors that mediate spontaneous migration beneath bone marrow stromal cells. Blood. 1999;94(11):3658–67.
Burger M, Hartmann T, Krome M, Rawluk J, Tamamura H, Fujii N, et al. Small peptide inhibitors of the CXCR4 chemokine receptor (CD184) antagonize the activation, migration, and antiapoptotic responses of CXCL12 in chronic lymphocytic leukemia B cells. Blood. 2005 Sep 1;106(5):1824–30.
Harzschel A, Zucchetto A, Gattei V, Hartmann TN. VLA-4 expression and activation in B cell malignancies: functional and clinical aspects. Int J Mol Sci. 2020 Mar 23:21(6):2206.
Sivina M, Hartmann E, Vasyutina E, Boucas JM, Breuer A, Keating MJ, et al. Stromal cells modulate TCL1 expression, interacting AP-1 components and TCL1-targeting micro-RNAs in chronic lymphocytic leukemia. Leukemia. 2012 Mar 5;26(8):1812–20.
Vom Stein AF, Rebollido-Rios R, Lukas A, Koch M, von Lom A, Reinartz S, et al. LYN kinase programs stromal fibroblasts to facilitate leukemic survival via regulation of c-JUN and THBS1. Nat Commun. 2023 Mar 10;14(1):1330.
Allen CD, Okada T, Cyster JG. Germinal-center organization and cellular dynamics. Immunity. 2007 Aug;27(2):190–202.
Krzysiek R, Lefèvre EA, Zou W, Foussat A, Bernard J, Portier A, et al. Antigen receptor engagement selectively induces macrophage inflammatory protein-1 alpha (MIP-1 alpha) and MIP-1 beta chemokine production in human B cells. J Immunol. 1999 Apr 15;162(8):4455–63.
Hartmann EM, Rudelius M, Burger JA, Rosenwald A. CCL3 chemokine expression by chronic lymphocytic leukemia cells orchestrates the composition of the microenvironment in lymph node infiltrates. Leuk Lymphoma. 2016;57(3):563–71.
Sivina M, Xiao L, Kim E, Vaca A, Chen SS, Keating MJ, et al. CXCL13 plasma levels function as a biomarker for disease activity in patients with chronic lymphocytic leukemia. Leukemia. 2021 Jun;35(6):1610–20.
Vardi A, Vlachonikola E, Karypidou M, Stalika E, Bikos V, Gemenetzi K, et al. Restrictions in the T-cell repertoire of chronic lymphocytic leukemia: high-throughput immunoprofiling supports selection by shared antigenic elements. Leukemia. 2017 Jul;31(7):1555–61.
Pizzolo G, Chilosi M, Ambrosetti A, Semenzato G, Fiore-Donati L, Perona G. Immunohistologic study of bone marrow involvement in B-chronic lymphocytic leukemia. Blood. 1983;62(6):1289–96.
Granziero L, Ghia P, Circosta P, Gottardi D, Strola G, Geuna M, et al. Survivin is expressed on CD40 stimulation and interfaces proliferation and apoptosis in B-cell chronic lymphocytic leukemia. Blood. 2001 May 1;97(9):2777–83.
Ghia P, Strola G, Granziero L, Geuna M, Guida G, Sallusto F, et al. Chronic lymphocytic leukemia B cells are endowed with the capacity to attract CD4+, CD40L+ T cells by producing CCL22. Eur J Immunol. 2002 May;32(5):1403–13.
Patten PE, Buggins AG, Richards J, Wotherspoon A, Salisbury J, Mufti GJ, et al. CD38 expression in chronic lymphocytic leukemia is regulated by the tumor microenvironment. Blood. 2008 May 15;111(10):5173–81.
Roessner PM, Seiffert M. T-cells in chronic lymphocytic leukemia: guardians or drivers of disease? Leukemia. Leukemia. 2020 Aug;34(8):2012–24.
Yin Q, Sivina M, Robins H, Yusko E, Vignali M, O’Brien S, et al. Ibrutinib therapy increases T cell repertoire diversity in patients with chronic lymphocytic leukemia. J Immunol. 2017 Feb 15;198(4):1740–7.
Ramsay AG, Johnson AJ, Lee AM, Gorgun G, Le Dieu R, Blum W, et al. Chronic lymphocytic leukemia T cells show impaired immunological synapse formation that can be reversed with an immunomodulating drug. J Clin Invest. 2008 Jul;118(7):2427–37.
Patten PEM, Ferrer G, Chen SS, Kolitz JE, Rai KR, Allen SL, et al. A detailed analysis of parameters supporting the engraftment and growth of chronic lymphocytic leukemia cells in immune-deficient mice. Front Immunol. 2021;12:627020.
Hofbauer JP, Heyder C, Denk U, Kocher T, Holler C, Trapin D, et al. Development of CLL in the TCL1 transgenic mouse model is associated with severe skewing of the T-cell compartment homologous to human CLL. Leukemia. 2011 Sep;25(9):1452–8.
Vardi A, Vlachonikola E, Papazoglou D, Psomopoulos F, Kotta K, Ioannou N, et al. T-cell dynamics in chronic lymphocytic leukemia under different treatment modalities. Clin Cancer Res. 2020 Sep 15;26(18):4958–69.
de Weerdt I, Hofland T, de Boer R, Dobber JA, Dubois J, van Nieuwenhuize D, et al. Distinct immune composition in lymph node and peripheral blood of CLL patients is reshaped during venetoclax treatment. Blood Adv. 2019 Sep 10;3(17):2642–52.
Vaca AM, Ioannou N, Sivina M, Vlachonikola E, Clise-Dwyer K, Kim E, et al. Activation and expansion of T-follicular helper cells in chronic lymphocytic leukemia nurselike cell co-cultures. Leukemia. 2022 May;36(5):1324–35.
Tandon B, Swerdlow SH, Hasserjian RP, Surti U, Gibson SE. Chronic lymphocytic leukemia/small lymphocytic lymphoma: another neoplasm related to the B-cell follicle?Leuk Lymphoma. 2015;56(12):3378–86.
Pedersen IM, Kitada S, Leoni LM, Zapata JM, Karras JG, Tsukada N, et al. Protection of CLL B cells by a follicular dendritic cell line is dependent on induction of Mcl-1. Blood. 2002 Sep 1;100(5):1795–801.
Heinig K, Gatjen M, Grau M, Stache V, Anagnostopoulos I, Gerlach K, et al. Access to follicular dendritic cells is a pivotal step in murine chronic lymphocytic leukemia B-cell activation and proliferation. Cancer Discov. 2014 Dec;4(12):1448–65.
Ponader S, Chen SS, Buggy JJ, Balakrishnan K, Gandhi V, Wierda WG, et al. The Bruton tyrosine kinase inhibitor PCI-32765 thwarts chronic lymphocytic leukemia cell survival and tissue homing in vitro and in vivo. Blood. 2012 Feb 2;119(5):1182–9.
Brown JR, Byrd JC, Coutre SE, Benson DM, Flinn IW, Wagner-Johnston ND, et al. Idelalisib, an inhibitor of phosphatidylinositol 3-kinase p110δ, for relapsed/refractory chronic lymphocytic leukemia. Blood. 2014 May 29;123(22):3390–7.
Stein H, Bonk A, Tolksdorf G, Lennert K, Rodt H, Gerdes J. Immunohistologic analysis of the organization of normal lymphoid tissue and non-Hodgkin’s lymphomas. J Histochem Cytochem. 1980 Aug;28(8):746–60.
Burger JA, Li KW, Keating MJ, Sivina M, Amer AM, Garg N, et al. Leukemia cell proliferation and death in chronic lymphocytic leukemia patients on therapy with the BTK inhibitor ibrutinib. JCI insight. 2017 Jan 26;2(2):e89904.
Fais F, Ghiotto F, Hashimoto S, Sellars B, Valetto A, Allen SL, et al. Chronic lymphocytic leukemia B cells express restricted sets of mutated and unmutated antigen receptors. J Clin Invest. 1998;102(8):1515–25.
Burger JA, Barr PM, Robak T, Owen C, Ghia P, Tedeschi A, et al. Long-term efficacy and safety of first-line ibrutinib treatment for patients with CLL/SLL: 5 years of follow-up from the phase 3 RESONATE-2 study. Leukemia. 2020;34:787–98.
Damle RN, Ghiotto F, Valetto A, Albesiano E, Fais F, Yan XJ, et al. B-cell chronic lymphocytic leukemia cells express a surface membrane phenotype of activated, antigen-experienced B lymphocytes. Blood. 2002 Jun 1;99(11):4087–93.
Mockridge CI, Potter KN, Wheatley I, Neville LA, Packham G, Stevenson FK. Reversible anergy of sIgM-mediated signaling in the two subsets of CLL defined by VH-gene mutational status. Blood. 2007 May 15;109(10):4424–31.
Ten Hacken E, Sivina M, Kim E, O’Brien S, Wierda WG, Ferrajoli A, et al. Functional differences between IgM and IgD signaling in chronic lymphocytic leukemia. J Immunol. 2016 Sep 15;197(6):2522–31.
Lanham S, Hamblin T, Oscier D, Ibbotson R, Stevenson F, Packham G. Differential signaling via surface IgM is associated with VH gene mutational status and CD38 expression in chronic lymphocytic leukemia. Blood. 2003 Feb 1;101(3):1087–93.
Chen L, Apgar J, Huynh L, Dicker F, Giago-McGahan T, Rassenti L, et al. ZAP-70 directly enhances IgM signaling in chronic lymphocytic leukemia. Blood. 2005 Mar 1;105(5):2036–41.
Messmer BT, Albesiano E, Efremov DG, Ghiotto F, Allen SL, Kolitz J, et al. Multiple distinct sets of stereotyped antigen receptors indicate a role for antigen in promoting chronic lymphocytic leukemia. J Exp Med. 2004 Aug 16;200(4):519–25.
Lanemo Myhrinder A, Hellqvist E, Sidorova E, Soderberg A, Baxendale H, Dahle C, et al. A new perspective: molecular motifs on oxidized LDL, apoptotic cells, and bacteria are targets for chronic lymphocytic leukemia antibodies. Blood. 2008 Apr 1;111(7):3838–48.
Hoogeboom R, van Kessel KP, Hochstenbach F, Wormhoudt TA, Reinten RJ, Wagner K, et al. A mutated B cell chronic lymphocytic leukemia subset that recognizes and responds to fungi. J Exp Med. 2013 Jan 14;210(1):59–70.
Chu CC, Catera R, Zhang L, Didier S, Agagnina BM, Damle RN, et al. Many chronic lymphocytic leukemia antibodies recognize apoptotic cells with exposed nonmuscle myosin heavy chain IIA: implications for patient outcome and cell of origin. Blood. 2010 May 13;115(19):3907–15.
Kostareli E, Hadzidimitriou A, Stavroyianni N, Darzentas N, Athanasiadou A, Gounari M, et al. Molecular evidence for EBV and CMV persistence in a subset of patients with chronic lymphocytic leukemia expressing stereotyped IGHV4-34 B-cell receptors. Leukemia. 2009 May;23(5):919–24.
Preud’homme JL, Seligmann M. Anti-human immunoglobulin G activity of membrane-bound monoclonal immunoglobulin M in lymphoproliferative disorders. Proc Natl Acad Sci USA. 1972 Aug;69(8):2132–5.
Preud’homme JL, Seligmann M. Surface bound immunoglobulins as a cell marker in human lymphoproliferative diseases. Blood. 1972 Dec;40(6):777–94.
Kostareli E, Gounari M, Janus A, Murray F, Brochet X, Giudicelli V, et al. Antigen receptor stereotypy across B-cell lymphoproliferations: the case of IGHV4-59/IGKV3-20 receptors with rheumatoid factor activity. Leukemia. 2012 May;26(5):1127–31.
Duhren-von Minden M, Ubelhart R, Schneider D, Wossning T, Bach MP, Buchner M, et al. Chronic lymphocytic leukaemia is driven by antigen-independent cell-autonomous signalling. Nature. 2012 Sep 13;489(7415):309–12.
Minici C, Gounari M, Ubelhart R, Scarfo L, Duhren-von Minden M, Schneider D, et al. Distinct homotypic B-cell receptor interactions shape the outcome of chronic lymphocytic leukaemia. Nat Commun. 2017 Jun 9;8:15746.
Mazzarello AN, Gentner-Gobel E, Duhren-von Minden M, Tarasenko TN, Nicolo A, Ferrer G, et al. B cell receptor isotypes differentially associate with cell signaling, kinetics, and outcome in chronic lymphocytic leukemia. J Clin Invest. 2022 Jan 18;132(2):e149308.
Woyach JA, Ruppert AS, Guinn D, Lehman A, Blachly JS, Lozanski A, et al. BTKC481S-Mediated resistance to ibrutinib in chronic lymphocytic leukemia. J Clin Oncol. 2017 May 01;35(13):1437–43.
Blombery P, Thompson ER, Lew TE, Tiong IS, Bennett R, Cheah CY, et al. Enrichment of BTK Leu528Trp mutations in patients with CLL on zanubrutinib: potential for pirtobrutinib cross-resistance. Blood Adv. 2022 Oct 25;6(20):5589–92.
Wang E, Mi X, Thompson MC, Montoya S, Notti RQ, Afaghani J, et al. Mechanisms of resistance to noncovalent bruton’s tyrosine kinase inhibitors. N Engl J Med. 2022 Feb 24;386(8):735–43.
Stamatopoulos K, Agathangelidis A, Rosenquist R, Ghia P. Antigen receptor stereotypy in chronic lymphocytic leukemia. Leukemia. 2017 Feb;31(2):282–91.
Maity PC, Bilal M, Koning MT, Young M, van Bergen CAM, Renna V, et al. IGLV3-21 * 01 is an inherited risk factor for CLL through the acquisition of a single-point mutation enabling autonomous BCR signaling. Proc Natl Acad Sci USA. 2020 Feb 25;117(8):4320–7.
Jaramillo S, Agathangelidis A, Schneider C, Bahlo J, Robrecht S, Tausch E, et al. Prognostic impact of prevalent chronic lymphocytic leukemia stereotyped subsets: analysis within prospective clinical trials of the German CLL Study Group (GCLLSG). Haematologica. 2020 Nov 1;105(11):2598–607.
Tobin G, Thunberg U, Johnson A, Eriksson I, Soderberg O, Karlsson K, et al. Chronic lymphocytic leukemias utilizing the VH3-21 gene display highly restricted Vlambda2-14 gene use and homologous CDR3s: implicating recognition of a common antigen epitope. Blood. 2003 Jun 15;101(12):4952–7.
Tobin G, Thunberg U, Karlsson K, Murray F, Laurell A, Willander K, et al. Subsets with restricted immunoglobulin gene rearrangement features indicate a role for antigen selection in the development of chronic lymphocytic leukemia. Blood. 2004 Nov 1;104(9):2879–85.
Widhopf GF2nd, Rassenti LZ, Toy TL, Gribben JG, Wierda WG, Kipps TJ. Chronic lymphocytic leukemia B cells of more than 1% of patients express virtually identical immunoglobulins. Blood. 2004 Oct 15;104(8):2499–504.
Stamatopoulos K, Belessi C, Moreno C, Boudjograh M, Guida G, Smilevska T, et al. Over 20% of patients with chronic lymphocytic leukemia carry stereotyped receptors: pathogenetic implications and clinical correlations. Blood. 2007 Jan 1;109(1):259–70.
Murray F, Darzentas N, Hadzidimitriou A, Tobin G, Boudjogra M, Scielzo C, et al. Stereotyped patterns of somatic hypermutation in subsets of patients with chronic lymphocytic leukemia: implications for the role of antigen selection in leukemogenesis. Blood. 2008 Feb 1;111(3):1524–33.
Darzentas N, Hadzidimitriou A, Murray F, Hatzi K, Josefsson P, Laoutaris N, et al. A different ontogenesis for chronic lymphocytic leukemia cases carrying stereotyped antigen receptors: molecular and computational evidence. Leukemia. 2010 Jan;24(1):125–32.
Agathangelidis A, Darzentas N, Hadzidimitriou A, Brochet X, Murray F, Yan XJ, et al. Stereotyped B-cell receptors in one-third of chronic lymphocytic leukemia: a molecular classification with implications for targeted therapies. Blood. 2012 May 10;119(19):4467–75.
Agathangelidis A, Chatzidimitriou A, Gemenetzi K, Giudicelli V, Karypidou M, Plevova K, et al. Higher-order connections between stereotyped subsets: implications for improved patient classification in CLL. Blood. 2021;137(10):1365–76.
Briney B, Inderbitzin A, Joyce C, Burton DR. Commonality despite exceptional diversity in the baseline human antibody repertoire. Nature. 2019 Feb;566(7744):393–7.
Richardson SJ, Matthews C, Catherwood MA, Alexander HD, Carey BS, Farrugia J, et al. ZAP-70 expression is associated with enhanced ability to respond to migratory and survival signals in B-cell chronic lymphocytic leukemia (B-CLL). Blood. 2006 May 1;107(9):3584–92.
Nagasawa T, Kikutani H, Kishimoto T. Molecular cloning and structure of a pre-B-cell growth-stimulating factor. Proc Natl Acad Sci USA. 1994;91(6):2305–9.
Bennett F, Rawstron A, Plummer M, de Tute R, Moreton P, Jack A, et al. B-cell chronic lymphocytic leukaemia cells show specific changes in membrane protein expression during different stages of cell cycle. Br J Haematol. 2007 Nov;139(4):600–4.
Quiroga MP, Balakrishnan K, Kurtova AV, Sivina M, Keating MJ, Wierda WG, et al. B-cell antigen receptor signaling enhances chronic lymphocytic leukemia cell migration and survival: specific targeting with a novel spleen tyrosine kinase inhibitor, R406. Blood. 2009 Jul 30;114(5):1029–37.
Vlad A, Deglesne PA, Letestu R, Saint-Georges S, Chevallier N, Baran-Marszak F, et al. Down-regulation of CXCR4 and CD62L in chronic lymphocytic leukemia cells is triggered by B-cell receptor ligation and associated with progressive disease. Cancer Res. 2009 Aug 15;69(16):6387–95.
Chen L, Widhopf G, Huynh L, Rassenti L, Rai KR, Weiss A, et al. Expression of ZAP-70 is associated with increased B-cell receptor signaling in chronic lymphocytic leukemia. Blood. 2002 Dec 15;100(13):4609–14.
Niedermeier M, Hennessy BT, Knight ZA, Henneberg M, Hu J, Kurtova AV, et al. Isoform-selective phosphoinositide 3'-kinase inhibitors inhibit CXCR4 signaling and overcome stromal cell-mediated drug resistance in chronic lymphocytic leukemia: a novel therapeutic approach. Blood. 2009 May 28;113(22):5549–57.
Cinamon G, Zachariah MA, Lam OM, Foss FWJr, Cyster JG. Follicular shuttling of marginal zone B cells facilitates antigen transport. Nat Immunol. 2008 Jan;9(1):54–62.
Muller G, Hopken UE, Lipp M. The impact of CCR7 and CXCR5 on lymphoid organ development and systemic immunity. Immunol Rev. 2003 Oct;195:117–35.
Forster R, Mattis AE, Kremmer E, Wolf E, Brem G, Lipp M. A putative chemokine receptor, BLR1, directs B cell migration to defined lymphoid organs and specific anatomic compartments of the spleen. Cell. 1996;87(6):1037–47.
Mueller SN, Germain RN. Stromal cell contributions to the homeostasis and functionality of the immune system. Nat Rev Immunol. 2009 Sep;9(9):618–29.
Allen CD, Ansel KM, Low C, Lesley R, Tamamura H, Fujii N, et al. Germinal center dark and light zone organization is mediated by CXCR4 and CXCR5. Nat Immunol. 2004 Sep;5(9):943–52.
Bajenoff M, Egen JG, Koo LY, Laugier JP, Brau F, Glaichenhaus N, et al. Stromal cell networks regulate lymphocyte entry, migration, and territoriality in lymph nodes. Immunity. 2006 Dec;25(6):989–1001.
Allen CD, Okada T, Tang HL, Cyster JG. Imaging of germinal center selection events during affinity maturation. Science. 2007 Jan 26;315(5811):528–31.
Schwickert TA, Lindquist RL, Shakhar G, Livshits G, Skokos D, Kosco-Vilbois MH, et al. In vivo imaging of germinal centres reveals a dynamic open structure. Nature. 2007 Mar 1;446(7131):83–7.
Ansel KM, Harris RB, Cyster JG. CXCL13 is required for B1 cell homing, natural antibody production, and body cavity immunity. Immunity. 2002 Jan;16(1):67–76.
Kurtova AV, Tamayo AT, Ford RJ, Burger JA. Mantle cell lymphoma cells express high levels of CXCR4, CXCR5, and VLA-4 (CD49d): importance for interactions with the stromal microenvironment and specific targeting. Blood. 2009 May 7;113(19):4604–13.
Trentin L, Cabrelle A, Facco M, Carollo D, Miorin M, Tosoni A, et al. Homeostatic chemokines drive migration of malignant B cells in patients with non-Hodgkin lymphomas. Blood. 2004 Jul 15;104(2):502–8.
Schall TJ, Bacon K, Camp RD, Kaspari JW, Goeddel DV. Human macrophage inflammatory protein alpha (MIP-1 alpha) and MIP-1 beta chemokines attract distinct populations of lymphocytes. J Exp Med. 1993 Jun 1;177(6):1821–6.
Alizadeh A, Eisen M, Davis RE, Ma C, Sabet H, Tran T, et al. The lymphochip: a specialized cDNA microarray for the genomic-scale analysis of gene expression in normal and malignant lymphocytes. Cold Spring Harb Symp Quant Biol. 1999;64:71–8.
Eberlein J, Nguyen TT, Victorino F, Golden-Mason L, Rosen HR, Homann D. Comprehensive assessment of chemokine expression profiles by flow cytometry. J Clin Invest. 2010 Mar 1;120(3):907–23.
Shaffer AL, Yu X, He Y, Boldrick J, Chan EP, Staudt LM. BCL-6 represses genes that function in lymphocyte differentiation, inflammation, and cell cycle control. Immunity. 2000 Aug;13(2):199–212.
Palacios F, Moreno P, Morande P, Abreu C, Correa A, Porro V, 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. Blood. 2010 Jun 3;115(22):4488–96.
Hoellenriegel J, Meadows SA, Sivina M, Wierda WG, Kantarjian H, Keating MJ, et al. The phosphoinositide 3'-kinase delta inhibitor, CAL-101, inhibits B-cell receptor signaling and chemokine networks in chronic lymphocytic leukemia. Blood. 2011 Sep 29;118(13):3603–12.
Zucchetto A, Tripodo C, Benedetti D, Deaglio S, Gaidano G, Del Poeta G, et al. Monocytes/macrophages but not T lymphocytes are the major targets of the CCL3/CCL4 chemokines produced by CD38(+)CD49d(+) chronic lymphocytic leukaemia cells. Br J Haematol. 2010 Jul;150(1):111–3.
Tamkun JW, DeSimone DW, Fonda D, Patel RS, Buck C, Horwitz AF, et al. Structure of integrin, a glycoprotein involved in the transmembrane linkage between fibronectin and actin. Cell. 1986 Jul 18;46(2):271–82.
Yonekawa K, Harlan JM. Targeting leukocyte integrins in human diseases. J Leukoc Biol. 2005 Feb;77(2):129–40.
Butcher EC, Picker LJ. Lymphocyte homing and homeostasis. Science. 1996;272(5258):60–6.
Springer TA. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell. 1994;76(2):301–14.
Matsunaga T, Takemoto N, Sato T, Takimoto R, Tanaka I, Fujimi A, et al. Interaction between leukemic-cell VLA-4 and stromal fibronectin is a decisive factor for minimal residual disease of acute myelogenous leukemia. Nat Med. 2003 Sep;9(9):1158–65.
Chan PY, Aruffo A. VLA-4 integrin mediates lymphocyte migration on the inducible endothelial cell ligand VCAM-1 and the extracellular matrix ligand fibronectin. J Biol Chem. 1993 Nov 25;268(33):24655–64.
Burger JA, Zvaifler NJ, Tsukada N, Firestein GS, Kipps TJ. Fibroblast-like synoviocytes support B-cell pseudoemperipolesis via a stromal cell-derived factor-1- and CD106 (VCAM-1)-dependent mechanism. J Clin Invest. 2001;107(3):305–15.
Zucchetto A, Vaisitti T, Benedetti D, Tissino E, Bertagnolo V, Rossi D, et al. The CD49d/CD29 complex is physically and functionally associated with CD38 in B-cell chronic lymphocytic leukemia cells. Leukemia. 2012 Jun;26(6):1301–12.
de Rooij MF, Kuil A, Geest CR, Eldering E, Chang BY, Buggy JJ, et al. The clinically active BTK inhibitor PCI-32765 targets B-cell receptor- and chemokine-controlled adhesion and migration in chronic lymphocytic leukemia. Blood. 2012 Mar 15;119(11):2590–4.
Shanafelt TD, Geyer SM, Bone ND, Tschumper RC, Witzig TE, Nowakowski GS, et al. CD49d expression is an independent predictor of overall survival in patients with chronic lymphocytic leukaemia: a prognostic parameter with therapeutic potential. Br J Haematol. 2008 Mar;140(5):537–46.
Friedberg JW, Sharman J, Sweetenham J, Johnston PB, Vose JM, Lacasce A, et al. Inhibition of Syk with fostamatinib disodium has significant clinical activity in non-Hodgkin lymphoma and chronic lymphocytic leukemia. Blood. 2010 Apr 1;115(13):2578–85.
Chiorazzi N, Rai KR, Ferrarini M. Chronic lymphocytic leukemia. N Engl J Med Overseas Ed. 2005 Feb 24;352(8):804–15.
Burger JA, Ghia P, Rosenwald A, Caligaris-Cappio F. The microenvironment in mature B-cell malignancies: a target for new treatment strategies. Blood. 2009 Oct 15;114(16):3367–75.