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Commentary Free access | 10.1172/JCI79189
1Department of Laboratory Medicine, University of California, San Francisco, San Francisco, California, USA.
2Department of Haematology, University of Cambridge, Cambridge, United Kingdom.
Address correspondence to: Markus Müschen, Department of Laboratory Medicine, Box 0134, University of California, San Francisco, San Francisco, California 94143-0134, USA. Phone: 415.502.0388; E-mail: markus.muschen@ucsf.edu.
Find articles by Swaminathan, S. in: JCI | PubMed | Google Scholar
1Department of Laboratory Medicine, University of California, San Francisco, San Francisco, California, USA.
2Department of Haematology, University of Cambridge, Cambridge, United Kingdom.
Address correspondence to: Markus Müschen, Department of Laboratory Medicine, Box 0134, University of California, San Francisco, San Francisco, California 94143-0134, USA. Phone: 415.502.0388; E-mail: markus.muschen@ucsf.edu.
Find articles by Müschen, M. in: JCI | PubMed | Google Scholar
Published November 10, 2014 - More info
It has recently been demonstrated that memory B cells can reenter and reengage germinal center (GC) reactions, opening the possibility that multi-hit lymphomagenesis gradually occurs throughout life during successive immunological challenges. Here, we investigated this scenario in follicular lymphoma (FL), an indolent GC-derived malignancy. We developed a mouse model that recapitulates the FL hallmark t(14;18) translocation, which results in constitutive activation of antiapoptotic protein B cell lymphoma 2 (BCL2) in a subset of B cells, and applied a combination of molecular and immunofluorescence approaches to track normal and t(14;18)+ memory B cells in human and BCL2-overexpressing B cells in murine lymphoid tissues. BCL2-overexpressing B cells required multiple GC transits before acquiring FL-associated developmental arrest and presenting as GC B cells with constitutive activation–induced cytidine deaminase (AID) mutator activity. Moreover, multiple reentries into the GC were necessary for the progression to advanced precursor stages of FL. Together, our results demonstrate that protracted subversion of immune dynamics contributes to early dissemination and progression of t(14;18)+ precursors and shapes the systemic presentation of FL patients.
Stéphanie Sungalee, Emilie Mamessier, Ester Morgado, Emilie Grégoire, Philip Z. Brohawn, Christopher A. Morehouse, Nathalie Jouve, Céline Monvoisin, Cédric Menard, Guilhaume Debroas, Mustapha Faroudi, Violaine Mechin, Jean-Marc Navarro, Charlotte Drevet, Franziska C. Eberle, Lionel Chasson, Fannie Baudimont, Stéphane J. Mancini, Julie Tellier, Jean-Michel Picquenot, Rachel Kelly, Paolo Vineis, Philippe Ruminy, Bruno Chetaille, Elaine S. Jaffe, Claudine Schiff, Jean Hardwigsen, David A. Tice, Brandon W. Higgs, Karin Tarte, Bertrand Nadel, Sandrine Roulland
Memory B cells are a dynamic subset of the mature B cell population that in some cases can reenter germinal centers (GCs) in response to iterative infections. Such a reactivation can lead to accumulation of genetic lesions in these cells, potentially from repetitive activation of the B cell mutator enzyme AID. Normal memory B cells do not survive repeated reentries into GCs. In this issue, Sungalee et al. demonstrate that memory B cells harboring the oncogenic BCL2:IGH translocation, which results in constitutive BCL2 expression, survive multiple GC entries upon repetitive immunization. Through these multiple GC reentries, the hallmark BCL2:IGH translocation enables AID-induced hypermutation and propagates clonal evolution toward malignant follicular lymphoma.
Follicular lymphoma (FL) is an indolent subtype of non-Hodgkin’s lymphoma (1) that clonally evolves over decades (2) before presenting as overt disease. In almost all cases of FL, the t(14;18)(q32;q21) translocation, which places the oncogene BCL2 under the control of the immunoglobulin heavy chain (IGH) enhancer, represents an initial genetic event (3–5). The juxtaposition of BCL2 to the JH cluster of the IGH locus confers a survival advantage on developmentally arrested germinal center (GC) B cells (6–8). Normal GC B cells do not express BCL2 and are characterized by a proapoptotic program of gene expression that includes cell surface death receptor FAS, tumor protein TP53, and BCL2-associated X (BAX) (9). Therefore, GC B cells are destined to die (9, 10) unless they are rescued by survival signals emanating from a B cell receptor (BCR) with high affinity to antigen. BCL2 expression is triggered in memory B cells that arise from a GC reaction and increases the half-life of these cells (11). As a consequence, transgenic mice that overexpress BCL2 in the hematopoietic compartment display a marked reduction of B cell apoptosis in GCs as compared with that seen in WT controls (12). Affinity maturation in these transgenic mice results in a memory B cell compartment with reduced stringency in the selection of high-affinity BCRs (12). These findings suggest that constitutive expression of BCL2 as the result of the t(14;18)(q32;q21) translocation fundamentally alters GC and memory B cell dynamics.
Despite constitutive BCL2 expression, cooperating genetic lesions are required for malignant transformation of B cells into full-blown FL (7, 8, 13, 14). The t(14;18)(q32;q21) translocation occurs at a low frequency in normal B cells in about 70% of healthy individuals and increases with age (15–18). Individuals in whom t(14;18)(q32;q21) translocation has occurred display developmentally arrested B cells (FL precursors) that have transitioned through the GC and have imprints of AID activity, namely somatic hypermutation (SHM) and class switch recombination (CSR) (16, 18). Full-blown FL arises from these atypical B cells decades later (2). The long latency period observed in FL development indicates a prolonged process of clonal evolution. Such a protracted clonal evolutionary process was previously demonstrated in a subset of pre–B cell acute lymphoblastic leukemia (ALL) patients who harbor the ets variant 6 runt-related transcription factor 1 (ETV6-RUNX1) translocation (19–21). Although the ETV6-RUNX1 rearrangement arises in utero (20), less than 1% of children who carry this rearrangement develop full-blown leukemia, which requires postnatal acquisition of secondary lesions in the preleukemic clone (21).
Only recently have studies begun to elucidate the mechanisms responsible for the long latency period in FL evolution. Multiple studies have shown that IgM+ memory B cells get reactivated and can reenter the GC upon antigenic recall (22–24). Importantly, GC reentry is restricted to the IgM+ memory B cell subset and does not occur in IgG+ memory B cells (22, 23). In this issue, Sungalee and colleagues reveal that the iterative GC reentry of t(14;18)(q32;q21)-carrying IgM+ memory B cells upon chronic immunization is the central driver of follicular lymphomagenesis (25).
Sungalee and colleagues developed murine models that recapitulate the genesis of FL by elegantly mimicking the sporadic occurrence of the t(14;18)(q32;q21) translocation in humans (25). BCL2tracer mice harbor an engineered human BCL2 transgene that must undergo rearrangement to be transcribed and is highly expressed in B cells upon activation of the RAG recombinases (25). The same BCL2 transgene could also be transduced via retrovirus into BM precursors, which could then be transplanted into irradiated mice. In humans, the t(14;18)(q32;q21) breakpoint combines features of RAG-mediated V(D)J recombination and AID-dependent CpG targeting and typically occurs in pro–/pre–B cells (26). This feature of the t(14;18)(q32;q21) translocation makes the RAG-dependent BCL2tracer model developed by Sungalee et al. physiologically relevant to human disease. Moreover, these models could be tweaked to study perturbations in the frequency of BCL2-expressing B cells in different lymphoid organs. The authors also investigated immunization-induced bidirectional transitions between GC and memory B cells in response to high expression levels of BCL2 (25).
Sungalee and colleagues determined that BCL2-expressing memory B cells are able to reenter the GC following repeated antigen challenges. As demonstrated by the BCL2tracer mouse model, a small number of initial GC transits is insufficient for the accumulation of abnormal BCL2-expressing B cells and the induction of FL (25). Therefore, the authors proposed and validated that chronic or repetitive antigen exposure is required to induce multiple iterative GC entries and genetic instability. Only BCL2-expressing memory B cells underwent repetitive GC transits by evading apoptosis (ref. 25 and Figure 1). The authors found that these cells also failed to differentiate after GC passage and continued to express IgM on the surface, thereby continuously accumulating in the IgM+ memory B cell pool (ref. 25 and Figure 1). The impaired differentiation, longer half-life (11), and IgM expression allowed BCL2hi memory B cells to iteratively reenter the GC at multiple cycles of antigenic recall. Moreover, these features also conferred a survival advantage for these BCL2-expressing B cells compared with normal B cells in the lymphoid follicle (ref. 25 and Figure 1).
Memory B cell dynamics in the multistep development of follicular lymphoma. (A) Normal and t(14;18)+ naive B cells both enter into GC reactions upon immunization. GC B cells undergo SHM and CSR. Owing to constitutive BCL2 expression, t(14;18)+ cells have a survival advantage over normal GC B cells; therefore, BCL2-overexpressing t(14;18)+ cells are positively selected into the IgM+ memory B cell pool and are capable of GC reentry upon reexposure to antigen. With every successive cycle of GC reentry, t(14;18)+ cells outgrow their healthy counterparts. (B) Decades of iterative GC reentries cause an accumulation of abnormal t(14;18)+ cells in the GC (termed FLIS). This process produces a continuous output of t(14;18)+ IgM+ memory B cells, ultimately leading to overt FL.
Healthy B cells were repeatedly weeded out by subsequent returns to the GC, leaving the developmentally blocked BCL2hi B cells to colonize the GC and cause FL in situ (FLIS) (Figure 1). Some of these BCL2hi B cells also disseminated into peripheral lymphoid tissues and were termed FL-like cells (FLLCs). Concordantly, Sungalee and colleagues observed wide dissemination of atypical t(14;18)(q32;q21)+ B cells in lymphoid tissues from healthy individuals who had no obvious symptoms of FL (25). FLIS and FLLCs both resembled GC B cells and have a BCL2+/ BCL6+/lo phenotype (25). These benign precursors eventually evolve over several decades (up to 20 years) to give rise to full-blown FL (13, 27).
Repeated GC transits carry the risk of genetic instability through repetitive exposure to AID expression (28); therefore, lymphomas arising from the GC, such as FLs, display a high degree of DNA damage resulting from SHM and CSR (14, 29–31). The findings by Sungalee et al. (25) build on these observations. Using whole-exome sequencing, the authors found evidence of increased mutations, possibly resulting from AID activity, in both GCs and memory B cells overexpressing BCL2 as compared with empty vector–transduced controls. Moreover, Sungalee and colleagues interrogated their clonal evolution model in humans by comparing the intraclonal variation (ICV) in t(14;18)(q32;q21)+ memory B cell clones with that in normal memory B cell clones. In agreement with their earlier findings, ICV was higher in the t(14;18)(q32;q21)+ memory B cell clones with an increased rate of SHM and CSR as compared with that in normal counterparts in the same individual (25). Thus, the t(14;18)(q32;q21) translocation supports multiple iterative reentries of IgM+ memory B cells into the GC compartment upon chronic immunization and thereby promotes AID-induced hypermutation. Cooperation between these two processes facilitates the malignant transformation of t(14;18)(q32;q21)+ B cells into overt FL (Figure 1).
A number of recent studies have shown that chronic and repetitive immune responses trigger reactivation and reentry of IgM+ memory B cells into the GC (22–24). This was unexpected, because earlier studies using genetic removal of a cognate antigen demonstrated that long-lived memory B cells do not need “reminders,” such as antigen persistence and reentry into GCs. Memory B cells that have evolved high-affinity antibodies are highly valuable to the adaptive immune system and therefore were thought to be exempt from GC reentry and the risk of permanent deletion from the repertoire (32). A possible explanation for this paradox lies in the coevolution of antibodies and antigens. For example, viral, parasitic, and bacterial antigens often self-mutate to evade host immune defenses (33–36). Such antigenic coevolution necessitates multiple reentries of memory B cells into GCs to “update” the repertoire and raise robust and high-affinity antibodies against genetically evolving pathogens. Sungalee et al. elegantly demonstrate that this normal physiological process can be subverted by t(14;18)(q32;q21) translocation and ultimately cause FL (25).
The “chronic infection model,” which links memory B cell dynamics and follicular lymphomagenesis, has several important implications. First, it provides an excellent example of the initiation of lymphoid malignancies from defects in normal physiological processes, which is exemplified by the memory B cell dynamics demonstrated by Sungalee et al. Second, it dispels the theory that constitutive BCL2 expression is sufficient to allow FL progression. Third, the model supports the increased incidence of disease with age. Fourth, the presence of AID activity in t(14;18)(q32;q21)+ cells explains FL evolution at a molecular level. Finally, the observations by Sungalee et al. provide proof of principle for previous epidemiological studies suggesting that delayed, recurrent, and chronic infections predispose humans to developing B lymphoid malignancies (37–41).
In light of the findings of Sungalee and colleagues (25), it may be worthwhile to investigate whether these studies can be extended to further examine other B lymphoid malignancies that require additional genetic lesions for transformation. Studies in this direction may unveil a previously unidentified mechanism by which infection leads to clonal evolution in certain subgroups of pediatric pre–B cell leukemias. Such investigations are ongoing for the ETV6-RUNX1 subgroup of pre–B cell ALL, in which Greaves and Wiemels have proposed that delayed pathogen exposure resulting in chronic and damaging immune responses during early childhood may predispose children carrying this rearrangement to overt leukemia (the so-called “delayed infections hypothesis,” refs. 37, 38).
This work is supported by grants from the National Cancer Institute (NCI), NIH (R01CA137060, R01CA139032, R01CA157644, R01CA169458, and R01CA172558, to M. Müschen); Translational Research Program grants from the Leukemia and Lymphoma Society (6132-09, 6097-10, and 6221-12); the William Lawrence and Blanche Hughes Foundation; and the California Institute for Regenerative Medicine (CIRM) (TR2-01816, to M. Müschen). M. Müschen is a Scholar of the Leukemia and Lymphoma Society and a Senior Investigator of the Wellcome Trust.
Address correspondence to: Markus Müschen, Department of Laboratory Medicine, Box 0134, University of California, San Francisco, San Francisco, California 94143-0134, USA. Phone: 415.502.0388; E-mail: markus.muschen@ucsf.edu.
Conflict of interest: The authors have declared that no conflict of interest exists.
Reference information: J Clin Invest. 2014;124(12):5095–5098. doi:10.1172/JCI79189.
See the related article at Germinal center reentries of BCL2-overexpressing B cells drive follicular lymphoma progression.