IL7Rα Signaling Prevents Premature Expression of AID In Human Pre-B Cells: Implications for Clonal Evolution of Childhood Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 26-26
Author(s):  
Srividya Swaminathan ◽  
Lars Klemm ◽  
Soo-mi Kweon ◽  
Anthony Ford ◽  
Klaus Schwarz ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Cell Clone ◽  
Lymphoblastic Leukemia ◽  
Clonal Evolution ◽  
Cell Receptor ◽  
B Cell Development ◽  
B Cell Receptor ◽  
Childhood All

Abstract Abstract 26 Background: Childhood acute lymphoblastic leukemia (ALL) typically arises from a pre-leukemic pre-B cell clone, which was established in utero (Greaves and Wiemels, 2003). This led to a scenario, in which the initial prenatal lesion is followed by a series of additional transforming events, which ultimately cause malignant transformation of a pre-B cell clone. For instance, the TEL-AML1 gene rearrangement defines the most frequent type of childhood ALL and is detected in ∼1% cord blood samples compared to the cumulative risk for TEL-AML1 ALL at 1:14,000. These findings support the notion that covert pre-leukemic clones are frequent but only a small minority of them develop into frank pre-B leukemia after critical secondary genetic lesions were acquired. The postnatal mechanism(s) that drive the evolution of the fetal pre-leukemic clone towards childhood ALL are not known. Hypothesis: We have recently demonstrated that aberrant somatic hypermutation activity of AID propagates progression of CML into lymphoid blast crisis (Klemm et al., 2009) and clonal evolution of acute lymphoblastic leukemia (Gruber et al., 2010). Here we test the hypothesis that premature expression of AID in human pre-B cells promotes the acquisition of secondary genetic lesions and propagates the clonal evolution of a pre-leukemic pre-B cell towards childhood ALL. Results: We performed a comprehensive analysis of human B cell development in bone marrow samples from two children carrying deleterious mutations of the IL7RA gene encoding one chain of the human IL7 receptor. As opposed to normal human pre-B cells, pre-B cells from IL7RA-mutant patients carried somatically mutated immunoglobulin genes. Premature hypermutation in IL7Rα-deficient pre-B cells was consistent with aberrant expression of AID in these cells. This led to the hypothesis that signaling via IL7Rα suppresses premature activation of AID-dependent hypermutation. To test this hypothesis, we stimulated mouse pre-B cells with LPS in the presence or absence of IL7, which is normally abundantly present in the bone marrow. While pre-B cells did not respond to LPS in the presence of IL7, IL7 withdrawal dramatically sensitized pre-B cells to LPS exposure: in the absence of IL7, LPS-stimulation of pre-B cells resulted in similar AID protein levels as in splenic germinal center B cells, where AID is normally active. We confirmed these observations studying pre-B cells from an AID-GFP reporter transgenic mouse strain. While LPS resulted in ∼2% AID-GFP+ cells in the presence of IL7, the fraction of AID-GFP+ cells increased to ∼45% when IL7 was removed. Since IL7Rα signaling involves Stat5 phosphorylation, we studied inducible deletion of both Stat5a and Stat5b in Stat5-fl/fl pre-B cells. Inducible deletion of Stat5a and Stat5b in pre-B cells had the same effect as IL7 withdrawal and led to transcriptional de-repression of AID. IL7Rα/Stat5 signaling likely involves negative regulation of FoxO3A via AKT since expression of a constitutively active FoxO3A mutant potentiated AID expression in pre-B cells. We next searched for a normal pre-B cell subset, in which loss of IL7Rα/Stat5 signaling occurs naturally. Since inducible activation of pre-B cell receptor signaling results in downregulation of IL7Rα surface expression, we tested pre-B cell receptor-positive stages of B cell development. Interestingly, AID mRNA levels were increased by >10-fold at the transition from IL7Rα-positive Fraction C’ pre-B cells to IL7Rα-negative Fraction D pre-B cells. Conclusion: AID is a tightly controlled mutator enzyme, which diversifies immunoglobulin genes upon antigen-encounter of germinal center B cells. The factors that prevent premature expression of AID in pre-germinal center stage B cells were not known. Here, we here we report a novel, IL7Rα/Stat5-dependent mechanism by which pre-B cells are rendered non-responsive to antigen-dependent upregulation of AID. Attenuation of the IL7Rα/Stat5 signal occurs naturally in Fraction D pre-B cells. As a consequence, Fraction D pre-B cells express significant levels of AID for a short time. We propose that Fraction D pre-B cells represent a subset of increased genetic vulnerability in the natural history of childhood ALL. Enlargement of the Fraction D pool or extension of the time window during which pre-B cells are at the Fraction D stage, may increase the risk to acquire secondary genetic lesions towards the development of childhood ALL. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Infectious Origins of Childhood Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 751-751
Author(s):  
Lars Klemm ◽  
Srividya Swaminathan ◽  
Anthony M Ford ◽  
Klaus Schwarz ◽  
David G. Schatz ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Cell Receptor ◽  
Cell Development ◽  
B Cell Development ◽  
Childhood All ◽  
Leukemic Clone ◽  
Germinal Center B Cells

Abstract Abstract 751 Background: In most cases, childhood acute lymphoblastic leukemia can be retraced to a recurrent genetic lesion in utero, which establishes a pre-leukemic clone. The TEL-AML1 fusion gene, for instance, arises prenatally and defines the most frequent subtype of childhood ALL. Strikingly, ∼1 of 100 healthy newborns carry a TEL-AML1 pre-leukemic clone, but only <1% of these children will eventually develop leukemia. Encounter of infectious antigen in B cell typically leads to activation of the mutator enzyme AID. While AID is required for class switch recombination and somatic hypermutation of immunoglobulin genes during affinity maturation of germinal center B cells, its premature activation may be deleterious. The underlying questions for this project were (1) how are B cells during their early development safeguarded from pre-mature AID expression and (2) whether pre-mature expression of AID in early B cell development is deleterious in the sense that it pre-disposes to the clonal evolution of a pre-leukemic B cell clone in the bone marrow. Results: We performed a comprehensive analysis of human B cell development in bone marrow samples from two children carrying deleterious mutations of the IL7RA gene encoding one chain of the human IL7 receptor. As opposed to normal human pre-B cells, pre-B cells from IL7RA-mutant patients carried somatically mutated immunoglobulin genes consistent with aberrant expression of AID in these cells. This led to the hypothesis that signaling via IL7Ra suppresses premature activation of AID-dependent hypermutation. To test this hypothesis, we stimulated mouse pre-B cells with LPS in the presence or absence of IL7, which is normally abundantly present in the bone marrow. While pre-B cells did not respond to LPS in the presence of IL7, IL7 withdrawal dramatically sensitized pre-B cells to LPS exposure: in the absence of IL7, LPS-stimulation of pre-B cells resulted in similar AID protein levels as in splenic germinal center B cells, where AID is normally active. We confirmed these observations studying pre-B cells from an AID-GFP reporter transgenic mouse strain. While LPS resulted in ∼2% AID-GFP+ cells in the presence of IL7, the fraction of AID-GFP+ cells increased to ∼45% when IL7 was removed. Since IL7Ra signaling involves Stat5 phosphorylation, we studied inducible Cre-mediated deletion of Stat5, which had the same effect as IL7 withdrawal and led to transcriptional de-repression of AID. IL7Ra/Stat5 signaling likely involves negative regulation of FoxO3A via AKT since expression of a constitutively active FoxO3A mutant potentiated AID expression in pre-B cells. We next searched for a normal pre-B cell subset, in which loss of IL7Ra/Stat5 signaling occurs naturally. Since inducible activation of pre-B cell receptor signaling results in downregulation of IL7Ra surface expression, we tested pre-B cell receptor-positive stages of B cell development. Interestingly, AID mRNA levels were increased by >10-fold at the transition from IL7Ra-positive Fraction C' pre-B cells to IL7Ra-negative Fraction D pre-B cells. Conclusion: AID is a tightly controlled mutator enzyme in mature germinal center B cells. The factors that prevent premature expression of AID during early B cell development were not known. Here, we here we report a novel, IL7Ra/Stat5-dependent mechanism by which pre-B cells are rendered non-responsive to antigen-dependent upregulation of AID. Attenuation of the IL7Ra/Stat5 signal occurs naturally in Fraction D pre-B cells. As a consequence, Fraction D pre-B cells express significant levels of AID for a short time. We propose that Fraction D pre-B cells represent a subset of increased genetic vulnerability in the natural history of childhood ALL. Disclosures: No relevant conflicts of interest to declare.

Cooperation Between Aid and the Rag1/Rag2 V(D)J Recombinase Drives Clonal Evolution of Childhood Acute Lymphoblastic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 519-519
Author(s):  
Srividya Swaminathan ◽  
Lars Klemm ◽  
Anthony M. Ford ◽  
Klaus Schwarz ◽  
Rafael Casellas ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Fusion Gene ◽  
Lymphoblastic Leukemia ◽  
Clonal Evolution ◽  
Cell Development ◽  
B Cell Development ◽  
Childhood All ◽  
Leukemic Clone ◽  
Late Stages

Abstract Abstract 519 Background and Rationale: In many cases, childhood acute lymphoblastic leukemia (ALL) can be retraced to a recurrent genetic lesion in utero which establishes a pre-leukemic clone. The TEL-AML1 fusion gene for instance, arises prenatally and defines the most frequent subtype of childhood ALL. Strikingly, ∼1 of 100 healthy newborns carry a TEL-AML1 pre-leukemic clone, but only less than 1% of these children eventually develop leukemia. Encounter of infectious antigen leads to activation of the mutator enzyme AID in mature B cells. While AID is required for somatic hypermutation and class switching during late stages of B cell development, its pre-mature activation may be deleterious. The underlying questions for this project were: (1) how are B cells safeguarded from pre-mature AID expression during their early development and (2) whether pre-mature AID expression during early B cell development is deleterious in the sense that it promotes the clonal evolution of a pre-leukemic B cell clone in the bone marrow. Results: Studying gene expression in a clinical trial for children with high risk pre-B ALL (COG P9906; n=207), we found that high expression levels of AID at the time of diagnosis is predictive of poor overall survival and a higher frequency of leukemia relapse. These findings suggest that AID may be a contributing factor to the clonal evolution of childhood pre-B ALL. Previous work by Michael Lieber's group proposed cooperation of AID and the V(D)J recombinase Rag1/Rag2 as a key mechanism leading to the acquisition of chromosomal translocations in human B cell malignancies (Tsai et al., 2008). Activity of Rag1/Rag2 V(D)J recombinase and AID is segregated to early and late stages of B cell development, respectively. However, we found that experimental withdrawal of IL7 receptor (IL7R) signaling in pre-B cells not only activates Rag1/Rag2 expression and V(D)J recombinase but also rendered pre-B cells responsive to antigen (LPS) encounter with strong upregulation of AID. We found that upon withdrawal of IL7, transcription of AID and Rag1/Rag2 is activated by the same elements through a Pten/FoxO-dependent pathway. To test whether IL7R signaling also negatively regulates AID activation in human pre-B cells, we performed a comprehensive analysis of human B cell development in bone marrow samples from two children carrying deleterious mutations of the IL7RA and IL2RG genes encoding the two chains of the human IL7R. As opposed to normal human pre-B cells, pre-B cells from IL7RA and IL2RG-mutant patients carried somatically mutated immunoglobulin genes consistent with aberrant expression of AID in these cells. Based on these observations, we propose that Fraction D pre-B cells represent a subset of increased genetic vulnerability owing to concomitant expression of both AID and Rag1/Rag2. To test whether the vulnerability of Fraction D pre-B cells is relevant in the clonal evolution of childhood ALL, we challenged pre-B cells carrying the TEL-AML1 fusion gene with 5 consecutive cycles of IL7 withdrawal (−IL7) and LPS stimulation (+LPS). To distinguish between the potential contribution of AID and Rag1/Rag2 to secondary genetic lesions, -IL7/+LPS-challenges were performed with wildtype pre-B cells, AID−/−, Rag1−/− and AID−/− Rag1−/− double knockout pre-B cells. TEL-AML1-bearing pre-B cells were labeled with firefly luciferase and then 25 million cells were injected into 7 recipient animals per group. While wildtype TEL-AML1 pre-B cells that went through 5 rounds of -IL7/+LPS-challenge caused leukemia in all recipient mice, TEL-AML1 pre-B cells lacking either AID or Rag1 failed to give rise to full-blown leukemia in transplant recipients. Conclusion: While one in 100 newborns carry the TEL-AML1 fusion molecule, the mechanisms that lead to the acquisition of critical secondary genetic lesions are not known. Here, we report a novel, IL7R/Stat5-dependent mechanism by which pre-B cells are rendered non-responsive to LPS-dependent upregulation of AID. We propose that Fraction D pre-B cells represent a subset of increased natural genetic vulnerability in the context of concomitant activativity of AID and Rag1/Rag2. Frequent exposure to infectious antigens (e.g. LPS) in this constellation may propagate clonal evolution towards full-blown leukemia. Disclosures: No relevant conflicts of interest to declare.

Mechanisms of Pre-B Cell Receptor-Inactivation In Acute Lymphoblastic Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 147-147
Author(s):  
Cihangir Duy ◽  
Daniel Nowak ◽  
Lars Klemm ◽  
Rahul Nahar ◽  
Carina Ng ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Lymphoblastic Leukemia ◽  
Cell Receptor ◽  
Signaling Molecules ◽  
B Cell Receptor ◽  
Dominant Negative ◽  
Leukemia Cells ◽  
Immunoglobulin Gene ◽  
Receptor Inactivation

Abstract Abstract 147 Background: We recently established that the pre-B cell receptor functions as a tumor suppressor in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL). The pre-B cell receptor promotes differentiation of normal pre-B cells and couples the immunoglobulin μ -chain to activating tyrosine kinases (e.g. SYK) via linker molecules (e.g. BLNK). In virtually all cases of Ph+ ALL, pre-B cell receptor function is compromised and its reconstitution induces rapid cell cycle arrest. However, genomic deletions in pre-B cell receptor pathway are rare and the mechanisms of inactivation are not known. Here we report that pre-B cell receptor inactivation occurs at multiple levels and involves at least four different mechanisms, namely (1) deleterious immunoglobulin gene rearrangement, (2) defective splicing of pre-B cell receptor signaling molecules, (3) expression of dominant-negative PAX5 fusion genes and (4) overexpression of inhibitory signaling molecules. Result: (1) Studying progressive transformation of pre-B cells in BCR-ABL1-transgenic mice, we observed that surface expression of the immunoglobulin μ -chain was downregulated after 60 days of age, which was a prerequisite for the onset of full-blown leukemia. While the repertoire of immunoglobulin gene rearrangements was polyclonal in wildtype pre-B cells, BCR-ABL1-transgenic pre-B cells show clonal expansions, which are derived from one ancestral productive immunoglobulin gene rearrangement in the transformed pre-B cell. However, the ancestral immunoglobulin gene rearrangements were rendered non-functional through deleterious secondary rearrangements. Likewise, in 47 of 57 cases of primary human Ph+ ALL, we detected traces of pre-B cell receptor-inactivation through secondary deleterious recombination events at the immunoglobulin μ -chain locus. (2) We studied pre-B cell receptor signaling molecules in primary human pre-B cells and 10 patient-derived Ph+ ALL samples by Western blotting and RT-PCR. As opposed to normal bone marrow pre-B cells, in all 10 cases of Ph+ ALL defective splice variants of the SYK tyrosine kinase and its linker molecule BLNK were found. Sequence analysis revealed a frequent 4 bp slippage during SYK pre-mRNA splicing which resulted in a truncated protein lacking the kinase domain, as confirmed by Western blot. To study the functional significance of defective Syk expression in Ph+ ALL cells, we transformed pre-B cells from Syk-fl/fl mice with BCR-ABL1 and deleted the Syk kinase using tamoxifen-inducible Cre. As opposed to Syk-fl/fl leukemia cells, inducible ablation of Syk rendered the leukemia cells insensitive to forced expression of the pre-B cell receptor. Multiple defective transcript variants of BLNK were found that all lacked exon 16 encoding the central part of the BLNK SH2 domain. In the absence of exon 16, BLNK splice variants were detached from the pre-B cell receptor and function in a dominant-negative way as they reduce Ca2+-mobilization in response to pre-B cell receptor stimulation. In a titration experiment, BLNK−/− leukemia cells were reconstituted with full-length and exon 16-deficient BLNK. Dominant-negative BLNK interfered with pre-B cell receptor-mediated tumor suppression at a ratio of 0.1 relative to full-length BLNK. Of note, we found somatic mutations within the splice site of exon 16 in 2 of 6 primary Ph+ ALL cases. (3) Ph+ ALL cells often carry chromosomal translocations leading to the expression of dominant-negative PAX5-fusion molecules. In a systematic gene expression analysis, we observed that ectopic expression of the dominant-negative PAX5-C20orf112 fusion led to downregulation of immunoglobulin μ -chain and the signaling molecules including SYK and BLNK. As a consequence, Ca2+-mobilization in response to pre-B cell receptor stimulation was significantly diminished. (4) Correction of defective immunoglobulin-μ chain and BLNK expression results in compensatory overexpression of a broad array of inhibitory signaling molecules. These molecules share an ITIM signaling motif, which attenuates pre-B cell receptor signal transduction through recruitment of inhibitory phosphatases. Conclusion: Even though loss of pre-B cell receptor function represents the uniform outcome of a diverse spectrum of lesions, individual Ph+ ALL subclones exhibit a complex pattern of shared and distinct defects involving one or more of these 4 mechanisms. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Essential Role of Phospholipase Cγ2 in Early B-Cell Development and Myc-Mediated Lymphomagenesis

2006 ◽  
Vol 26 (24) ◽  
pp. 9364-9376 ◽  
Author(s):  
Renren Wen ◽  
Yuhong Chen ◽  
Li Bai ◽  
Guoping Fu ◽  
James Schuman ◽  
...  
Keyword(s):  
B Cells ◽  
Transgenic Mice ◽  
B Cell ◽  
Cell Receptor ◽  
Cell Development ◽  
B Cell Development ◽  
B Cell Receptor ◽  
Wild Type ◽  
Interleukin 7

ABSTRACT Phospholipase Cγ2 (PLCγ2) is a critical signaling effector of the B-cell receptor (BCR). Here we show that PLCγ2 deficiency impedes early B-cell development, resulting in an increase of B220+ CD43+ BP-1+ CD24hi pre-BCR+ large pre-B cells. PLCγ2 deficiency impairs pre-BCR-mediated functions, leading to enhanced interleukin-7 (IL-7) signaling and elevated levels of RAGs in the selected large pre-B cells. Consequently, PLCγ2 deficiency renders large pre-B cells susceptible to transformation, resulting in dramatic acceleration of Myc-induced lymphomagenesis. PLCγ2 −/− Eμ-Myc transgenic mice mainly develop lymphomas of B220+ CD43+ BP-1+ CD24hi pre-BCR+ large pre-B-cell origin, which are uncommon in wild-type Eμ-Myc transgenics. Furthermore, lymphomas from PLCγ2 −/− Eμ-Myc transgenic mice exhibited a loss of p27Kip1 and often displayed alterations in Arf or p53. Thus, PLCγ2 plays an important role in pre-BCR-mediated early B-cell development, and its deficiency leads to markedly increased pools of the most at-risk large pre-B cells, which display hyperresponsiveness to IL-7 and express high levels of RAGs, making them prone to secondary mutations and Myc-induced malignancy.

scholarly journals Rapid Emergence of T Follicular Helper and Germinal Center B Cells Following Antiretroviral Therapy in Advanced HIV Disease

2021 ◽  
Vol 12 ◽  
Author(s):  
Chun-Shu Wong ◽  
Clarisa M. Buckner ◽  
Silvia Lucena Lage ◽  
Luxin Pei ◽  
Felipe L. Assis ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
Antiretroviral Therapy ◽  
B Cell ◽  
Germinal Center ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Cd4 T Cell ◽  
Follicular Helper ◽  
Cell Counts

Low nadir CD4 T-cell counts in HIV+ patients are associated with high morbidity and mortality and lasting immune dysfunction, even after antiretroviral therapy (ART). The early events of immune recovery of T cells and B cells in severely lymphopenic HIV+ patients have not been fully characterized. In a cohort of lymphopenic (CD4 T-cell count &lt; 100/µL) HIV+ patients, we studied mononuclear cells isolated from peripheral blood (PB) and lymph nodes (LN) pre-ART (n = 40) and 6-8 weeks post-ART (n = 30) with evaluation of cellular immunophenotypes; histology on LN sections; functionality of circulating T follicular helper (cTfh) cells; transcriptional and B-cell receptor profile on unfractionated LN and PB samples; and plasma biomarker measurements. A group of 19 healthy controls (HC, n = 19) was used as a comparator. T-cell and B-cell lymphopenia was present in PB pre-ART in HIV+ patients. CD4:CD8 and CD4 T- and B-cell PB subsets partly normalized compared to HC post-ART as viral load decreased. Strikingly in LN, ART led to a rapid decrease in interferon signaling pathways and an increase in Tfh, germinal center and IgD-CD27- B cells, consistent with histological findings of post-ART follicular hyperplasia. However, there was evidence of cTfh cells with decreased helper capacity and of limited B-cell receptor diversification post-ART. In conclusion, we found early signs of immune reconstitution, evidenced by a surge in LN germinal center cells, albeit limited in functionality, in HIV+ patients who initiate ART late in disease.

scholarly journals BCR Affinity Influences T-B Interactions and B Cell Development in Secondary Lymphoid Organs

2021 ◽  
Vol 12 ◽  
Author(s):  
Alec J. Wishnie ◽  
Tzippora Chwat-Edelstein ◽  
Mary Attaway ◽  
Bao Q. Vuong
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Receptor ◽  
Cell Development ◽  
B Cell Development ◽  
Affinity Maturation ◽  
B Cell Receptor ◽  
Specific Cell ◽  
High Affinity ◽  
Tfh Cells

B cells produce high-affinity immunoglobulins (Igs), or antibodies, to eliminate foreign pathogens. Mature, naïve B cells expressing an antigen-specific cell surface Ig, or B cell receptor (BCR), are directed toward either an extrafollicular (EF) or germinal center (GC) response upon antigen binding. B cell interactions with CD4+ pre-T follicular helper (pre-Tfh) cells at the T-B border and effector Tfh cells in the B cell follicle and GC control B cell development in response to antigen. Here, we review recent studies demonstrating the role of B cell receptor (BCR) affinity in modulating T-B interactions and the subsequent differentiation of B cells in the EF and GC response. Overall, these studies demonstrate that B cells expressing high affinity BCRs preferentially differentiate into antibody secreting cells (ASCs) while those expressing low affinity BCRs undergo further affinity maturation or differentiate into memory B cells (MBCs).

Flow Cytometric Diagnosis of the Cell Lineage and Developmental Stage of Acute Lymphoblastic Leukemia by Novel Monoclonal Antibodies Specific to Human Pre–B-Cell Receptor

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4317-4324 ◽  
Author(s):  
Keiko Tsuganezawa ◽  
Nobutaka Kiyokawa ◽  
Yoshinobu Matsuo ◽  
Fujiko Kitamura ◽  
Noriko Toyama-Sorimachi ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
B Cell ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Cell Lineage ◽  
Cell Receptor ◽  
B Cell Development ◽  
B Cell Receptor ◽  
Cytoplasmic Staining ◽  
Flow Cytometric

Abstract Three novel monoclonal antibodies (MoAbs) have been established that recognize distinct epitopes of a human pre–B-cell receptor (pre-BCR) composed of a μ heavy (μH) chain and a λ5/VpreB surrogate light (SL) chain. HSL11 reacts with λ5 whereas HSL96 reacts with VpreB. Intriguingly, HSL2 does not bind to each component of the pre-BCR but does bind to the completely assembled pre-BCR complex. Flow cytometric analyses with cytoplasmic staining of a panel of human cell lines showed that HSL11 and HSL96 specifically stained cell lines derived from the pro–B and pre–B-cell stages of B-cell development. In contrast, HSL2 stained exclusively cell lines derived from the pre–B-cell stage. These results prompted us to explore the possibility of clinical application of these MoAbs for the determination of the cell lineage and developmental stage of acute lymphoblastic leukemia (ALL). Whereas none of mature B-lineage ALLs (B-ALLs), T-lineage ALLs (T-ALLs), and acute myeloid leukemias analyzed were stained in the cytoplasm with these three MoAbs, the vast majority of non–B- and non–T-ALLs (53 out of 56 cases) were found positive for either λ5, Vpre-B, or both in their cytoplasm. Among these 53 cytoplasmic SL chain-positive ALLs, 19 cases were also positive for cytoplasmic μH chain, indicative of pre–B-cell origin. Interestingly, 6 out of these 19 pre–B-ALL cases were found negative for cytoplasmic staining with HSL2. From these results, we propose a novel classification of B-ALL in which five subtypes are defined on the basis of the differential expression of SL chain, μH chain, pre-BCR, and light chain along the B-cell development.

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