pre-BII Cells Research Articles

Profiling the impact of anti-human CD20 monoclonal antibodies on lymphocyte B cell subsets and their precursors in the bone marrow and in lymphoid tissues in an immunocompromised mouse engrafted with human cells

Ofatumumab (OFA) and ocrelizumab (OCRE) are two anti-CD20 monoclonal antibodies approved for the treatment of relapsing forms of multiple sclerosis due to their ability to deplete B lymphocytes. The aim of this study was to investigate the impact of these anti-hCD20 antibodies on B lymphocyte subsets in the circulation and in primary and secondary lymphoid organs in an immune system humanized mouse model (immunocompromised Rag2−/−Il2rg−/−CD47−/−) engrafted with human CD34+ hematopoietic stem cells. Three months after humanization, mice, which present adaptive immune cells only of human origin, were treated with OFA (0.3 mg/Kg; day 1, 3 and 5), or OCRE (10 mg/kg; day 1) or saline. Seven days after the last injection a robust (>90 %) decrease of circulating human CD20+ B lymphocytes was observed in both OFA- and OCRE-treated mice. A partial replenishment of B lymphocytes was detectable in blood 36 days from the last injection in OFA-treated mice, while no B lymphocytes could be detected in OCRE-treated mice up to 65 days post injection. Bone marrow profiling showed that during hCD20+ B cell depletion and replenishment, OCRE-treated mice preserved only preB-I cells in the bone marrow, while the bone marrow of OFA-treated mice presented both preB-I as well as preB-II cells, with the latter subset being the one closest to differentiate into immature B cells. These data together with changes in B cell distribution in other tissues suggest that ofatumumab preserve BM niches, critical for B lymphocyte replenishment, limiting potential side effects of the treatment associated with the increased risk of infection.

Pax5 Heterozygosity Affects B-Cell Differentiation and Generates a Deregulated Precursor Population in the Bone Marrow

Introduction: PAX5 is a well-known master regulator for B-cell development, commitment and identity. Hence, germline or somatic deregulation of PAX5 facilitates the development of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Previously, our group has described B-cell development abnormalities in three families carrying PAX5 germline variants ( Escudero et al., Leukemia, 2022) as well as established a link between Pax5 heterozygosity and infection exposure, thereby recapitulating the incomplete penetrance of the human BCP-ALL ( Martin-Lorenzo et al., Cancer Discovery, 2015). Nevertheless, the molecular single-cell characteristics of the susceptible precursor population mediated by reduced Pax5 levels is still poorly understood. Methods: Here, we deeply characterized the susceptible precursor population in Pax5+/- mice utilizing single-cell RNA Sequencing (scRNA-Seq), multicolor flow cytometry analyses and in-vivo transplantation models. Results: Our data show, that Pax5 heterozygosity leads to an aberrant pre-BII population (B220 +CD19 +IgM -CD25 +) in the bone marrow (BM) of Pax5+/- mice (p=0.0026, Student's t-test) (Fig. 1A). This population is stable over time and characterized by higher CD25 and IL-7-Receptor expression (p<0.0001, Student's t-test). Furthermore, bulk RNA-Sequencing of the pre-BII population revealed B-cell receptor (BCR) signaling as one of the top downregulated pathways (including CD79a/b, Lyn and CD72), while DNA replication and cell cycle signaling pathways were upregulated in Pax5+/- vs. Wildtype (WT) pre-BII cells. ScRNA-Seq analyses confirmed these results (Fig. 1B) and further showed that pre-BII cells of Pax5+/- mice are skewed toward the less commonly used lambda light chain BCR-rearrangements. The observed delay in the transition to IgM-positivity was additionally validated in a murine in-vivo transplantation model. Here, after 72 hours pre-BII cells from Pax5+/- mice preferably homed to the BM, while matured transplanted WT cells were predominantly found in the spleen (p=0.0023 and p=0.0140, respectively, Student's t-test). To depict the full spectrum of malignant transformation based on the described deregulated precursor B-cell population, we additionally performed scRNA-Seq on different stages of Pax5-mediated BCP-ALL evolution. Our data show that in a first step, arrested pre-leukemic cells of Pax5+/- mice lose their B-cell identity and display high Myc levels. Sequentially, full blown BCP-ALL arises in a second step after acquisition of additional oncogenic driver mutations (including Jak1 and Jak3). Interestingly, although the analyzed BCP-ALLs of Pax5+/- mice were all based on the same predisposition, they greatly varied in their transcriptional profile and cell-cycle state, depending on their oncogenic 2 nd hit. Conclusion: In summary, we deeply characterized how reduced Pax5 transcriptional activity in the BM generates a predisposed precursor B-cell environment, which is susceptibility for malignant transformation. These findings are important for understanding the molecular mechanisms to prevent or treat a significant proportion of childhood BCP-ALLs.

IL-7 receptor signaling drives human B-cell progenitor differentiation and expansion

AbstractAlthough absence of interleukin-7 (IL-7) signaling completely abrogates T and B lymphopoiesis in mice, patients with severe combined immunodeficiency caused by mutations in the IL-7 receptor α chain (IL-7Rα) still generate peripheral blood B cells. Consequently, human B lymphopoiesis has been thought to be independent of IL-7 signaling. Using flow cytometric analysis and single-cell RNA sequencing of bone marrow samples from healthy controls and patients who are IL-7Rα deficient, in combination with in vitro modeling of human B-cell differentiation, we demonstrate that IL-7R signaling plays a crucial role in human B lymphopoiesis. IL-7 drives proliferation and expansion of early B-cell progenitors but not of pre-BII large cells and has a limited role in the prevention of cell death. Furthermore, IL-7 guides cell fate decisions by enhancing the expression of BACH2, EBF1, and PAX5, which jointly orchestrate the specification and commitment of early B-cell progenitors. In line with this observation, early B-cell progenitors of patients with IL-7Rα deficiency still expressed myeloid-specific genes. Collectively, our results unveil a previously unknown role for IL-7 signaling in promoting the B-lymphoid fate and expanding early human B-cell progenitors while defining important differences between mice and humans. Our results have implications for hematopoietic stem cell transplantation strategies in patients with T− B+ severe combined immunodeficiency and provide insights into the role of IL-7R signaling in leukemogenesis.

P306: PAX5 DEFICIENCY AND GERMLINE SUSCEPTIBILITY TO PEDIATRIC LEUKEMIA

Background: Somatic mutations affecting the PAX5 gene are common in pediatric B-cell acute lymphoblastic leukemia (B-ALL). However, germline PAX5 mutations are extremely rare. While it is known that PAX5 acts as an indispensable master regulator of proliferation and differentiation in B-cells, the pre-leukemic setting mediated by reduced PAX5 levels is still poorly understood. Aims: To investigate the genetic and immunological effect of inherited PAX5 germline mutations in humans and to characterize the effect of Pax5 heterozygosity on B-cell development utilizing single-cell RNA Sequencing in mice. Methods: Aiming to understand the etiology of childhood BCP-ALL in the setting of reduced Pax5 levels, we used 9-color FACS staining and scRNA/bulk RNA-Sequencing, as well as whole exome Sequencing technology. Results: Here, we describe two new families with inherited PAX5 germline variants affecting amino acid 183 and B-ALL development, including one family with a novel amino acid substitution p.Gly183Arg. Exome analysis of both families together with one family harboring PAX5 p.Gly183Ser previously reported by Auer et al., (Leukemia, 2014) yielded no additional common germline variants that could explain the incomplete penetrance in the observed leukemia development. A study of the respective B-cell differentiation comparing PBMCs from unrelated non-carriers, healthy carriers, and patients revealed a significant decrease of intermittent B-cells and memory B-cells in carriers and patients compared to non-carriers (Student’s T-test; p<0.05 and p<0.01, respectively). Finally, immunological studies performed using bone marrow samples from a patient and an unrelated control showed a reduction in the number of mature B cells, suggesting a block of the B-cell differentiation at the pre-B stage in the patient. In line, a comprehensive flow cytometry analysis of the B-cell development in the bone marrow of healthy Pax5 heterozygous (Pax5+/-) mice, showed a robust and age independent enrichment of the pre-BII population (B220+CD19+IgM-CD25+) compared to their Wildtype (WT) littermates (Student’s t-test; p=0.003). Bulk RNA-Sequencing of sorted preB-II cells (3 WT vs. 3 Pax5+/- mice) revealed deregulations in major B-cell receptor (BCR) signaling components including downregulation of Cd79a/b, Lyn, MTor and various ITIMs. Subsequent scRNA-Sequencing (4 WT vs. 4 Pax5+/- mice, pool of 20,000 cells hashtag labeled, with 50,000 reads/cell and VDJ-rearrangement analysis) confirmed a delayed BCR assembly and showed a skewing of light chain rearrangements towards the lambda isotype in Pax5+/- mice. Summary/Conclusion: Overall, our data provide evidence for B-cell dysregulations mediated by reduced Pax5 levels in the germline of mice and men. Ultimately these characterizations will help to advance the understanding of molecular mechanisms and susceptible populations associated with B-ALL. In turn, these findings are important to address the far more frequent somatic PAX5 mutations with the goal to prevent or treat a significant proportion of childhood leukemias.

Pax5 Heterozygosity Delays BCR Expression in Preb-II Cells and Generates a Preleukemic Population

Introduction: In B-cells, PAX5 acts as an indispensable master regulator of proliferation and differentiation. In accordance, germline or somatic deregulation of PAX5 facilitates the development of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). In this regard, our group has previously established a link between Pax5 and infection exposure in a Pax5+/- mouse model, recapitulating the human disease and providing an explanation for the unique age peak between 2-6 years in pediatric BCP-ALL (Martin-Lorenzo et al., Cancer Discovery, 2015). Nevertheless, the preleukemic setting mediated by reduced Pax5 levels is still poorly understood. Here, we deeply characterized the preleukemic population in Pax5+/- mice utilizing single-cell RNA Sequencing (scRNA-Seq) and multicolor flow cytometry (FACS) analyses.Methods: Aiming to understand the etiology of childhood BCP-ALL in the setting of reduced Pax5 levels, we used 9-color FACS staining and scRNA/bulk RNA-Seq to pinpoint and elucidate a preleukemic population in Pax5+/- mice, which was further investigated after challenges with external stimuli.Results: A comprehensive flow cytometry staining depicting all B-cell developmental stages within the murine bone marrow (BM) showed a robust enrichment of the pre-BII population (B220 +CD19 +IgM -CD25 +) in healthy Pax5+/- mice compared to their wildtype (WT) littermates (Student's t-test; p=0.003). The increased preB-II population could be detected across all ages (3, 6 and 9 months) and the individual cells displayed a higher mean fluorescence intensity (MFI) of CD25 and IL7-Receptor (Student's t-test p=0.0001 and p=0.036, respectively), with almost complete absence of BP-1 (Student's t-test p=0.0001). Bulk RNA-Seq of sorted preB-II populations (3 WT vs. 3 Pax5+/- mice) revealed massive deregulations in major B-cell receptor (BCR) signaling components including downregulation of Cd79a/b, Lyn, MTor and various ITIMs, with concomitant upregulation of Jak3 in Pax5+/- vs WT preB-II cells, which is in line with somatic Jak-Stat activation in infection-driven Pax5+/- leukemias.Additional in-depth characterization of the enriched preB-II population via scRNA-Seq (3 WT vs. 3 Pax5+/- mice, pool of 10,000 cells hashtag labeled, with 50,000 reads/cell and VDJ-rearrangement analysis) furthermore delineated defined sub-developmental stages within the preB-II compartment. In line with the bulk RNA-Seq data, reduced Pax5 levels led to delayed BCR assembly and an arrest of Pax5+/- preB-II cells shortly before BCR expression within the most mature clusters.Finally, challenging Pax5+/- mice with vesicular stomatitis virus (VSV) confirmed the preleukemic identity of this preB-II population, which transformed to a full blown leukemia after additional loss of the second Pax5 allele.Conclusion: In summary, in mice, reduced Pax5 levels generate a preleukemic preB-II population in the bone marrow, which delays BCR rearrangement and confers susceptibility for malignant transformation through infection exposure to drive BCP-ALL development. These findings are important for understanding the molecular mechanisms to prevent or treat a significant proportion of childhood BCP-ALLs. DisclosuresNo relevant conflicts of interest to declare.

Single cell RNA-seq identifies the origins of heterogeneity in efficient cell transdifferentiation and reprogramming.

Forced transcription factor expression can transdifferentiate somatic cells into other specialised cell types or reprogram them into induced pluripotent stem cells (iPSCs) with variable efficiency. To better understand the heterogeneity of these processes, we used single-cell RNA sequencing to follow the transdifferentation of murine pre-B cells into macrophages as well as their reprogramming into iPSCs. Even in these highly efficient systems, there was substantial variation in the speed and path of fate conversion. We predicted and validated that these differences are inversely coupled and arise in the starting cell population, with Mychigh large pre-BII cells transdifferentiating slowly but reprogramming efficiently and Myclow small pre-BII cells transdifferentiating rapidly but failing to reprogram. Strikingly, differences in Myc activity predict the efficiency of reprogramming across a wide range of somatic cell types. These results illustrate how single cell expression and computational analyses can identify the origins of heterogeneity in cell fate conversion processes.

Normal B-Cell Gene Expression Signatures Classifies Chronic Lymphocytic Leukemia into Distinct Subtypes - Indication of Plasticity

Chronic lymphocytic leukemia (CLL) is the most common blood cancer in adults and known for its heterogeneous disease course. The nature of CLL calls for new studies of pathogenetic variables validated by prognostic impact. Here we propose a new biological classification system of CLL that associates normal B cell subsets gene expression (GE) signatures (BAGS) with GE in CLL.We used fluorescence activated cell sorting, GE profiling, and statistical modeling to generate a B-cell associated gene signature (BAGS) classifier for pre-BI, pre-BII, immature, naive, memory, and plasma cells harvested from healthy bone marrow samples during cardiac surgery.We used the Gene Expression Omnibus (GEO) to identify 8 independent CLL cohorts with available microarray data generated on compatible array platforms. Altogether, the study included 1239 patients, who were untreated at sampling for microarray analysis.The classifier was established using regularized multinomial regression on median-centered GE profiles from the normal B cell subsets and consisted of 184 genes with 27-54 genes per subtype. Each patient was assigned to a BAGS subtype (pre-BI, pre-BII, immature, naive, memory, and plasmacell) according to the highest probability score of the classifier. The minimum probability threshold for confident classification was chosen such that 15% remained unclassified.BAGS subtype frequencies in the CLL cohorts were analyzed using Fisher's exact test and revealed a distribution pattern that did not vary significantly between the 8 cohorts (P = 0.22) (Fig 1). Most samples were classified as memory (28.1-45.5%), followed by naive (15.2-32.3%), pre-BI (10.0-17.6 %), immature (0.0-8.7%), plasmacell (1.8-4.6%), and pre-BII (0.0-3.9%).To validate the prognostic impact, our primary endpoint was time to treatment start and was evaluated using the Kaplan Meier method using available data (n = 68). We saw a significant shorter time to treatment start amongst patient with a pre-germinal center CLL subtype (pre-BI, pre-BII, immature, and naive) compared to post-germinal center subtypes (memory and plasmacell) (P = 0.03) (Fig 2).Next, BAGS subtype was associated with the IgVH mutational status (n = 222), which showed that a larger proportion of post-germinal center subtypes had mutated IgVH genes (73.4%), whereas pre-germinal center subtypes were just as likely to have unmutated IgVH genes (48.8%) as mutated IgVH genes (52.2%).Finally, to investigate biological differences between the BAGS subtypes, we identified differentially expressed genes (DEG) in each subtype versus the rest. Fisher's method was used to combine p-values for statistically significant DEGs in a metaanalyis including all cohorts. Of known therapeutic CLL targets, CD20 and CD52 were both significantly upregulated in the memory BAGS subtype compared to the rest.Using a phenotypic cell of origin approach, our findings show that CLL samples display end-stage phenotypes resembling the whole spectrum of normal B-cells and indicate transcriptional plasticity as a trait that may contribute to the heterogeneity observed in CLL. [Display omitted] [Display omitted] DisclosuresEl-Galaly:Roche: Consultancy, Other: travel funding.

Mechanisms of clonal evolution in childhood acute lymphoblastic leukemia.

Childhood acute lymphoblastic leukemia can often be retraced to a pre-leukemic clone carrying a prenatal genetic lesion. Postnatally acquired mutations then drive clonal evolution towards overt leukemia. RAG1-RAG2 and AID enzymes, the diversifiers of immunoglobulin genes, are strictly segregated to early and late stages of B-lymphopoiesis, respectively. Here, we identified small pre-BII cells as a natural subset of increased genetic vulnerability owing to concurrent activation of these enzymes. Consistent with epidemiological findings on childhood ALL etiology, susceptibility to genetic lesions during B-lymphopoiesis at the large to small pre-BII transition is exacerbated by abnormal cytokine signaling and repetitive inflammatory stimuli. We demonstrate that AID and RAG1-RAG2 drive leukemic clonal evolution with repeated exposure to inflammatory stimuli, paralleling chronic infections in childhood.

Pre-B cell receptor binding to galectin-1 modifies galectin-1/carbohydrate affinity to modulate specific galectin-1/glycan lattice interactions.

Galectins are glycan-binding proteins involved in various biological processes including cell/cell interactions. During B-cell development, bone marrow stromal cells secreting galectin-1 (GAL1) constitute a specific niche for pre-BII cells. Besides binding glycans, GAL1 is also a pre-B cell receptor (pre-BCR) ligand that induces receptor clustering, the first checkpoint of B-cell differentiation. The GAL1/pre-BCR interaction is the first example of a GAL1/unglycosylated protein interaction in the extracellular compartment. Here we show that GAL1/pre-BCR interaction modifies GAL1/glycan affinity and particularly inhibits binding to LacNAc containing epitopes. GAL1/pre-BCR interaction induces local conformational changes in the GAL1 carbohydrate-binding site generating a reduction in GAL1/glycan affinity. This fine tuning of GAL1/glycan interactions may be a strategic mechanism for allowing pre-BCR clustering and pre-BII cells departure from their niche. Altogether, our data suggest a novel mechanism for a cell to modify the equilibrium of the GAL1/glycan lattice involving GAL1/unglycosylated protein interactions.

A Multiple Myeloma Classification System That Associates Normal Bone Marrow B-Cell Subset Phenotypes with Disease Stage and Prognosis

IntroductionToday’s diagnostic tests for multiple myeloma reflect the criteria of the updated WHO classification based on biological, morphological and clinical heterogeneity. It has been the concept behind the present study to extend our current biological classification and provide a new tool to generate insight into the stage of clonal differentiation and oncogenesis.The goal of the present study was to generate B-cell subset associated gene signatures (BAGS) from the BM hierarchy used to assign individual phenotypic cell of origin (pCOO) subtypes in patients and validate its pathogenetic impact by prognostic evaluation.MethodsBone marrows (BM, n=7) were harvested from sternum during cardiac surgery. B-cell subsets were phenotyped by Euroflow standard for multiparametric flow cytometry and FACS-sorted for microarray analysis on the Human Exon 1.0 ST Arrays platform. This combination allowed us to generate five BAGS for PreBI, PreBII, immature (Im), naïve (N), memory (M) B-cells, and plasma cells (PC) of normal BM. The BAGS classifier was based on all median-centred probe sets from the data set by regularized multinomial regression with 6 discrete outcomes corresponding to each B-cell subset and the elastic net penalty using the algorithm implemented in the R-package glmnet24. Each clinical data set was probe-set-wise adjusted to have a zero median and the same variance as in the BM data set. The associated cell of origin subset for each patient in each data set was predicted by the BAGS classifier by assigning the class with the highest predicted probability score above 0.45 and otherwise UC. All statistical analyses were done with R version 3.0.2. Survival analysis was performed by the Kaplan-Meier method and log-rank tests. Clinical data sets were from University of Arkansas for Medical Sciences UAMS (n=559), the Dana Farber Cancer Institute DFCI (n=170) both available on the GEO website.ResultsFirst, we verified the quality of the sampled B-cell subsets based on the expression patterns of differentiation marker genes. Next, we constructed the BAGS-classifier provided by 38-68 gene probe sets (n), capable of assigning samples to each of the six subtypes of PreBI (n=38), PreBII (n=82), Im (n=71), N (n=68), M (n=52) and PC (n=43).Second, we assigned individual myeloma cases in the 2 patient cohorts including a total of 729 myeloma patients. The resultant assignments generated patient BAGS subtypes with diagnostic frequencies of 0,5-0,6 % preBI, 7-8 % preBII, 27-30 % Im, 8-9 % N, 36-37 % M, 1-5 % PC and 15-16 % UC subtypes. The frequencies were not different between the cohorts.Third, the BAGS subtypes was associated significantly with overall survival (P = 8.6 x 10-5) for high dose melphalan and autologous stem cell transplanted UAMS patients1, as illustrated in Figure 1. The most significant impact was observed within the PreB-II (light blue) and M (acid green) subtypes conferred with significant inferior prognosis compared to the Im (amethyst), N (apple green) and PC (blue) subtypes. The PreB-II and M subtypes in the UAMS cohort were correlated to the ISS stage I-III with 33%, 49% and 69% of the cases, respectively.Fourth, we compared the BAGS subtypes and the TC classification2 with no observed correlations (results not shown).ConclusionsOur data support a new COO defined BAGS classification based on the normal BM B-cell subset phenotypes with impact on staging and prognosis in multiple myeloma and a new diagnostic platform, which may result in more effective disease management.References 1) Zhan F, Huang Y, Colla S et al. The molecular classification of multiple myeloma. Blood. 2006 Sep 15;108(6):2020-8. 2) Bergsagel PL, Kuehl WM, Zhan F, Sawyer J, Barlogie B, Shaughnessy J Jr. Cyclin D dysregulation: an early and unifying pathogenic event in multiple myeloma. Blood. 2005 Jul 1;106(1):296-303. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The BLNK adaptor protein has a nonredundant role in human B-cell differentiation

Expression of the pre-B-cell receptor (pre-BCR) by pre-BII cells constitutes a crucial checkpoint in B-cell differentiation. Mutations that affect the pre-B-cell receptor result in early B-cell differentiation blockades that lead to primary B-cell immunodeficiencies. BLNK adaptor protein has a key role in the pre-B-cell receptor signaling cascade, as illustrated by the abnormal B-cell development in the 4 patients with BLNK gene defects reported to date. However, the BLNK protein's precise function in human B-cell differentiation has not been completely specified. B-cell development, including IgVH and Vk chain repertoires analysis, was studied in the bone marrow of a new case of BLNK deficiency in vitro and in vivo. Here, we report on a patient with agammaglobulinemia, with a total absence of circulating B cells. We detected a homozygous mutation in BLNK, which leads to the complete abrogation of BLNK protein expression. In the bone marrow, we identified a severe differentiation blockade at the pre-BI- to pre-BII-cell transition. IgVH gene rearrangements and selection of the IgH repertoire were normal, whereas the patient's pre-BI cells showed very restricted usage of the IgVκ repertoire. Complementation of bone marrow progenitors from the patient with the BLNK gene and transplantation into NOD/SCID/γcko mice allowed the complete restoration of B-cell differentiation and a normal usage of the IgVκ genes.

B-Cell Progenitors and Precursors Change Their Microenvironment in Fetal Liver During Early Development

The microenvironments, in which B lymphocytes develop in fetal liver, are largely still unknown. Among the nonhematopoietic cells, we have identified and FACS-separated two subpopulations, CD45(-) TER119(-) VCAM-1(+) cells that are either CD105(high) LYVE-1(high) or CD105(low) ALCAM(high) . Immunohistochemical analyses find three of four c-Kit(+) IL-7Rα(+) B220(low) CD19(-) SLC(-) B progenitors in contact with vascular endothelial-type LYVE-1(high) cells on embryonic day 13.5. One day later c-Kit(+) IL-7Rα(+) cells develop to CD19(- and +) , SLC-expressing, DHJH-rearranged pre/pro and pro/preB-I cells. Less than 10% are still in contact with LYVE-1(high) cells, but half of them are now in contact with mesenchymally derived ALCAM(high) liver cells. All of these ALCAM(high) cells, but not the LYVE-1(high) cells produce IL-7 and CXCL12, while both produce CXCL10. Progenitors and pro/preB-I cells are chemoattracted in vitro toward CXCL10 and 12, suggesting that lymphoid progenitors with Ig gene loci in germline configuration enter the developing fetal liver at E13.5 from vascular endothelium, attracted by CXCL10, and then migrate within a day to an ALCAM(high) liver cell microenvironment, differentiating to DHJH-rearranging, surrogate light chain-expressing pre/proB and pro/preB-I cells, attracted by CXCL10 and 12. Between E15.5 and E16.5 preB-I cells expand 10-fold in continued contact with ALCAM(high) cells and begin VH- to DHJH-rearrangements in further differentiated c-Kit(-) IL-7Rα(-) preBII cells. STEM Cells 2013;31:2800-2812.

Structural Basis for Galectin-1-dependent Pre-B Cell Receptor (Pre-BCR) Activation

During B cell differentiation in the bone marrow, the expression and activation of the pre-B cell receptor (pre-BCR) constitute crucial checkpoints for B cell development. Both constitutive and ligand-dependent pre-BCR activation modes have been described. The pre-BCR constitutes an immunoglobulin heavy chain (Igμ) and a surrogate light chain composed of the invariant λ5 and VpreB proteins. We previously showed that galectin-1 (GAL1), produced by bone marrow stromal cells, is a pre-BCR ligand that induces receptor clustering, leading to efficient pre-BII cell proliferation and differentiation. GAL1 interacts with the pre-BCR via the unique region of λ5 (λ5-UR). Here, we investigated the solution structure of a minimal λ5-UR motif that interacts with GAL1. This motif adopts a stable helical conformation that docks onto a GAL1 hydrophobic surface adjacent to its carbohydrate binding site. We identified key hydrophobic residues from the λ5-UR as crucial for the interaction with GAL1 and for pre-BCR clustering. These residues involved in GAL1-induced pre-BCR activation are different from those essential for autonomous receptor activation. Overall, our results indicate that constitutive and ligand-induced pre-BCR activation could occur in a complementary manner.

B Cell Development and the Splenic Microenvironment in Dlk-1 Deficient Mice

AbstractAbstract 1251 Background:DLK-1 is a transmembrane and secreted protein that plays a crucial role in normal B cell development and differentiation. In a previous study we showed that DLK-1 knockout mice (Dlk1−/− mice (KO)) have distinct differences in B cell fractions in the spleen and bone marrow compared to wild-type Dlk1+/+ (WT) mice. KO mice showed a decrease in follicular (FO) B cells and an increase in the size of the marginal zone (MZ) and number of MZ B cells in the spleen of 8 week old mice. Furthermore, there was an exaggerated primary T-dependent antigen-specific humoral immune response. The mechanisms underlying the changes in splenic B cell fractions between KO and WT mice are not yet clear. It has been suggested that stromal microenvironmental cells form distinct cellular niches that influence different stages of B cell development. Alterations in these stromal niches due to absence of DLK-1 may be an underlying cause of the observed splenic B cell fraction alterations. It was previously shown that Galectin-1 (GAL1) plays an important role in the bone marrow microenvironment and affects proliferation and differentiation of normal mouse pre-BII cells (Blood April 21, 2011). This study is designed to investigate the splenic stromal cells in DLK-1 deficient mice as a step toward understanding these B cell alterations, and to test whether the stromal cell derived-GAL1 influences splenic B-cell development. Methods:For detection of B cell fractions, a multicolor flow cytometry panel consisting of anti-IgD/IgM/CD23/CD93/CD45R/CD21/CD35 was used. B cell fractions were identified as follows: MZ (CD23neg-low, CD21high), FO (CD23high, CD21intermediate), Tr1 (CD23neg, CD21neg, AA4.1pos, B220pos), Tr2 (CD23pos, AA4.1pos, B220pos). For detection of stromal cell fractions, an anti-CD11c/Ter119/CD19/Gr-1/Tie-2/CD45/CD31/CD117/CD34 panel was used. Stromal cells were identified as follows: CD45neg, lineage− (Ter119, CD19, GR1, CD11c, and CD34), CD117neg, CD34neg, Tie2neg. Two stromal cell fractions were detected: CD31 +and CD31−. Galectin-1 expression was evaluated on CD31+ and CD31−stroma cell fractions. An eight color flow cytometric panel consisting of antibody directed to MHC II, CD11c, Gr-1, CD8a, B220, and CD11b was used to evaluate myeloid, lymphoid and plasmacytoid dendritic cell fractions. Results:Our data showed an increase in MZ B-cells (p<0.005) and a decrease in FO B cells (p<0.005) in the KO mice. When looking at the splenic stromal cells, we found an increase in the CD31+ fraction and a decrease in the CD31- fraction in KO mice compared to WT mice (p<0.005). We observed that the CD31 + stromal cell expressed high galectin-1 (GAL1) levels compared to CD31− stroma cells. Also there was an increased percentage of myeloid dendritic cells in KO compared to WT (p< 0.05). Conclusion:We have shown that absence of DLK-1 leads to alterations in splenic stromal populations and that these may play an important role in altered B cell populations and function observed in DLK-1-deficient mice. We are investigating whether the CD31+ GAL1 +stroma cell is a key stroma cell that plays a critical role in splenic B cell development and is responsible for the B-lineage differences seen in DLK-1-deficient mice. Our observation that there is an increase in the myeloid dendritic cell fraction may explain the exaggerated primary immune response seen in the KO mice. These data show that DLK-1-deficiency leads to several changes in the splenic cellular microenvironment and suggest that these changes contribute to alterations in B-cell development and function. Disclosures:No relevant conflicts of interest to declare.

Pre-B Cell Receptor-Mediated Activation of BCL6 Induces Pre-B Cell Quiescence Through Transcriptional Repression of MYC

Abstract 1406 Background:Pre-B cell receptor signaling is critical to induce initial expansion of the pool of differentiating B cell precursors but also mediates subsequent cell cycle exit and quiescence. Initial cell surface expression of the pre-B cell receptor induces a series of tyrosine phosphorylation events resulting in MYC and CCND2-mediated proliferation. After 2–5 divisions, however, large pre-BII (Fraction C’) cells exit cell cycle to become resting, small pre-BII cells (Fraction D). While the signaling pathway resulting in pre-B cell receptor-induced proliferation is well understood, the mechanism by which pre-BII cells exit cell cycle, however, is currently unclear. Results:This checkpoint and cell cycle exit at the Fraction C'-D transition is critical for immunoglobulin light chain gene recombination and to prevent malignant transformation into acute lymphoblastic leukemia. Here we demonstrate that inducible activation of pre-B cell receptor signaling recapitulates the initial proliferative burst followed by cell cycle exit, which is characterized by strong upregulation of BCL6 and subsequent loss of MYC and CCND2 expression. ChIP-on-chip analysis revealed that the transcriptional repressor BCL6 is directly recruited to the MYC promoter. Inducible activation of pre-B cell receptor signaling in pre-B cells from BCL6-deficient mice failed to induce cell cycle exit and quiescence. We conclude that activation of BCL6 downstream of the pre-B cell receptor is crucial for the induction of quiescence at the Fraction C'-D checkpoint. As expected, inducible activation of BCL6 downstream of the pre-B cell receptor results in transcriptional repression of MYC and CCND2. Overexpression of MYC prevented pre-B cell receptor/BCL6-induced cell cycle exit. Conclusion:Hence, pre-B cell receptor signaling induces cellular quiescence of Fraction C' pre-B cells through activation of BCL6 and BCL6-mediated transcriptional repression of MYC and CCND2. Disclosures:No relevant conflicts of interest to declare.

Galectins and microenvironmental niches during hematopoiesis

Galectins, a family of evolutionarily conserved glycan-binding proteins, are involved in the regulation of multiple cellular processes (e.g. immunity, apoptosis, cellular signaling, development, angiogenesis and cellular growth) and diseases (e.g. chronic inflammation, autoimmunity, cancer, infection). We discuss here how galectins contribute to the development of specialized microenvironmental niches during hematopoiesis. An expanding set of data strengthens a role of galectins in hematopoietic differentiation, particularly by setting specific interactions between hematopoietic and stromal cells: galectin-5 is found in reticulocytes and erythroblastic islands suggesting a major role during erythropoiesis; galectin-1 and 3 are involved in thymocyte apoptosis, signaling and intrathymic migration; galectin-1 plays critical roles in pre-BII cells development. Moreover, expression of galectins-1 and 10 are differentially expressed during T-regulatory cell development. Various galectins (3, 4, 5, 9) have been reported to be regulated during myelopoiesis and traffic into intracellular compartments, dictating the cellular distribution of specific glycoproteins and glycosphingolipids. The abundance of galectins in both extracellular and intracellular compartments, their multifunctional properties and ability to form supramolecular signaling complexes with specific glycoconjugates, make these glycan-binding proteins excellent candidates to mediate interactions between hematopoietic cells and the stromal microenvironment. Their secretion by one of the cellular partners can modulate adhesive properties by cross-linking specific glycoconjugates present on stromal or hematopoietic cells, by favoring the formation of synapses or by creating glycoprotein lattices on the surface of different cell types. Their divergent specificities and affinities for various glycoproteins contribute to the multiplicity of their cellular interactions.

Pre-B cell receptor–mediated activation of BCL6 induces pre-B cell quiescence through transcriptional repression of MYC

AbstractInitial cell surface expression of the pre-B cell receptor induces proliferation. After 2 to 5 divisions, however, large pre-BII (Fraction C‘) cells exit cell cycle to become resting, small pre-BII cells (Fraction D). The mechanism by which pre-BII cells exit cell cycle, however, is currently unclear. The checkpoint at the Fraction C‘-D transition is critical for immunoglobulin light chain gene recombination and to prevent malignant transformation into acute lymphoblastic leukemia. Here we demonstrate that inducible activation of pre-B cell receptor signaling induces cell-cycle exit through up-regulation of the transcriptional repressor BCL6. Inducible activation of BCL6 downstream of the pre-B cell receptor results in transcriptional repression of MYC and CCND2. Hence, pre-B cell receptor-mediated activation of BCL6 limits pre-B cell proliferation and induces cellular quiescence at the small pre-BII (Fraction D) stage.

Galectin-1–expressing stromal cells constitute a specific niche for pre-BII cell development in mouse bone marrow

AbstractIn the bone marrow (BM), stromal cells constitute a supportive tissue indispensable for the generation of pro-B/pre-BI, pre-BII, and immature B lymphocytes. IL-7–producing stromal cells constitute a cellular niche for pro-B/pre-BI cells, but no specific stromal cell microenvironment was identified for pre-BII cells expressing a functional pre-B cell receptor (pre-BCR). However expression of the pre-BCR represents a crucial checkpoint during B-cell development. We recently demonstrated that the stromal cell derived-galectin1 (GAL1) is a ligand for the pre-BCR, involved in the proliferation and differentiation of normal mouse pre-BII cells. Here we show that nonhematopoietic osteoblasts and reticular cells in the BM express GAL1. We observed that pre-BII cells, unlike the other B-cell subsets, were specifically localized in close contact with GAL1+ reticular cells. We also determined that IL-7+ and GAL1+ cells represent 2 distinct mesenchymal populations with different BM localization. These results demonstrate the existence of a pre-BII specific stromal cell niche and indicate that early B cells move from IL-7+ to GAL1+ supportive BM niches during their development.

Premature replacement of μ with α immunoglobulin chains impairs lymphopoiesis and mucosal homing but promotes plasma cell maturation

Sequentially along B cell differentiation, the different classes of membrane Ig heavy chains associate with the Ig alpha/Ig beta heterodimer within the B cell receptor (BCR). Whether each Ig class conveys specific signals adapted to the corresponding differentiation stage remains debated. We investigated the impact of the forced expression of an IgA-class receptor throughout murine B cell differentiation by knocking in the human C alpha Ig gene in place of the S mu region. Despite expression of a functional BCR, homozygous mutant mice showed a partial developmental blockade at the pro-B/pre-BI and large pre-BII cell stages, with decreased numbers of small pre-BII cells. Beyond this stage, peripheral B cell compartments of reduced size developed and allowed specific antibody responses, whereas mature cells showed constitutive activation and a strong commitment to plasma cell differentiation. Secreted IgA correctly assembled into polymers, associated with the murine J chain, and was transported into secretions. In heterozygous mutants, cells expressing the IgA allele competed poorly with those expressing IgM from the wild-type allele and were almost undetectable among peripheral B lymphocytes, notably in gut-associated lymphoid tissues. Our data indicate that the IgM BCR is more efficient in driving early B cell education and in mucosal site targeting, whereas the IgA BCR appears particularly suited to promoting activation and differentiation of effector plasma cells.

Impaired B-cell development at the pre-BII-cell stage in galectin-1–deficient mice due to inefficient pre-BII/stromal cell interactions

AbstractActivation of the pre-B-cell receptor (pre-BCR) in the bone marrow depends on both tonic and ligand-induced signaling and leads to pre-BII-cell proliferation and differentiation. Using normal mouse bone marrow pre-BII cells, we demonstrate that the ligand-induced pre-BCR activation depends on pre-BCR/galectin-1/integrin interactions leading to pre-BCR clustering at the pre-BII/stromal cell synapse. In contrast, heparan sulfates, shown to be pre-BCR ligands in mice, are not implicated in pre-BCR relocalization. Inhibition of pre-BCR/galectin-1/integrin interactions has functional consequences, since pre-BII-cell proliferation and differentiation are impaired in an in vitro B-cell differentiation assay, without affecting cellular apoptosis. Most strikingly, although galectin-1–deficient mice do not show an apparent B-cell phenotype, the kinetics of de novo B-cell reconstitution after hydroxyurea treatment indicates a specific delay in pre-BII-cell recovery due to a decrease in pre-BII-cell differentiation and proliferation. Thus, although it remains possible that the pre-BCR interacts with other ligands, these results highlight the role played by the stromal cell–derived galectin-1 for the efficient development of normal pre-BII cells and suggest the existence of pre-BII–specific stromal cell niches in normal bone marrow.