Analyses of IGHV gene mutations in chronic lymphocytic leukemia (CLL) have had a major impact on the prognostication and treatment of this disease. A hallmark of IGHV-mutation status is that it very rarely changes clonally over time. Nevertheless, targeted and deep DNA sequencing of IGHV-IGHD-IGHJ regions has revealed intraclonal heterogeneity. We used a DNA sequencing approach that achieves considerable depth and minimizes artefacts and amplification bias to identify IGHV-IGHD-IGHJ subclones in patients with prolonged temporal follow-up. Our findings extend previous studies, revealing intraclonal IGHV-IGHD-IGHJ diversification in almost all CLL clones. Also, they indicate that some subclones with additional IGHV-IGHD-IGHJ mutations can become a large fraction of the leukemic burden, reaching numerical criteria for monoclonal B-cell lymphocytosis. Notably, the occurrence and complexity of post-transformation IGHV-IGHD-IGHJ heterogeneity and the expansion of diversified subclones are similar among U-CLL and M-CLL patients. The molecular characteristics of the mutations present in the parental, clinically dominant CLL clone (CDC) differed from those developing post-transformation (post-CDC). Post-CDC mutations exhibit significantly lower fractions of mutations bearing signatures of activation induced deaminase (AID) and of error-prone repair by Polη, and most of the mutations were not ascribable to those enzymes. Additionally, post-CDC mutations displayed a lower percentage of nucleotide transitions compared with transversions that was also not like the action of AID. Finally, the post-CDC mutations led to significantly lower ratios of replacement to silent mutations in VH CDRs and higher ratios in VH FRs, distributions different from mutations found in normal B-cell subsets undergoing an AID-mediated process. Based on these findings, we propose that post-transformation mutations in CLL cells either reflect a dysfunctional standard somatic mutational process or point to the action of another mutational process not previously associated with IG V gene loci. If the former option is the case, post-CDC mutations could lead to a lesser dependence on antigen dependent BCR signaling and potentially a greater influence of off-target, non-IG genomic mutations. Alternatively, the latter activity could add a new stimulatory survival/growth advantage mediated by the BCR through structurally altered FRs, such as that occurring by superantigen binding and stimulation.
The adult immune system consists of cells that emerged at various times during ontogeny. We aimed to define the relationship between developmental origin and composition of the adult B cell pool during unperturbed hematopoiesis. Lineage tracing stratified murine adult B cells based on the timing of output, revealing that a substantial portion originated within a restricted neonatal window. In addition to B-1a cells, early-life time-stamped B cells included clonally interrelated IgA plasma cells in the gut and bone marrow. These were actively maintained by B cell memory within gut chronic germinal centers and contained commensal microbiota reactivity. Neonatal rotavirus infection recruited recurrent IgA clones that were distinct from those arising by infection with the same antigen in adults. Finally, gut IgA plasma cells arose from the same hematopoietic progenitors as B-1a cells during ontogeny. Thus, a complex layer of neonatally imprinted B cells confer unique antibody responses later in life.
Although chronic lymphocytic leukemia (CLL) is diagnosed by identifying a circulating B-cell clone that exceeds 5x106/μL, additional distinct clones (ADC) have been identified in various studies. Notably, the numbers of ADC documented in these studies has increased as the various technologies evolved. To better define the frequency and the characteristics of ADCs in CLL, we used a Next Generation Sequencing (NGS) platform that affords high sequencing depth along with steps that limit overcounting to analyze IGHV-IGHD-IGHJ gene rearrangements in circulating CD5+ B cells from 57 patients. Notably, all patients had at least one ADC, in addition to the clinically relevant clone (CRC)., in 46 patients for whom lymphocyte count data were available, 44 had at least one ADC above the threshold of 1 B cell/μL, and remarkably, the average number of ADCs was 12 per patient. Notably, in two patients, the predominant ADC (pADC) qualified clinically as a separate CLL clone and in the in the remaining cases as low/high-count monoclonal B-cell lymphocytosis (MBL) clones. Moreover, in 11 patients studied longitudinally, pADCs were persistent and often increased in number. ADCs in patients with CLL exhibited fourfold more stereotyped IGHV-IGHD-IGH rearrangements than found in CD5+ B cells from healthy individuals, and IGHV use, somatic mutations, and Ig isotype distribution was similar between pADCs and CRCs. Thus, finding multiple expanded clones within the CD5+ B cells is the rule in patients with CLL, indicating that leukemogenesis is a multiclonal process that likely involves competition among B cells with special BCR features.
The LIN28B RNA binding protein exhibits an ontogenically restricted expression pattern and is a key molecular regulator of fetal and neonatal B lymphopoiesis. It enhances the positive selection of CD5+ immature B cells early in life through amplifying the CD19/PI3K/c-MYC pathway and is sufficient to reinitiate self-reactive B-1a cell output when ectopically expressed in the adult. In this study, interactome analysis in primary B cell precursors showed direct binding by LIN28B to numerous ribosomal protein transcripts, consistent with a regulatory role in cellular protein synthesis. Induction of LIN28B expression in the adult setting is sufficient to promote enhanced protein synthesis during the small Pre-B and immature B cell stages, but not during the Pro-B cell stage. This stage dependent effect was dictated by IL-7 mediated signaling, which masked the impact of LIN28B through an overpowering stimulation on the c-MYC/protein synthesis axis in Pro-B cells. Importantly, elevated protein synthesis was a distinguishing feature between neonatal and adult B cell development that was critically supported by endogenous Lin28b expression early in life. Finally, we used a ribosomal hypomorphic mouse model to demonstrate that subdued protein synthesis is specifically detrimental for neonatal B lymphopoiesis and the output of B-1a cells, without affecting B cell development in the adult. Taken together, we identify elevated protein synthesis as a defining requirement for early-life B cell development that critically depends on Lin28b . Our findings offer new mechanistic insights into the layered formation of the complex adult B cell repertoire.
Chronic Lymphocytic Leukemia (CLL) is characterized by the accumulation of monoclonal CD5+ B cells with low surface immunoglobulins (IG). About 40% of CLL clones utilize quasi-identical B cell receptors, defined as stereotyped BCR. CLL-like stereotyped-IG rearrangements are present in normal B cells as a part of the public IG repertoire. In this study, we collected details on the representation and features of CLL-like stereotyped-IG in the IGH repertoire of B-cell subpopulations purified from the peripheral blood of nine healthy donors. The B-cell subpopulations were also fractioned according to the expression of surface CD5 molecules and IG light chain, IGκ and IGλ. IG rearrangements, obtained by high throughput sequencing, were scanned for the presence of CLL-like stereotyped-IG. CLL-like stereotyped-IG did not accumulate preferentially in the CD5+ B cells, nor in specific B-cell subpopulations or the CD5+ cell fraction thereof, and their distribution was not restricted to a single IG light chain type. CLL-like stereotyped-IG shared with the corresponding CLL stereotype rearrangements the IGHV mutational status. Instead, for other features such as IGHV genes and frequency, CLL stereotyped-IGs presented a CLL-like subset specific behavior which could, or could not, be consistent with CLL stereotyped-IGs. Therefore, as opposed to the immuno-phenotype, the features of the CLL stereotyped-IG repertoire suggest a CLL stereotyped subset-specific ontogeny. Overall, these findings suggest that the immune-genotype can provide essential details in tracking and defining the CLL cell of origin.
Abstract Progression of chronic lymphocytic leukemia (CLL) results from the expansion of a small fraction of proliferating leukemic B cells. When comparing the global gene expression of recently divided CLL cells with that of previously divided cells, we found higher levels of genes involved in regulating gene expression. One of these was the oncogene Musashi 2 (MSI2), an RNA-binding protein that induces or represses translation. While there is an established role for MSI2 in normal and malignant stem cells, much less is known about its expression and role in CLL. Here we report for the first time ex vivo and in vitro experiments that MSI2 protein levels are higher in dividing and recently divided leukemic cells and that downregulating MSI2 expression or blocking its function eliminates primary human and murine CLL and mature myeloid cells. Notably, mature T cells and hematopoietic stem and progenitor cells are not affected. We also confirm that higher MSI2 levels correlate with poor outcome markers, shorter time-to-first-treatment, and overall survival. Thus, our data highlight an important role for MSI2 in CLL-cell survival and proliferation and associate MSI2 with poor prognosis in CLL patients. Collectively, these findings pinpoint MSI2 as a potentially valuable therapeutic target in CLL.
Abstract B-cell chronic lymphocytic leukemia (B-CLL) clones express BCR specific for microbes and/or apoptotic cells and elevated TLR-9. In patients, B-CLL clonal expansion occurs within lymphoid tissues. We recently reported that CpG DNA (ODN-2006) and IL-15, found within stromal cells of CLL-infiltrated lymphoid tissues, manifest synergy in promoting in vitro B-CLL growth (J. Immunol 195:901–923, 2015). This study explores the mechanism responsible by using purified B-CLL from peripheral blood to assess the temporal requirements for IL-15; effect of ODN on expression of IL-15 receptors (IL-15Rα and CD122 (IL-2/15Rβ)); and IL-15-induced activation of AKT and STAT5. Experiments with CFSE-labeled B-CLL show that IL-15 is critical during a 20–36 h window after ODN priming. Neutralizing mAbs to IL-15 or CD122 fully abrogate IL-15-driven growth when added during this interval. RT-PCR/immunofluorescence studies show that ODN triggers rapid increases in mRNA/protein for IL-15Rα and CD122 that are blocked by IkBα inhibitor. Furthermore, ODN-primed B-CLL exposed to IL-15 manifest elevated pAKT(Ser473) and pSTAT5(Tyr694) levels. Both PI-3K/AKT and STAT5 pathways are functionally relevant, as indicated by growth abrogation by pharmacologic inhibitors, LY294002, pimozide or STAT5 Inh II. Importantly, activated STAT5 levels are high in cycling CFSE-labeled B-CLL; furthermore, extended divisions were blocked by delayed (day 4) administration of STAT5 inhibitors or neutralizing mAbs to IL-15/CD122. Together, findings show that ODN + IL-15-driven growth represents coordinated early functions of TLR-9 and CD122 as well as later CD122 signaling during clonal expansion. This synergy may help drive in vivo growth of B-CLL clones in patients.
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