Enhanced protein synthesis is a defining requirement for neonatal B cell development.

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.

CLL stereotyped B-cell receptor immunoglobulin sequences are recurrent in the B-cell repertoire of healthy individuals: Apparent lack of central and early peripheral tolerance censoring.

Introduction: The leukemic cells of patients with chronic lymphocytic leukemia (CLL) are often unique, expressing remarkably similar IGHV-IGHD-IGHJ gene rearrangements, "stereotyped BCRs". The B-cell receptors (BCRs) on CLL cells are also distinctive in often deriving from autoreactive B lymphocytes, leading to the assumption of a defect in immune tolerance. Results: Using bulk and single-cell immunoglobulin heavy and light chain variable domain sequencing, we enumerated CLL stereotype-like IGHV-IGHD-IGHJ sequences (CLL-SLS) in B cells from cord blood (CB) and adult peripheral blood (PBMC) and bone marrow (BM of healthy donors. CLL-SLS were found at similar frequencies among CB, BM, and PBMC, suggesting that age does not influence CLL-SLS levels. Moreover, the frequencies of CLL-SLS did not differ among B lymphocytes in the BM at early stages of development, and only re-circulating marginal zone B cells contained significantly higher CLL-SLS frequencies than other mature B-cell subpopulations. Although we identified CLL-SLS corresponding to most of the CLL major stereotyped subsets, CLL-SLS frequencies did not correlate with those found in patients. Interestingly, in CB samples, half of the CLL-SLS identified were attributed to two IGHV-mutated subsets. We also found satellite CLL-SLS among the same normal samples, and they were also enriched in naïve B cells but unexpectedly, these were ~10-fold higher than standard CLL-SLS. In general, IGHV-mutated CLL-SLS subsets were enriched among antigen-experienced B-cell subpopulations, and IGHV-unmutated CLL-SLS were found mostly in antigen-inexperienced B cells. Nevertheless, CLL-SLS with an IGHV-mutation status matching that of CLL clones varied among the normal B-cell subpopulations, suggesting that specific CLL-SLS could originate from distinct subpopulations of normal B cells. Lastly, using single-cell DNA sequencing, we identified paired IGH and IGL rearrangements in normal B lymphocytes resembling those of stereotyped BCRs in CLL, although some differed from those in patients based on IG isotype or somatic mutation. Discussion: CLL-SLS are present in normal B-lymphocyte populations at all stages of development. Thus, despite their autoreactive profile they are not deleted by central tolerance mechanisms, possibly because the level of autoreactivity is not registered as dangerous by deletion mechanisms or because editing of L-chain variable genes occurred which our experimental approach could not identify.

A self-sustaining layer of early-life-origin B cells drives steady-state IgA responses in the adult gut.

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.

Characterizing Features of Human Circulating B Cells Carrying CLL-Like Stereotyped Immunoglobulin Rearrangements.

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.

Developmental cues license megakaryocyte priming in murine hematopoietic stem cells.

The fetal-to-adult switch in hematopoietic stem cell (HSC) behavior is characterized by alterations in lineage output and entry into deep quiescence. Here we identify the emergence of megakaryocyte (Mk)-biased HSCs as an event coinciding with this developmental switch. Single-cell chromatin accessibility analysis reveals a ubiquitous acquisition of Mk lineage priming signatures in HSCs during the fetal-to-adult transition. These molecular changes functionally coincide with increased amplitude of early Mk differentiation events after acute inflammatory insult. Importantly, we identify LIN28B, known for its role in promoting fetal-like self-renewal, as an insulator against the establishment of an Mk-biased HSC pool. LIN28B protein is developmentally silenced in the third week of life, and its prolonged expression delays emergency platelet output in young adult mice. We propose that developmental regulation of Mk priming may represent a switch for HSCs to toggle between prioritizing self-renewal in the fetus and increased host protection in postnatal life.

B cell receptor isotypes differentially associate with cell signaling, kinetics, and outcome in chronic lymphocytic leukemia.

In chronic lymphocytic leukemia (CLL), the B cell receptor (BCR) plays a critical role in disease development and progression, as indicated by the therapeutic efficacy of drugs blocking BCR signaling. However, the mechanism(s) underlying BCR responsiveness are not completely defined. Selective engagement of membrane IgM or IgD on CLL cells, each coexpressed by more than 90% of cases, leads to distinct signaling events. Since both IgM and IgD carry the same antigen-binding domains, the divergent actions of the receptors are attributed to differences in immunoglobulin (Ig) structure or the outcome of signal transduction. We showed that IgM, not IgD, level and organization associated with CLL-cell birth rate and the type and consequences of BCR signaling in humans and mice. The latter IgM-driven effects were abrogated when BCR signaling was inhibited. Collectively, these studies demonstrated a critical, selective role for IgM in BCR signaling and B cell fate decisions, possibly opening new avenues for CLL therapy.

Post-Transformation IGHV-IGHD-IGHJ Mutations in Chronic Lymphocytic Leukemia B Cells: Implications for Mutational Mechanisms and Impact on Clinical Course.

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.

Myeloid-derived suppressor cell subtypes differentially influence T-cell function, T-helper subset differentiation, and clinical course in CLL.

Cancer pathogenesis involves the interplay of tumor- and microenvironment-derived stimuli. Here we focused on the influence of an immunomodulatory cell type, myeloid-derived suppressor cells (MDSCs), and their lineage-related subtypes on autologous T lymphocytes. Although MDSCs as a group correlated with an immunosuppressive Th repertoire and worse clinical course, MDSC subtypes (polymorphonuclear, PMN-MDSC, and monocytic, M-MDSCs) were often functionally discordant. In vivo, PMN-MDSCs existed in higher numbers, correlated with different Th-subsets, and more strongly associated with poor clinical course than M-MDSCs. In vitro, PMN-MDSCs were more efficient at blocking T-cell growth and promoted Th17 differentiation. Conversely, in vitro M-MDSCs varied in their ability to suppress T-cell proliferation, due to the action of TNFα, and promoted a more immunostimulatory Th compartment. Ibrutinib therapy impacted MDSCs differentially as well, since after initiating therapy, PMN-MDSC numbers progressively declined, whereas M-MDSC numbers were unaffected, leading to a set of less immunosuppressive Th cells. Consistent with this, clinical improvement based on decreasing CLL-cell numbers correlated with the decrease in PMN-MDSCs. Collectively, the data support a balance between PMN-MDSC and M-MDSC numbers and function influencing CLL disease course.

Developmental changes in the rules for B cell selection.

The autoimmune checkpoint during B cell maturation eliminates self-antigen reactive specificities from the mature B cell repertoire. However, an exception to this rule is illustrated by B-1 cells, an innate-like self-reactive B cell subset that is positively selected into the mature B cell pool in a self-antigen-driven fashion. The mechanisms by which B-1 cells escape central tolerance have puzzled the field for decades. A key clue comes from their restricted developmental window during fetal and neonatal life. Here we use B-1 cells as a prototypic early life derived B cell subset to explore developmental changes in the constraints of B cell selection. We discuss recent advancements in the understanding of the molecular program, centered around the RNA binding protein Lin28b, that licenses self-reactive B-1 cell output during ontogeny. Finally, we speculate on the possible link between the unique rules of early life B cell tolerance and the establishment of B cell - microbial mutualism to propose an integrated model for how developmental and environmental cues come together to create a protective layer of B cell memory involved in neonatal immune imprinting.

Musashi 2 influences chronic lymphocytic leukemia cell survival and growth making it a potential therapeutic target.

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.

B-1 Cells Carry the Memory of Neonatal Immune Imprinting.

Group A Streptococcus is a common pathogen that elicits a protective humoral response against the cell wall component GlcNAc. In this issue of Immunity, New et al. demonstrate the ability of long-lived B-1 cells to be programmed by microbial colonization and early life immunization to uniquely incorporate GlcNAc reactivity in mice, establishing their critical role in mediating neonatal immune imprinting.

Lin28b controls a neonatal to adult switch in B cell positive selection.

The ability of B-1 cells to become positively selected into the mature B cell pool, despite being weakly self-reactive, has puzzled the field since its initial discovery. Here, we explore changes in B cell positive selection as a function of developmental time by exploiting a link between CD5 surface levels and the natural occurrence of self-reactive B cell receptors (BCRs) in BCR wild-type mice. We show that the heterochronic RNA binding protein Lin28b potentiates a neonatal mode of B cell selection characterized by enhanced overall positive selection in general and the developmental progression of CD5+ immature B cells in particular. Lin28b achieves this by amplifying the CD19/PI3K/c-Myc positive feedback loop, and ectopic Lin28b expression restores both positive selection and mature B cell numbers in CD19-/- adult mice. Thus, the temporally restricted expression of Lin28b relaxes the rules for B cell selection during ontogeny by modulating tonic signaling. We propose that this neonatal mode of B cell selection represents a cell-intrinsic cue to accelerate the de novo establishment of the adaptive immune system and incorporate a layer of natural antibody-mediated immunity throughout life.

Low-dose irradiated mesenchymal stromal cells break tumor defensive properties in vivo.

Solid tumors, including gliomas, still represent a challenge to clinicians and first line treatments often fail, calling for new paradigms in cancer therapy. Novel strategies to overcome tumor resistance are mainly represented by multi-targeted approaches, and cell vector-based therapy is one of the most promising treatment modalities under development. Here, we show that mouse bone marrow-derived mesenchymal stromal cells (MSCs), when primed with low-dose irradiation (irMSCs), undergo changes in their immunogenic and angiogenic capacity and acquire anti-tumoral properties in a mouse model of glioblastoma (GBM). Following grafting in GL261 glioblastoma, irMSCs migrate extensively and selectively within the tumor and infiltrate predominantly the peri-vascular niche, leading to rejection of established tumors and cure in 29% of animals. The therapeutic radiation dose window is narrow, with effects seen between 2 and 15 Gy, peaking at 5 Gy. A single low-dose radiation decreases MSCs inherent immune suppressive properties in vitro as well as shapes their immune regulatory ability in vivo. Intra-tumorally grafted irMSCs stimulate the immune system and decrease immune suppression. Additionally, irMSCs enhance peri-tumoral reactive astrocytosis and display anti-angiogenic properties. Hence, the present study provides strong evidence for a therapeutic potential of low-dose irMSCs in cancer as well as giving new insight into MSC biology and applications.

Novel Method for High-Throughput Full-Length IGHV-D-J Sequencing of the Immune Repertoire from Bulk B-Cells with Single-Cell Resolution.

Efficient and accurate high-throughput DNA sequencing of the adaptive immune receptor repertoire (AIRR) is necessary to study immune diversity in healthy subjects and disease-related conditions. The high complexity and diversity of the AIRR coupled with the limited amount of starting material, which can compromise identification of the full biological diversity makes such sequencing particularly challenging. AIRR sequencing protocols often fail to fully capture the sampled AIRR diversity, especially for samples containing restricted numbers of B lymphocytes. Here, we describe a library preparation method for immunoglobulin sequencing that results in an exhaustive full-length repertoire where virtually every sampled B-cell is sequenced. This maximizes the likelihood of identifying and quantifying the entire IGHV-D-J repertoire of a sample, including the detection of rearrangements present in only one cell in the starting population. The methodology establishes the importance of circumventing genetic material dilution in the preamplification phases and incorporates the use of certain described concepts: (1) balancing the starting material amount and depth of sequencing, (2) avoiding IGHV gene-specific amplification, and (3) using Unique Molecular Identifier. Together, this methodology is highly efficient, in particular for detecting rare rearrangements in the sampled population and when only a limited amount of starting material is available.