Early Generated B-1-Derived B Cells Have the Capacity To Progress To Become Mantle Cell Lymphoma-like Neoplasia in Aged Mice.

In mice, fetal/neonatal B-1 cell development generates murine CD5+ B cells (B1a) with autoreactivity. We analyzed B1a cells at the neonatal stage in a VH11/D/JH knock-in mouse line (VH11t) that generates an autoreactive antiphosphatidylcholine BCR. Our study revealed that antiphosphatidylcholine B1a cells develop in liver, mature in spleen, and distribute in intestine/colon, mesenteric lymph node (mLN), and body cavity as the outcome of B-1 cell development before B-2 cell development. Throughout life, self-renewing B-1 B1a cells circulate through intestine, mesenteric vessel, and blood. The body cavity-deposited B1a cells also remigrate. In old age, some B1a cells proceed to monoclonal B cell lymphocytosis. When neonatal B-1 B1a cells express an antithymocyte/Thy-1 autoreactivity (ATA) BCR transgene in the C.B17 mouse background, ATA B cells increase in PBL and strongly develop lymphomas in aging mice that feature splenomegaly and mLN hyperplasia with heightened expression of CD11b, IL-10, and activated Stat3. At the adult stage, ATA B cells were normally present in the mantle zone area, including in intestine. Furthermore, frequent association with mLN hyperplasia suggests the influence by intestinal microenvironment on lymphoma development. When cyclin D1 was overexpressed by the Eµ-cyclin D1 transgene, ATA B cells progressed to further diffused lymphoma in aged mice, including in various lymph nodes with accumulation of IgMhiIgDloCD5+CD23-CD43+ cells, resembling aggressive human mantle cell lymphoma. Thus, our findings reveal that early generated B cells, as an outcome of B-1 cell development, can progress to become lymphocytosis, lymphoma, and mantle cell lymphoma-like neoplasia in aged mice.

Natural anti-intestinal goblet cell autoantibody production from marginal zone B cells.

Expression of a germline VH3609/D/JH2 IgH in mice results in the generation of B1 B cells with anti-thymocyte/Thy-1 glycoprotein autoreactivity by coexpression of Vk21-5/Jk2 L chain leading to production of serum IgM natural autoantibody. In these same mice, the marginal zone (MZ) B cell subset in spleen shows biased usage of a set of Ig L chains different from B1 B cells, with 30% having an identical Vk19-17/Jk1 L chain rearrangement. This VH3609/Vk19-17 IgM is reactive with intestinal goblet cell granules, binding to the intact large polymatrix form of mucin 2 glycoprotein secreted by goblet cells. Analysis of a µκ B cell AgR (BCR) transgenic (Tg) mouse with this anti-goblet cell/mucin2 autoreactive (AGcA) specificity demonstrates that immature B cells expressing the Tg BCR become MZ B cells in spleen by T cell-independent BCR signaling. These Tg B cells produce AGcA as the predominant serum IgM, but without enteropathy. Without the transgene, AGcA autoreactivity is low but detectable in the serum of BALB/c and C.B17 mice, and this autoantibody is specifically produced by the MZ B cell subset. Thus, our findings reveal that AGcA is a natural autoantibody associated with MZ B cells.

Pla2g2a promotes innate Th2-type immunity lymphocytes to increase B1a cells.

Newborns require early generation of effective innate immunity as a primary physiological mechanism for survival. The neonatal Lin28+Let7- developmental pathway allows increased generation of Th2-type cells and B1a (B-1 B) cells compared to adult cells and long-term maintenance of these initially generated innate cells. For initial B1a cell growth from the neonatal to adult stage, Th2-type IL-5 production from ILC2s and NKT2 cells is important to increase B1a cells. The Th17 increase is dependent on extracellular bacteria, and increased bacteria leads to lower Th2-type generation. Secreted group IIA-phospholipase A2 (sPLA2-IIA) from the Pla2g2a gene can bind to gram-positive bacteria and degrade bacterial membranes, controlling microbiota in the intestine. BALB/c mice are Pla2g2a+, and express high numbers of Th2-type cells and B1a cells. C57BL/6 mice are Pla2g2a-deficient and distinct from the SLAM family, and exhibit fewer NKT2 cells and fewer B1a cells from the neonatal to adult stage. We found that loss of Pla2g2a in the BALB/c background decreased IL-5 from Th2-type ILC2s and NKT2s but increased bacterial-reactive NKT17 cells and MAIT cells, and decreased the number of early-generated B1a cells and MZ B cells and the CD4/CD8 T cell ratio. Low IL-5 by decreased Th2-type cells in Pla2g2a loss led to low early-generated B1a cell growth from the neonatal to adult stage. In anti-thymocyte/Thy-1 autoreactive µκ transgenic (ATAµκ Tg) Pla2g2a+ BALB/c background C.B17 mice generated NKT2 cells that continuously control CD1d+ B1 B cells through old aging and lost CD1d in B1 B cells generating strong B1 ATA B cell leukemia/lymphoma. Pla2g2a-deficient ATAµκTg C57BL/6 mice suppressed the initial B1a cell increase, with low/negative spontaneous leukemia/lymphoma generation. These data confirmed that the presence of Pla2g2a to control bacteria is important to allow the neonatal to adult stage. Pla2g2a promotes innate Th2-type immunity lymphocytes to increase early generated B1a cells.

Positive selection of anti-thy-1 autoreactive B-1 cells and natural serum autoantibody production independent from bone marrow B cell development.

A natural serum autoantibody specific for the Thy-1 glycoprotein (anti-Thy-1 autoantibody [ATA]) is produced by B-1 cells that are positively selected by self-antigen. Here, using ATA micro kappa transgenic mice we show that cells with this B cell receptor are negatively selected during bone marrow (BM) development. In a Thy-1 null environment, BM ATA B cells progress to a normal follicular stage in spleen. However, in a self-antigen-positive environment, development is arrested at an immature stage in the spleen, concomitant with induction of CD5. Such cells are tolerant and short-lived, different from B-1. Nonetheless, ATA-positive selection was evident by self-antigen-dependent high serum ATA production, comprising approximately 90% of serum immunoglobulin M in ATA micro kappa mice. Splenectomy did not eliminate ATA production and transfer of tolerant splenic B cells did not induce it. These findings demonstrate that B-1 positive selection, resulting in the production of natural serum ATA, arises independently from the major pathway of BM B cell development and selection.

Crucial Role of Increased Arid3a at the Pre-B and Immature B Cell Stages for B1a Cell Generation.

The Lin28b+Let7- axis in fetal/neonatal development plays a role in promoting CD5+ B1a cell generation as a B-1 B cell developmental outcome. Here we identify the Let7 target, Arid3a, as a crucial molecular effector of the B-1 cell developmental program. Arid3a expression is increased at pro-B cell stage and markedly increased at pre-B and immature B cell stages in the fetal/neonatal liver B-1 development relative to that in the Lin28b-Let7+ adult bone marrow (BM) B-2 cell development. Analysis of B-lineage restricted Lin28b transgenic (Tg) mice, Arid3a knockout and Arid3a Tg mice, confirmed that increased Arid3a allows B cell generation without requiring surrogate light chain (SLC) associated pre-BCR stage, and prevents MHC class II cell expression at the pre-B and newly generated immature B cell stages, distinct from pre-BCR dependent B development with MHC class II in adult BM. Moreover, Arid3a plays a crucial role in supporting B1a cell generation. The increased Arid3a leads higher Myc and Bhlhe41, and lower Siglec-G and CD72 at the pre-B and immature B cell stages than normal adult BM, to allow BCR signaling induced B1a cell generation. Arid3a-deficiency selectively blocks the development of B1a cells, while having no detectable effect on CD5- B1b, MZ B, and FO B cell generation resembling B-2 development outcome. Conversely, enforced expression of Arid3a by transgene is sufficient to promote the development of B1a cells from adult BM. Under the environment change between birth to adult, altered BCR repertoire in increased B1a cells occurred generated from adult BM. However, crossed with B1a-restricted VH/D/J IgH knock-in mice allowed to confirm that SLC-unassociated B1a cell increase and CLL/lymphoma generation can occur in aged from Arid3a increased adult BM. These results confirmed that in fetal/neonatal normal mice, increased Arid3a at the pre-B cell and immature B cell stages is crucial for generating B1a cells together with the environment for self-ligand reactive BCR selection, B1a cell maintenance, and potential for development of CLL/Lymphoma in aged mice.

NLR Nod1 signaling promotes survival of BCR-engaged mature B cells through up-regulated Nod1 as a positive outcome.

Although B cell development requires expression of the B cell antigen receptor (BCR), it remains unclear whether engagement of self-antigen provides a positive impact for most B cells. Here, we show that BCR engagement by self-ligand during development in vivo results in up-regulation of the Nod-like receptor member Nod1, which recognizes the products of intestinal commensal bacteria. In anti-thymocyte/Thy-1 autoreactive BCR knock-in mice lacking self-Thy-1 ligand, immunoglobulin light chain editing occurred, generating B cells with up-regulated Nod1, including follicular and marginal zone B cells with natural autoreactivity. This BCR editing with increased Nod1 resulted in preferential survival. In normal adult mice, most mature B cells are enriched for Nod1 up-regulated cells, and signaling through Nod1 promotes competitive survival of mature B cells. These findings demonstrate a role for microbial products in promoting survival of mature B cells through up-regulated Nod1, providing a positive effect of BCR engagement on development of most B cells.

Early generated B1 B cells with restricted BCRs become chronic lymphocytic leukemia with continued c-Myc and low Bmf expression.

In mice, generation of autoreactive CD5+ B cells occurs as a consequence of BCR signaling induced by (self)-ligand exposure from fetal/neonatal B-1 B cell development. A fraction of these cells self-renew and persist as a minor B1 B cell subset throughout life. Here, we show that transfer of early generated B1 B cells from Eµ-TCL1 transgenic mice resulted in chronic lymphocytic leukemia (CLL) with a biased repertoire, including stereotyped BCRs. Thus, B1 B cells bearing restricted BCRs can become CLL during aging. Increased anti-thymocyte/Thy-1 autoreactive (ATA) BCR cells in the B1 B cell subset by transgenic expression yielded spontaneous ATA B-CLL/lymphoma incidence, enhanced by TCL1 transgenesis. In contrast, ATA B-CLL did not develop from other B cell subsets, even when the identical ATA BCR was expressed on a Thy-1 low/null background. Thus, both a specific BCR and B1 B cell context were important for CLL progression. Neonatal B1 B cells and their CLL progeny in aged mice continued to express moderately up-regulated c-Myc and down-regulated proapoptotic Bmf, unlike most mature B cells in the adult. Thus, there is a genetic predisposition inherent in B-1 development generating restricted BCRs and self-renewal capacity, with both features contributing to potential for progression to CLL.

B cells generated by B-1 development can progress to chronic lymphocytic leukemia.

B cells generated early during fetal/neonatal B-1 development in mice include autoreactive cells with detectable CD5 upregulation induced by B cell receptor (BCR) signaling (B1a cells). A fraction of B1a cells are maintained by self-renewal for life, with the potential risk of dysregulated growth and progression to chronic lymphocytic leukemia (CLL)/lymphoma during aging. In studies using the Eµ-hTCL1 transgenic mouse system, it became clear that this B1a subset has a higher potential than other B cell subsets for progression to CLL. We have generated several autoreactive germline BCR gene models to compare B cells generated under conditions of natural exposure to autoantigen. Analysis of the mice has been key in understanding the importance of the BCR and BCR signaling for generating different B cell subsets and for investigating the cellular origin of B-CLL.

Evidence of marginal-zone B cell-positive selection in spleen.

Antigen receptor-mediated signaling is critical for the development and survival of B cells. However, it has not been established whether B cell development requires a signal from self-ligand engagement at the immature stage, a process known as "positive selection." Here, using a monoclonal B cell receptor (BCR) mouse line, specific for the self-Thy-1/CD90 glycoprotein, we demonstrate that BCR crosslinking by low-dose self-antigen promotes survival of immature B cells in culture. In spleen, an increase in BCR signaling strength, induced by low-dose self-antigen, directed naive immature B cells to mature, not into the default follicular B cell fate, but instead into the marginal-zone B cell subset. These data indicate that positive selection can occur in developing B cells and that BCR signal strength is a key factor in deciding between two functionally distinct mature B cell compartments in the microenvironment of the spleen.