Masayuki Kuraoka

Metabolic Reprogramming Is Required for Antibody Production That Is Suppressed in Anergic but Exaggerated in Chronically BAFF-Exposed B Cells

B cell activation leads to proliferation and Ab production that can protect from pathogens or promote autoimmunity. Regulation of cell metabolism is essential to support the demands of lymphocyte growth and effector function and may regulate tolerance. In this study, we tested the regulation and role of glucose uptake and metabolism in the proliferation and Ab production of control, anergic, and autoimmune-prone B cells. Control B cells had a balanced increase in lactate production and oxygen consumption following activation, with proportionally increased glucose transporter Glut1 expression and mitochondrial mass upon either LPS or BCR stimulation. This contrasted with metabolic reprogramming of T cells, which had lower glycolytic flux when resting but disproportionately increased this pathway upon activation. Importantly, tolerance greatly affected B cell metabolic reprogramming. Anergic B cells remained metabolically quiescent, with only a modest increase in glycolysis and oxygen consumption with LPS stimulation. B cells chronically stimulated with elevated BAFF, however, rapidly increased glycolysis and Ab production upon stimulation. Induction of glycolysis was critical for Ab production, as glycolytic inhibition with the pyruvate dehydrogenase kinase inhibitor dichloroacetate sharply suppressed B cell proliferation and Ab secretion in vitro and in vivo. Furthermore, B cell–specific deletion of Glut1 led to reduced B cell numbers and impaired Ab production in vivo. Together, these data show that activated B cells require Glut1-dependent metabolic reprogramming to support proliferation and Ab production that is distinct from T cells and that this glycolytic reprogramming is regulated in tolerance.

IL-1R Type I-Dependent Hemopoietic Stem Cell Proliferation Is Necessary for Inflammatory Granulopoiesis and Reactive Neutrophilia

Infections and inflammation trigger neutrophilias that are supported by a hematopoietic program of accelerated granulopoiesis known as emergency granulopoiesis. The intrinsic factors that drive reactive neutrophilias and emergency granulopoiesis have been inferred but not demonstrated. Here, we show that alum cannot elicit reactive neutrophilias in IL-1R type I (IL-1RI)−/− mice, whereas other inflammatory responses, including eosinophilia and Ab production, remain intact. Analysis of this specific impairment revealed an unanticipated role for IL-1RI in supporting increased proliferation by granulocyte/macrophage progenitors and, surprisingly, multipotent progenitors and hematopoietic stem cells (HSC). Indeed, HSC and multipotent progenitor proliferative responses were most suppressed in IL-1RI−/− mice, suggesting a critical role for their proliferation in inflammatory granulopoiesis. Whereas IL-1 drives increased HSC proliferation directly in vitro, IL-1RI expression by radiation-resistant host cells was both necessary and sufficient for alum-induced HSC, multipotent progenitor, and granulocyte/macrophage progenitor proliferation and reactive neutrophilias in radiation chimeric mice. Thus, IL-1 plays a necessary, but indirect, role in the support of alum-induced neutrophilias by expanding both pluripotent and myeloid progenitor compartments to accelerate granulopoiesis.

Activation-induced cytidine deaminase mediates central tolerance in B cells

The Aicda gene product, activation-induced cytidine deaminase (AID), initiates somatic hypermutation, class-switch recombination, and gene conversion of Ig genes by the deamination of deoxycytidine, followed by error-prone mismatch- or base-excision DNA repair. These processes are crucial for the generation of genetically diverse, high affinity antibody and robust humoral immunity, but exact significant genetic damage and promote cell death. In mice, physiologically significant AID expression was thought to be restricted to antigen-activated, mature B cells in germinal centers. We now demonstrate that low levels of AID in bone marrow immature and transitional B cells suppress the development of autoreactivity. Aicda−/− mice exhibit significantly increased serum autoantibody and reduced capacity to purge autoreactive immature and transitional B cells. In vitro, AID deficient immature/transitional B cells are significantly more resistant to anti-IgM–induced apoptosis than their normal counterparts. Thus, early AID expression plays a fundamental and unanticipated role in purging self-reactive immature and transitional B cells during their maturation in the bone marrow.

Activation-Induced Cytidine Deaminase Expression and Activity in the Absence of Germinal Centers: Insights into Hyper-IgM Syndrome

Somatic hypermutation normally occurs as a consequence of the expression of activation-induced cytidine deaminase (AID) by Ag-activated, mature B cells during T cell-dependent germinal center responses. Nonetheless, despite their inability to express CD154 and initiate GC responses, patients with type 1 hyper-IgM syndrome (HIGM1) support populations of IgM+IgD+CD27+ B cells that express mutated Ig genes. The origin of these mutated B cells is unknown; the IgM+IgD+CD27+ cells do not express AID and appear to acquire mutations independent of stringent selection by Ag. Here, we demonstrate that immature/transitional 1 B cells from the bone marrow of CD154-deficient mice express AID and acquire Ig mutations that lack the hallmarks of antigenic selection via BCR signaling. Comparable levels of AID expression was found in developmentally immature B cells recovered from murine fetal liver and from human immature/transitional 1 B cells recovered from umbilical cord blood. AID expression in human fetal liver was also robust, approaching that of human tonsil tissue and the human germinal center B cell line, Ramos. These observations led us to conclude that AID expression in developing human B cells is the origin of the mutated IgM+IgD+CD27+ B cells present in HIGM1 patients, and we propose that both mice and humans share a latent, AID-dependent pathway for the preimmune diversification of B lymphocytes that is more prominent in chicken, sheep, and rabbits.

Natural IgM Is Produced by CD5− Plasma Cells That Occupy a Distinct Survival Niche in Bone Marrow

Natural IgM is constitutively present in the serum, where it aids in the early control of viral and bacterial expansions. Natural IgM also plays a significant role in the prevention of autoimmune disease by promoting the clearance of cellular debris. Nevertheless, the origins of natural IgM have not been precisely defined. Previous studies focused on the role of CD5+ B1 cells in the production of natural IgM, but we show in this article that a discrete population of CD5− IgM plasmablasts and plasma cells in the bone marrow (BM) produces the majority of serum IgM in resting mice. These Ab-secreting cells (ASC) originate from peritoneal cavity–resident cells, because transfer of peritoneal cells completely restores serum IgM and the specific compartment of BM ASC in Rag1-deficient mice. We show that BM natural IgM ASC arise from a fetal-lineage progenitor that is neither B1a nor B1b, and that this IgM ASC compartment contains a substantial fraction of long-lived plasma cells that do not occupy the IgG plasma cell survival niche in the BM; instead, they are supported by IL-5. In summary, we identified the primary source of natural IgM and showed that these ASC are maintained long-term in a unique survival niche within the BM.

AID expression during B-cell development: searching for answers

Expression of activation-induced cytidine deaminase (AID) by germinal center (GC) B cells drives the processes of immunoglobulin (Ig) somatic hypermutation (SHM) and class switch recombination (CSR) necessary for the generation of high affinity IgG serum antibody and the memory B-cell compartment. Increasing evidence indicates that AID is also expressed at low levels in developing B cells but to date, this early, developmentally regulated AID expression has no known function. Does the timing and extent of AID expression in developmentally immature, non-GC B cells provide clues to reveal its physiologic role?

The human fetal lymphocyte lineage: identification by CD27 and LIN28B expression in B cell progenitors.

CD27, a member of the TNFR superfamily, is used to identify human memory B cells. Nonetheless, CD27+ B cells are present in patients with HIGM1 syndrome who are unable to generate GCs or memory B cells. CD27+IgD+ fetal B cells are present in umbilical cord blood, and CD27 may also be a marker of the human B1‐like B cells. To define the origin of naïve CD27+IgD+ human B cells, we studied B cell development in both fetal and adult tissues. In human FL, most CD19+ cells coexpressed CD10, a marker of human developing B cells. Some CD19+CD10+ B cells expressed CD27, and these fetal CD27+ cells were present in the pro‐B, pre‐B, and immature/transitional B cell compartments. Lower frequencies of phenotypically identical cells were also identified in adult BM. CD27+ pro‐B, pre‐B, and immature/transitional B cells expressed recombination activating gene‐1, terminal deoxynucleotidyl transferase and Vpre‐B mRNA comparably to their CD27− counterparts. CD27+ and CD27− developing B cells showed similar Ig heavy chain gene usage with low levels of mutations, suggesting that CD27+ developing B cells are distinct from mutated memory B cells. Despite these similarities, CD27+ developing B cells differed from CD27− developing B cells by their increased expression of LIN28B, a transcription factor associated with the fetal lymphoid lineages of mice. Furthermore, CD27+ pro‐B cells efficiently generated IgM+IgD+ immature/transitional B cells in vitro. Our observations suggest that CD27 expression during B cell development identifies a physiologic state or lineage for human B cell development distinct from the memory B cell compartment.

TSC1 Promotes B Cell Maturation but Is Dispensable for Germinal Center Formation.

Accumulating evidence indicates that the tuberous sclerosis complex 1 (TSC1), a tumor suppressor that acts by inhibiting mTOR signaling, plays an important role in the immune system. We report here that TSC1 differentially regulates mTOR complex 1 (mTORC1) and mTORC2/Akt signaling in B cells. TSC1 deficiency results in the accumulation of transitional-1 (T1) B cells and progressive losses of B cells as they mature beyond the T1 stage. Moreover, TSC1KO mice exhibit a mild defect in the serum antibody responses or rate of Ig class-switch recombination after immunization with a T-cell-dependent antigen. In contrast to a previous report, we demonstrate that both constitutive Peyer’s patch germinal centers (GCs) and immunization-induced splenic GCs are unimpaired in TSC1-deficient (TSC1KO) mice and that the ratio of GC B cells to total B cells is comparable in WT and TSC1KO mice. Together, our data demonstrate that TSC1 plays important roles for B cell development, but it is dispensable for GC formation and serum antibody responses.

Memory B Cells that Cross-React with Group 1 and Group 2 Influenza A Viruses Are Abundant in Adult Human Repertoires

Summary Human B cell antigen‐receptor (BCR) repertoires reflect repeated exposures to evolving influenza viruses; new exposures update the previously generated B cell memory (Bmem) population. Despite structural similarity of hemagglutinins (HAs) from the two groups of influenza A viruses, cross‐reacting antibodies (Abs) are uncommon. We analyzed Bmem compartments in three unrelated, adult donors and found frequent cross‐group BCRs, both HA‐head directed and non‐head directed. Members of a clonal lineage from one donor had a BCR structure similar to that of a previously described Ab, encoded by different gene segments. Comparison showed that both Abs contacted the HA receptor‐binding site through long heavy‐chain third complementarity determining regions. Affinities of the clonal‐lineage BCRs for historical influenza‐virus HAs from both group 1 and group 2 viruses suggested that serial responses to seasonal influenza exposures had elicited the lineage and driven affinity maturation. We propose that appropriate immunization regimens might elicit a comparably broad response. Graphical Abstract Figure. No Caption available. HighlightsHuman Bmem cells were found with BCRs cross‐reactive for influenza A groups 1 and 2Cross‐group Bmem cells were abundant, unlike cross‐group serum antibodiesStructures of HA receptor‐site directed, cross‐group antibodies showed key contactsCross‐group lineage antibodies were similar to a genetically unrelated antibody &NA; Hemagglutinins (HAs) from the two influenza A subtype groups have similar structures, but cross‐reacting serum antibodies are rare. McCarthy et al. nonetheless found, in three donors, abundant cross‐group B cell receptors (BCRs), many with epitopes on the HA head. Members of one clonal lineage had a BCR structure similar to that of a previously described, genetically unrelated antibody. Serial responses to seasonal influenza appear to have elicited the lineage and driven affinity maturation. Appropriate immunization regimens might elicit comparable responses.

Enhanced Antibody Responses to an HIV-1 Membrane-Proximal External Region Antigen in Mice Reconstituted with Cultured Lymphocytes

We have shown that the protective HIV-1 Ab, 2F5, avidly reacts with a conserved mammalian self-Ag, kynureninase, and that the development of B cells specific for the 2F5 epitope is constrained by immunological tolerance. These observations suggest that the capacity to mount Ab responses to the 2F5 epitope is mitigated by tolerance, but such capacity may be latent in the pretolerance and/or anergic B cell pools. In this study, we use B cell tetramer reagents to track the frequencies of B cells that recognize the HIV-1 2F5 epitope (SP62): in C57BL/6 mice, SP62-binding transitional B cells are readily identified in bone marrow but are lost during subsequent development. Unsurprisingly then, immunization with SP62 immunogen does not elicit significant humoral responses in normal C57BL/6 mice. Reconstitution of Rag1null mice with normal congenic B cells that have matured in vitro restores the capacity to mount significant serum Ab and germinal center responses to this HIV-1 epitope. These B cell cultures are permissive for the development of autoreactive B cells and support the development of SP62-specific B cell compartments normally lost in 2F5 Ab knockin mice. The recovery of humoral responses to the 2F5/SP62 epitope of HIV-1 by reconstitution with B cells containing forbidden, autoreactive clones provides direct evidence that normal C57BL/6 mice latently possess the capacity to generate humoral responses to a conserved, neutralizing HIV-1 epitope.