Targeting BMI-1 in B cells restores effective humoral immune responses and controls chronic viral infection.

Ineffective antibody-mediated responses are a key characteristic of chronic viral infection. However, our understanding of the intrinsic mechanisms that drive this dysregulation are unclear. Here, we identify that targeting the epigenetic modifier BMI-1 in mice improves humoral responses to chronic lymphocytic choriomeningitis virus. BMI-1 was upregulated by germinal center B cells in chronic viral infection, correlating with changes to the accessible chromatin landscape, compared to acute infection. B cell-intrinsic deletion of Bmi1 accelerated viral clearance, reduced splenomegaly and restored splenic architecture. Deletion of Bmi1 restored c-Myc expression in B cells, concomitant with improved quality of antibody and coupled with reduced antibody-secreting cell numbers. Specifically, BMI-1-deficiency induced antibody with increased neutralizing capacity and enhanced antibody-dependent effector function. Using a small molecule inhibitor to murine BMI-1, we could deplete antibody-secreting cells and prohibit detrimental immune complex formation in vivo. This study defines BMI-1 as a crucial immune modifier that controls antibody-mediated responses in chronic infection.

Predicting plasma cell retention and loss over a lifetime.

Plasma cells (PCs) rely on external survival cues for persistence, which limits the size of the PC pool. How, then, are new specificities incorporated into a saturated system? In this issue of Immunity, Simons and Karin put forward a mathematical framework to explain PC retention that makes testable predictions about steady-state lifespan structure, withstands tests based on accrual and displaceability, and accounts for lifespan stratification with specificity.

Intrinsically determined turnover underlies broad heterogeneity in plasma-cell lifespan.

Antibodies produced by antibody-secreting plasma cells (ASCs) underlie multiple forms of long-lasting immunity. Here we examined the mechanisms regulating ASC turnover and persistence using a genetic reporter to time-stamp ASCs. This approach revealed ASC lifespans as heterogeneous and falling on a continuum, with only a small fraction surviving for >60 days. ASC longevity past 60 days was independent of isotype but correlated with a phenotype that developed progressively and ultimately associated with an underlying "long-lived" ASC (LL ASC)-enriched transcriptional program. While some of the differences between LL ASCs and other ASCs appeared to be acquired with age, other features were shared with some younger ASCs, such as high CD138 and CD93. Turnover was unaffected by altered ASC production, arguing against competition for niches as a major driver of turnover. Thus, ASC turnover is set by intrinsic lifespan limits, with steady-state population dynamics governed by niche vacancy rather than displacement.

Interleukin-21, acting beyond the immunological synapse, independently controls T follicular helper and germinal center B cells.

Germinal centers (GCs), transient structures within B cell follicles and central to affinity maturation, require the coordinated behavior of T and B cells. IL-21, a pleiotropic T cell-derived cytokine, is key to GC biology through incompletely understood mechanisms. By genetically restricting production and receipt of IL-21 in vivo, we reveal how its independent actions on T and B cells combine to regulate the GC. IL-21 established the magnitude of the GC B cell response by promoting CD4+ T cell expansion and differentiation in a dose-dependent manner and with paracrine activity. Within GC, IL-21 specifically promoted B cell centroblast identity and, when bioavailability was high, plasma cell differentiation. Critically, these actions may occur irrespective of cognate T-B interactions, making IL-21 a general promoter of growth as distinct to a mediator of affinity-driven selection via synaptic delivery. This promiscuous activity of IL-21 explains the consequences of IL-21 deficiency on antibody-based immunity.

The BTG2-PRMT1 module limits pre-B cell expansion by regulating the CDK4-Cyclin-D3 complex.

Developing pre-B cells in the bone marrow alternate between proliferation and differentiation phases. We found that protein arginine methyl transferase 1 (PRMT1) and B cell translocation gene 2 (BTG2) are critical components of the pre-B cell differentiation program. The BTG2-PRMT1 module induced a cell-cycle arrest of pre-B cells that was accompanied by re-expression of Rag1 and Rag2 and the onset of immunoglobulin light chain gene rearrangements. We found that PRMT1 methylated cyclin-dependent kinase 4 (CDK4), thereby preventing the formation of a CDK4-Cyclin-D3 complex and cell cycle progression. Moreover, BTG2 in concert with PRMT1 efficiently blocked the proliferation of BCR-ABL1-transformed pre-B cells in vitro and in vivo. Our results identify a key molecular mechanism by which the BTG2-PRMT1 module regulates pre-B cell differentiation and inhibits pre-B cell leukemogenesis.

Transcriptional profiling of mouse B cell terminal differentiation defines a signature for antibody-secreting plasma cells.

When B cells encounter an antigen, they alter their physiological state and anatomical localization and initiate a differentiation process that ultimately produces antibody-secreting cells (ASCs). We have defined the transcriptomes of many mature B cell populations and stages of plasma cell differentiation in mice. We provide a molecular signature of ASCs that highlights the stark transcriptional divide between B cells and plasma cells and enables the demarcation of ASCs on the basis of location and maturity. Changes in gene expression correlated with cell-division history and the acquisition of permissive histone modifications, and they included many regulators that had not been previously implicated in B cell differentiation. These findings both highlight and expand the core program that guides B cell terminal differentiation and the production of antibodies.

Mcl-1 is essential for the survival of plasma cells.

The long-term survival of plasma cells is entirely dependent on signals derived from their environment. These extrinsic factors presumably induce and sustain the expression of antiapoptotic proteins of the Bcl-2 family. It is uncertain whether there is specificity among Bcl-2 family members in the survival of plasma cells and whether their expression is linked to specific extrinsic factors. We found here that deletion of the gene encoding the antiapoptotic protein Mcl-1 in plasma cells resulted in rapid depletion of this population in vivo. Furthermore, we found that the receptor BCMA was needed to establish high expression of Mcl-1 in bone marrow but not spleen plasma cells and that establishing this survival pathway preceded the component of plasma cell differentiation that depends on the transcriptional repressor Blimp-1. Our results identify a critical role for Mcl-1 in the maintenance of plasma cells.

The development and fate of follicular helper T cells defined by an IL-21 reporter mouse.

Germinal centers require CD4⁺ follicular helper T cells (TFH cells), whose hallmark is expression of the transcriptional repressor Bcl-6, the chemokine receptor CXCR5 and interleukin 21 (IL-21). To track the development and fate of TFH cells, we generated an IL-21 reporter mouse by introducing sequence encoding green fluorescent protein (GFP) into the Il21 locus; these mice had expression of IL-21­GFP in CD4⁺CXCR5⁺PD-1⁺ TFH cells. IL-21­GFP⁺ TFH cells were multifunctional helper cells that coexpressed several cytokines, including interferon-g (IFN-g), IL-2 and IL-4. TFH cells proliferated and gave rise to transferrable memory cells with plasticity, which differentiated after recall into conventional effector helper T cells and TFH cells. Thus, we demonstrated that TFH cells were not terminally differentiated but instead retained the flexibility to be recruited into other helper T cell subsets and nonlymphoid tissues.

IL-21 has a critical role in establishing germinal centers by amplifying early B cell proliferation.

The proliferation and differentiation of antigen-specific B cells, including the generation of germinal centers (GC), are prerequisites for long-lasting, antibody-mediated immune protection. Affinity for antigen determines B cell recruitment, proliferation, differentiation, and competitiveness in the response, largely through determining access to T cell help. However, how T cell-derived signals contribute to these outcomes is incompletely understood. Here, we report how the signature cytokine of follicular helper T cells, IL-21, acts as a key regulator of the initial B cell response by accelerating cell cycle progression and the rate of cycle entry, increasing their contribution to the ensuing GC. This effect occurs over a wide range of initial B cell receptor affinities and correlates with elevated AKT and S6 phosphorylation. Moreover, the resultant increased proliferation can explain the IL-21-mediated promotion of plasma cell differentiation. Collectively, our data establish that IL-21 acts from the outset of a T cell-dependent immune response to increase cell cycle progression and fuel cyclic re-entry of B cells, thereby regulating the initial GC size and early plasma cell output.

How intrinsic and extrinsic regulators of plasma cell survival might intersect for durable humoral immunity.

Plasma cells (PC) are key to protective immunity because they secrete antibodies. Surviving for periods ranging from days to decades in mammals, PC possess varying survival times that cannot be entirely stochastic or extrinsically set, as presumed half-lives vary with antigenic specificity. Here, we review the signals that impart survival potential to PC. These include signals provided during formation, and signals experienced once generated and embedded in the so-called long-lived niche. These signals all feed into survival by maintaining PC expression of MCL1, potentially synergistically with influences of other BCL2 family members. Herein, we propose that each formed PC has a capacity to respond to extrinsic cues that sets an upper maximum to its lifespan, but survival is also affected by variable availability of signals provided in BM survival niches. PC survival thus becomes a function of immunogen characteristics and niche anatomy, determined by the weighted survival benefit ascribed to each involved factor. Most factors, such as supporting cell types and secreted proteins, are predicted to influence survival times varying temporally by orders of magnitude, rather than absolute PC abundances measured at a single time, which may account for the variation in PC lifespan evident in the literature.

Germinal center B and follicular helper T cells: siblings, cousins or just good friends?

Humoral immunity requires interaction between specialized populations of B cells and CD4(+) T cells, called follicular helper T cells (T(FH) cells), in the germinal center (GC) to produce memory B cells and long-lived plasma cells. Molecular crosstalk between GC B cells and T(FH) cells influences the survival, proliferation and differentiation of each cell type. This pairing of GC B cells and T(FH) cells also occurs at the transcriptional level as the Bcl-6­IRF4­Blimp-1 axis, which is crucial for B cell differentiation, is also essential for the T(FH) cell identity. Less is known about the memory B cells that arise from the GC pool, as they seem to be distinctly 'programmed' on the basis of their antigen receptor affinity to enter the long-lived memory pool.

CD137L and CD4 T cells limit BCL6-expressing pre-germinal center B cell expansion and BCL6-driven B cell malignancy.

Aberrant expression of the proto-oncogene BCL6 is a driver of tumorigenesis in diffuse large B cell lymphoma (DLBCL). Mice overexpressing BCL6 from the B cell-specific immunoglobulin heavy chain µ intron promoter (Iµ-Bcl6Tg/+ ) develop B cell lymphomas with features typical of human DLBCL. While the development of B cell lymphoma in these mice is tightly controlled by T cells, the mechanisms of this immune surveillance are poorly understood. Here we show that CD4 T cells contribute to the control of lymphoproliferative disease in lymphoma-prone Iµ-Bcl6Tg/+ mice. We reveal that this CD4 T cell immuno-surveillance requires signaling by the co-stimulatory molecule CD137 ligand (CD137L; also known as 4-1BBL), which may promote the transition of pre-malignant B cells with an activated phenotype into the germinal center stage via reverse signaling, preventing their hazardous accumulation. Thus, CD137L-mediated CD4 T cell immuno-surveillance adds another layer of protection against B cell malignancy to that provided by CD8 T cell cytotoxicity.

Feeling a little SYK after mixing BAFF with BCR.

In this issue of Immunity, Schweighoffer et al. (2013) report that full BAFF responsiveness in B cells requires the tyrosine kinase Syk and that BAFF may co-opt components of the B cell receptor in transmitting its survival signal.

Display of Native Antigen on cDC1 That Have Spatial Access to Both T and B Cells Underlies Efficient Humoral Vaccination.

Follicular dendritic cells and macrophages have been strongly implicated in presentation of native Ag to B cells. This property has also occasionally been attributed to conventional dendritic cells (cDC) but is generally masked by their essential role in T cell priming. cDC can be divided into two main subsets, cDC1 and cDC2, with recent evidence suggesting that cDC2 are primarily responsible for initiating B cell and T follicular helper responses. This conclusion is, however, at odds with evidence that targeting Ag to Clec9A (DNGR1), expressed by cDC1, induces strong humoral responses. In this study, we reveal that murine cDC1 interact extensively with B cells at the border of B cell follicles and, when Ag is targeted to Clec9A, can display native Ag for B cell activation. This leads to efficient induction of humoral immunity. Our findings indicate that surface display of native Ag on cDC with access to both T and B cells is key to efficient humoral vaccination.

Hhex regulates murine lymphoid progenitor survival independently of Stat5 and Cdkn2a.

The transcription factor Hhex (hematopoietically expressed homeobox gene) is critical for development of multiple lymphoid lineages beyond the common lymphoid progenitor. In addition, Hhex regulates hematopoietic stem cell (HSC) self-renewal, emergency hematopoiesis, and acute myeloid leukemia initiation and maintenance. Hhex mediates its effects on HSCs and acute myeloid leukemia stem cells via repression of the Cdkn2a tumor suppressor locus. However, we report here that loss of Cdkn2a does not rescue the failure of lymphoid development caused by loss of Hhex. As loss of Hhex causes apoptosis of lymphoid progenitors associated with impaired Bcl2 expression and defective Stat5b signaling, we tested the effects of rescuing these pathways using transgenic mice. Expression of the anti-apoptotic factor Bcl2, but not activated Stat5, rescued the development of T-, B-, and NK-cell lineages in the absence of Hhex. These results indicate that Bcl2 expression, but not Stat5b signaling or loss of Cdkn2a, can overcome the lymphoid deficiencies caused by the absence of Hhex, suggesting that the primary role of this transcription factor is to promote survival of lymphoid progenitors during early lymphoid development.

Self-reactive and polyreactive B cells are generated and selected in the germinal center during γ-herpesvirus infection.

Immune responses against certain viruses are accompanied by auto-antibody production although the origin of these infection-associated auto-antibodies is unclear. Here, we report that murine γ-herpesvirus 68 (MHV68)-induced auto-antibodies are derived from polyreactive B cells in the germinal center (GC) through the activity of short-lived plasmablasts. The analysis of recombinant antibodies from MHV68-infected mice revealed that about 40% of IgG+ GC B cells were self-reactive, with about half of them being polyreactive. On the other hand, virion-reactive clones accounted for only a minor proportion of IgG+ GC B cells, half of which also reacted with self-antigens. The self-reactivity of most polyreactive clones was dependent on somatic hypermutation (SHM), but this was dispensable for the reactivity of virus mono-specific clones. Furthermore, both virus-mono-specific and polyreactive clones were selected to differentiate to B220lo CD138+ plasma cells (PCs). However, the representation of GC-derived polyreactive clones was reduced and that of virus-mono-specific clones was markedly increased in terminally differentiated PCs as compared to transient plasmablasts. Collectively, our findings demonstrate that, during acute MHV68 infection, self-reactive B cells are generated through SHM and selected for further differentiation to short-lived plasmablasts but not terminally differentiated PCs.

Dynein light chain regulates adaptive and innate B cell development by distinctive genetic mechanisms.

Mechanistic differences in the development and function of adaptive, high-affinity antibody-producing B-2 cells and innate-like, "natural" antibody-producing B-1a cells remain poorly understood. Here we show that the multi-functional dynein light chain (DYNLL1/LC8) plays important roles in the establishment of B-1a cells in the peritoneal cavity and in the ongoing development of B-2 lymphoid cells in the bone marrow of mice. Epistasis analyses indicate that Dynll1 regulates B-1a and early B-2 cell development in a single, linear pathway with its direct transcriptional activator ASCIZ (ATMIN/ZNF822), and that the two genes also have complementary functions during late B-2 cell development. The B-2 cell defects caused by loss of DYNLL1 were associated with lower levels of the anti-apoptotic protein BCL-2, and could be supressed by deletion of pro-apoptotic BIM which is negatively regulated by both DYNLL1 and BCL-2. Defects in B cell development caused by loss of DYNLL1 could also be partially suppressed by a pre-arranged SWHEL Igm-B cell receptor transgene. In contrast to the rescue of B-2 cell numbers, the B-1a cell deficiency in Dynll1-deleted mice could not be suppressed by the loss of Bim, and was further compounded by the SWHEL transgene. Conversely, oncogenic MYC expression, which is synthetic lethal with Dynll1 deletion in B-2 cells, did not further reduce B-1a cell numbers in Dynll1-defcient mice. Finally, we found that the ASCIZ-DYNLL1 axis was also required for the early-juvenile development of aggressive MYC-driven and p53-deficient B cell lymphomas. These results identify ASCIZ and DYNLL1 as the core of a transcriptional circuit that differentially regulates the development of the B-1a and B-2 B lymphoid cell lineages and plays a critical role in lymphomagenesis.

BAFF, IL-4 and IL-21 separably program germinal center-like phenotype acquisition, BCL6 expression, proliferation and survival of CD40L-activated B cells in vitro.

A B cell culture system using BAFF, IL-4 and IL-21 was recently developed that generates B cells with phenotypic and functional characteristics of in vivo-generated germinal center (GC) B cells. Here, we observe discrete influences of each exogenous signal on the expansion and differentiation of a CD40L-activated B cell pool. IL-4 was expressly necessary, but neither BAFF nor IL-21 was required for B cell acquisition of the GC B cell phenotypes of peanut agglutinin binding and loss of CD38 and IgD expression. Both IL-4 and IL-21 enhanced cell cycle entry upon initial activation dose-dependently, and did so additively. Importantly, while both cytokines acted in concert to increase overall BCL6 expression amounts, IL-21 exposure uniquely caused a small proportion of cells to attain a higher level of BCL6 expression, reminiscent of in vivo GC B cells. In contrast, BAFF supported survival of a fraction of memory-like B cells in extended cultures after removal of surrogate T cell-help signals. Thus, by separably programming proliferation, survival and GC phenotype acquisition, IL-4, BAFF and IL-21 drive distinct components of activated B cell fate.

Innate Allorecognition Results in Rapid Accumulation of Monocyte-Derived Dendritic Cells.

Although the mechanisms governing the innate recognition of pathogen-associated molecular patterns have been well defined, how allogeneic cellular stimuli evoke innate responses remains less so. In this article, we report that upon i.v. transfer (to avoid major iatrogenic interference), allogeneic but not syngeneic leukocytes could induce a rapid (after 1 d) accumulation of host monocyte-derived dendritic cells (moDCs) without any increase in conventional DCs. This occurred in various donor-host strain combinations, did not require MHC mismatch, and could be induced by various donor cell types including B cells, T cells, or NK cells. Using RAG(-/-)γc(-/-) and scid γc(-/-)mice with different MHC, we found that the presence of either donor or host lymphoid cells was required. Alloinduced moDC accumulation was significantly reduced when splenocytes from mice deficient in NK cells by genetic ablation were used as donors. A major component of this moDC accumulation appears to be recruitment. Our findings provide new insights into how the innate and adaptive immune system may interact during allogeneic encounters and thus transplant rejection.