BCL6 controls contact-dependent help delivery during follicular T-B cell interactions.

BCL6 is required for development of follicular T helper (Tfh) cells to support germinal center (GC) formation. However, it is not clear what unique functions programmed by BCL6 can explain its absolute essentiality in T cells for GC formation. We found that ablation of one Bcl6 allele did not appreciably alter early T cell activation and follicular localization but inhibited GC formation and Tfh cell maintenance. BCL6 impinged on Tfh calcium signaling and also controlled Tfh entanglement with and CD40L delivery to B cells. Amounts of BCL6 protein and nominal frequencies of Tfh cells markedly changed within hours after strengths of T-B cell interactions were altered in vivo, while CD40L overexpression rectified both defective GC formation and Tfh cell maintenance because of the BCL6 haploinsufficiency. Our results reveal BCL6 functions in Tfh cells that are essential for GC formation and suggest that BCL6 helps maintain Tfh cell phenotypes in a T cell non-autonomous manner.

Multifaceted Effects of Antigen Valency on B Cell Response Composition and Differentiation In Vivo.

How antigen valency affects B cells in vivo during immune responses is not well understood. Here, using HIV immunogens with defined valencies ranging from 1 to 60, we investigated the role of antigen valency during different phases of B cell responses in vivo. Highly multimerized immunogens preferentially rapidly activated cognate B cells, with little affinity discrimination. This led to strong early induction of the transcription factors IRF4 (interferon regulatory factor 4) and Bcl6, driving both early extrafollicular plasma cell and germinal center responses, in a CD4+ T-cell-dependent manner, involving B cells with a broad range of affinities. Low-valency antigens induced smaller effector B cell responses, with preferential recruitment of high-affinity B cells. Thus, antigen valency has multifaceted effects on B cell responses and can dictate affinity thresholds and competitive landscapes for B cells in vivo, with implications for vaccine design.

A Thpok-Directed Transcriptional Circuitry Promotes Bcl6 and Maf Expression to Orchestrate T Follicular Helper Differentiation.

The generation of high-affinity neutralizing antibodies, the objective of most vaccine strategies, occurs in B cells within germinal centers (GCs) and requires rate-limiting "help" from follicular helper CD4+ T (Tfh) cells. Although Tfh differentiation is an attribute of MHC II-restricted CD4+ T cells, the transcription factors driving Tfh differentiation, notably Bcl6, are not restricted to CD4+ T cells. Here, we identified a requirement for the CD4+-specific transcription factor Thpok during Tfh cell differentiation, GC formation, and antibody maturation. Thpok promoted Bcl6 expression and bound to a Thpok-responsive region in the first intron of Bcl6. Thpok also promoted the expression of Bcl6-independent genes, including the transcription factor Maf, which cooperated with Bcl6 to mediate the effect of Thpok on Tfh cell differentiation. Our findings identify a transcriptional program that links the CD4+ lineage with Tfh differentiation, a limiting factor for efficient B cell responses, and suggest avenues to optimize vaccine generation.

Unique Immune Cell Coactivators Specify Locus Control Region Function and Cell Stage.

Locus control region (LCR) functions define cellular identity and have critical roles in diseases such as cancer, although the hierarchy of structural components and associated factors that drive functionality are incompletely understood. Here we show that OCA-B, a B cell-specific coactivator essential for germinal center (GC) formation, forms a ternary complex with the lymphoid-enriched OCT2 and GC-specific MEF2B transcription factors and that this complex occupies and activates an LCR that regulates the BCL6 proto-oncogene and is uniquely required by normal and malignant GC B cells. Mechanistically, through OCA-B-MED1 interactions, this complex is required for Mediator association with the BCL6 promoter. Densely tiled CRISPRi screening indicates that only LCR segments heavily bound by this ternary complex are essential for its function. Our results demonstrate how an intimately linked complex of lineage- and stage-specific factors converges on specific and highly essential enhancer elements to drive the function of a cell-type-defining LCR.

A p85α-osteopontin axis couples the receptor ICOS to sustained Bcl-6 expression by follicular helper and regulatory T cells.

Follicular helper T cells (TFH cells) and follicular regulatory T cells (TFR cells) regulate the quantity and quality of humoral immunity. Although both cell types express the costimulatory receptor ICOS and require the transcription factor Bcl-6 for their differentiation, the ICOS-dependent pathways that coordinate their responses are not well understood. Here we report that activation of ICOS in CD4(+) T cells promoted interaction of the p85α regulatory subunit of the signaling kinase PI(3)K and intracellular osteopontin (OPN-i), followed by translocation of OPN-i to the nucleus, its interaction with Bcl-6 and protection of Bcl-6 from ubiquitin-dependent proteasome degradation. Post-translational protection of Bcl-6 by OPN-i was essential for sustained responses of TFH cells and TFR cells and regulation of the germinal center B cell response to antigen. Thus, the p85α-OPN-i axis represents a molecular bridge that couples activation of ICOS to Bcl-6-dependent functional differentiation of TFH cells and TFR cells; this suggests new therapeutic avenues to manipulate the responses of these cells.

IL-7 coordinates proliferation, differentiation and Tcra recombination during thymocyte ß-selection.

Signaling via the pre-T cell antigen receptor (pre-TCR) and the receptor Notch1 induces transient self-renewal (ß-selection) of TCRß(+) CD4(-)CD8(-) double-negative stage 3 (DN3) and DN4 progenitor cells that differentiate into CD4(+)CD8(+) double-positive (DP) thymocytes, which then rearrange the locus encoding the TCR α-chain (Tcra). Interleukin 7 (IL-7) promotes the survival of TCRß(-) DN thymocytes by inducing expression of the pro-survival molecule Bcl-2, but the functions of IL-7 during ß-selection have remained unclear. Here we found that IL-7 signaled TCRß(+) DN3 and DN4 thymocytes to upregulate genes encoding molecules involved in cell growth and repressed the gene encoding the transcriptional repressor Bcl-6. Accordingly, IL-7-deficient DN4 cells lacked trophic receptors and did not proliferate but rearranged Tcra prematurely and differentiated rapidly. Deletion of Bcl6 partially restored the self-renewal of DN4 cells in the absence of IL-7, but overexpression of BCL2 did not. Thus, IL-7 critically acts cooperatively with signaling via the pre-TCR and Notch1 to coordinate proliferation, differentiation and Tcra recombination during ß-selection.

Lineage-specific functions of Bcl-6 in immunity and inflammation are mediated by distinct biochemical mechanisms.

The transcription factor Bcl-6 orchestrates germinal center (GC) reactions through its actions in B cells and T cells and regulates inflammatory signaling in macrophages. Here we found that genetic replacement with mutated Bcl6 encoding Bcl-6 that cannot bind corepressors to its BTB domain resulted in disruption of the formation of GCs and affinity maturation of immunoglobulins due to a defect in the proliferation and survival of B cells. In contrast, loss of function of the BTB domain had no effect on the differentiation and function of follicular helper T cells or that of other helper T cell subsets. Bcl6-null mice had a lethal inflammatory phenotype, whereas mice with a mutant BTB domain had normal healthy lives with no inflammation. The repression of inflammatory responses by Bcl-6 in macrophages was accordingly independent of the repressor function of the BTB domain. Bcl-6 thus mediates its actions through lineage-specific biochemical functions.

Looping around Bcl6 in Germinal Center to Sharpen B Cell Immunity.

We are beginning to understand the function of 3D genome architecture in the immune system. In this issue, Bunting et al. (2016) reported massive multi-layer genome reorganization from naive B cells to germinal center B cells, centered on a locus control region of Bcl6.

The Integrin LFA-1 Controls T Follicular Helper Cell Generation and Maintenance.

T follicular helper (Tfh) cells are a CD4+ T cell subset critical for long-lived humoral immunity. We hypothesized that integrins play a decisive role in Tfh cell biology. Here we show that Tfh cells expressed a highly active form of leukocyte function-associated antigen-1 (LFA-1) that was required for their survival within the germinal center niche. In addition, LFA-1 promoted expression of Bcl-6, a transcriptional repressor critical for Tfh cell differentiation, and inhibition of LFA-1 abolished Tfh cell generation and prevented protective humoral immunity to intestinal helminth infection. Furthermore, we demonstrated that expression of Talin-1, an adaptor protein that regulates LFA-1 affinity, dictated Tfh versus Th2 effector cell differentiation. Collectively, our results define unique functions for LFA-1 in the Tfh cell effector program and suggest that integrin activity is important in lineage decision-making events in the adaptive immune system.

Multi-tiered Reorganization of the Genome during B Cell Affinity Maturation Anchored by a Germinal Center-Specific Locus Control Region.

During the humoral immune response, B cells undergo a dramatic change in phenotype to enable antibody affinity maturation in germinal centers (GCs). Using genome-wide chromosomal conformation capture (Hi-C), we found that GC B cells undergo massive reorganization of the genomic architecture that encodes the GC B cell transcriptome. Coordinate expression of genes that specify the GC B cell phenotype-most prominently BCL6-was achieved through a multilayered chromatin reorganization process involving (1) increased promoter connectivity, (2) formation of enhancer networks, (3) 5' to 3' gene looping, and (4) merging of gene neighborhoods that share active epigenetic marks. BCL6 was an anchor point for the formation of GC-specific gene and enhancer loops on chromosome 3. Deletion of a GC-specific, highly interactive locus control region upstream of Bcl6 abrogated GC formation in mice. Thus, large-scale and multi-tiered genomic three-dimensional reorganization is required for coordinate expression of phenotype-driving gene sets that determine the unique characteristics of GC B cells.

Molecular mechanisms that control the expression and activity of Bcl-6 in TH1 cells to regulate flexibility with a TFH-like gene profile.

The transcription factors T-bet and Bcl-6 are required for the establishment of a T helper type 1 cell (T(H)1 cell) and follicular helper T cell (T(FH) cell) gene-expression profile, respectively. Here we found that high concentrations of interleukin 2 (IL-2) inhibited Bcl-6 expression in polarized T(H)1 cells. Mechanistically, the low concentrations of Bcl-6 normally found in effector T(H)1 cells did not repress its target genes because a T-bet-Bcl-6 complex masked the Bcl-6 DNA-binding domain. T(H)1 cells increased their Bcl-6/T-bet ratio in response to limiting IL-2 conditions, which allowed excess Bcl-6 to repress its direct target Prdm1 (which encodes the transcriptional repressor Blimp-1). The Bcl-6-dependent repression of Blimp-1 effectively induced a partial T(FH) profile because Blimp-1 directly repressed a subset of T(FH) signature genes, including Cxcr5. Thus, IL-2-signaling regulates the Bcl-6-Blimp-1 axis in T(H)1 cells to maintain flexibility with a T(FH) cell-like gene profile.

The proto-oncogene MYC is required for selection in the germinal center and cyclic reentry.

After antigenic challenge, B cells enter the dark zone (DZ) of germinal centers (GCs) to proliferate and hypermutate their immunoglobulin genes. Mutants with greater affinity for the antigen are positively selected in the light zone (LZ) to either differentiate into plasma and memory cells or reenter the DZ. The molecular circuits that govern positive selection in the GC are not known. We show here that the GC reaction required biphasic regulation of expression of the cell-cycle regulator c-Myc that involved its transient induction during early GC commitment, its repression by Bcl-6 in DZ B cells and its reinduction in B cells selected for reentry into the DZ. Inhibition of c-Myc in vivo led to GC collapse, which indicated an essential role for c-Myc in GCs. Our results have implications for the mechanism of GC selection and the role of c-Myc in lymphomagenesis.

TGF-ß and retinoic acid induce the microRNA miR-10a, which targets Bcl-6 and constrains the plasticity of helper T cells.

Distinct CD4(+) T cell subsets are critical for host defense and immunoregulation. Although these subsets can act as terminally differentiated lineages, they have been increasingly noted to demonstrated plasticity. MicroRNAs are factors that control T cell stability and plasticity. Here we report that naturally occurring regulatory T cells (T(reg) cells) had high expression of the microRNA miR-10a and that miR-10a was induced by retinoic acid and transforming growth factor-ß (TGF-ß) in inducible T(reg) cells. By simultaneously targeting the transcriptional repressor Bcl-6 and the corepressor Ncor2, miR-10a attenuated the phenotypic conversion of inducible T(reg) cells into follicular helper T cells. We also found that miR-10a limited differentiation into the T(H)17 subset of helper T cells and therefore represents a factor that can fine-tune the plasticity and fate of helper T cells.

Vaccine-elicited CD4 T cells prevent the deletion of antiviral B cells in chronic infection.

Chronic viral infections subvert protective B cell immunity. An early type I interferon (IFN-I)-driven bias to short-lived plasmablast differentiation leads to clonal deletion, so-called "decimation," of antiviral memory B cells. Therefore, prophylactic countermeasures against decimation remain an unmet need. We show that vaccination-induced CD4 T cells prevented the decimation of naïve and memory B cells in chronically lymphocytic choriomeningitis virus (LCMV)-infected mice. Although these B cell responses were largely T independent when IFN-I was blocked, preexisting T help assured their sustainability under conditions of IFN-I-driven inflammation by instructing a germinal center B cell transcriptional program. Prevention of decimation depended on T cell-intrinsic Bcl6 and Tfh progeny formation. Antigen presentation by B cells, interactions with antigen-specific T helper cells, and costimulation by CD40 and ICOS were also required. Importantly, B cell-mediated virus control averted Th1-driven immunopathology in LCMV-challenged animals with preexisting CD4 T cell immunity. Our findings show that vaccination-induced Tfh cells represent a cornerstone of effective B cell immunity to chronic virus challenge, pointing the way toward more effective B cell-based vaccination against persistent viral diseases.

A BATF-ling connection between B cells and follicular helper T cells.

The transcription factor BATF directly regulates key components of the formation and function of follicular helper T cells and antibody class switching in B cells.

The transcription factor BATF controls the global regulators of class-switch recombination in both B cells and T cells.

The transcription factor BATF controls the differentiation of interleukin 17 (IL-17)-producing helper T cells (T(H)17 cells) by regulating expression of the transcription factor RORγt itself and RORγt target genes such as Il17. Here we report the mechanism by which BATF controls in vivo class-switch recombination (CSR). In T cells, BATF directly controlled expression of the transcription factors Bcl-6 and c-Maf, both of which are needed for development of follicular helper T cells (T(FH) cells). Restoring T(FH) cell activity to Batf(-/-) T cells in vivo required coexpression of Bcl-6 and c-Maf. In B cells, BATF directly controlled the expression of both activation-induced cytidine deaminase (AID) and of germline transcripts of the intervening heavy-chain region and constant heavy-chain region (I(H)-C(H)). Thus, BATF functions at multiple hierarchical levels in two cell types to globally regulate switched antibody responses in vivo.

Monocyte-Derived Dendritic Cells (moDCs) Differentiate into Bcl6+ Mature moDCs to Promote Cyclic di-GMP Vaccine Adjuvant-Induced Memory TH Cells in the Lung.

Induction of lung mucosal immune responses is highly desirable for vaccines against respiratory infections. We recently showed that monocyte-derived dendritic cells (moDCs) are responsible for lung IgA induction. However, the dendritic cell subset inducing lung memory TH cells is unknown. In this study, using conditional knockout mice and adoptive cell transfer, we found that moDCs are essential for lung mucosal responses but are dispensable for systemic vaccine responses. Next, we showed that mucosal adjuvant cyclic di-GMP differentiated lung moDCs into Bcl6+ mature moDCs promoting lung memory TH cells, but they are dispensable for lung IgA production. Mechanistically, soluble TNF mediates the induction of lung Bcl6+ moDCs. Our study reveals the functional heterogeneity of lung moDCs during vaccination and paves the way for an moDC-targeting vaccine strategy to enhance immune responses on lung mucosa.

CD49d marks Th1 and Tfh-like antigen-specific CD4+ T cells during Plasmodium chabaudi infection.

Upon activation, specific CD4+ T cells up-regulate the expression of CD11a and CD49d, surrogate markers of pathogen-specific CD4+ T cells. However, using T-cell receptor transgenic mice specific for a Plasmodium antigen, termed PbT-II, we found that activated CD4+ T cells develop not only to CD11ahiCD49dhi cells, but also to CD11ahiCD49dlo cells during acute Plasmodium infection. CD49dhi PbT-II cells, localized in the red pulp of spleens, expressed transcription factor T-bet and produced IFN-γ, indicating that they were type 1 helper T (Th1)-type cells. In contrast, CD49dlo PbT-II cells resided in the white pulp/marginal zones and were a heterogeneous population, with approximately half of them expressing CXCR5 and a third expressing Bcl-6, a master regulator of follicular helper T (Tfh) cells. In adoptive transfer experiments, both CD49dhi and CD49dlo PbT-II cells differentiated into CD49dhi Th1-type cells after stimulation with antigen-pulsed dendritic cells, while CD49dhi and CD49dlo phenotypes were generally maintained in mice infected with Plasmodium chabaudi. These results suggest that CD49d is expressed on Th1-type Plasmodium-specific CD4+ T cells, which are localized in the red pulp of the spleen, and can be used as a marker of antigen-specific Th1 CD4+ T cells, rather than that of all pathogen-specific CD4+ T cells.

CD11c+ T-bet+ B Cells Require IL-21 and IFN-γ from Type 1 T Follicular Helper Cells and Intrinsic Bcl-6 Expression but Develop Normally in the Absence of T-bet.

CD11c+ T-bet+ B cells generated during ehrlichial infection require CD4+ T cell help and IL-21 signaling for their development, but the exact T cell subset required had not been known. In this study, we show in a mouse model of Ehrlichia muris that type 1 T follicular helper (TFH1) cells provide help to CD11c+ T-bet+ B cells via the dual secretion of IL-21 and IFN-γ in a CD40/CD40L-dependent manner. TFH1 cell help was delivered in two phases: IFN-γ signals were provided early in infection, whereas CD40/CD40L help was provided late in infection. In contrast to T-bet+ T cells, T-bet+ B cells did not develop in the absence of B cell-intrinsic Bcl-6 but were generated in the absence of T-bet. T-bet-deficient memory B cells were largely indistinguishable from their wild-type counterparts, although they no longer underwent switching to IgG2c. These data suggest that a primary function of T-bet in B cells during ehrlichial infection is to promote appropriate class switching, not lineage specification. Thus, CD11c+ memory B cells develop normally without T-bet but require Bcl-6 and specialized help from dual cytokine-producing TFH1 cells.

Transcription factors IRF8 and PU.1 are required for follicular B cell development and BCL6-driven germinal center responses.

The IRF and Ets families of transcription factors regulate the expression of a range of genes involved in immune cell development and function. However, the understanding of the molecular mechanisms of each family member has been limited due to their redundancy and broad effects on multiple lineages of cells. Here, we report that double deletion of floxed Irf8 and Spi1 (encoding PU.1) by Mb1-Cre (designated DKO mice) in the B cell lineage resulted in severe defects in the development of follicular and germinal center (GC) B cells. Class-switch recombination and antibody affinity maturation were also compromised in DKO mice. RNA-seq (sequencing) and ChIP-seq analyses revealed distinct IRF8 and PU.1 target genes in follicular and activated B cells. DKO B cells had diminished expression of target genes vital for maintaining follicular B cell identity and GC development. Moreover, our findings reveal that expression of B-cell lymphoma protein 6 (BCL6), which is critical for development of germinal center B cells, is dependent on IRF8 and PU.1 in vivo, providing a mechanism for the critical role for IRF8 and PU.1 in the development of GC B cells.