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Memory B cells (MBCs) are essential for long-lived humoral immunity. However, the transcription factors involved in MBC differentiation are poorly defined. Here, using single-cell RNA sequencing analysis, we identified a population of germinal center (GC) B cells in the process of differentiating into MBCs. Using an inducible CRISPR–Cas9 screening approach, we identified the hematopoietically expressed homeobox protein Hhex as a transcription factor regulating MBC differentiation. The corepressor Tle3 was also identified in the screen and was found to interact with Hhex to promote MBC development. Bcl-6 directly repressed Hhex in GC B cells. Reciprocally, Hhex-deficient MBCs exhibited increased Bcl6 expression and reduced expression of the Bcl-6 target gene Bcl2 . Overexpression of Bcl-2 was able to rescue MBC differentiation in Hhex-deficient cells. We also identified Ski as an Hhex-induced transcription factor involved in MBC differentiation. These findings establish an important role for Hhex–Tle3 in regulating the transcriptional circuitry governing MBC differentiation. Generation of memory B cells is crucial for protective immunity to infectious agents. Cyster and colleagues show that the transcription factor Hhex interacting with Tle3 promotes memory B cell generation.

B cells and follicular helper T cells in B cell follicles can act as important reservoirs for chronic infection by viruses such as HIV or EBV. Yu and colleagues show that a specialized subpopulation of cytotoxic T cells can enter the B cell follicles to eliminate such virus-infected cells. During unresolved infections, some viruses escape immunological control and establish a persistant reservoir in certain cell types, such as human immunodeficiency virus (HIV), which persists in follicular helper T cells (T FH cells), and Epstein-Barr virus (EBV), which persists in B cells. Here we identified a specialized group of cytotoxic T cells (T C cells) that expressed the chemokine receptor CXCR5, selectively entered B cell follicles and eradicated infected T FH cells and B cells. The differentiation of these cells, which we have called 'follicular cytotoxic T cells' (T FC cells), required the transcription factors Bcl6, E2A and TCF-1 but was inhibited by the transcriptional regulators Blimp1, Id2 and Id3. Blimp1 and E2A directly regulated Cxcr5 expression and, together with Bcl6 and TCF-1, formed a transcriptional circuit that guided T FC cell development. The identification of T FC cells has far-reaching implications for the development of strategies to control infections that target B cells and T FH cells and to treat B cell–derived malignancies.

Genes that drive the proliferation, survival, invasion and metastasis of malignant cells have been identified for many human cancers 1 – 4 . Independent studies have identified cell death pathways that eliminate cells for the good of the organism 5 , 6 . The coexistence of cell death pathways with driver mutations suggests that the cancer driver could be rewired to activate cell death using chemical inducers of proximity (CIPs). Here we describe a new class of molecules called transcriptional/epigenetic CIPs (TCIPs) that recruit the endogenous cancer driver, or a downstream transcription factor, to the promoters of cell death genes, thereby activating their expression. We focused on diffuse large B cell lymphoma, in which the transcription factor B cell lymphoma 6 (BCL6) is deregulated 7 . BCL6 binds to the promoters of cell death genes and epigenetically suppresses their expression 8 . We produced TCIPs by covalently linking small molecules that bind BCL6 to those that bind to transcriptional activators that contribute to the oncogenic program, such as BRD4. The most potent molecule, TCIP1, increases binding of BRD4 by 50% over genomic BCL6-binding sites to produce transcriptional elongation at pro-apoptotic target genes within 15 min, while reducing binding of BRD4 over enhancers by only 10%, reflecting a gain-of-function mechanism. TCIP1 kills diffuse large B cell lymphoma cell lines, including chemotherapy-resistant, TP53 -mutant lines, at EC 50 of 1–10 nM in 72 h and exhibits cell-specific and tissue-specific effects, capturing the combinatorial specificity inherent to transcription. The TCIP concept also has therapeutic applications in regulating the expression of genes for regenerative medicine and developmental disorders. A new class of molecules can recruit downstream transcription factors or endogenous cancer drivers to cell death promoters and activate the expression of these genes.

T follicular helper (T FH ) cells are a distinct type of CD4 + T cells that are essential for most antibody and B lymphocyte responses. T FH cell regulation and dysregulation is involved in a range of diseases. Bcl-6 is the lineage-defining transcription factor of T FH cells and its activity is essential for T FH cell differentiation and function. However, how Bcl-6 controls T FH biology has largely remained unclear, at least in part due to the intrinsic challenges of connecting repressors to gene upregulation in complex cell types with multiple possible differentiation fates. Multiple competing models were tested here by a series of experimental approaches to determine that Bcl-6 exhibits negative autoregulation and controls pleiotropic attributes of T FH differentiation and function, including migration, costimulation, inhibitory receptors and cytokines, via multiple repressor-of-repressor gene circuits. Bcl-6 is the signature transcription factor for T FH cells. Crotty and colleagues provide a comprehensive transcriptional map depicting the regulatory circuitry controlled by Bcl-6 in determining T FH cell fate and function.

Diffuse large B cell lymphoma (DLBCL) is the most common B cell non-Hodgkin lymphoma and remains incurable in around 40% of patients. Efforts to sequence the coding genome identified several genes and pathways that are altered in this disease, including potential therapeutic targets 1 – 5 . However, the non-coding genome of DLBCL remains largely unexplored. Here we show that active super-enhancers are highly and specifically hypermutated in 92% of samples from individuals with DLBCL, display signatures of activation-induced cytidine deaminase activity, and are linked to genes that encode B cell developmental regulators and oncogenes. As evidence of oncogenic relevance, we show that the hypermutated super-enhancers linked to the BCL6 , BCL2 and CXCR4 proto-oncogenes prevent the binding and transcriptional downregulation of the corresponding target gene by transcriptional repressors, including BLIMP1 (targeting BCL6 ) and the steroid receptor NR3C1 (targeting BCL2 and CXCR4 ). Genetic correction of selected mutations restored repressor DNA binding, downregulated target gene expression and led to the counter-selection of cells containing corrected alleles, indicating an oncogenic dependency on the super-enhancer mutations. This pervasive super-enhancer mutational mechanism reveals a major set of genetic lesions deregulating gene expression, which expands the involvement of known oncogenes in DLBCL pathogenesis and identifies new deregulated gene targets of therapeutic relevance. Active super-enhancers are highly and specifically hypermutated in 92% of diffuse large B cell lymphoma samples and display signatures of activation-induced cytidine deaminase activity, leading to the dysregulation of genes encoding B cell developmental regulators and oncogenes.

The molecular control of germinal center selection is still being determined. Dalla-Favera and colleagues show that the cell-cycle regulator c-Myc is essential for B cell selection and reentry into the germinal center. 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.

IL-7 is known to control the survival of immature DN thymocytes before β-selection. Guidos and colleagues show that during β-selection, IL-7 controls the growth and differentiation of thymocytes, in part by repressing the transcription factor Bcl-6. Signaling via the pre–T cell antigen receptor (pre-TCR) and the receptor Notch1 induces transient self-renewal (β-selection) of TCRβ + 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 TCRβ − DN thymocytes by inducing expression of the pro-survival molecule Bcl-2, but the functions of IL-7 during β-selection have remained unclear. Here we found that IL-7 signaled TCRβ + 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 β-selection.

Humoral immunity is necessary for controlling viral infection. Ballesteros-Tato and colleagues show that development of follicular regulatory T cells is prevented by high concentrations of interleukin 2 at the peak of viral infection, but resumes at later time points to suppress autoantibody production. Interleukin 2 (IL-2) promotes Foxp3 + regulatory T (T reg ) cell responses, but inhibits T follicular helper (T FH ) cell development. However, it is not clear how IL-2 affects T follicular regulatory (T FR ) cells, a cell type with properties of both T reg and T FH cells. Using an influenza infection model, we found that high IL-2 concentrations at the peak of the infection prevented T FR cell development by a Blimp-1-dependent mechanism. However, once the immune response resolved, some T reg cells downregulated CD25, upregulated Bcl-6 and differentiated into T FR cells, which then migrated into the B cell follicles to prevent the expansion of self-reactive B cell clones. Thus, unlike its effects on conventional T reg cells, IL-2 inhibits T FR cell responses.

Oncolytic viruses (OVs) emerge as a promising cancer immunotherapy. However, the temporal impact on tumor cells and the tumor microenvironment, and the nature of anti-tumor immunity post-therapy remain largely unclear. Here we report that CD4 + T cells are required for durable tumor control in syngeneic murine models of glioblastoma multiforme after treatment with an oncolytic herpes simplex virus (oHSV) engineered to express IL-12. The upregulated MHCII on residual tumor cells facilitates programmed polyfunctional CD4 + T cells for tumor control and for recall responses. Mechanistically, the proper ratio of Bcl-6 to T-bet in CD4 + T cells navigates their enhanced anti-tumor capacity, and a reciprocal IL6ra-Bcl-6 regulatory axis in a memory CD4 + T-cell subset, which requires MHCII signals from reprogrammed tumor cells, tumor-infiltrating and resident myeloid cells, is necessary for the prolonged response. These findings uncover an OV-induced tumor/myeloid-CD4 + T-cell partnership, leading to long-term anti-tumor immune memory, and improved OV therapeutic efficacy. Oncolytic herpes simplex virus−1 (oHSV) can boost anti-tumor immune responses in gliomas. Here the authors report that CD4 + T cells are required for the therapeutic activity of an oHSV-engineered to express IL-12 in preclinical glioblastoma models.

The transcription factor BATF is known to control switched antibody responses. Murphy and colleagues show that BATF functions at multiple hierarchical levels in follicular helper T cells and B cells to regulate these responses. 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 .