BRWD1 establishes epigenetic states for germinal center initiation, maintenance, and function.

Germinal center (GC) B cells segregate into three subsets that compartmentalize the antagonistic molecular programs of selection, proliferation, and somatic hypermutation. In bone marrow, the epigenetic reader BRWD1 orchestrates and insulates the sequential stages of cell proliferation and Igk recombination. We hypothesized BRWD1 might play similar insulative roles in the periphery. In Brwd1 -/- follicular B cells, GC initiation and class switch recombination following immunization were inhibited. In contrast, in Brwd1 -/- GC B cells there was admixing of chromatin accessibility across GC subsets and transcriptional dysregulation including induction of inflammatory pathways. This global molecular GC dysregulation was associated with specific defects in proliferation, affinity maturation, and tolerance. These data suggest that GC subset identity is required for some but not all GC-attributed functions. Furthermore, these data demonstrate a central role for BRWD1 in orchestrating epigenetic transitions at multiple steps along B cell developmental and activation pathways.

The PTPN2/PTPN1 inhibitor ABBV-CLS-484 unleashes potent anti-tumour immunity.

Immune checkpoint blockade is effective for some patients with cancer, but most are refractory to current immunotherapies and new approaches are needed to overcome resistance1,2. The protein tyrosine phosphatases PTPN2 and PTPN1 are central regulators of inflammation, and their genetic deletion in either tumour cells or immune cells promotes anti-tumour immunity3-6. However, phosphatases are challenging drug targets; in particular, the active site has been considered undruggable. Here we present the discovery and characterization of ABBV-CLS-484 (AC484), a first-in-class, orally bioavailable, potent PTPN2 and PTPN1 active-site inhibitor. AC484 treatment in vitro amplifies the response to interferon and promotes the activation and function of several immune cell subsets. In mouse models of cancer resistant to PD-1 blockade, AC484 monotherapy generates potent anti-tumour immunity. We show that AC484 inflames the tumour microenvironment and promotes natural killer cell and CD8+ T cell function by enhancing JAK-STAT signalling and reducing T cell dysfunction. Inhibitors of PTPN2 and PTPN1 offer a promising new strategy for cancer immunotherapy and are currently being evaluated in patients with advanced solid tumours (ClinicalTrials.gov identifier NCT04777994 ). More broadly, our study shows that small-molecule inhibitors of key intracellular immune regulators can achieve efficacy comparable to or exceeding that of antibody-based immune checkpoint blockade in preclinical models. Finally, to our knowledge, AC484 represents the first active-site phosphatase inhibitor to enter clinical evaluation for cancer immunotherapy and may pave the way for additional therapeutics that target this important class of enzymes.

The endogenous repertoire harbors self-reactive CD4+ T cell clones that adopt a follicular helper T cell-like phenotype at steady state.

The T cell repertoire of healthy mice and humans harbors self-reactive CD4+ conventional T (Tconv) cells capable of inducing autoimmunity. Using T cell receptor profiling paired with in vivo clonal analysis of T cell differentiation, we identified Tconv cell clones that are recurrently enriched in non-lymphoid organs following ablation of Foxp3+ regulatory T (Treg) cells. A subset of these clones was highly proliferative in the lymphoid organs at steady state and exhibited overt reactivity to self-ligands displayed by dendritic cells, yet were not purged by clonal deletion. These clones spontaneously adopted numerous hallmarks of follicular helper T (TFH) cells, including expression of Bcl6 and PD-1, exhibited an elevated propensity to localize within B cell follicles at steady state, and produced interferon-γ in non-lymphoid organs following sustained Treg cell depletion. Our work identifies a naturally occurring population of self-reactive TFH-like cells and delineates a previously unappreciated fate for self-specific Tconv cells.

IRF4 expression by lung dendritic cells drives acute but not Trm cell-dependent memory Th2 responses.

Expression of the transcription factor interferon regulatory factor 4 (IRF4) is required for the development of lung conventional DCs type 2 (cDC2s) that elicit Th2 responses, yet how IRF4 functions in lung cDC2s throughout the acute and memory allergic response is not clear. Here, we used a mouse model that loses IRF4 expression after lung cDC2 development to demonstrate that mice with IRF4-deficient DCs display impaired memory responses to allergen. This defect in the memory response was a direct result of ineffective Th2 induction and impaired recruitment of activated effector T cells to the lung after sensitization. IRF4-deficient DCs demonstrated defects in their migration to the draining lymph node and in T cell priming. Finally, T cells primed by IRF4-competent DCs mediated potent memory responses independently of IRF4-expressing DCs, demonstrating that IRF4-expressing DCs are not necessary during the memory response. Thus, IRF4 controlled a program in mature DCs governing Th2 priming and effector responses, but IRF4-expressing DCs were dispensable during tissue-resident memory T cell-dependent memory responses.

Asymmetrical forward and reverse developmental trajectories determine molecular programs of B cell antigen receptor editing.

During B lymphopoiesis, B cell progenitors progress through alternating and mutually exclusive stages of clonal expansion and immunoglobulin (Ig) gene rearrangements. Great diversity is generated through the stochastic recombination of Ig gene segments encoding heavy and light chain variable domains. However, this commonly generates autoreactivity. Receptor editing is the predominant tolerance mechanism for self-reactive B cells in the bone marrow (BM). B cell receptor editing rescues autoreactive B cells from negative selection through renewed light chain recombination first at Igκ then Igλ loci. Receptor editing depends on BM microenvironment cues and key transcription factors such as NF-κB, FOXO, and E2A. The specific BM factor required for receptor editing is unknown. Furthermore, how transcription factors coordinate these developmental programs to promote usage of the λ chain remains poorly defined. Therefore, we used two mouse models that recapitulate pathways by which Igλ light chain-positive B cells develop. The first has deleted J kappa (Jκ) genes and hence models Igλ expression resulting from failed Igκ recombination (Igκdel). The second models autoreactivity by ubiquitous expression of a single-chain chimeric anti-Igκ antibody (κ-mac). Here, we demonstrated that autoreactive B cells transit asymmetric forward and reverse developmental trajectories. This imparted a unique epigenetic landscape on small pre-B cells, which opened chromatin to transcription factors essential for Igλ recombination. The consequences of this asymmetric developmental path were both amplified and complemented by CXCR4 signaling. These findings reveal how intrinsic molecular programs integrate with extrinsic signals to drive receptor editing.

Compartments and Connections Within the Germinal Center.

Protective high affinity antibody responses emerge through an orchestrated developmental process that occurs in germinal centers (GCs). While GCs have been appreciated since 1930, a wealth of recent progress provides new insights into the molecular and cellular dynamics governing humoral immunity. In this review, we highlight advances that demonstrate that fundamental GC B cell function, selection, proliferation and SHM occur within distinct cell states. The resulting new model provides new opportunities to understand the evolution of immunity in infectious, autoimmune and neoplastic diseases.

Novel specialized cell state and spatial compartments within the germinal center.

Within germinal centers (GCs), complex and highly orchestrated molecular programs must balance proliferation, somatic hypermutation and selection to both provide effective humoral immunity and to protect against genomic instability and neoplastic transformation. In contrast to this complexity, GC B cells are canonically divided into two principal populations, dark zone (DZ) and light zone (LZ) cells. We now demonstrate that, following selection in the LZ, B cells migrated to specialized sites within the canonical DZ that contained tingible body macrophages and were sites of ongoing cell division. Proliferating DZ (DZp) cells then transited into the larger DZ to become differentiating DZ (DZd) cells before re-entering the LZ. Multidimensional analysis revealed distinct molecular programs in each population commensurate with observed compartmentalization of noncompatible functions. These data provide a new three-cell population model that both orders critical GC functions and reveals essential molecular programs of humoral adaptive immunity.

CXCR4 signaling directs Igk recombination and the molecular mechanisms of late B lymphopoiesis.

In B lymphopoiesis, activation of the pre-B cell antigen receptor (pre-BCR) is associated with both cell cycle exit and Igk recombination. Yet how the pre-BCR mediates these functions remains unclear. Here, we demonstrate that the pre-BCR initiates a feed-forward amplification loop mediated by the transcription factor interferon regulatory factor 4 and the chemokine receptor C-X-C motif chemokine receptor 4 (CXCR4). CXCR4 ligation by C-X-C motif chemokine ligand 12 activates the mitogen-activated protein kinase extracellular-signal-regulated kinase, which then directs the development of small pre- and immature B cells, including orchestrating cell cycle exit, pre-BCR repression, Igk recombination and BCR expression. In contrast, pre-BCR expression and escape from interleukin-7 have only modest effects on B cell developmental transcriptional and epigenetic programs. These data show a direct and central role for CXCR4 in orchestrating late B cell lymphopoiesis. Furthermore, in the context of previous findings, our data provide a three-receptor system sufficient to recapitulate the essential features of B lymphopoiesis in vitro.

Quantifying in situ adaptive immune cell cognate interactions in humans.

Two-photon excitation microscopy (TPEM) has revolutionized the understanding of adaptive immunity. However, TPEM usually requires animal models and is not amenable to the study of human disease. The recognition of antigen by T cells requires cell contact and is associated with changes in T cell shape. We postulated that by capturing these features in fixed tissue samples, we could quantify in situ adaptive immunity. Therefore, we used a deep convolutional neural network to identify fundamental distance and cell-shape features associated with cognate help (cell-distance mapping (CDM)). In mice, CDM was comparable to TPEM in discriminating cognate T cell-dendritic cell (DC) interactions from non-cognate T cell-DC interactions. In human lupus nephritis, CDM confirmed that myeloid DCs present antigen to CD4+ T cells and identified plasmacytoid DCs as an important antigen-presenting cell. These data reveal a new approach with which to study human in situ adaptive immunity broadly applicable to autoimmunity, infection, and cancer.

BRWD1 orchestrates epigenetic landscape of late B lymphopoiesis.

Transcription factor (TF) networks determine cell fate in hematopoiesis. However, how TFs cooperate with other regulatory mechanisms to instruct transcription remains poorly understood. Here we show that in small pre-B cells, the lineage restricted epigenetic reader BRWD1 closes early development enhancers and opens the enhancers of late B lymphopoiesis to TF binding. BRWD1 regulates over 7000 genes to repress proliferative and induce differentiation programs. However, BRWD1 does not regulate the expression of TFs required for B lymphopoiesis. Hypogammaglobulinemia patients with BRWD1 mutations have B-cell transcriptional profiles and enhancer landscapes similar to those observed in Brwd1-/- mice. These data indicate that, in both mice and humans, BRWD1 is a master orchestrator of enhancer accessibility that cooperates with TF networks to drive late B-cell development.

Regulated Capture of Vκ Gene Topologically Associating Domains by Transcription Factories.

Expression of vast repertoires of antigen receptors by lymphocytes, with each cell expressing a single receptor, requires stochastic activation of individual variable (V) genes for transcription and recombination. How this occurs remains unknown. Using single-cell RNA sequencing (scRNA-seq) and allelic variation, we show that individual pre-B cells monoallelically transcribe divergent arrays of Vκ genes, thereby opening stochastic repertoires for subsequent Vκ-Jκ recombination. Transcription occurs upon translocation of Vκ genes to RNA polymerase II arrayed on the nuclear matrix in transcription factories. Transcription is anchored by CTCF-bound sites or E2A-loaded Vκ promotors and continues over large genomic distances delimited only by topological associating domains (TADs). Prior to their monoallelic activation, Vκ loci are transcriptionally repressed by cyclin D3, which prevents capture of Vκ gene containing TADs by transcription factories. Cyclin D3 also represses protocadherin, olfactory, and other monoallelically expressed genes, suggesting a widely deployed mechanism for coupling monoallelic gene activation with cell cycle exit.

Inflammatory Changes in Bone Marrow Microenvironment Associated with Declining B Lymphopoiesis.

B lymphopoiesis arrests precipitously in rabbits such that by 2-4 mo of age, before sexual maturity, little to no B lymphopoiesis occurs in the bone marrow (BM). Previously, we showed that in mice, adipocytes inhibit B lymphopoiesis in vitro by inducing inflammatory myeloid cells, which produce IL-1ß. In this study, we characterized rabbit BM after the arrest of B lymphopoiesis and found a dramatic increase in fat, increased CD11b+ myeloid cells, and upregulated expression of the inflammatory molecules, IL-1ß and S100A9, by the myeloid cells. We added BM fat, CD11b+ myeloid cells, and recombinant S100A9 to B lymphopoiesis cultures and found that they inhibited B lymphopoiesis and enhanced myelopoiesis. Unlike IL-1ß, which inhibits B lymphopoiesis by acting on early lymphoid progenitors, S100A9 inhibits B lymphopoiesis by acting on myeloid cells and promoting the release of inflammatory molecules, including IL-1ß. Many molecules produced by adipocytes activate the NLRP3 inflammasome, and the NLRP3 inhibitor, glibenclamide, restored B lymphopoiesis and minimized induction of myeloid cells induced by adipocyte-conditioned medium in vitro. We suggest that fat provides an inflammatory microenvironment in the BM and promotes/activates myeloid cells to produce inflammatory molecules such as IL-1ß and S100A9, which negatively regulate B lymphopoiesis.

Bone marrow fat and the decline of B lymphopoiesis in rabbits.

B lymphopoiesis is necessary to generate a diverse pool of naïve B cells that are able to respond to a broad spectrum of antigens during immune responses to pathogens and to vaccination. Rabbits have been utilized for many years to generate high affinity monoclonal and polyclonal antibodies. Specific antibodies generated in rabbits have greatly advanced scientific discoveries, but the unique qualities of rabbit B cell development have been underappreciated. Unlike in humans and mice, where B lymphopoiesis declines in mid to late life, B lymphopoiesis in rabbits arrests early in life, between 2 and 4 months of age. This review focuses on the early loss of B cell development in rabbits and the contribution of the bone marrow microenvironment to this process. We also propose directions for future research in this area, and discuss how the rabbit can be used as a model to understand the decline of B lymphopoiesis that occurs in humans late in life. Such studies will be important for developing therapeutics targeted to prevent and/or reverse declining B lymphopoiesis in the elderly, as well as boosting immunity and antibody responses after infection or vaccination.

Inhibition of B Lymphopoiesis by Adipocytes and IL-1-Producing Myeloid-Derived Suppressor Cells.

B lymphopoiesis declines with age, and this decline correlates with increased adipose tissue in the bone marrow (BM). Also, adipocyte-derived factors are known to inhibit B lymphopoiesis. Using cocultures of mouse BM cells with OP9 stromal cells, we found that adipocyte-conditioned medium induces the generation of CD11b(+)Gr1(+) myeloid cells, which inhibit B cell development in vitro. Adipocyte-conditioned medium-induced CD11b(+)Gr1(+) cells express Arg1 (arginase) and Nos2 (inducible NO synthase) and suppress CD4(+) T cell proliferation, indicating that these cells are myeloid-derived suppressor cells (MDSCs). Blocking arginase and inducible NO synthase did not restore B lymphopoiesis, indicating that inhibition is not mediated by these molecules. Transwell and conditioned-medium experiments showed that MDSCs inhibit B lymphopoiesis via soluble factors, and by cytokine array we identified IL-1 as an important factor. Addition of anti-IL-1 Abs restored B lymphopoiesis in BM cultures containing MDSCs, showing that MDSC inhibition of B lymphopoiesis is mediated by IL-1. By treating hematopoietic precursors with IL-1, we found that multipotent progenitors are targets of IL-1. This study uncovers a novel function for MDSCs to inhibit B lymphopoiesis through IL-1. We suggest that inflammaging contributes to a decline of B lymphopoiesis in aged individuals, and furthermore, that MDSCs and IL-1 provide therapeutic targets for restoration of B lymphopoiesis in aged and obese individuals.