Unraveling the diversity and functions of tissue-resident plasma cells.

Antibody-secreting plasma cells (PCs) are generated in secondary lymphoid organs but are reported to reside in an emerging range of anatomical sites. Analysis of the transcriptome of different tissue-resident (Tr)PC populations revealed that they each have their own transcriptional signature indicative of functional adaptation to the host tissue environment. In contrast to expectation, all TrPCs were extremely long-lived, regardless of their organ of residence, with longevity influenced by intrinsic factors like the immunoglobulin isotype. Analysis at single-cell resolution revealed that the bone marrow is unique in housing a compendium of PCs generated all over the body that retain aspects of the transcriptional program indicative of their tissue of origin. This study reveals that extreme longevity is an intrinsic property of TrPCs whose transcriptome is imprinted by signals received both at the site of induction and within the tissue of residence.

IKAROS and AIOLOS directly regulate AP-1 transcriptional complexes and are essential for NK cell development.

Ikaros transcription factors are essential for adaptive lymphocyte function, yet their role in innate lymphopoiesis is unknown. Using conditional genetic inactivation, we show that Ikzf1/Ikaros is essential for normal natural killer (NK) cell lymphopoiesis and IKZF1 directly represses Cish, a negative regulator of interleukin-15 receptor resulting in impaired interleukin-15 receptor signaling. Both Bcl2l11 and BIM levels, and intrinsic apoptosis were increased in Ikzf1-null NK cells, which in part accounts for NK lymphopenia as both were restored to normal levels when Ikzf1 and Bcl2l11 were co-deleted. Ikzf1-null NK cells presented extensive transcriptional alterations with reduced AP-1 transcriptional complex expression and increased expression of Ikzf2/Helios and Ikzf3/Aiolos. IKZF1 and IKZF3 directly bound AP-1 family members and deletion of both Ikzf1 and Ikzf3 in NK cells resulted in further reductions in Jun/Fos expression and complete loss of peripheral NK cells. Collectively, we show that Ikaros family members are important regulators of apoptosis, cytokine responsiveness and AP-1 transcriptional activity.

Liver Immune Profiling Reveals Pathogenesis and Therapeutics for Biliary Atresia.

Biliary atresia (BA) is a severe cholangiopathy that leads to liver failure in infants, but its pathogenesis remains to be fully characterized. By single-cell RNA profiling, we observed macrophage hypo-inflammation, Kupffer cell scavenger function defects, cytotoxic T cell expansion, and deficiency of CX3CR1+effector T and natural killer (NK) cells in infants with BA. More importantly, we discovered that hepatic B cell lymphopoiesis did not cease after birth and that tolerance defects contributed to immunoglobulin G (IgG)-autoantibody accumulation in BA. In a rhesus-rotavirus induced BA model, depleting B cells or blocking antigen presentation ameliorated liver damage. In a pilot clinical study, we demonstrated that rituximab was effective in depleting hepatic B cells and restoring the functions of macrophages, Kupffer cells, and T cells to levels comparable to those of control subjects. In summary, our comprehensive immune profiling in infants with BA had educed that B-cell-modifying therapies may alleviate liver pathology.

The cis-Regulatory Atlas of the Mouse Immune System.

A complete chart of cis-regulatory elements and their dynamic activity is necessary to understand the transcriptional basis of differentiation and function of an organ system. We generated matched epigenome and transcriptome measurements in 86 primary cell types that span the mouse immune system and its differentiation cascades. This breadth of data enable variance components analysis that suggests that genes fall into two distinct classes, controlled by either enhancer- or promoter-driven logic, and multiple regression that connects genes to the enhancers that regulate them. Relating transcription factor (TF) expression to the genome-wide accessibility of their binding motifs classifies them as predominantly openers or closers of local chromatin accessibility, pinpointing specific cis-regulatory elements where binding of given TFs is likely functionally relevant, validated by chromatin immunoprecipitation sequencing (ChIP-seq). Overall, this cis-regulatory atlas provides a trove of information on transcriptional regulation through immune differentiation and a foundational scaffold to define key regulatory events throughout the immunological genome.

Blockade of the co-inhibitory molecule PD-1 unleashes ILC2-dependent antitumor immunity in melanoma.

Group 2 innate lymphoid cells (ILC2s) are essential to maintain tissue homeostasis. In cancer, ILC2s can harbor both pro-tumorigenic and anti-tumorigenic functions, but we know little about their underlying mechanisms or whether they could be clinically relevant or targeted to improve patient outcomes. Here, we found that high ILC2 infiltration in human melanoma was associated with a good clinical prognosis. ILC2s are critical producers of the cytokine granulocyte-macrophage colony-stimulating factor, which coordinates the recruitment and activation of eosinophils to enhance antitumor responses. Tumor-infiltrating ILC2s expressed programmed cell death protein-1, which limited their intratumoral accumulation, proliferation and antitumor effector functions. This inhibition could be overcome in vivo by combining interleukin-33-driven ILC2 activation with programmed cell death protein-1 blockade to significantly increase antitumor responses. Together, our results identified ILC2s as a critical immune cell type involved in melanoma immunity and revealed a potential synergistic approach to harness ILC2 function for antitumor immunotherapies.

A new lymphoid-primed progenitor marked by Dach1 downregulation identified with single cell multi-omics.

A classical view of blood cell development is that multipotent hematopoietic stem and progenitor cells (HSPCs) become lineage-restricted at defined stages. Lin-c-Kit+Sca-1+Flt3+ cells, termed lymphoid-primed multipotent progenitors (LMPPs), have lost megakaryocyte and erythroid potential but are heterogeneous in their fate. Here, through single-cell RNA sequencing, we identify the expression of Dach1 and associated genes in this fraction as being coexpressed with myeloid/stem genes but inversely correlated with lymphoid genes. Through generation of Dach1-GFP reporter mice, we identify a transcriptionally and functionally unique Dach1-GFP- subpopulation within LMPPs with lymphoid potential with low to negligible classic myeloid potential. We term these 'lymphoid-primed progenitors' (LPPs). These findings define an early definitive branch point of lymphoid development in hematopoiesis and a means for prospective isolation of LPPs.

Selective deployment of transcription factor paralogs with submaximal strength facilitates gene regulation in the immune system.

In multicellular organisms, duplicated genes can diverge through tissue-specific gene expression patterns, as exemplified by highly regulated expression of RUNX transcription factor paralogs with apparent functional redundancy. Here we asked what cell-type-specific biologies might be supported by the selective expression of RUNX paralogs during Langerhans cell and inducible regulatory T cell differentiation. We uncovered functional nonequivalence between RUNX paralogs. Selective expression of native paralogs allowed integration of transcription factor activity with extrinsic signals, while non-native paralogs enforced differentiation even in the absence of exogenous inducers. DNA binding affinity was controlled by divergent amino acids within the otherwise highly conserved RUNT domain and evolutionary reconstruction suggested convergence of RUNT domain residues toward submaximal strength. Hence, the selective expression of gene duplicates in specialized cell types can synergize with the acquisition of functional differences to enable appropriate gene expression, lineage choice and differentiation in the mammalian immune system.

Transcription-factor-mediated supervision of global genome architecture maintains B cell identity.

Recent studies have elucidated cell-lineage-specific three-dimensional genome organization; however, how such specific architecture is established or maintained is unclear. We hypothesized that lineage-defining transcription factors maintain cell identity via global control of genome organization. These factors bind many genomic sites outside of the genes that they directly regulate and thus are potentially implicated in three-dimensional genome organization. Using chromosome-conformation-capture techniques, we show that the transcription factor Paired box 5 (Pax5) is critical for the establishment and maintenance of the global lineage-specific architecture of B cells. Pax5 was found to supervise genome architecture throughout B cell differentiation, until the plasmablast stage, in which Pax5 is naturally silenced and B cell-specific genome structure is lost. Crucially, Pax5 did not rely on ongoing transcription to organize the genome. These results implicate sequence-specific DNA-binding proteins in global genome organization to establish and maintain lineage fidelity.

Transcriptional Networks Driving Dendritic Cell Differentiation and Function.

Dendritic cells (DCs) are the sentinels of the immune system, sensing a diverse array of pathogens to stimulate a robust and appropriate immune response. To initiate responses to highly disparate challenges, DCs have diversified into multiple phenotypically, anatomically, and functionally distinct cell types. As a result of the application of new single-cell technologies, the full extent of this diversity, as well as the developmental relationships of the DC lineages, is currently undergoing reassessment. Here, we review the cellular and molecular evidence that underpins current models of DC differentiation and functional diversification in the murine and human systems. We discuss these models in the context of the diversity revealed by single-cell studies and propose that understanding DC identity will require defining the regulatory interactions that control gene expression in these cells.

Blimp-1 controls plasma cell function through the regulation of immunoglobulin secretion and the unfolded protein response.

Plasma cell differentiation requires silencing of B cell transcription, while it establishes antibody-secretory function and long-term survival. The transcription factors Blimp-1 and IRF4 are essential for the generation of plasma cells; however, their function in mature plasma cells has remained elusive. We found that while IRF4 was essential for the survival of plasma cells, Blimp-1 was dispensable for this. Blimp-1-deficient plasma cells retained their transcriptional identity but lost the ability to secrete antibody. Blimp-1 regulated many components of the unfolded protein response (UPR), including XBP-1 and ATF6. The overlap in the functions of Blimp-1 and XBP-1 was restricted to that response, with Blimp-1 uniquely regulating activity of the kinase mTOR and the size of plasma cells. Thus, Blimp-1 was required for the unique physiological ability of plasma cells that enables the secretion of protective antibody.

Multifunctional role of the transcription factor Blimp-1 in coordinating plasma cell differentiation.

The transcription factor Blimp-1 is necessary for the generation of plasma cells. Here we studied its functions in plasmablast differentiation by identifying regulated Blimp-1 target genes. Blimp-1 promoted the migration and adhesion of plasmablasts. It directly repressed genes encoding several transcription factors and Aicda (which encodes the cytidine deaminase AID) and thus silenced B cell-specific gene expression, antigen presentation and class-switch recombination in plasmablasts. It directly activated genes, which led to increased expression of the plasma cell regulator IRF4 and proteins involved in immunoglobulin secretion. Blimp-1 induced the transcription of immunoglobulin genes by controlling the 3' enhancers of the loci encoding the immunoglobulin heavy chain (Igh) and κ-light chain (Igk) and, furthermore, regulated the post-transcriptional expression switch from the membrane-bound form of the immunoglobulin heavy chain to its secreted form by activating Ell2 (which encodes the transcription-elongation factor ELL2). Notably, Blimp-1 recruited chromatin-remodeling and histone-modifying complexes to regulate its target genes. Hence, many essential functions of plasma cells are under the control of Blimp-1.

A molecular threshold for effector CD8(+) T cell differentiation controlled by transcription factors Blimp-1 and T-bet.

T cell responses are guided by cytokines that induce transcriptional regulators, which ultimately control differentiation of effector and memory T cells. However, it is unknown how the activities of these molecular regulators are coordinated and integrated during the differentiation process. Using genetic approaches and transcriptional profiling of antigen-specific CD8(+) T cells, we reveal a common program of effector differentiation that is regulated by IL-2 and IL-12 signaling and the combined activities of the transcriptional regulators Blimp-1 and T-bet. The loss of both T-bet and Blimp-1 leads to abrogated cytotoxic function and ectopic IL-17 production in CD8(+) T cells. Overall, our data reveal two major overlapping pathways of effector differentiation governed by the availability of Blimp-1 and T-bet and suggest a model for cytokine-induced transcriptional changes that combine, quantitatively and qualitatively, to promote robust effector CD8(+) T cell differentiation.

A Regulatory Circuit Controlling the Dynamics of NFκB cRel Transitions B Cells from Proliferation to Plasma Cell Differentiation.

Humoral immunity depends on efficient activation of B cells and their subsequent differentiation into antibody-secreting cells (ASCs). The transcription factor NFκB cRel is critical for B cell proliferation, but incorporating its known regulatory interactions into a mathematical model of the ASC differentiation circuit prevented ASC generation in simulations. Indeed, experimental ectopic cRel expression blocked ASC differentiation by inhibiting the transcription factor Blimp1, and in wild-type (WT) cells cRel was dynamically repressed during ASC differentiation by Blimp1 binding the Rel locus. Including this bi-stable circuit of mutual cRel-Blimp1 antagonism into a multi-scale model revealed that dynamic repression of cRel controls the switch from B cell proliferation to ASC generation phases and hence the respective cell population dynamics. Our studies provide a mechanistic explanation of how dysregulation of this bi-stable circuit might result in pathologic B cell population phenotypes and thus offer new avenues for diagnostic stratification and treatment.

Transcription Factor PU.1 Promotes Conventional Dendritic Cell Identity and Function via Induction of Transcriptional Regulator DC-SCRIPT.

Dendritic cells (DCs) are can be broadly divided into conventional (cDC) and plasmacytoid (pDC) subsets. Despite the importance of this lineage diversity, its genetic basis is not fully understood. We found that conditional ablation of the Ets-family transcription factor PU.1 in DC-restricted progenitors led to increased pDC production at the expense of cDCs. PU.1 controlled many of the cardinal functions of DCs, such as antigen presentation by cDCs and type I interferon production by pDCs. Conditional ablation of PU.1 de-repressed the pDC transcriptional signature in cDCs. The combination of genome-wide mapping of PU.1 binding and gene expression analysis revealed a key role for PU.1 in maintaining cDC identity through the induction of the transcriptional regulator DC-SCRIPT. PU.1 activated DC-SCRIPT expression, which in turn promoted cDC formation, particularly of cDC1s, and repressed pDC development. Thus, cDC identity is regulated by a transcriptional node requiring PU.1 and DC-SCRIPT.

MYB orchestrates T cell exhaustion and response to checkpoint inhibition.

CD8+ T cells that respond to chronic viral infections or cancer are characterized by the expression of inhibitory receptors such as programmed cell death protein 1 (PD-1) and by the impaired production of cytokines. This state of restrained functionality-which is referred to as T cell exhaustion1,2-is maintained by precursors of exhausted T (TPEX) cells that express the transcription factor T cell factor 1 (TCF1), self-renew and give rise to TCF1- exhausted effector T cells3-6. Here we show that the long-term proliferative potential, multipotency and repopulation capacity of exhausted T cells during chronic infection are selectively preserved in a small population of transcriptionally distinct CD62L+ TPEX cells. The transcription factor MYB is not only essential for the development of CD62L+ TPEX cells and maintenance of the antiviral CD8+ T cell response, but also induces functional exhaustion and thereby prevents lethal immunopathology. Furthermore, the proliferative burst in response to PD-1 checkpoint inhibition originates exclusively from CD62L+ TPEX cells and depends on MYB. Our findings identify CD62L+ TPEX cells as a stem-like population that is central to the maintenance of long-term antiviral immunity and responsiveness to immunotherapy. Moreover, they show that MYB is a transcriptional orchestrator of two fundamental aspects of exhausted T cell responses: the downregulation of effector function and the long-term preservation of self-renewal capacity.

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.

The transcriptional regulators IRF4, BATF and IL-33 orchestrate development and maintenance of adipose tissue-resident regulatory T cells.

Foxp3(+) regulatory T (Treg) cells in visceral adipose tissue (VAT-Treg cells) are functionally specialized tissue-resident cells that prevent obesity-associated inflammation and preserve insulin sensitivity and glucose tolerance. Their development depends on the transcription factor PPAR-γ; however, the environmental cues required for their differentiation are unknown. Here we show that interleukin 33 (IL-33) signaling through the IL-33 receptor ST2 and myeloid differentiation factor MyD88 is essential for development and maintenance of VAT-Treg cells and sustains their transcriptional signature. Furthermore, the transcriptional regulators BATF and IRF4 were necessary for VAT-Treg differentiation through direct regulation of ST2 and PPAR-γ expression. IL-33 administration induced vigorous population expansion of VAT-Treg cells, which tightly correlated with improvements in metabolic parameters in obese mice. Human omental adipose tissue Treg cells also showed high ST2 expression, suggesting an evolutionarily conserved requirement for IL-33 in VAT-Treg cell homeostasis.

Transcription Factor Theft-PU.1 Caught Red-Handed.

The mechanistic understanding of gene-expression regulation is still evolving. In this issue of Immunity, Hosokawa et al. (2018) reveal that PU.1 represses transcription indirectly during early T cell development by "stealing" other regulators such as Runx1 and Satb1 from their DNA binding sites.

Effector Regulatory T Cell Differentiation and Immune Homeostasis Depend on the Transcription Factor Myb.

FoxP3-expressing regulatory T (Treg) cells are essential for maintaining immune homeostasis. Activated Treg cells undergo further differentiation into an effector state that highly expresses genes critical for Treg cell function, although how this process is coordinated on a transcriptional level is poorly understood. Here, we demonstrate that mice lacking the transcription factor Myb in Treg cells succumbed to a multi-organ inflammatory disease. Myb was specifically expressed in, and required for the differentiation of, thymus-derived effector Treg cells. The combination of transcriptome and genomic footprint analyses revealed that Myb directly regulated a large proportion of the gene expression specific to effector Treg cells, identifying Myb as a critical component of the gene regulatory network controlling effector Treg cell differentiation and function.