Lrp1 is essential for lethal Rift Valley fever hepatic disease in mice.

Rift Valley fever virus (RVFV) is an emerging arbovirus found in Africa. While RVFV is pantropic and infects many cells and tissues, viral replication and necrosis within the liver play a critical role in mediating severe disease. The low-density lipoprotein receptor-related protein 1 (Lrp1) is a recently identified host factor for cellular entry and infection by RVFV. The biological significance of Lrp1, including its role in hepatic disease in vivo, however, remains to be determined. Because Lrp1 has a high expression level in hepatocytes, we developed a mouse model in which Lrp1 is specifically deleted in hepatocytes to test how the absence of liver Lrp1 expression affects RVF pathogenesis. Mice lacking Lrp1 expression in hepatocytes showed minimal RVFV replication in the liver, longer time to death, and altered clinical signs toward neurological disease. In contrast, RVFV infection levels in other tissues showed no difference between the two genotypes. Therefore, Lrp1 is essential for RVF hepatic disease in mice.

Population dynamics and gene regulation of T cells in response to chronic antigen stimulation.

T cells are activated by antigen and co-stimulatory receptor signaling and undergo robust proliferation and differentiation into effector cells with protective function. Such quantitatively and qualitatively amplified T cell responses are effective in controlling acute infection and are followed by contraction of the effector population and the formation of resting memory T cells for enhanced protection against previously experienced antigens. However, in the face of persistent antigen during chronic viral infection, in autoimmunity, or in the tumor microenvironment, T cells exhibit distinct responses relative to those in acute insult in several aspects, including reduced clonal expansion and impaired effector function associated with inhibitory receptor expression, a state known as exhaustion. Nevertheless, their responses to chronic infection and tumors are sustained through the establishment of hierarchical heterogeneity, which preserves the duration of the response by generating newly differentiated effector cells. In this review, we highlight recent findings on distinct dynamics of T cell responses under "exhausting" conditions and the roles of the transcription factors that support attenuated yet long-lasting T cell responses as well as the establishment of dysfunctional states.

Divergent clonal differentiation trajectories of T cell exhaustion.

Chronic antigen exposure during viral infection or cancer promotes an exhausted T cell (Tex) state with reduced effector function. However, whether all antigen-specific T cell clones follow the same Tex differentiation trajectory remains unclear. Here, we generate a single-cell multiomic atlas of T cell exhaustion in murine chronic viral infection that redefines Tex phenotypic diversity, including two late-stage Tex subsets with either a terminal exhaustion (Texterm) or a killer cell lectin-like receptor-expressing cytotoxic (TexKLR) phenotype. We use paired single-cell RNA and T cell receptor sequencing to uncover clonal differentiation trajectories of Texterm-biased, TexKLR-biased or divergent clones that acquire both phenotypes. We show that high T cell receptor signaling avidity correlates with Texterm, whereas low avidity correlates with effector-like TexKLR fate. Finally, we identify similar clonal differentiation trajectories in human tumor-infiltrating lymphocytes. These findings reveal clonal heterogeneity in the T cell response to chronic antigen that influences Tex fates and persistence.

Antigen-specific depletion of CD4+ T cells by CAR T cells reveals distinct roles of higher- and lower-affinity TCRs during autoimmunity.

Both higher- and lower-affinity self-reactive CD4+ T cells are expanded in autoimmunity; however, their individual contribution to disease remains unclear. We addressed this question using peptide-MHCII chimeric antigen receptor (pMHCII-CAR) T cells to specifically deplete peptide-reactive T cells in mice. Integration of improvements in CAR engineering with TCR repertoire analysis was critical for interrogating in vivo the role of TCR affinity in autoimmunity. Our original MOG35-55 pMHCII-CAR, which targeted only higher-affinity TCRs, could prevent the induction of experimental autoimmune encephalomyelitis (EAE). However, pMHCII-CAR enhancements to pMHCII stability, as well as increased survivability via overexpression of a dominant-negative Fas, were required to target lower-affinity MOG-specific T cells and reverse ongoing clinical EAE. Thus, these data suggest a model in which higher-affinity autoreactive T cells are required to provide the "activation energy" for initiating neuroinflammatory injury, but lower-affinity cells are sufficient to maintain ongoing disease.

Oropouche orthobunyavirus infection is mediated by the cellular host factor Lrp1.

Oropouche orthobunyavirus (OROV; Peribunyaviridae) is a mosquito-transmitted virus that causes widespread human febrile illness in South America, with occasional progression to neurologic effects. Host factors mediating the cellular entry of OROV are undefined. Here, we show that OROV uses the host protein low-density lipoprotein-related protein 1 (Lrp1) for efficient cellular infection. Cells from evolutionarily distinct species lacking Lrp1 were less permissive to OROV infection than cells with Lrp1. Treatment of cells with either the high-affinity Lrp1 ligand receptor-associated protein (RAP) or recombinant ectodomain truncations of Lrp1 significantly reduced OROV infection. In addition, chimeric vesicular stomatitis virus (VSV) expressing OROV glycoproteins (VSV-OROV) bound to the Lrp1 ectodomain in vitro. Furthermore, we demonstrate the biological relevance of the OROV-Lrp1 interaction in a proof-of-concept mouse study in which treatment of mice with RAP at the time of infection reduced tissue viral load and promoted survival from an otherwise lethal infection. These results with OROV, along with the recent finding of Lrp1 as an entry factor for Rift Valley fever virus, highlight the broader significance of Lrp1 in cellular infection by diverse bunyaviruses. Shared strategies for entry, such as the critical function of Lrp1 defined here, provide a foundation for the development of pan-bunyaviral therapeutics.

Ablation of cDC2 development by triple mutations within the Zeb2 enhancer.

The divergence of the common dendritic cell progenitor1-3 (CDP) into the conventional type 1 and type 2 dendritic cell (cDC1 and cDC2, respectively) lineages4,5 is poorly understood. Some transcription factors act in the commitment of already specified progenitors-such as BATF3, which stabilizes Irf8 autoactivation at the +32 kb Irf8 enhancer4,6-but the mechanisms controlling the initial divergence of CDPs remain unknown. Here we report the transcriptional basis of CDP divergence and describe the first requirements for pre-cDC2 specification. Genetic epistasis analysis7 suggested that Nfil3 acts upstream of Id2, Batf3 and Zeb2 in cDC1 development but did not reveal its mechanism or targets. Analysis of newly generated NFIL3 reporter mice showed extremely transient NFIL3 expression during cDC1 specification. CUT&RUN and chromatin immunoprecipitation followed by sequencing identified endogenous NFIL3 binding in the -165 kb Zeb2 enhancer8 at three sites that also bind the CCAAT-enhancer-binding proteins C/EBPα and C/EBPß. In vivo mutational analysis using CRISPR-Cas9 targeting showed that these NFIL3-C/EBP sites are functionally redundant, with C/EBPs supporting and NFIL3 repressing Zeb2 expression at these sites. A triple mutation of all three NFIL3-C/EBP sites ablated Zeb2 expression in myeloid, but not lymphoid progenitors, causing the complete loss of pre-cDC2 specification and mature cDC2 development in vivo. These mice did not generate T helper 2 (TH2) cell responses against Heligmosomoides polygyrus infection, consistent with cDC2 supporting TH2 responses to helminths9-11. Thus, CDP divergence into cDC1 or cDC2 is controlled by competition between NFIL3 and C/EBPs at the -165 kb Zeb2 enhancer.

BCL6-dependent TCF-1+ progenitor cells maintain effector and helper CD4+ T cell responses to persistent antigen.

Soon after activation, CD4+ T cells are segregated into BCL6+ follicular helper (Tfh) and BCL6- effector (Teff) T cells. Here, we explored how these subsets are maintained during chronic antigen stimulation using the mouse chronic LCMV infection model. Using single cell-transcriptomic and epigenomic analyses, we identified a population of PD-1+ TCF-1+ CD4+ T cells with memory-like features. TCR clonal tracing and adoptive transfer experiments demonstrated that these cells have self-renewal capacity and continue to give rise to both Teff and Tfh cells, thus functioning as progenitor cells. Conditional deletion experiments showed Bcl6-dependent development of these progenitors, which were essential for sustaining antigen-specific CD4+ T cell responses to chronic infection. An analogous CD4+ T cell population developed in draining lymph nodes in response to tumors. Our study reveals the heterogeneity and plasticity of CD4+ T cells during persistent antigen exposure and highlights their population dynamics through a stable, bipotent intermediate state.

Hobit confers tissue-dependent programs to type 1 innate lymphoid cells.

Identification of type 1 innate lymphoid cells (ILC1s) has been problematic. The transcription factor Hobit encoded by Zfp683 has been proposed as a major driver of ILC1 programs. Using Zfp683 reporter mice, we showed that correlation of Hobit expression with ILC1s is tissue- and context-dependent. In liver and intestinal mucosa, Zfp683 expression correlated well with ILC1s; in salivary glands, Zfp683 was coexpressed with the natural killer (NK) master transcription factors Eomes and TCF1 in a unique cell population, which we call ILC1-like NK cells; during viral infection, Zfp683 was induced in conventional NK cells of spleen and liver. The impact of Zfp683 deletion on ILC1s and NK cells was also multifaceted, including a marked decrease in granzyme- and interferon-gamma (IFNγ)-producing ILC1s in the liver, slightly fewer ILC1s and more Eomes+ TCF1+ ILC1-like NK cells in salivary glands, and only reduced production of granzyme B by ILC1 in the intestinal mucosa. NK cell-mediated control of viral infection was unaffected. We conclude that Hobit has two major impacts on ILC1s: It sustains liver ILC1 numbers, while promoting ILC1 functional maturation in other tissues by controlling TCF1, Eomes, and granzyme expression.

Lrp1 is a host entry factor for Rift Valley fever virus.

Rift Valley fever virus (RVFV) is a zoonotic pathogen with pandemic potential. RVFV entry is mediated by the viral glycoprotein (Gn), but host entry factors remain poorly defined. Our genome-wide CRISPR screen identified low-density lipoprotein receptor-related protein 1 (mouse Lrp1/human LRP1), heat shock protein (Grp94), and receptor-associated protein (RAP) as critical host factors for RVFV infection. RVFV Gn directly binds to specific Lrp1 clusters and is glycosylation independent. Exogenous addition of murine RAP domain 3 (mRAPD3) and anti-Lrp1 antibodies neutralizes RVFV infection in taxonomically diverse cell lines. Mice treated with mRAPD3 and infected with pathogenic RVFV are protected from disease and death. A mutant mRAPD3 that binds Lrp1 weakly failed to protect from RVFV infection. Together, these data support Lrp1 as a host entry factor for RVFV infection and define a new target to limit RVFV infections.

Unexpected suppression of tumorigenesis by c-MYC via TFAP4-dependent restriction of stemness in B lymphocytes.

The proliferative burst of B lymphocytes is essential for antigen receptor repertoire diversification during the development and selective expansion of antigen-specific clones during immune responses. High proliferative activity inevitably promotes oncogenesis, the risk of which is further elevated in B lymphocytes by endogenous gene rearrangement and somatic mutations. However, B-cell-derived cancers are rare, perhaps owing to putative intrinsic tumor-suppressive mechanisms. We show that c-MYC facilitates B-cell proliferation as a protumorigenic driver and unexpectedly coengages counteracting tumor suppression through its downstream factor TFAP4. TFAP4 is mutated in human lymphoid malignancies, particularly in >10% of Burkitt lymphomas, and reduced TFAP4 expression was associated with poor survival of patients with MYC-high B-cell acute lymphoblastic leukemia. In mice, insufficient TFAP4 expression accelerated c-MYC-driven transformation of B cells. Mechanistically, c-MYC suppresses the stemness of developing B cells by inducing TFAP4 and restricting self-renewal of proliferating B cells. Thus, the pursuant transcription factor cascade functions as a tumor suppressor module that safeguards against the transformation of developing B cells.

Identification of a T-bethi Quiescent Exhausted CD8 T Cell Subpopulation That Can Differentiate into TIM3+CX3CR1+ Effectors and Memory-like Cells.

Persistent Ag induces a dysfunctional CD8 T cell state known as "exhaustion" characterized by PD-1 expression. Nevertheless, exhausted CD8 T cells retain functionality through continued differentiation of progenitor into effector cells. However, it remains ill-defined how CD8 T cell effector responses are sustained in situ. In this study, we show using the mouse chronic lymphocytic choriomeningitis virus infection model that CX3CR1+ CD8 T cells contain a T-bet-dependent TIM3-PD-1lo subpopulation that is distinct from the TIM3+CX3CR1+PD-1+ proliferative effector subset. The TIM3-CX3CR1+ cells are quiescent and express a low but significant level of the transcription factor TCF-1, demonstrating similarity to TCF-1hi progenitor CD8 T cells. Furthermore, following the resolution of lymphocytic choriomeningitis virus viremia, a substantial proportion of TCF-1+ memory-like CD8 T cells show evidence of CX3CR1 expression during the chronic phase of the infection. Our results suggest a subset of the CX3CR1+ exhausted population demonstrates progenitor-like features that support the generation of the CX3CR1+ effector pool from the TCF-1hi progenitors and contribute to the memory-like pool following the resolution of viremia.

Differential usage of transcriptional repressor Zeb2 enhancers distinguishes adult and embryonic hematopoiesis.

The transcriptional repressor ZEB2 regulates development of many cell fates among somatic, neural, and hematopoietic lineages, but the basis for its requirement in these diverse lineages is unclear. Here, we identified a 400-basepair (bp) region located 165 kilobases (kb) upstream of the Zeb2 transcriptional start site (TSS) that binds the E proteins at several E-box motifs and was active in hematopoietic lineages. Germline deletion of this 400-bp region (Zeb2Δ-165mice) specifically prevented Zeb2 expression in hematopoietic stem cell (HSC)-derived lineages. Zeb2Δ-165 mice lacked development of plasmacytoid dendritic cells (pDCs), monocytes, and B cells. All macrophages in Zeb2Δ-165 mice were exclusively of embryonic origin. Using single-cell chromatin profiling, we identified a second Zeb2 enhancer located at +164-kb that was selectively active in embryonically derived lineages, but not HSC-derived ones. Thus, Zeb2 expression in adult, but not embryonic, hematopoiesis is selectively controlled by the -165-kb Zeb2 enhancer.

Thymic development of gut-microbiota-specific T cells.

Humans and their microbiota have coevolved a mutually beneficial relationship in which the human host provides a hospitable environment for the microorganisms and the microbiota provides many advantages for the host, including nutritional benefits and protection from pathogen infection1. Maintaining this relationship requires a careful immune balance to contain commensal microorganisms within the lumen while limiting inflammatory anti-commensal responses1,2. Antigen-specific recognition of intestinal microorganisms by T cells has previously been described3,4. Although the local environment shapes the differentiation of effector cells3-5 it is unclear how microbiota-specific T cells are educated in the thymus. Here we show that intestinal colonization in early life leads to the trafficking of microbial antigens from the intestine to the thymus by intestinal dendritic cells, which then induce the expansion of microbiota-specific T cells. Once in the periphery, microbiota-specific T cells have pathogenic potential or can protect against related pathogens. In this way, the developing microbiota shapes and expands the thymic and peripheral T cell repertoire, allowing for enhanced recognition of intestinal microorganisms and pathogens.

Bromodomain protein BRD4 directs and sustains CD8 T cell differentiation during infection.

In response to infection, pathogen-specific CD8 T cells differentiate into functionally diverse effector and memory T cell populations critical for resolving disease and providing durable immunity. Through small-molecule inhibition, RNAi studies, and induced genetic deletion, we reveal an essential role for the chromatin modifier and BET family member BRD4 in supporting the differentiation and maintenance of terminally fated effector CD8 T cells during infection. BRD4 bound diverse regulatory regions critical to effector T cell differentiation and controlled transcriptional activity of terminal effector-specific super-enhancers in vivo. Consequentially, induced deletion of Brd4 or small molecule-mediated BET inhibition impaired maintenance of a terminal effector T cell phenotype. BRD4 was also required for terminal differentiation of CD8 T cells in the tumor microenvironment in murine models, which we show has implications for immunotherapies. Taken together, these data reveal an unappreciated requirement for BRD4 in coordinating activity of cis regulatory elements to control CD8 T cell fate and lineage stability.

Open conformation of tetraspanins shapes interaction partner networks on cell membranes.

Tetraspanins, including CD53 and CD81, regulate a multitude of cellular processes through organizing an interaction network on cell membranes. Here, we report the crystal structure of CD53 in an open conformation poised for partner interaction. The large extracellular domain (EC2) of CD53 protrudes away from the membrane surface and exposes a variable region, which is identified by hydrogen-deuterium exchange as the common interface for CD53 and CD81 to bind partners. The EC2 orientation in CD53 is supported by an extracellular loop (EC1). At the closed conformation of CD81, however, EC2 disengages from EC1 and rotates toward the membrane, thereby preventing partner interaction. Structural simulation shows that EC1-EC2 interaction also supports the open conformation of CD81. Disrupting this interaction in CD81 impairs the accurate glycosylation of its CD19 partner, the target for leukemia immunotherapies. Moreover, EC1 mutations in CD53 prevent the chemotaxis of pre-B cells toward a chemokine that supports B-cell trafficking and homing within the bone marrow, a major CD53 function identified here. Overall, an open conformation is required for tetraspanin-partner interactions to support myriad cellular processes.

Strength of tonic T cell receptor signaling instructs T follicular helper cell-fate decisions.

T follicular helper (TFH) cells are critical in adaptive immune responses to pathogens and vaccines; however, what drives the initiation of their developmental program remains unclear. Studies suggest that a T cell antigen receptor (TCR)-dependent mechanism may be responsible for the earliest TFH cell-fate decision, but a critical aspect of the TCR has been overlooked: tonic TCR signaling. We hypothesized that tonic signaling influences early TFH cell development. Here, two murine TCR-transgenic CD4+ T cells, LLO56 and LLO118, which recognize the same antigenic peptide presented on major histocompatibility complex molecules but experience disparate strengths of tonic signaling, revealed low tonic signaling promotes TFH cell differentiation. Polyclonal T cells paralleled these findings, with naive Nur77 expression distinguishing TFH cell potential. Two mouse lines were also generated to both increase and decrease tonic signaling strength, directly establishing an inverse relationship between tonic signaling strength and TFH cell development. Our findings elucidate a central role for tonic TCR signaling in early TFH cell-lineage decisions.

PD-1 Signaling Promotes Control of Chronic Viral Infection by Restricting Type-I-Interferon-Mediated Tissue Damage.

Immune responses are essential for pathogen elimination but also cause tissue damage, leading to disease or death. However, it is unclear how the host immune system balances control of infection and protection from the collateral tissue damage. Here, we show that PD-1-mediated restriction of immune responses is essential for durable control of chronic LCMV infection in mice. In contrast to responses in the chronic phase, PD-1 blockade in the subacute phase of infection paradoxically results in viral persistence. This effect is associated with damage to lymphoid architecture and subsequently decreases adaptive immune responses. Moreover, this tissue damage is type I interferon dependent, as sequential blockade of the interferon receptor and PD-1 pathways prevents immunopathology and enhances control of infection. We conclude that PD-1-mediated suppression is required as an immunoregulatory mechanism for sustained responses to chronic viral infection by antagonizing type-I interferon-dependent immunopathology.

Acetate Promotes T Cell Effector Function during Glucose Restriction.

Competition for nutrients like glucose can metabolically restrict T cells and contribute to their hyporesponsiveness during cancer. Metabolic adaptation to the surrounding microenvironment is therefore key for maintaining appropriate cell function. For instance, cancer cells use acetate as a substrate alternative to glucose to fuel metabolism and growth. Here, we show that acetate rescues effector function in glucose-restricted CD8+ T cells. Mechanistically, acetate promotes histone acetylation and chromatin accessibility and enhances IFN-γ gene transcription and cytokine production in an acetyl-CoA synthetase (ACSS)-dependent manner. Ex vivo acetate treatment increases IFN-γ production by exhausted T cells, whereas reducing ACSS expression in T cells impairs IFN-γ production by tumor-infiltrating lymphocytes and tumor clearance. Thus, hyporesponsive T cells can be epigenetically remodeled and reactivated by acetate, suggesting that pathways regulating the use of substrates alternative to glucose could be therapeutically targeted to promote T cell function during cancer.

Regulation of metabolic supply and demand during B cell activation and subsequent differentiation.

B cell activation and differentiation are associated with marked changes in proliferative and effector functions. Each stage of B cell differentiation thus has unique metabolic demands. New studies have provided insight on how nutrient uptake and usage by B cells are regulated by B cell receptor signals, autophagy, mammalian target of rapamycin, and transcriptional control of transporters and rate-limiting enzymes. A recurring theme is that these pathways play distinct roles ranging from survival to antibody production, depending on the B cell fate. We review recently published data that define how these pathways control metabolic flux in B cells, with a particular emphasis on genetic and in vivo evidence. We further discuss how lessons from T cells can guide future directions.