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Antibody agonists trigger immune receptor signaling through local exclusion of receptor-type protein tyrosine phosphatases.
Antibodies can block immune receptor engagement or trigger the receptor machinery to initiate signaling. We hypothesized that antibody agonists trigger signaling by sterically excluding large receptor-type protein tyrosine phosphatases (RPTPs) such as CD45 from sites of receptor engagement. An agonist targeting the costimulatory receptor CD28 produced signals that depended on antibody immobilization and were sensitive to the sizes of the receptor, the RPTPs, and the antibody itself. Although both the agonist and a non-agonistic anti-CD28 antibody locally excluded CD45, the agonistic antibody was more effective. An anti-PD-1 antibody that bound membrane proximally excluded CD45, triggered Src homology 2 domain-containing phosphatase 2 recruitment, and suppressed systemic lupus erythematosus and delayed-type hypersensitivity in experimental models. Paradoxically, nivolumab and pembrolizumab, anti-PD-1-blocking antibodies used clinically, also excluded CD45 and were agonistic in certain settings. Reducing these agonistic effects using antibody engineering improved PD-1 blockade. These findings establish a framework for developing new and improved therapies for autoimmunity and cancer.
Harnessing Single-Cell Technologies in the Search for New Therapies for Diamond-Blackfan Anemia Syndrome.
The emergence of multiomic single-cell technologies over the last decade has led to improved insights into both normal hematopoiesis and its perturbation in a variety of hematological disorders. Diamond-Blackfan anemia (DBA) syndrome is one such disorder where single-cell assays have helped to delineate the cellular and molecular defects underlying the disease. DBA is caused by heterozygous loss-of-function germline variants in genes encoding ribosomal proteins (RPs). Despite the widespread role of ribosomes in hematopoiesis, the most frequent and severe cytopenia in DBA is anemia. In this review we discussed how single-cell studies, including clonogenic cell culture assays, fluorescence-activated cell sorting (FACS) and single-cell RNA sequencing (scRNA-seq), have led to insights into the pathogenesis of DBA. The main therapies are regular blood transfusions, glucocorticoids, or hematopoietic stem cell transplantation (HSCT) but all are associated with significant morbidity and mortality. We will therefore outline how single-cell studies can inform new therapies for DBA. Furthermore, we discussed how DBA serves as a useful model for understanding normal erythropoiesis in terms of its cellular hierarchy, molecular regulation during homeostasis, and response to "stress."
Aldehyde-mediated inhibition of asparagine biosynthesis has implications for diabetes and alcoholism.
Patients with alcoholism and type 2 diabetes manifest altered metabolism, including elevated aldehyde levels and unusually low asparagine levels. We show that asparagine synthetase B (ASNS), the only human asparagine-forming enzyme, is inhibited by disease-relevant reactive aldehydes, including formaldehyde and acetaldehyde. Cellular studies show non-cytotoxic amounts of reactive aldehydes induce a decrease in asparagine levels. Biochemical analyses reveal inhibition results from reaction of the aldehydes with the catalytically important N-terminal cysteine of ASNS. The combined cellular and biochemical results suggest a possible mechanism underlying the low asparagine levels in alcoholism and diabetes. The results will stimulate research on the biological consequences of the reactions of aldehydes with nucleophilic residues.
Hepcidin-Mediated Hypoferremia Disrupts Immune Responses to Vaccination and Infection.
BACKGROUND: How specific nutrients influence adaptive immunity is of broad interest. Iron deficiency is the most common micronutrient deficiency worldwide and imparts a significant burden of global disease; however, its effects on immunity remain unclear. METHODS: We used a hepcidin mimetic and several genetic models to examine the effect of low iron availability on T cells in vitro and on immune responses to vaccines and viral infection in mice. We examined humoral immunity in human patients with raised hepcidin and low serum iron caused by mutant TMPRSS6. We tested the effect of iron supplementation on vaccination-induced humoral immunity in piglets, a natural model of iron deficiency. FINDINGS: We show that low serum iron (hypoferremia), caused by increased hepcidin, severely impairs effector and memory responses to immunizations. The intensified metabolism of activated lymphocytes requires the support of enhanced iron acquisition, which is facilitated by IRP1/2 and TFRC. Accordingly, providing extra iron improved the response to vaccination in hypoferremic mice and piglets, while conversely, hypoferremic humans with chronically increased hepcidin have reduced concentrations of antibodies specific for certain pathogens. Imposing hypoferremia blunted the T cell, B cell, and neutralizing antibody responses to influenza virus infection in mice, allowing the virus to persist and exacerbating lung inflammation and morbidity. CONCLUSIONS: Hypoferremia, a well-conserved physiological innate response to infection, can counteract the development of adaptive immunity. This nutrient trade-off is relevant for understanding and improving immune responses to infections and vaccines in the globally common contexts of iron deficiency and inflammatory disorders. FUNDING: Medical Research Council, UK.
Germinal Center B Cells Replace Their Antigen Receptors in Dark Zones and Fail Light Zone Entry when Immunoglobulin Gene Mutations are Damaging.
Adaptive immunity involves the development of bespoke antibodies in germinal centers (GCs) through immunoglobulin somatic hypermutation (SHM) in GC dark zones (DZs) and clonal selection in light zones (LZs). Accurate selection requires that cells fully replace surface B cell receptors (BCRs) following SHM, but whether this happens before LZ entry is not clear. We found that most GC B cells degrade pre-SHM receptors before leaving the DZ, and that B cells acquiring crippling mutations during SHM rarely reached the LZ. Instead, apoptosis was triggered preferentially in late G1, a stage wherein cells with functional BCRs re-entered cell cycle or reduced surface expression of the chemokine receptor CXCR4 to enable LZ migration. Ectopic expression of the anti-apoptotic gene Bcl2 was not sufficient for cells with damaging mutations to reach the LZ, suggesting that BCR-dependent cues may actively facilitate the transition. Thus, BCR replacement and pre-screening in DZs prevents the accumulation of clones with non-functional receptors and facilitates selection in the LZ.
Germinal centers output clonally diverse plasma cell populations expressing high- and low-affinity antibodies.
Germinal centers (GCs) form in lymph nodes after immunization or infection to facilitate antibody affinity maturation and memory and plasma cell (PC) development. PC differentiation is thought to involve stringent selection for GC B cells expressing the highest-affinity antigen receptors, but how this plays out during complex polyclonal responses is unclear. We combine temporal lineage tracing with antibody characterization to gain a snapshot of PCs developing during influenza infection. GCs co-mature B cell clones with antibody affinities spanning multiple orders of magnitude; however, each generates PCs with similar efficiencies, including weak binders. Within lineages, PC selection is not restricted to variants with the highest-affinity antibodies. Differentiation is commonly associated with proliferative expansion to produce "nodes" of identical PCs. Immunization-induced GCs generate fewer PCs but still of low- and high-antibody affinities. We propose that generating low-affinity antibody PCs reflects an evolutionary compromise to facilitate diverse serum antibody responses.
Expression of the Plasma Cell Transcriptional Regulator Blimp-1 by Dark Zone Germinal Center B Cells During Periods of Proliferation.
Long-lived plasma cells (PCs) develop in germinal centers (GCs) by the differentiation of affinity matured B cells. Antibody affinity maturation involves iterative rounds of somatic hypermutation in dark zones (DZs) and selection in light zones (LZs), however the details of where, when and how PC commitment occurs are not well-understood. Fate bifurcation at the time of selection is one possibility, with the very highest affinity GC B cells differentiating as an alternative to DZ re-entry. However, how this model fits with a need to also retain these clones in the response is not clear. Here, we show that subsets of bona fide DZ cells express the plasma cell master regulator Blimp-1 at low levels during periods of proliferation. Ex vivo culture experiments demonstrate that these cells are not yet committed to plasma cell differentiation but that they may be sensitized to go down that route. Contrary to models in which T cells directly select GC B cells to begin expressing Blimp-1, we found that expression of this transcriptional regulator occurred even when follicular helper T cells were ablated. We speculate that Blimp-1 may be induced during proliferation in the DZ, and that as such single selected cells might give rise to both GC and post-GC progeny.
Scoring a HAT-Trick against Lymphoma.
The genes encoding the histone acetyltransferases (HATs) CREBB-binding protein (CREBBP) and EP300 are commonly mutated in germinal-center-derived B cell lymphomas, and their inactivation is thought to contribute to lymphomagenesis. In this issue of Immunity, Meyer et al. (2019) demonstrate that the somatic inactivation of one histone modifying enzyme might leave lymphomas uniquely sensitive to antagonists of the other.
Apoptotic cell fragments locally activate tingible body macrophages in the germinal center.
Germinal centers (GCs) that form within lymphoid follicles during antibody responses are sites of massive cell death. Tingible body macrophages (TBMs) are tasked with apoptotic cell clearance to prevent secondary necrosis and autoimmune activation by intracellular self antigens. We show by multiple redundant and complementary methods that TBMs derive from a lymph node-resident, CD169-lineage, CSF1R-blockade-resistant precursor that is prepositioned in the follicle. Non-migratory TBMs use cytoplasmic processes to chase and capture migrating dead cell fragments using a "lazy" search strategy. Follicular macrophages activated by the presence of nearby apoptotic cells can mature into TBMs in the absence of GCs. Single-cell transcriptomics identified a TBM cell cluster in immunized lymph nodes which upregulated genes involved in apoptotic cell clearance. Thus, apoptotic B cells in early GCs trigger activation and maturation of follicular macrophages into classical TBMs to clear apoptotic debris and prevent antibody-mediated autoimmune diseases.
Secondary influenza challenge triggers resident memory B cell migration and rapid relocation to boost antibody secretion at infected sites.
Resident memory B (BRM) cells develop and persist in the lungs of influenza-infected mice and humans; however, their contribution to recall responses has not been defined. Here, we used two-photon microscopy to visualize BRM cells within the lungs of influenza -virus immune and reinfected mice. Prior to re-exposure, BRM cells were sparsely scattered throughout the tissue, displaying limited motility. Within 24 h of rechallenge, these cells increased their migratory capacity, localized to infected sites, and subsequently differentiated into plasma cells. Alveolar macrophages mediated this process, in part by inducing expression of chemokines CXCL9 and CXCL10 from infiltrating inflammatory cells. This led to the recruitment of chemokine receptor CXCR3-expressing BRM cells to infected regions and increased local antibody concentrations. Our study uncovers spatiotemporal mechanisms that regulate lung BRM cell reactivation and demonstrates their capacity to rapidly deliver antibodies in a highly localized manner to sites of viral replication.
Competition for refueling rather than cyclic reentry initiation evident in germinal centers.
Antibody affinity maturation occurs in germinal centers (GCs) through iterative rounds of somatic hypermutation and proliferation in dark zones (DZs) and selection in light zones (LZs). GC B cells exit cell cycle a number of hours before entering LZs; therefore, continued participation in responses requires that they subsequently reenter cell cycle and move back to DZs, a process known as cyclic reentry. Affinity enhancements are thought to arise by B cells having to compete to initiate cyclic reentry each time they enter LZs, with T cell help being a major determinant; however, direct proof is lacking. Using Fucci2 mice, we confirmed an association between B cell receptor affinity and the first step of cyclic reentry, S phase initiation from a resting LZ state. However, neither T cell ablation nor MHCII deletion prevented resting LZ cells from reentering cell cycle, and this late G1-S transition was also not detectably restricted by competition. In contrast, using BATF induction as exemplar, we found that T cells "refueled" LZ cells in an affinity-dependent manner that was limited by both competition and cells' intrinsic antigen-acquiring abilities. Therefore, cyclic reentry initiation and B cell refueling are independently regulated in GCs, which may contribute to permitting cells of different competencies to be sustained alongside each other and allow T cell support to be provided across a dynamic range commensurate with affinity. We speculate that this less binary selection mechanism could help GCs nurture complex antibody maturation pathways and support the clonal diversity required for countering fast-evolving pathogens.
Ubiquitin-mediated fluctuations in MHC class II facilitate efficient germinal center B cell responses.
Antibody affinity maturation occurs in germinal centers (GCs) through iterative rounds of somatic hypermutation and selection. Selection involves B cells competing for T cell help based on the amount of antigen they capture and present on their MHC class II (MHCII) proteins. How GC B cells are able to rapidly and repeatedly transition between mutating their B cell receptor genes and then being selected shortly after is not known. We report that MHCII surface levels and degradation are dynamically regulated in GC B cells. Through ectopic expression of a photoconvertible MHCII-mKikGR chimeric gene, we found that individual GC B cells differed in the rates of MHCII protein turnover. Fluctuations in surface MHCII levels were dependent on ubiquitination and the E3 ligase March1. Increases in March1 expression in centroblasts correlated with decreases in surface MHCII levels, whereas CD83 expression in centrocytes helped to stabilize MHCII at that stage. Defects in MHCII ubiquitination caused GC B cells to accumulate greater amounts of a specific peptide-MHCII (pMHCII), suggesting that MHCII turnover facilitates the replacement of old complexes. We propose that pMHCII complexes are periodically targeted for degradation in centroblasts to favor the presentation of recently acquired antigens, thereby promoting the fidelity and efficiency of selection.
The microRNA cluster miR-17∼92 promotes TFH cell differentiation and represses subset-inappropriate gene expression.
Follicular helper T cells (TFH cells) are the prototypic helper T cell subset specialized to enable B cells to form germinal centers (GCs) and produce high-affinity antibodies. We found that expression of microRNAs (miRNAs) by T cells was essential for TFH cell differentiation. More specifically, we show that after immunization of mice with protein, the miRNA cluster miR-17∼92 was critical for robust differentiation and function of TFH cells in a cell-intrinsic manner that occurred regardless of changes in proliferation. In a viral infection model, miR-17∼92 restrained the expression of genes 'inappropriate' to the TFH cell subset, including the direct miR-17∼92 target Rora. Removal of one Rora allele partially 'rescued' the inappropriate gene signature in miR-17∼92-deficient TFH cells. Our results identify the miR-17∼92 cluster as a critical regulator of T cell-dependent antibody responses, TFH cell differentiation and the fidelity of the TFH cell gene-expression program.
Pathways of memory CD8+ T-cell development.
CD8(+) T-cell responses must have at least two components, a replicative cell type that proliferates in the secondary lymphoid tissue and that is responsible for clonal expansion, and cytotoxic cells with effector functions that mediate the resolution of the infection in the peripheral tissues. To confer memory, the response must also generate replication-competent T cells that persist in the absence of antigen after the primary infection is cleared. The current models of memory differentiation differ in regards to whether or not memory CD8(+) T cells acquire effector functions during their development. In this review we discuss the existing models for memory development and the consequences that the recent finding that memory CD8(+) T cells may express granzyme B during their development has for them. We propose that memory CD8(+) T cells represent a self-renewing population of T cells that may acquire effector functions but that do not lose the naïve-like attributes of lymphoid homing, antigen-independent persistence or the capacity for self-renewal.
Germinal centers: programmed for affinity maturation and antibody diversification.
The seminal discovery by Eisen that antibodies undergo improvements in antigen-binding affinity over the course of an immune response led to a long running search for the underlying mechanism. Germinal centers in lymphoid organs are now recognized to be critically involved in this phenomenon, known as antibody affinity maturation. As well as improving in affinity for specific epitopes, some antibody responses maintain or even increase their breadth of antigen-recognition over time. This has led to another intense line of research aimed at understanding how broadly neutralizing anti-pathogen responses are generated. Recent work indicates that germinal centers also play an important role in the diversification process. We discuss current understanding of how germinal centers are programmed to support both affinity maturation and antibody diversification.