Trafficking of B cell antigen in lymph nodes.

The clonal selection theory first proposed by Macfarlane Burnet is a cornerstone of immunology (1). At the time, it revolutionized the thinking of immunologists because it provided a simple explanation for lymphocyte specificity, immunological memory, and elimination of self-reactive clones (2). The experimental demonstration by Nossal & Lederberg (3) that B lymphocytes bear receptors for a single antigen raised the central question of where B lymphocytes encounter antigen. This question has remained mostly unanswered until recently. Advances in techniques such as multiphoton intravital microscopy (4, 5) have provided new insights into the trafficking of B cells and their antigen. In this review, we summarize these advances in the context of our current view of B cell circulation and activation.

Clonal Evolution of Autoreactive Germinal Centers.

Germinal centers (GCs) are the primary sites of clonal B cell expansion and affinity maturation, directing the production of high-affinity antibodies. This response is a central driver of pathogenesis in autoimmune diseases, such as systemic lupus erythematosus (SLE), but the natural history of autoreactive GCs remains unclear. Here, we present a novel mouse model where the presence of a single autoreactive B cell clone drives the TLR7-dependent activation, expansion, and differentiation of other autoreactive B cells in spontaneous GCs. Once tolerance was broken for one self-antigen, autoreactive GCs generated B cells targeting other self-antigens. GCs became independent of the initial clone and evolved toward dominance of individual clonal lineages, indicating affinity maturation. This process produced serum autoantibodies to a breadth of self-antigens, leading to antibody deposition in the kidneys. Our data provide insight into the maturation of the self-reactive B cell response, contextualizing the epitope spreading observed in autoimmune disease.

Haplosufficiency of MHCII in autoreactive germinal center B cells.

Utilizing an autoimmune bone marrow chimera model we determined that B cells depend critically on MHCII expression for participation in the germinal center, but cells displaying a 50% reduction in surface MHCII compete efficiently with their wild-type counterparts. This provides insights into the requirements for germinal center participation.

Characterization of DNA-protein complexes by nanoparticle tracking analysis and their association with systemic lupus erythematosus.

Nanotechnology enables investigations of single biomacromolecules, but technical challenges have limited the application in liquid biopsies, for example, blood plasma. Nonetheless, tools to characterize single molecular species in such samples represent a significant unmet need with the increasing appreciation of the physiological importance of protein structural changes at nanometer scale. Mannose-binding lectin (MBL) is an oligomeric plasma protein and part of the innate immune system through its ability to activate complement. MBL also serves a role as a scavenger for cellular debris, especially DNA. This may link functions of MBL with several inflammatory diseases in which cell-free DNA now appears to play a role, but mechanistic insight has been lacking. By making nanoparticle tracking analysis possible in human plasma, we now show that superoligomeric structures of MBL form nanoparticles with DNA. These oligomers correlate with disease activity in systemic lupus erythematosus patients. With the direct quantification of the hydrodynamic radius, calculations following the principles of Taylor dispersion in the blood stream connect the size of these complexes to endothelial inflammation, which is among the most important morbidities in lupus. Mechanistic insight from an animal model of lupus supported that DNA-stabilized superoligomers stimulate the formation of germinal center B cells and drive loss of immunological tolerance. The formation involves an inverse relationship between the concentration of MBL superoligomers and antibodies to double-stranded DNA. Our approach implicates the structure of DNA-protein nanoparticulates in the pathobiology of autoimmune diseases.

Capture of influenza by medullary dendritic cells via SIGN-R1 is essential for humoral immunity in draining lymph nodes.

A major pathway for B cell acquisition of lymph-borne particulate antigens relies on antigen capture by subcapsular sinus macrophages of the lymph node. Here we tested whether this mechanism is also important for humoral immunity to inactivated influenza virus. By multiple approaches, including multiphoton intravital imaging, we found that antigen capture by sinus-lining macrophages was important for limiting the systemic spread of virus but not for the generation of influenza-specific humoral immunity. Instead, we found that dendritic cells residing in the lymph node medulla use the lectin receptor SIGN-R1 to capture lymph-borne influenza virus and promote humoral immunity. Thus, our results have important implications for the generation of durable humoral immunity to viral pathogens through vaccination.

Seeing the Confetti Colors in a New Light Utilizing Flow Cytometry and Imaging Flow Cytometry.

Stochastic multicolor transgenic labeling systems, such as the Brainbow reporters, have emerged as powerful tools in lineage tracing experiments. Originally designed for large-scale mapping of neuronal projections in densely populated tissues, they have been repurposed for diverse uses. The Brainbow 2.1-derived Confetti reporter was used, for example, to define stem cell clonality and dynamics in crypts of the intestinal mucosa, T-cell clonality, microglial heterogeneity, and B-cell clonal evolution in germinal centers. Traditionally, read-outs have relied on imaging in situ, providing information about cellular localization within tissue stroma. However, recent applications of the technique have moved into hematopoietically derived motile cell types, for example, T and B lymphocytes and their progeny, creating an unmet need to survey larger populations of cells ex vivo to determine labeling densities or skews in color representation over time to read-out clonal expansion and selection effects. Originally designed for imaging methods, these reporters encode information in the spectral properties of fluorophores and their subcellular localization, making them poorly suited to traditional flow cytometry analyses. The advent of high-content imaging and imaging flow cytometry have recently closed the gap between flow cytometry and imaging. We analyzed a 10-color biallelic Confetti reporter using flow and imaging flow cytometry. Beyond its use as a high-throughput method for measuring reporter labeling densities and color distributions over time, it also opens the door to new avenues of research relying on similar read-outs, for example, tumor heterogeneity and clonal dynamics. © 2020 International Society for Advancement of Cytometry.

T follicular regulatory cells: Guardians of the germinal centre?

It is a central tenet of the clonal selection theory, that lymphocyte repertoires are tolerized to self-antigens during their ontogeny. Germinal centres are the sites in secondary lymphoid tissues where B cells undergo affinity maturation and class-switching to produce high-affinity antibodies. This process is crucial, both in our ability to mount protective humoral responses to infections and to vaccinations, but it is also involved in untoward reactions to self-antigens, which underlie autoimmunity. The process of affinity maturation poses a significant challenge to tolerance, as the random nature of somatic hypermutation can introduce novel reactivities. Therefore, it has been a long-standing idea that mechanisms must exist which limit the emergence of autoreactivity at the germinal centre level. One of these mechanisms is the requirement for linked recognition, which imposes on B cells a dependence on centrally tolerant T follicular helper cells. However, as linked recognition can be bypassed by adduct formation of autoantigenic complexes, it has been an appealing notion that there should be an additional layer of dominant mechanisms regulating emergence of autoreactive specificities. About a decade ago, this notion was addressed by the discovery of a novel subset of T regulatory cells localizing to the germinal centre and regulating germinal centre B-cell responses. Here, we detail the progress that has been made towards characterizing this T follicular regulatory cell subset and understanding the functions of these 'guardians of the germinal centre'.

Complement activation, regulation, and molecular basis for complement-related diseases.

The complement system is an essential element of the innate immune response that becomes activated upon recognition of molecular patterns associated with microorganisms, abnormal host cells, and modified molecules in the extracellular environment. The resulting proteolytic cascade tags the complement activator for elimination and elicits a pro-inflammatory response leading to recruitment and activation of immune cells from both the innate and adaptive branches of the immune system. Through these activities, complement functions in the first line of defense against pathogens but also contributes significantly to the maintenance of homeostasis and prevention of autoimmunity. Activation of complement and the subsequent biological responses occur primarily in the extracellular environment. However, recent studies have demonstrated autocrine signaling by complement activation in intracellular vesicles, while the presence of a cytoplasmic receptor serves to detect complement-opsonized intracellular pathogens. Furthermore, breakthroughs in both functional and structural studies now make it possible to describe many of the intricate molecular mechanisms underlying complement activation and the subsequent downstream events, as well as its cross talk with, for example, signaling pathways, the coagulation system, and adaptive immunity. We present an integrated and updated view of complement based on structural and functional data and describe the new roles attributed to complement. Finally, we discuss how the structural and mechanistic understanding of the complement system rationalizes the genetic defects conferring uncontrolled activation or other undesirable effects of complement.

Endogenous Natural Complement Inhibitor Regulates Cardiac Development.

Congenital heart defects are a major cause of perinatal mortality and morbidity, affecting >1% of all live births in the Western world, yet a large fraction of such defects have an unknown etiology. Recent studies demonstrated surprising dual roles for immune-related molecules and their effector mechanisms during fetal development and adult homeostasis. In this article, we describe the function of an endogenous complement inhibitor, mannan-binding lectin (MBL)-associated protein (MAp)44, in regulating the composition of a serine protease-pattern recognition receptor complex, MBL-associated serine protease (MASP)-3/collectin-L1/K1 hetero-oligomer, which impacts cardiac neural crest cell migration. We used knockdown and rescue strategies in zebrafish, a model allowing visualization and assessment of heart function, even in the presence of severe functional defects. Knockdown of embryonic expression of MAp44 caused impaired cardiogenesis, lowered heart rate, and decreased cardiac output. These defects were associated with aberrant neural crest cell behavior. We found that MAp44 competed with MASP-3 for pattern recognition molecule interaction, and knockdown of endogenous MAp44 expression could be rescued by overexpression of wild-type MAp44. Our observations provide evidence that immune molecules are centrally involved in the orchestration of cardiac tissue development.

Complement activation by ligand-driven juxtaposition of discrete pattern recognition complexes.

Defining mechanisms governing translation of molecular binding events into immune activation is central to understanding immune function. In the lectin pathway of complement, the pattern recognition molecules (PRMs) mannan-binding lectin (MBL) and ficolins complexed with the MBL-associated serine proteases (MASP)-1 and MASP-2 cleave C4 and C2 to generate C3 convertase. MASP-1 was recently found to be the exclusive activator of MASP-2 under physiological conditions, yet the predominant oligomeric forms of MBL carry only a single MASP homodimer. This prompted us to investigate whether activation of MASP-2 by MASP-1 occurs through PRM-driven juxtaposition on ligand surfaces. We demonstrate that intercomplex activation occurs between discrete PRM/MASP complexes. PRM ligand binding does not directly escort the transition of MASP from zymogen to active enzyme in the PRM/MASP complex; rather, clustering of PRM/MASP complexes directly causes activation. Our results support a clustering-based mechanism of activation, fundamentally different from the conformational model suggested for the classical pathway of complement.

Co-complexes of MASP-1 and MASP-2 associated with the soluble pattern-recognition molecules drive lectin pathway activation in a manner inhibitable by MAp44.

The lectin pathway of complement is an integral component of innate immunity. It is activated upon binding of mannan-binding lectin (MBL) or ficolins (H-, L-, and M-ficolin) to suitable ligand patterns on microorganisms. MBL and ficolins are polydisperse homo-oligomeric molecules, found in complexes with MBL-associated serine proteases (MASP-1, -2, and -3) and MBL-associated proteins (MAp19 and MAp44). This scenario is far more complex than the well-defined activation complex of the classical pathway, C1qC1r(2)C1s(2), and the composition of the activating complexes of the lectin pathway is ill defined. We and other investigators recently demonstrated that both MASP-1 and MASP-2 are crucial to lectin pathway activation. MASP-1 transactivates MASP-2 and, although MASP-1 also cleaves C2, MASP-2 cleaves both C4 and C2, allowing formation of the C3 convertase, C4bC2a. Juxtaposition of MASP-1 and MASP-2 during activation must be required for transactivation. We previously presented a possible scenario, which parallels that of the classical pathway, in which MASP-1 and MASP-2 are found together in the same MBL or ficolin complex. In this study, we demonstrate that, although MASPs do not directly form heterodimers, the addition of MBL or ficolins allows the formation of MASP-1-MASP-2 co-complexes. We find that such co-complexes have a functional role in activating complement and are present in serum at varying levels, impacting on the degree of complement activation. This raises the novel possibility that MAp44 may inhibit complement, not simply by brute force displacement of MASP-2 from MBL or ficolins, but by disruption of co-complexes, hence impairing transactivation. We present support for this contention.

Mitochondria and the lectin pathway of complement.

Mitochondria, the powerhouses of our cells, are remnants of a eubacterial endosymbiont. Notwithstanding the evolutionary time that has passed since the initial endosymbiotic event, mitochondria have retained many hallmarks of their eubacterial origin. Recent studies have indicated that during perturbations of normal homeostasis, such as following acute trauma leading to massive necrosis and release of mitochondria, the immune system might mistake symbiont for enemy and initiate an inappropriate immune response. The innate immune system is the first line of defense against invading microbial pathogens, and as such is the primary suspect in the recognition of mitochondria-derived danger-associated molecular patterns and initiation of an aberrant response. Conversely, innate immune mechanisms are also central to noninflammatory clearance of innocuous agents. Here we investigated the role of a central humoral component of innate immunity, the lectin pathway of complement, in recognition of mitochondria in vitro and in vivo. We found that the soluble pattern recognition molecules, mannan-binding lectin (MBL), L-ficolin, and M-ficolin, were able to recognize mitochondria. Furthermore, MBL in complex with MBL-associated serine protease 2 (MASP-2) was able to activate the lectin pathway and deposit C4 onto mitochondria, suggesting that these molecules are involved either in homeostatic clearance of mitochondria or in induction of untoward inflammatory reactions. We found that following mitochondrial challenge, C3 was consumed in vivo in the absence of overt inflammation, indicating a potential role of complement in noninflammatory clearance of mitochondria. Thus, we report here the first indication of involvement of the lectin pathway in mitochondrial immune handling.

Disease-causing mutations in genes of the complement system.

Recent studies have revealed profound developmental consequences of mutations in genes encoding proteins of the lectin pathway of complement activation, a central component of the innate immune system. Apart from impairment of immunity against microorganisms, it is known that hereditary deficiencies of this system predispose one to autoimmune conditions. Polymorphisms in complement genes are linked to, for example, atypical hemolytic uremia and age-dependent macular degeneration. The complement system comprises three convergent pathways of activation: the classical, the alternative, and the lectin pathway. The recently discovered lectin pathway is less studied, but polymorphisms in the plasma pattern-recognition molecule mannan-binding lectin (MBL) are known to impact its level, and polymorphisms in the MBL-associated serine protease-2 (MASP-2) result in defects of complement activation. Recent studies have described roles outside complement and immunity of another MBL-associated serine protease, MASP-3, in the etiology of 3MC syndrome, an autosomal-recessive disorder involving a spectrum of developmental features, including characteristic facial dysmorphism. Syndrome-causing mutations were identified in MASP1, encoding MASP-3 and two additional proteins, MASP-1 and MAp44. Furthermore, an association was discovered between 3MC syndrome and mutations in COLEC11, encoding CL-K1, another molecule of the lectin pathway. The findings were confirmed in zebrafish, indicating that MASP-3 and CL-K1 underlie an evolutionarily conserved pathway of embryonic development. Along with the discovery of a role of C1q in pruning synapses in mice, these recent advances point toward a broader role of complement in development. Here, we compare the functional immunologic consequences of "conventional" complement deficiencies with these newly described developmental roles.

Complement C4 maintains peripheral B-cell tolerance in a myeloid cell dependent manner.

The factors that allow self-reactive B cells to escape negative selection and become activated remain poorly defined. Using a BCR knock-in mouse strain, we identify a pathway by which B-cell selection to nucleolar self-antigens is complement dependent. Deficiency in complement component C4 led to a breakdown in the elimination of autoreactive B-cell clones at the transitional stage, characterized by a relative increase in their response to a range of stimuli, entrance into follicles, and a greater propensity to form self-reactive GCs. Using mixed BM chimeras, we found that the myeloid compartment was sufficient to restore negative selection in the autoreactive mice. A model is proposed in which in the absence of complement C4, inappropriate clearance of apoptotic debris promotes chronic activation of myeloid cells, allowing the maturation and activation of self-reactive B-cell clones leading to increased spontaneous formation of GCs.

Mannan-binding lectin-associated serine protease (MASP)-1 is crucial for lectin pathway activation in human serum, whereas neither MASP-1 nor MASP-3 is required for alternative pathway function.

The lectin pathway of complement is an important component of innate immunity. Its activation has been thought to occur via recognition of pathogens by mannan-binding lectin (MBL) or ficolins in complex with MBL-associated serine protease (MASP)-2, followed by MASP-2 autoactivation and cleavage of C4 and C2 generating the C3 convertase. MASP-1 and MASP-3 are related proteases found in similar complexes. MASP-1 has been shown to aid MASP-2 convertase generation by auxiliary C2 cleavage. In mice, MASP-1 and MASP-3 have been reported to be central also to alternative pathway function through activation of profactor D and factor B. In this study, we present functional studies based on a patient harboring a nonsense mutation in the common part of the MASP1 gene and hence deficient in both MASP-1 and MASP-3. Surprisingly, we find that the alternative pathway in this patient functions normally, and is unaffected by reconstitution with MASP-1 and MASP-3. Conversely, we find that the patient has a nonfunctional lectin pathway, which can be restored by MASP-1, implying that this component is crucial for complement activation. We show that, although MASP-2 is able to autoactivate under artificial conditions, MASP-1 dramatically increases lectin pathway activity at physiological conditions through direct activation of MASP-2. We further demonstrate that MASP-1 and MASP-2 can associate in the same MBL complex, and that such cocomplexes are found in serum, providing a scenario for transactivation of MASP-2. Hence, in functional terms, it appears that MASP-1 and MASP-2 act in a manner analogous to that of C1r and C1s of the classical pathway.

The follicular dendritic cell: At the germinal center of autoimmunity?

Autoimmune diseases strain healthcare systems worldwide as their incidence rises, and current treatments put patients at risk for infections. An increased understanding of autoimmune diseases is required to develop targeted therapies that do not impair normal immune function. Many autoimmune diseases present with autoantibodies, which drive local or systemic inflammation. This indicates the presence of autoreactive B cells that have escaped tolerance. An important step in the development of autoreactive B cells is the germinal center (GC) reaction, where they undergo affinity maturation toward cognate self-antigen. Follicular dendritic cells (FDCs) perform the essential task of antigen presentation to B cells during the affinity maturation process. However, in recent years, it has become clear that FDCs play a much more active role in regulation of GC processes. Here, we evaluate the biology of FDCs in the context of autoimmune disease, with the goal of informing future therapeutic strategies.

Recombinant expression of the autocatalytic complement protease MASP-1 is crucially dependent on co-expression with its inhibitor, C1 inhibitor.

MASP-1 is a protease of the lectin pathway of complement. It is homologous with MASP-2, previously thought both necessary and sufficient for lectin pathway activation. Recently MASP-1 has taken centre stage with the observation that it is crucial to the activation of MASP-2 and thus central to complement activation. Numerous additional functions have been suggested for MASP-1 and its importance is obvious. Yet, thorough analyses of proteolytic activities and physiological roles in the human scenario have been hampered by difficulties in purifying or producing full-length human MASP-1. We present the successful expression of full-length recombinant human MASP-1 entirely in the zymogen form in a mammalian expression system. We found that the catalytic activity of MASP-1 suppresses its expression through rapid auto-activation and auto-degradation. This auto-degradation was not inhibited by the addition of inhibitors to the culture medium, and it was subsequently found to occur intracellularly. Numerous mutations aimed at attenuating auto-activation or preventing auto-degradation failed to rescue expression, as did also attempts at stabilizing the protease by co-expression with MBL or ficolins or expression in hepatocyte cell lines, representing the natural site of synthesis. The active protease was finally produced through co-expression with the serine protease inhibitor C1 inhibitor. We demonstrate that the expressed protease is capable of binding MBL and auto-activating, and is catalytically active. We have generalized the concept to the expression also of MASP-2 entirely in its zymogen form and with improved yields. We suggest a general advantage of expressing aggressive, autocatalytic proteases with their cognate inhibitors.

Epigenetic Silencing of LRP2 Is Associated with Dedifferentiation and Poor Survival in Multiple Solid Tumor Types.

More than 80% of human cancers originate in epithelial tissues. Loss of epithelial cell characteristics are hallmarks of tumor development. Receptor-mediated endocytosis is a key function of absorptive epithelial cells with importance for cellular and organismal homeostasis. LRP2 (megalin) is the largest known endocytic membrane receptor and is essential for endocytosis of various ligands in specialized epithelia, including the proximal tubules of the kidney, the thyroid gland, and breast glandular epithelium. However, the role and regulation of LRP2 in cancers that arise from these tissues has not been delineated. Here, we examined the expression of LRP2 across 33 cancer types in The Cancer Genome Atlas. As expected, the highest levels of LRP2 were found in cancer types that arise from LRP2-expressing absorptive epithelial cells. However, in a subset of tumors from these cancer types, we observed epigenetic silencing of LRP2. LRP2 expression showed a strong inverse correlation to methylation of a specific CpG site (cg02361027) in the first intron of the LRP2 gene. Interestingly, low expression of LRP2 was associated with poor patient outcome in clear cell renal cell carcinoma, papillary renal cell carcinoma, mesothelioma, papillary thyroid carcinoma, and invasive breast carcinoma. Furthermore, loss of LRP2 expression was associated with dedifferentiated histological and molecular subtypes of these cancers. These observations now motivate further studies on the functional role of LRP2 in tumors of epithelial origin and the potential use of LRP2 as a cancer biomarker.

B cell MHC haplotype affects follicular inclusion, germinal center participation and plasma cell differentiation in a mouse model of lupus.

Introduction: MHC class II molecules are essential for appropriate immune responses against pathogens but are also implicated in pathological responses in autoimmune diseases and transplant rejection. Previous studies have shed light on the systemic contributions of MHC haplotypes to the development and severity of autoimmune diseases. In this study, we addressed the B cell intrinsic MHC haplotype impact on follicular inclusion, germinal center (GC) participation and plasma cell (PC) differentiation in the context of systemic lupus erythematosus (SLE). Methods: We leveraged the 564Igi mouse model which harbors a B cell receptor knock-in from an autoreactive B cell clone recognizing ribonuclear components, including double-stranded DNA (dsDNA). This model recapitulates the central hallmarks of the early stages of SLE. We compared 564Igi heterozygous offspring on either H2b/b, H2b/d, or H2d/d background. Results: This revealed significantly higher germinal center (GC) B cell levels in the spleens of H2b/b and H2b/d as compared to H2d/d (p<0.0001) mice. In agreement with this, anti-dsDNA-antibody levels were higher in H2b/b and H2b/d than in H2d/d (p<0.0001), with H2b/b also being higher compared to H2b/d (p<0.01). Specifically, these differences held true both for autoantibodies derived from the knock-in clone and from wild-type (WT) derived clones. In mixed chimeras where 564Igi H2b/b, H2b/d and H2d/d cells competed head-to-head in the same environment, we observed a significantly higher inclusion of H2b/b cells in GC and PC compartments relative to their representation in the B cell repertoire, compared to H2b/d and H2d/d cells. Furthermore, in mixed chimeras in which WT H2b/b and WT H2d/d cells competed for inclusion in GCs associated with an epitope spreading process, H2b/b cells participated to a greater extent and contributed more robustly to the PC compartment. Finally, immature WT H2b/b cells had a higher baseline of BCRs with an autoreactive idiotype and were subject to more stringent negative selection at the transitional stage. Discussion: Taken together, our findings demonstrate that B cell intrinsic MHC haplotype governs their capacity for participation in the autoreactive response at multiple levels: follicular inclusion, GC participation, and PC output. These findings pinpoint B cells as central contributors to precipitation of autoimmunity.

Antigen footprint governs activation of the B cell receptor.

Antigen binding by B cell receptors (BCR) on cognate B cells elicits a response that eventually leads to production of antibodies. However, it is unclear what the distribution of BCRs is on the naïve B cell and how antigen binding triggers the first step in BCR signaling. Using DNA-PAINT super-resolution microscopy, we find that most BCRs are present as monomers, dimers, or loosely associated clusters on resting B cells, with a nearest-neighbor inter-Fab distance of 20-30 nm. We leverage a Holliday junction nanoscaffold to engineer monodisperse model antigens with precision-controlled affinity and valency, and find that the antigen exerts agonistic effects on the BCR as a function of increasing affinity and avidity. Monovalent macromolecular antigens can activate the BCR at high concentrations, whereas micromolecular antigens cannot, demonstrating that antigen binding does not directly drive activation. Based on this, we propose a BCR activation model determined by the antigen footprint.