Validation of a murine proteome-wide phage display library for identification of autoantibody specificities.

Autoimmunity is characterized by loss of tolerance to tissue-specific as well as systemic antigens, resulting in complex autoantibody landscapes. Here, we introduce and extensively validate the performance characteristics of a murine proteome-wide library for phage display immunoprecipitation and sequencing (PhIP-seq) in profiling mouse autoantibodies. This library was validated using 7 genetically distinct mouse lines across a spectrum of autoreactivity. Mice deficient in antibody production (Rag2-/- and µMT) were used to model nonspecific peptide enrichments, while cross-reactivity was evaluated using anti-ovalbumin B cell receptor-restricted OB1 mice as a proof of principle. The PhIP-seq approach was then utilized to interrogate 3 distinct autoimmune disease models. First, serum from Lyn-/- IgD+/- mice with lupus-like disease was used to identify nuclear and apoptotic bleb reactivities. Second, serum from nonobese diabetic (NOD) mice, a polygenic model of pancreas-specific autoimmunity, was enriched in peptides derived from both insulin and predicted pancreatic proteins. Lastly, Aire-/- mouse sera were used to identify numerous autoantigens, many of which were also observed in previous studies of humans with autoimmune polyendocrinopathy syndrome type 1 carrying recessive mutations in AIRE. These experiments support the use of murine proteome-wide PhIP-seq for antigenic profiling and autoantibody discovery, which may be employed to study a range of immune perturbations in mouse models of autoimmunity profiling.

Molecular basis for potent B cell responses to antigen displayed on particles of viral size.

Multivalent viral epitopes induce rapid, robust and T cell-independent humoral immune responses, but the biochemical basis for such potency remains incompletely understood. We take advantage of a set of liposomes of viral size engineered to display affinity mutants of the model antigen (Ag) hen egg lysozyme. Particulate Ag induces potent 'all-or-none' B cell responses that are density dependent but affinity independent. Unlike soluble Ag, particulate Ag induces signal amplification downstream of the B cell receptor by selectively evading LYN-dependent inhibitory pathways and maximally activates NF-κB in a manner that mimics T cell help. Such signaling induces MYC expression and enables even low doses of particulate Ag to trigger robust B cell proliferation in vivo in the absence of adjuvant. We uncover a molecular basis for highly sensitive B cell responses to viral Ag display that is independent of encapsulated nucleic acids and is not merely accounted for by avidity and B cell receptor cross-linking.

Validation of a murine proteome-wide phage display library for the identification of autoantibody specificities.

Autoimmunity is characterized by loss of tolerance to tissue-specific as well as systemic antigens, resulting in complex autoantibody landscapes. Here, we introduce and extensively validate the performance characteristics of a murine proteome-wide library for phage display immunoprecipitation and sequencing (PhIP-seq), to profile mouse autoantibodies. This system and library were validated using seven genetic mouse models across a spectrum of autoreactivity. Mice deficient in antibody production (Rag2-/- and µMT) were used to model non-specific peptide enrichments, while cross-reactivity was evaluated using anti-ovalbumin B cell receptor (BCR)-restricted OB1 mice as a proof of principle. The PhIP-seq approach was then utilized to interrogate three distinct autoimmune disease models. First, serum from Lyn-/- IgD+/- mice with lupus-like disease was used to identify nuclear and apoptotic bleb reactivities, lending support to the hypothesis that apoptosis is a shared origin of these antigens. Second, serum from non-obese diabetic (NOD) mice, a polygenic model of pancreas-specific autoimmunity, enriched peptides derived from both insulin and predicted pancreatic proteins. Lastly, Aire-/- mouse sera were used to identify numerous auto-antigens, many of which were also observed in previous studies of humans with autoimmune polyendocrinopathy syndrome type 1 (APS1) carrying recessive mutations in AIRE. Among these were peptides derived from Perilipin-1, a validated autoimmune biomarker of generalized acquired lipodystrophy in humans. Autoreactivity to Perilipin-1 correlated with lymphocyte infiltration in adipose tissue and underscores the approach in revealing previously unknown specificities. These experiments support the use of murine proteome-wide PhIP-seq for antigenic profiling and autoantibody discovery, which may be employed to study a range of immune perturbations in mouse models of autoimmunity.

Molecular basis for potent B cell responses to antigen displayed on particles of viral size.

Although it has long been appreciated that multivalent antigens - and particularly viral epitope display - produce extremely rapid, robust, and T-independent humoral immune responses, the biochemical basis for such potency has been incompletely understood. Here we take advantage of a set of neutral liposomes of viral size that are engineered to display affinity mutants of the model antigen (Ag) hen egg lysozyme at precisely varied density. We show that particulate Ag display by liposomes induces highly potent B cell responses that are dose-and density-dependent but affinity-independent. Titrating dose of particulate, but not soluble, Ag reveals bimodal Erk phosphorylation and cytosolic calcium increases. Particulate Ag induces signal amplification downstream of the B cell receptor (BCR) by selectively evading LYN-dependent inhibitory pathways, but in vitro potency is independent of CD19. Importantly, Ag display on viral-sized particles signals independently of MYD88 and IRAK1/4, but activates NF- κ B robustly in a manner that mimics T cell help. Together, such biased signaling by particulate Ag promotes MYC expression and reduces the threshold required for B cell proliferation relative to soluble Ag. These findings uncover a molecular basis for highly sensitive B cell response to viral Ag display and remarkable potency of virus-like particle vaccines that is not merely accounted for by avidity and BCR cross-linking, and is independent of the contribution of B cell nucleic acid-sensing machinery.

Interferon gamma constrains type 2 lymphocyte niche boundaries during mixed inflammation.

Allergic immunity is orchestrated by group 2 innate lymphoid cells (ILC2s) and type 2 helper T (Th2) cells prominently arrayed at epithelial- and microbial-rich barriers. However, ILC2s and Th2 cells are also present in fibroblast-rich niches within the adventitial layer of larger vessels and similar boundary structures in sterile deep tissues, and it remains unclear whether they undergo dynamic repositioning during immune perturbations. Here, we used thick-section quantitative imaging to show that allergic inflammation drives invasion of lung and liver non-adventitial parenchyma by ILC2s and Th2 cells. However, during concurrent type 1 and type 2 mixed inflammation, IFNγ from broadly distributed type 1 lymphocytes directly blocked both ILC2 parenchymal trafficking and subsequent cell survival. ILC2 and Th2 cell confinement to adventitia limited mortality by the type 1 pathogen Listeria monocytogenes. Our results suggest that the topography of tissue lymphocyte subsets is tightly regulated to promote appropriately timed and balanced immunity.

NR4A nuclear receptors in T and B lymphocytes: Gatekeepers of immune tolerance.

Random VDJ recombination early in T and B cell development enables the adaptive immune system to recognize a vast array of evolving pathogens via antigen receptors. However, the potential of such randomly generated TCRs and BCRs to recognize and respond to self-antigens requires layers of tolerance mechanisms to mitigate the risk of life-threatening autoimmunity. Since they were originally cloned more than three decades ago, the NR4A family of nuclear hormone receptors have been implicated in many critical aspects of immune tolerance, including negative selection of thymocytes, peripheral T cell tolerance, regulatory T cells (Treg), and most recently in peripheral B cell tolerance. In this review, we discuss important insights from many laboratories as well as our own group into the function and mechanisms by which this small class of primary response genes promotes self-tolerance and immune homeostasis to balance the need for host defense against the inherent risks posed by the adaptive immune system.

Negative feedback by NUR77/Nr4a1 restrains B cell clonal dominance during early T-dependent immune responses.

B cell clones compete for entry into and dominance within germinal centers (GCs), where the highest-affinity B cell receptors (BCRs) are selected. However, diverse and low-affinity B cells can enter and reside in GCs for extended periods. To reconcile these observations, we hypothesize that a negative feedback loop may operate within B cells to preferentially restrain high-affinity clones from monopolizing the early GC niche. Here, we report a role for the nuclear receptor NUR77/Nr4a1 in this process. We show that NUR77 expression scales with antigen stimulation and restrains B cell expansion. Although NUR77 is dispensable for regulating GC size when GCs are elicited in a largely clonal manner, it serves to curb immunodominance under conditions where diverse clonal populations must compete for a constrained niche. We propose that this is important to preserve early clonal diversity in order to limit holes in the post-immune repertoire and to optimize GC selection.

The BAFFling persistence of memory B cells.

Although BAFF/BLyS and its receptor, BAFFR, play critical roles in naive B cell survival, the pathways involved in the persistence of memory B cells are largely unknown. In this issue of JEM, two groups, Müller-Winkler et al. (https://doi.org/10.1084/jem.20191393) and Lau et al. (https://doi.org/10.1084/jem.20191167), take complementary approaches to identify an essential role for BAFFR in the survival of memory B cells.

NR4A nuclear receptors restrain B cell responses to antigen when second signals are absent or limiting.

Antigen stimulation (signal 1) triggers B cell proliferation and primes B cells to recruit, engage and respond to T cell help (signal 2). Failure to receive signal 2 within a defined time window results in B cell apoptosis, yet the mechanisms that enforce dependence on co-stimulation are incompletely understood. Nr4a1-3 encode a small family of orphan nuclear receptors that are rapidly induced by B cell antigen receptor stimulation. Here, we show that Nr4a1 and Nr4a3 play partially redundant roles to restrain B cell responses to antigen in the absence of co-stimulation and do so, in part, by repressing the expression of BATF and, consequently, MYC. The NR4A family also restrains B cell access to T cell help by repressing expression of the T cell chemokines CCL3 and CCL4, as well as CD86 and ICAM1. Such NR4A-mediated regulation plays a role specifically under conditions of competition for limiting T cell help.

Peripheral Tolerance Checkpoints Imposed by Ubiquitous Antigen Expression Limit Antigen-Specific B Cell Responses under Strongly Immunogenic Conditions.

A series of layered peripheral checkpoints maintain self-reactive B cells in an unresponsive state. Autoantibody production occurs when these checkpoints are breached; however, when and how this occurs is largely unknown. In particular, how self-reactive B cells are restrained during bystander inflammation in otherwise healthy individuals is poorly understood. A weakness has been the unavailability of methods capable of dissecting physiologically relevant B cell responses without the use of an engineered BCR. Resolving this will provide insights that decipher how this process goes awry during autoimmunity or could be exploited for therapy. In this study, we use a strong adjuvant to provide bystander innate and adaptive signals that promote B cell responsiveness in conjunction with newly developed B cell detection tools to study in detail the ways that peripheral tolerance mechanisms limit the expansion and function of self-reactive B cells activated under these conditions. We show that although self-reactive B cells are recruited into the germinal center, their development does not proceed, possibly because of rapid counterselection. Consequently, differentiation of plasma cells is blunted, and Ab responses are transient and devoid of affinity maturation. We propose this approach, and these tools can be more widely applied to track Ag-specific B cell responses to more disease-relevant Ags, without the need for BCR transgenic mice, in settings where tolerance pathways are compromised or have been genetically manipulated to drive stronger insights into the biology underlying B cell-mediated autoimmunity.

Transfer of antigen-encoding bone marrow under immune-preserving conditions deletes mature antigen-specific B cells in recipients and inhibits antigen-specific antibody production.

BACKGROUND AIMS: Pathological activation and collaboration of T and B cells underlies pathogenic autoantibody responses. Existing treatments for autoimmune disease cause non-specific immunosuppression, and induction of antigen-specific tolerance remains an elusive goal. Many immunotherapies aim to manipulate the T-cell component of T-B interplay, but few directly target B cells. One possible means to specifically target B cells is the transfer of gene-engineered BM that, once engrafted, gives rise to widespread specific and tolerogenic antigen expression within the hematopoietic system. METHODS: Gene-engineered bone marrow encoding ubiquitous ovalbumin expression was transferred after low-dose (300-cGy) immune-preserving irradiation. B-cell responsiveness was monitored by analyzing ovalbumin-specific antibody production after immunization with ovalbumin/complete Freund's adjuvant. Ovalbumin-specific B cells and their response to immunization were analyzed using multi-tetramer staining. When antigen-encoding bone marrow was transferred under immune-preserving conditions, cognate antigen-specific B cells were purged from the recipient's preexisting B-cell repertoire and the repertoire that arose after bone marrow transfer. RESULTS: OVA-specific B-cell deletion was apparent within the established host B-cell repertoire as well as that developing after gene-engineered bone marrow transfer. OVA-specific antibody production was substantially inhibited by transfer of OVA-encoding BM and activation of OVA-specific B cells, germinal center formation and subsequent OVA-specific plasmablast differentiation were all inhibited. Low levels of gene-engineered bone marrow chimerism were sufficient to limit antigen-specific antibody production. RESULTS: These data show that antigen-specific B cells within an established B-cell repertoire are susceptible to de novo tolerance induction, and this can be achieved by transfer of gene-engineered bone marrow. This adds further dimensions to the utility of antigen-encoding bone marrow transfer as an immunotherapeutic tool.

Synthetic Liposomal Mimics of Biological Viruses for the Study of Immune Responses to Infection and Vaccination.

Human viruses possess very complex supramolecular structures. Both icosahedral and enveloped viruses typically display an array of viral-encoded protein antigens at varied spatial densities on the viral particle surface. The viral nucleic acid genome, on the other hand, is encapsulated inside the viral particle. Although both the surface antigen and the interior nucleic acids could independently produce immunological responses, how B cells integrate these two types of signals and respond to a typical virus particle to initiate activation is not well understood at a molecular level. The study of these fundamental biological processes would benefit from the development of viral structural mimics that are well constructed to incorporate both quantitative and qualitative viral features for presentation to B cells. These novel tools would enable researchers to systematically dissect the underlying processes. Here we report the development of such particulate antigens based on liposomes engineered to display a model protein antigen, hen egg lysozyme (HEL). We developed methods to overexpress and purify various affinity mutants of HEL from E. coli. We conjugated the purified recombinant HEL proteins onto the surface of a virion-sized liposome in an orientation-specific manner at defined spatial densities and also encapsulated nucleic acid molecules into the interior of the liposome. Both the chemical conjugation of the HEL antigen on liposome surfaces and the encapsulation of nucleic acids were stable under physiologically relevant conditions. These liposomes elicited antigen-specific B-cell responses in vitro, which validate these supramolecular structures as a novel and effective approach to mimic and systematically isolate the role of essential viral features in directing the B-cell response to particulate antigens.

Tetramer-based identification of naïve antigen-specific B cells within a polyclonal repertoire.

Detecting naïve antigen-specific B cells can be challenging. Use of multiple, complementary tetramers with different fluorochromes enhances sensitivity and specificity allowing naïve antigen-specific B cells to be readily distinguished within a polyclonal repertoire. Activated, affinity-matured B cells, however, can be detected effectively using a single tetramer.

Re-educating immunity in respiratory allergies: the potential for hematopoietic stem cell-mediated gene therapy.

Respiratory allergies represent a significant disease burden worldwide affecting up to 300 million people globally. Medication and avoidance of known triggers do not address the underlying pathology. Traditional immunotherapies for allergy aim to reinstate immune homeostasis but require years of treatment and have poor long-term efficacy. Novel approaches, such as gene-engineered hematopoietic stem cell transplantation, induce profound antigen-specific tolerance in autoimmunity. Recent evidence shows this approach may also have therapeutic utility for allergy. Here, we review the mechanisms of antigen-specific tolerance and the potential of stem cell-mediated gene therapy to induce tolerance in allergic respiratory diseases.

Allergen-encoding bone marrow transfer inactivates allergic T cell responses, alleviating airway inflammation.

Memory Th2 cell responses underlie the development and perpetuation of allergic diseases. Because these states result from immune dysregulation, established Th2 cell responses represent a significant challenge for conventional immunotherapies. New approaches that overcome the detrimental effects of immune dysregulation are required. We tested whether memory Th2 cell responses were silenced using a therapeutic approach where allergen expression in DCs is transferred to sensitized recipients using BM cells as a vector for therapeutic gene transfer. Development of allergen-specific Th2 responses and allergen-induced airway inflammation was blocked by expression of allergen in DCs. Adoptive transfer studies showed that Th2 responses were inactivated by a combination of deletion and induction of T cell unresponsiveness. Transfer of BM encoding allergen expression targeted to DCs terminated, in an allergen-specific manner, Th2 responses in sensitized recipients. Importantly, when preexisting airway inflammation was present, there was effective silencing of Th2 cell responses, airway inflammation was alleviated, and airway hyperreactivity was reversed. The effectiveness of DC-targeted allergen expression to terminate established Th2 responses in sensitized animals indicates that exploiting cell-intrinsic T cell tolerance pathways could lead to development of highly effective immunotherapies.

Short-course rapamycin treatment enables engraftment of immunogenic gene-engineered bone marrow under low-dose irradiation to permit long-term immunological tolerance.

BACKGROUND: Application of genetically modified hematopoietic stem cells is increasingly mooted as a clinically relevant approach to protein replacement therapy, immune tolerance induction or conditions where both outcomes may be helpful. Hematopoietic stem and progenitor cell (HSPC)-mediated gene therapy often requires highly toxic pretransfer recipient conditioning to provide a 'niche' so that transferred HSPCs can engraft effectively and to prevent immune rejection of neoantigen-expressing engineered HSPCs. For widespread clinical application, reducing conditioning toxicity is an important requirement, but reduced conditioning can render neoantigen-expressing bone marrow (BM) and HSC susceptible to immune rejection if immunity is retained. METHODS: BM or HSPC-expressing OVA ubiquitously (actin.OVA) or targeted to MHC II+ cells was transferred using low-dose (300 cGy) total body irradiation. Recipients were administered rapamycin, cyclosporine or vehicle for 3 weeks commencing at BM transfer. Engraftment was determined using CD45 congenic donors and recipients. Induction of T-cell tolerance was tested by immunising recipients and analysing in-vivo cytotoxic T-lymphocyte (CTL) activity. The effect of rapamycin on transient effector function during tolerance induction was tested using an established model of tolerance induction where antigen is targeted to dendritic cells. RESULTS: Immune rejection of neoantigen-expressing BM and HSPCs after low-dose irradiation was prevented by a short course of rapamycin, but not cyclosporine, treatment. Whereas transient T-cell tolerance developed in recipients of OVA-expressing BM administered vehicle, only when engraftment of neoantigen-expressing BM was facilitated with rapamycin treatment did stable, long-lasting T-cell tolerance develop. Rapamycin inhibited transient effector function development during tolerance induction and inhibited development of CTL activity in recipients of OVA-expressing BM. CONCLUSIONS: Rapamycin acts to suppress acquisition of transient T-cell effector function during peripheral tolerance induction elicited by HSPC-encoded antigen. By facilitating engraftment, short-course rapamycin permits development of long-term stable T-cell tolerance.