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.

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.

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.

Quantitative and temporal requirements revealed for Zap70 catalytic activity during T cell development.

The catalytic activity of Zap70 is crucial for T cell antigen receptor (TCR) signaling, but the quantitative and temporal requirements for its function in thymocyte development are not known. Using a chemical-genetic system to selectively and reversibly inhibit Zap70 catalytic activity in a model of synchronized thymic selection, we showed that CD4(+)CD8(+) thymocytes integrate multiple, transient, Zap70-dependent signals over more than 36 h to reach a cumulative threshold for positive selection, whereas 1 h of signaling was sufficient for negative selection. Titration of Zap70 activity resulted in graded reductions in positive and negative selection but did not decrease the cumulative TCR signals integrated by positively selected OT-I cells, which revealed heterogeneity, even among CD4(+)CD8(+) thymocytes expressing identical TCRs undergoing positive selection.

An Integrated Signaling Threshold Initiates IgG Response toward Virus-like Immunogens.

Class-switched neutralizing Ab (nAb) production is rapidly induced upon many viral infections. However, due to the presence of multiple components in virions, the precise biochemical and biophysical signals from viral infections that initiate nAb responses remain inadequately defined. Using a reductionist system of synthetic virus-like structures, in this study, we show that a foreign protein on a virion-sized liposome can serve as a stand-alone danger signal to initiate class-switched nAb responses without T cell help or TLR but requires CD19. Introduction of internal nucleic acids (iNAs) obviates the need for CD19, lowers the epitope density (ED) required to elicit the Ab response, and transforms these structures into highly potent immunogens that rival conventional virus-like particles in their ability to elicit strong Ag-specific IgG. As early as day 5 after immunization, structures harboring iNAs and decorated with just a few molecules of surface Ag at doses as low as 100 ng induced all IgG subclasses of Ab in mice and reproduced the IgG2a/2c restriction that is long observed in live viral infections. These findings reveal a shared mechanism for the nAb response in mice. High ED is capable but not necessary for driving Ab secretion. Instead, even a few molecules of surface Ag, when combined with nucleic acids within these structures, can trigger strong IgG production. As a result, the signaling threshold for induction of IgG in individual B cells is set by dual signals originating from both ED on the surface and the presence of iNAs within viral particulate immunogens.

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.

Digital signaling and hysteresis characterize ras activation in lymphoid cells.

Activation of Ras proteins underlies functional decisions in diverse cell types. Two molecules, RasGRP and SOS, catalyze Ras activation in lymphocytes. Binding of active Ras to SOS' allosteric pocket markedly increases SOS' activity establishing a positive feedback loop for SOS-mediated Ras activation. Integrating in silico and in vitro studies, we demonstrate that digital signaling in lymphocytes (cells are "on" or "off") is predicated upon feedback regulation of SOS. SOS' feedback loop leads to hysteresis in the dose-response curve, which can enable a capacity to sustain Ras activation as stimuli are withdrawn and exhibit "memory" of past encounters with antigen. Ras activation via RasGRP alone is analog (graded increase in amplitude with stimulus). We describe how complementary analog (RasGRP) and digital (SOS) pathways act on Ras to efficiently convert analog input to digital output. Numerous predictions regarding the impact of our findings on lymphocyte function and development are noted.

ATP-competitive partial antagonists of the IRE1α RNase segregate outputs of the UPR.

The unfolded protein response (UPR) homeostatically matches endoplasmic reticulum (ER) protein-folding capacity to cellular secretory needs. However, under high or chronic ER stress, the UPR triggers apoptosis. This cell fate dichotomy is promoted by differential activation of the ER transmembrane kinase/endoribonuclease (RNase) IRE1α. We previously found that the RNase of IRE1α can be either fully activated or inactivated by ATP-competitive kinase inhibitors. Here we developed kinase inhibitors, partial antagonists of IRE1α RNase (PAIRs), that partially antagonize the IRE1α RNase at full occupancy. Biochemical and structural studies show that PAIRs promote partial RNase antagonism by intermediately displacing the helix αC in the IRE1α kinase domain. In insulin-producing ß-cells, PAIRs permit adaptive splicing of Xbp1 mRNA while quelling destructive ER mRNA endonucleolytic decay and apoptosis. By preserving Xbp1 mRNA splicing, PAIRs allow B cells to differentiate into immunoglobulin-producing plasma cells. Thus, an intermediate RNase-inhibitory 'sweet spot', achieved by PAIR-bound IRE1α, captures a desirable conformation for drugging this master UPR sensor/effector.

Self-reactivity on a spectrum: A sliding scale of peripheral B cell tolerance.

Efficient mechanisms of central tolerance, including receptor editing and deletion, prevent highly self-reactive B cell receptors (BCRs) from populating the periphery. Despite this, modest self-reactivity persists in (and may even be actively selected into) the mature B cell repertoire. In this review, we discuss new insights into mechanisms of peripheral B cell tolerance that restrain mature B cells from mounting inappropriate responses to endogenous antigens, and place recent work into historical context. In particular, we discuss new findings that have arisen from application of a novel in vivo reporter of BCR signaling, Nur77-eGFP, expression of which scales with the degree of self-reactivity in both monoclonal and polyclonal B cell repertoires. We discuss new and historical evidence that self-reactivity is not just tolerated, but actively selected into the peripheral repertoire. We review recent progress in understanding how dual expression of the IgM and IgD BCR isotypes on mature naive follicular B cells tunes responsiveness to endogenous antigen recognition, and discuss how this may be integrated with other features of clonal anergy. Finally, we discuss how expression of Nur77 itself couples chronic antigen stimulation with B cell tolerance.

Reporters of TCR signaling identify arthritogenic T cells in murine and human autoimmune arthritis.

How pathogenic cluster of differentiation 4 (CD4) T cells in rheumatoid arthritis (RA) develop remains poorly understood. We used Nur77-a marker of T cell antigen receptor (TCR) signaling-to identify antigen-activated CD4 T cells in the SKG mouse model of autoimmune arthritis and in patients with RA. Using a fluorescent reporter of Nur77 expression in SKG mice, we found that higher levels of Nur77-eGFP in SKG CD4 T cells marked their autoreactivity, arthritogenic potential, and ability to more readily differentiate into interleukin-17 (IL-17)-producing cells. The T cells with increased autoreactivity, nonetheless had diminished ex vivo inducible TCR signaling, perhaps reflective of adaptive inhibitory mechanisms induced by chronic autoantigen exposure in vivo. The enhanced autoreactivity was associated with up-regulation of IL-6 cytokine signaling machinery, which might be attributable, in part, to a reduced amount of expression of suppressor of cytokine signaling 3 (SOCS3)-a key negative regulator of IL-6 signaling. As a result, the more autoreactive GFPhi CD4 T cells from SKGNur mice were hyperresponsive to IL-6 receptor signaling. Consistent with findings from SKGNur mice, SOCS3 expression was similarly down-regulated in RA synovium. This suggests that despite impaired TCR signaling, autoreactive T cells exposed to chronic antigen stimulation exhibit heightened sensitivity to IL-6, which contributes to the arthritogenicity in SKG mice, and perhaps in patients with RA.

Optimal Development of Mature B Cells Requires Recognition of Endogenous Antigens.

It has long been appreciated that highly autoreactive BCRs are actively removed from the developing B cell repertoire by Ag-dependent receptor editing and deletion. However, there is persistent debate about whether mild autoreactivity is simply tolerated or positively selected into the mature B cell repertoire as well as at what stage, to what extent, under what conditions, and into which compartments this occurs. In this study, we describe two minor, trackable populations of B cells in B1-8i Ig transgenic mice that express the VH186.2 H chain and recognize a common foreign Ag (the hapten 4-hydroxy-3-nitrophenylacetyl) but differ in L chain expression. We use the Nur77-eGFP reporter of BCR signaling to define their reactivity toward endogenous Ags. The less autoreactive of these two populations is strongly counterselected during the development of mature B1a, follicular, and marginal zone B cells. By genetically manipulating the strength of BCR signal transduction via the titration of surface CD45 expression, we demonstrate that this B cell population is not negatively selected but instead displays characteristics of impaired positive selection. We demonstrate that mild self-reactivity improves the developmental fitness of B cell clones in the context of a diverse population of B cells, and positive selection by endogenous Ags shapes the mature B cell repertoire.

Nur77 Links Chronic Antigen Stimulation to B Cell Tolerance by Restricting the Survival of Self-Reactive B Cells in the Periphery.

Nur77 (Nr4a1) belongs to a small family of orphan nuclear receptors that are rapidly induced by BCR stimulation, yet little is known about its function in B cells. We have previously characterized a reporter of Nr4a1 transcription, Nur77-eGFP, in which GFP expression faithfully detects Ag encounter by B cells in vitro and in vivo. In this study, we report that Nur77 expression correlates with the degree of self-reactivity, counterselection, and anergy among individual B cell clones from two distinct BCR transgenic mouse models but is dispensable for all of these tolerance mechanisms. However, we identify a role for Nur77 in restraining survival of self-reactive B cells in the periphery under conditions of competition for a limited supply of the survival factor BAFF. We find that Nur77 deficiency results in the progressive accumulation of self-reactive B cells in the mature repertoire with age and is sufficient to break B cell tolerance in VH3H9 H chain transgenic mice. We thus propose that Nur77 is upregulated in self-reactive B cells in response to chronic Ag stimulation and selectively restricts the survival of these cells, gradually pruning self-reactivity from the mature repertoire to impose a novel layer of peripheral B cell tolerance.

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.

Tonic Signals: Why Do Lymphocytes Bother?

Since the 1990s it has been known that B and T lymphocytes exhibit low-level, constitutive signaling in the basal state (tonic signaling). These lymphocytes display a range of affinity for self, which in turn generates a range of tonic signaling. Surprisingly, what signaling pathways are active in the basal state and the functional relevance of the observed tonic signaling heterogeneity remain open questions today. Here we summarize what is known about the mechanistic and functional details of tonic signaling. We highlight recent advances that have increased our understanding of how the amount of tonic signal impacts immune function, describing novel tools that have moved the field forward and toward a molecular understanding of tonic signaling.

CD45-Csk phosphatase-kinase titration uncouples basal and inducible T cell receptor signaling during thymic development.

The kinase-phosphatase pair Csk and CD45 reciprocally regulate phosphorylation of the inhibitory tyrosine of the Src family kinases Lck and Fyn. T cell receptor (TCR) signaling and thymic development require CD45 expression but proceed constitutively in the absence of Csk. Here, we show that relative titration of CD45 and Csk expression reveals distinct regulation of basal and inducible TCR signaling during thymic development. Low CD45 expression is sufficient to rescue inducible TCR signaling and positive selection, whereas high expression is required to reconstitute basal TCR signaling and beta selection. CD45 has a dual positive and negative regulatory role during inducible but not basal TCR signaling. By contrast, Csk titration regulates basal but not inducible signaling. High physiologic expression of CD45 is thus required for two reasons-to downmodulate inducible TCR signaling during positive selection and to counteract Csk during basal TCR signaling.

IL-2 Modulates the TCR Signaling Threshold for CD8 but Not CD4 T Cell Proliferation on a Single-Cell Level.

Lymphocytes integrate Ag and cytokine receptor signals to make cell fate decisions. Using a specific reporter of TCR signaling that is insensitive to cytokine signaling, Nur77-eGFP, we identify a sharp, minimal threshold of cumulative TCR signaling required for proliferation in CD4 and CD8 T cells that is independent of both Ag concentration and affinity. Unexpectedly, IL-2 reduces this threshold in CD8 but not CD4 T cells, suggesting that integration of multiple mitogenic inputs may alter the minimal requirement for TCR signaling in CD8 T cells. Neither naive CD4 nor naive CD8 T cells are responsive to low doses of IL-2. We show that activated CD8 T cells become responsive to low doses of IL-2 more quickly than CD4 T cells, and propose that this relative delay in turn accounts for the differential effects of IL-2 on the minimal TCR signaling threshold for proliferation in these populations. In contrast to Nur77-eGFP, c-Myc protein expression integrates mitogenic signals downstream of both IL-2 and the TCR, yet marks an invariant minimal threshold of cumulative mitogenic stimulation required for cell division. Our work provides a conceptual framework for understanding the regulation of clonal expansion of CD8 T cells by subthreshold TCR signaling in the context of mitogenic IL-2 signals, thereby rendering CD8 T cells exquisitely dependent upon environmental cues. Conversely, CD4 T cell proliferation requires an invariant minimal intensity of TCR signaling that is not modulated by IL-2, thereby restricting responses to low-affinity or low-abundance self-antigens even in the context of an inflammatory milieu.

Endogenous antigen tunes the responsiveness of naive B cells but not T cells.

In humans, up to 75% of newly generated B cells and about 30% of mature B cells show some degree of autoreactivity. Yet, how B cells establish and maintain tolerance in the face of autoantigen exposure during and after development is not certain. Studies of model B-cell antigen receptor (BCR) transgenic systems have highlighted the critical role of functional unresponsiveness or 'anergy'. Unlike T cells, evidence suggests that receptor editing and anergy, rather than deletion, account for much of B-cell tolerance. However, it remains unclear whether the mature diverse B-cell repertoire of mice contains anergic autoreactive B cells, and if so, whether antigen was encountered during or after their development. By taking advantage of a reporter mouse in which BCR signalling rapidly and robustly induces green fluorescent protein expression under the control of the Nur77 regulatory region, antigen-dependent and antigen-independent BCR signalling events in vivo during B-cell maturation were visualized. Here we show that B cells encounter antigen during development in the spleen, and that this antigen exposure, in turn, tunes the responsiveness of BCR signalling in B cells at least partly by downmodulating expression of surface IgM but not IgD BCRs, and by modifying basal calcium levels. By contrast, no analogous process occurs in naive mature T cells. Our data demonstrate not only that autoreactive B cells persist in the mature repertoire, but that functional unresponsiveness or anergy exists in the mature B-cell repertoire along a continuum, a fact that has long been suspected, but never yet shown. These results have important implications for understanding how tolerance in T and B cells is differently imposed, and how these processes might go awry in disease.