Biology subjects

Zikherman, J.

Publications and source records attributed to Zikherman, J..

10 recordsLinked to original sources

bioRxiv2026

Virus-like antigen display delivers a stand-alone danger signal through the BCR that circumvents tolerance

How B cells discriminate self from foreign antigens remains a central question, given inherent autoreactivity of the mature B cell receptor (BCR) repertoire. Soluble antigen (sAg) induces tolerance, whereas patterned antigen display on virus-like particles (pAg) triggers robust B cell responses that can proceed without T cell help. Here, we show how this divergence arises early in BCR signaling. Unlike sAg, pAg can bypass a Lyn-dependent negative feedback loop to trigger digital signaling, such that ultra-low concentrations of pAg produce strong and sustained Ca2+ responses. Surprisingly, pAg drives maximal nuclear NF-{kappa}B but limited NFAT, whereas sAg does the opposite, reflecting differential production of diacylglycerol. Consequently, sAg induced an NFAT-dependent anergy program, whereas pAg evaded this state and instead engaged a cMyc-driven program that partially resembles a TLR-dependent danger response. Our findings reveal how proximal signaling directs distinct transcriptional fate to enable immunogenic B cell responses to virus-like antigen display.

immunology↗

bioRxiv2024

Transcriptional control of central T cell tolerance by NR4A family nuclear receptors

Although deletion of self-reactive thymocytes and their diversion into regulatory T cell (Treg) lineage are critical for immune tolerance and homeostasis, the molecular pathways that link antigen recognition to these fates are incompletely understood. The Nr4a nuclear hormone receptors are transcriptionally upregulated in response to TCR signaling in the thymus and are implicated in both deletion and diversion, but the mechanisms by which they operate are not clear. Redundancy among the family members and their requirement for Treg generation and maintenance have obscured their role in negative selection. Here we take advantage of competitive bone marrow chimeras and the OT-II/RIPmOVA model to demonstrate that Nr4a1 and Nr4a3 are essential for upregulation of Bcl2l11/BIM and negative selection by tissue-restricted model self-antigen (TRA). Moreover, we reveal that the Nr4a family is absolutely required for full induction of a broad transcriptional program triggered in self-reactive thymocytes by TRA recognition, and conserved across model systems and the natural repertoire. Importantly, both model self antigen-specific TCR Tg and polyclonal thymocytes lacking Nr4a1/3 that escape negative selection acquire an anergy-like program that persists in the periphery and is also evident among wild-type recent thymic emigrants (RTEs). We propose that the Nr4a family transduces TCR signals during thymic development to enforce the fates of highly self-reactive clones, mediating not only deletion and Treg diversion, but also contributing to a cell-intrinsic, persistent anergy-like program that may operate at the margins of canonical thymic tolerance mechanisms to restrain self-reactive T cells after thymic egress.

immunology↗

bioRxiv2024

An integrated signaling threshold initiates IgG response towards virus-like immunogens

Class-switched neutralizing antibody (nAb) production is rapidly induced upon many viral infections. However, due to the presence of multiple components in typical 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 (SVLS) containing minimal, highly purified biochemical components commonly found in enveloped viruses, here 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 in the absence of cognate T cell help or Toll-like receptor signaling but requires CD19, the antigen (Ag) coreceptor on B cells. 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 harbouring iNAs and decorated with just a few molecules of surface Ag at doses as low as 100 ng induced all IgG subclasses of Ab known in mice and reproduced the IgG2a/2c restriction that has been long observed in live viral infections. These findings reveal a shared mechanism for nAb response upon viral infection. High ED is capable but not necessary for driving Ab secretion in vivo. Instead, even a few molecules of surface Ag, when combined with nucleic acids within these structures, can trigger strong antiviral IgG production. As a result, the signaling threshold for the induction of neutralizing IgG is set by dual signals originating from both ED on the surface and the presence of iNAs within viral particulate immunogens. One-sentence summaryReconstitution of minimal viral signals necessary to initiate antiviral IgG

immunology↗

bioRxiv2023

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 MT) 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.

immunology↗

bioRxiv2023

Mechanisms of neutralizing antibody response probed using synthetic virus-like structures

The durability of an antibody (Ab) response is highly important for antiviral vaccines. However, due to the complex compositions of natural virions, the molecular determinants of Ab durability from viral infection or inactivated viral vaccines have been incompletely understood. Here we used a reductionist system of liposome-based virus-like structures to examine the durability of Abs in primary immune responses in mice. This system allowed us to independently vary fundamental viral attributes and to do so without additional adjuvants to model natural viruses. We show that a single injection of antigens (Ags) orderly displayed on a virion-sized liposome is sufficient to induce a long-lived neutralizing Ab (nAb) response. Introduction of internal nucleic acids dramatically modulates the magnitude of long-term Ab responses without alteration of the long-term kinetic trends. These Abs are characterized by exceptionally slow off-rates of [~]0.0005 s-1, which emerged as early as day 5 after injection and these off-rates are comparable to that of affinity-matured monoclonal Abs. A single injection of these structures at doses as low as 100 ng led to lifelong nAb production in BALB/c mice. Thus, a minimal virus-like immunogen can give rise to potent and long-lasting antiviral Abs in a primary response in mice without live infection. This has important implications for understanding both live viral infection and for optimized vaccine design.

immunology↗

bioRxiv2023

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-{kappa}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.

immunology↗

bioRxiv2021

Regulation of layered T cell tolerance mechanisms by the NR4A family is essential to preserve immune homeostasis and suppress autoimmunity

The NR4A family of orphan nuclear receptors (Nr4a1-3) plays redundant roles upstream of Foxp3 to establish and maintain Treg identity; deletion of multiple family members in the thymus results in Treg deficiency and a severe inflammatory disease. Consequently, it has been challenging to isolate the functions of this family in other immune cells. Here we take advantage of a competitive bone marrow chimera strategy, coupled with conditional genetic tools, to rescue Treg homeostasis and unmask such functions. Unexpectedly, chimeras harboring Nr4a1-/- Nr4a3-/- (DKO) bone marrow develop autoantibodies and a systemic inflammatory disease despite a replete Treg compartment of largely wild-type origin. This disease differs qualitatively from that seen with Treg-deficiency and is B cell-extrinsic. Negative selection of DKO thymocytes is profoundly impaired in a cell-intrinsic manner. Consistent with escape of self-reactive T cells into the periphery, DKO T cells with functional and phenotypic features of anergy accumulate in chimeric mice. Despite this, DKO T cells exhibit enhanced IL-2 production, implying a cell-intrinsic role for the NR4A family in peripheral T cell tolerance. These studies reveal roles for the NR4A family in multiple layered T cell tolerance mechanisms and demonstrate that each is essential to preserve immune homeostasis.

immunology↗

bioRxiv2020

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 (GC), where the highest affinity BCRs are selected. However, diverse and low affinity B cells can enter and reside in GCs for extended periods. To reconcile these observations, we hypothesized 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 previously showed that NUR77 expression scales with antigen stimulation and restrains B cell expansion when T cell help is limiting. Here we show that, although NUR77 is dispensable for regulating GC size when GC are elicited in a largely clonal manner, it serves to curb immunodominance under conditions where diverse clonal populations must compete for a constrained niche. Moreover, this is independent of B cell precursor frequency and reflects, at least in part, a B cell-intrinsic role for NUR77. We propose that this is important to preserve early B cell clonal diversity in order to limit holes in the post-immune repertoire and to optimize GC selection.

immunology↗

bioRxiv2020

A negative feedback loop mediated by the NR4A family of nuclear hormone receptors restrains expansion of B cells that receive signal one in the absence of signal two

Ag stimulation (signal 1) triggers B cell activation and proliferation, and primes B cells to recruit, engage, and respond to T cell help (signal 2). However, failure to receive signal 2 within a defined window of time results in an abortive round of proliferation, followed by anergy or apoptosis. Although the molecular basis of T cell help has been extensively dissected, the mechanisms that restrain Ag-stimulated B cells, and enforce dependence upon co-stimulation, are incompletely understood. Nr4a1-3 encode a small family of orphan nuclear receptors that are rapidly induced by B cell receptor (BCR) stimulation, yet little is known about their function in humoral immune responses. Here we use germline and conditional loss-of-function mouse models to show that Nr4a1 and Nr4a3 play partially redundant roles to restrain both the survival and proliferation of B cells that receive signal 1 in the absence of co-stimulatory signals, and do so in part by repressing expression of BATF and consequently c-MYC. Correspondingly, Ab responses to TI-2 immunogens are enhanced in the absence of Nr4a1, but are unaltered in response to immunogens that incorporate co-stimulatory signals. Unexpectedly, we also identify a role for the NR4A family in restraining B cell access to T cell help by repressing expression of the T cell chemokines CCL3/4, as well as CD86 and ICAM1, and show that this is relevant under conditions of competition for limiting T cell help. Our studies collectively reveal a novel negative feedback loop mediated by the NR4A family that increases B cell dependence upon T cell help and restrains strongly Ag-activated B cell clones from monopolizing limiting amounts of T cell help. We speculate that this imposes B cell tolerance and dampens immunodominance to facilitate preservation of clonal diversity during an immune response.

immunology↗

bioRxiv2018

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

How pathogenic 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 IL-17 producing cells. Moreover, the enhanced autoreactivity was associated with upregulation of IL-6 cytokine signaling machinery. As a result, the more autoreactive GFPhi CD4 T cells from SKGNur mice were hyper-responsive to IL-6. 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. Additionally, endogenous Nur77 protein expression was enriched in a subset of synovial CD4 T cells from arthritic joints in SKG mice, consistent with antigen-activated T cells. This was was extended to patients with seropositive RA, suggesting that pathogenic CD4 T cells are enriched in RA joints and are likely recognizing an intra-articular auto-antigen(s).

immunology↗