Biology subjects

Boscia, A. R.

Publications and source records attributed to Boscia, A. R..

5 recordsLinked to original sources

bioRxiv2025

Red Blood Cell Transfusion is a Non-Canonical Immune Stimulus Characterized by the Suboptimal Induction of CD4+ T Cell Help

Red blood cell (RBC) alloimmunization to non-ABO antigens is a major clinical complication for chronically transfused patients. When exposed to transfused RBCs carrying foreign antigens, some patients generate IgG antibodies that target these antigens, creating potential barriers to future transfusions. Interestingly, other patients produce only IgM antibodies against the same non-ABO antigens, which generally have fewer clinical consequences. Despite the stark differences in their impact, the factors regulating IgM versus IgG production in response to transfused RBCs remain poorly understood. This study explores the balance between IgM and IgG production following transfusion, comparing it to the well-characterized antibody response induced by vaccination in mouse models. By directly assessing antibody levels following RBC transfusion versus Alum-adjuvanted vaccination, we demonstrate that transfusion of RBCs expressing a model antigen is a relatively weak inducer of IgG class switching. Additionally, loss-of-function experiments using CD40L blockade and CD4 depletion confirmed that T cell help is essential for class switching after transfusion but has no effect on IgM production. Most notably, providing supra-physiological levels of T cell help enhanced class switching in a dose-dependent manner after transfusion, whereas vaccination-induced class switching remained unaffected. These findings support a model in which the limited IgG class switching following transfusion stems from suboptimal T cell help compared to vaccination. Furthermore, they suggest that transfusion activates T cells through a non-canonical pathway, distinct from the mechanisms driving immune responses to standard Alum vaccination.

immunology↗

bioRxiv2025

Base Editing Rescue of Seizures and SUDEP in SCN8A Developmental Epileptic Encephalopathy

SCN8A encodes the voltage-gated sodium channel Nav1.6 which plays a key role in facilitating neuronal excitability. Mutations in SCN8A, particularly gain-of-function missense variants, are associated with SCN8A developmental and epileptic encephalopathy (DEE), a severe epilepsy syndrome characterized by spontaneous seizures, movement disorders, cognitive dysfunction, and sudden unexpected death in epilepsy (SUDEP). The recurrent SCN8A variant R1872W destabilizes inactivation of the sodium channel, resulting in neuronal hyperexcitability and onset of seizures. Current treatments, including anti-seizure medications (ASMs) that broadly target sodium channels, are often ineffective in SCN8A DEE patients and are associated with significant side effects, highlighting the need for targeted therapies. In this study, we utilized base editing as a therapeutic strategy to correct the patient derived R1872W SCN8A variant. Using two engineered mammalian cell line screens, we identified several targeting constructs that successfully reverted the R1872W variant to the reference allele. Our most effective construct, a modified adenine base editor, along with a paired successful guide RNA, was selected and packaged within a dual PhP.eB-adeno-associated virus (AAV) delivery system. This dual AAV therapy, referred to as SCN8A-ABE, was administered to mice expressing the R1872W variant at P2. Treatment with SCN8A-ABE significantly increased survival of mice expressing R1872W and either significantly reduced or completely inhibited seizure occurrence. Assessment of editing efficiencies revealed approximately 30% conversion of the mutant tryptophan to wildtype arginine observable in RNA transcripts from hippocampal and cortex tissue. Electrophysiological recordings revealed a rescue of seizure-associated neuronal hyperexcitability and a suppression of the pathogenic sodium channel behavior in treated mice. Associated comorbidities, including movement disorders and anxiety-like behaviors, were also improved in treated mice. These findings demonstrate the profound potential of base editing as a targeted and effective therapeutic approach for SCN8A DEE, addressing the underlying genetic mutation driving the disease.

bioengineering↗

bioRxiv2025

Transfusion of allogenic murine HOD red blood cells preferentially induces low-affinity, short-lived IgG antibodies that are germinal center independent

Transfusion-induced anti-red blood cell (RBC) alloantibodies pose a significant risk to patients who require chronic transfusions. Anti-RBC alloantibodies can be remarkably short-lived (i.e. evanescent), leading to clinically relevant alloantibodies that are not detected in later pre-transfusion antibody screens. Subsequent transfusion of alloantigen-positive RBCs stimulates a rapid memory antibody response that may induce a delayed hemolytic transfusion reaction (DHTR), causing morbidity and occasional mortality in chronically transfused patients. It is unclear why transfusions favor evanescent antibody responses over long-lived antibodies typically observed upon infections and vaccinations. We therefore turned to the HOD mouse model of RBC alloimmunization to elucidate regulators of antibody persistence in response to allogenic transfusions. By following antibody responses over time in transfused mice, we found that HOD-specific alloantibodies rapidly decay within three months while vaccination-induced antibodies remain constant. Thus, the HOD model recapitulates RBC antibody evanescence. The rapid antibody evanescence suggests that transfusion is a poor inducer of germinal centers (GCs), specialized immunological structures where B cells differentiate into germinal center B (GC B) cells and undergo iterative rounds of affinity maturation, ultimately differentiating into long-lived plasma cells that can produce antibodies for decades. Consistent with this hypothesis, we failed to observe an increase in GC B cell formation in response to transfusion, and the majority of anti-RBC alloantibodies were low affinity when compared to vaccination. To formally test the functional requirement for GCs in anti-RBC alloantibody production, we employed two orthogonal approaches to disrupt GC formation: i) day 4 CD40L blockade and ii) genetic disruption of the GC-transcription factor BCL6 selectively in B cells. Both approaches fully blocked GC formation, yet anti-RBC alloantibody production was unchanged. Collectively, our data demonstrate that anti-HOD RBC alloantibodies are GC-independent, low affinity and short-lived. The GC-independence of HOD RBC IgG responses has important implications for understanding the cellular and molecular pathways that regulate the humoral immune response to transfused RBCs, potentially explaining anti-RBC alloantibody evanescence patterns in patients.

immunology↗

bioRxiv2024

Parvalbumin Interneuron Impairment Leads to Synaptic Transmission Deficits and Seizures in SCN8A Epileptic Encephalopathy

SCN8A epileptic encephalopathy (EE) is a severe epilepsy syndrome resulting from de novo mutations in the voltage-gated sodium channel Nav1.6, encoded by the gene SCN8A. Nav1.6 is expressed in both excitatory and inhibitory neurons, yet previous studies have primarily focused on the impact SCN8A mutations have on excitatory neuron function, with limited studies on the importance of inhibitory interneurons to seizure onset and progression. Inhibitory interneurons are critical in balancing network excitability and are known to contribute to the pathophysiology of other epilepsies. Parvalbumin (PV) interneurons are the most prominent inhibitory neuron subtype in the brain, making up about 40% of inhibitory interneurons. Notably, PV interneurons express high levels of Nav1.6. To assess the role of PV interneurons within SCN8A EE, we used two mouse models harboring patient-derived SCN8A gain-of-function mutations, Scn8aD/+, where the SCN8A mutation N1768D is expressed globally, and Scn8aW/+-PV, where the SCN8A mutation R1872W is selectively expressed in PV interneurons. Expression of the R1872W SCN8A mutation selectively in PV interneurons led to the development of spontaneous seizures in Scn8aW/+-PV mice and seizure-induced death, decreasing survival compared to wild-type. Electrophysiology studies showed that PV interneurons in Scn8aD/+ and Scn8aW/+-PV mice were susceptible to depolarization block, a state of action potential failure. Scn8aD/+ and Scn8aW/+-PV interneurons also exhibited increased persistent sodium current, a hallmark of SCN8A gain-of-function mutations that contributes to depolarization block. Evaluation of synaptic connections between PV interneurons and pyramidal cells showed an increase in synaptic transmission failure at high frequencies (80-120Hz) as well as an increase in synaptic latency in Scn8aD/+ and Scn8aW/+-PV interneurons. These data indicate a distinct impairment of synaptic transmission in SCN8A EE, potentially decreasing overall cortical network inhibition. Together, our novel findings indicate that failure of PV interneuron spiking via depolarization block along with frequency-dependent inhibitory synaptic impairment likely elicits an overall reduction in the inhibitory drive in SCN8A EE, leading to unchecked excitation and ultimately resulting in seizures and seizure-induced death.

neuroscience↗

bioRxiv2023

Class switching is differentially regulated in RBC alloimmunization and vaccination

BackgroundStudies of human patients have shown that most anti-RBC alloantibodies are IgG1 or IgG3 subclasses, though it is unclear why transfused RBCs preferentially drive these subclasses over others. Though mouse models allow for the mechanistic exploration of class-switching, previous studies of RBC alloimmunization in mice have focused more on the total IgG response than the relative distribution, abundance, or mechanism of IgG subclass generation. Given this major gap, we compared the IgG subclass distribution generated in response to transfused RBCs relative to protein in alum vaccination, and determined the role of STAT6 in their generation. Study Design and MethodsWT mice were either immunized with Alum/HEL-OVA or transfused with HOD RBCs and levels of anti-HEL IgG subtypes were measured using end-point dilution ELISAs. To study the role of STAT6 in IgG class-switching, we first generated and validated novel STAT6 KO mice using CRISPR/cas9 gene editing. STAT6 KO mice were then transfused with HOD RBCs or immunized with Alum/HEL-OVA, and IgG subclasses were quantified by ELISA. ResultsWhen compared to antibody responses to Alum/HEL-OVA, transfusion of HOD RBCs induced lower levels of IgG1, IgG2b and IgG2c but similar levels of IgG3. Class switching to most IgG subtypes remained largely unaffected in STAT6 deficient mice in response to HOD RBC transfusion, with the one exception being IgG2b. In contrast, STAT6 deficient mice showed altered levels of all IgG subtypes following Alum vaccination. DiscussionOur results show that anti-RBC class-switching occurs via alternate mechanisms when compared to the well-studied immunogen alum vaccination.

immunology↗