Biology subjects

Rawlings, D. J.

Publications and source records attributed to Rawlings, D. J..

16 recordsLinked to original sources

bioRxiv2026

CD19 CAR Engineered T Regulatory Cells Promote Durable Immune Tolerance in Experimental Systemic Lupus Erythematosus

While the immune reset induced by CD19 CAR T therapies has shown striking promise in systemic lupus erythematosus (SLE), the nature and duration of benefit mediated by B cell depletion remains unclear. Loss of tolerance in SLE is driven by extrafollicular and germinal center interactions between dendritic cells and self-reactive T and B cells. We hypothesized that CD19 CAR-engineered regulatory T cells (CAR19 EngTreg), capable of suppressing multiple autoimmune effector cell types, might promote long-term tolerance without producing high-levels of inflammatory mediators. To generate CAR19 EngTreg, we combined CD19 CAR delivery with homology directed repair at the FOXP3 locus to drive constitutive, stable FOXP3 expression in CD4 T cells. CAR19 EngTreg expressed canonical Treg markers; exhibited minimal inflammatory cytokine production; and blocked human B cell differentiation into antibody secreting plasma cells. In humanized mouse models, transfer of CAR19 EngTreg led to a reduction in B cell numbers with low inflammatory cytokine production relative to CD19 CAR Teff, and reduced tissue inflammation. In a short-term, in vivo imiquimod-induced inflammatory model, murine CAR19 EngTreg reduced skin pathology, spleen size, and inflammatory cytokines. Finally, in two distinct mouse models of lupus, CAR19 EngTreg persisted for >4 months leading to reduced autoimmune T and B effector populations, sustained reduction in multiple autoantibodies and improved renal function and histopathology. Thus, CAR19 EngTreg may offer a promising and durable therapeutic avenue for B cell-mediated autoimmune disorders capable of long-term regulation of both autoreactive B and T cells.

immunology↗

bioRxiv2026

Lymphatic networks enable diversification of B cell responses to vaccines

Vaccination generates heterogenous B cell responses that collectively provide protective immunity, yet how this diversity is established remains poorly understood. Here, we identify the draining lymphatic network as a spatial organizer of humoral immunity. Vaccine dispersal across interconnected draining lymph nodes (dLNs) establishes gradients of antigen and inflammation that create distinct B cell differentiation and selection environments. dLNs proximal to the vaccination site preferentially support antibody-secreting cells and clonally diverse germinal center (GC) responses. In contrast, distal dLNs impose more stringent selection, favoring clonally restricted GCs enriched for high-affinity B cells. Spatially distinct inflammatory environments further determine antibody isotype, linking LN position to antibody effector class. These spatial biases persist upon recall and can be reprogrammed through vaccine design, including nanoparticle properties and LN-directed targeting. Thus, lymphatic networks diversify B cell fate, repertoire and affinity selection, and antibody isotype, revealing a programmable axis for tuning humoral immunity through vaccine design.

immunology↗

bioRxiv2026

Antibody-dependent priming of spontaneous germinal centers by autoreactive B cells

Autoreactive germinal centers (GCs) are central to autoimmune pathogenesis, yet the mechanisms by which single autoreactive B cell clones prime systemic autoimmunity remain unclear. Using the 564Igi mixed chimera model, we demonstrate that autoreactive 564Igi B cells break tolerance in wild-type B cells through an unexpected mechanism independent of cognate T cell interactions. While B cell-intrinsic TLR7 signaling was essential for spontaneous GC formation, deletion of MHC class II, CD40, or CD80/86 on GC-priming 564Igi B cells failed to prevent GCs. Instead, CRISPR-mediated deletion of Prdm1 (encoding BLIMP-1) in 564Igi B cells ablated spontaneous GCs, implicating autoantibody production as the primary driver. These findings reveal that autoantibodies can initiate feed-forward mechanisms that propagate systemic autoimmunity, independent of B cell-intrinsic antigen presentation.

immunology↗

bioRxiv2025

Anti-malaria RTS,S/AS01 vaccine generates a limited pool of memory B cells expressing specificities associated with protection

RTS,S/AS01, an adjuvanted subunit malaria vaccine, induces protective but short-lived anti- Plasmodium falciparum circumsporozoite protein (CSP) antibody titers. To better understand the lack of sustained protection post-vaccination, we analyzed CSP-specific B cells over time in malaria-naive individuals following immunization with RTS,S/AS01. Longitudinal analyses of the cellular responses revealed a shift in the specificity of CSP-specific B cells over time. Early post- vaccine responses were dominated by plamsablasts and class-switched memory B cells (MBCs) specific for the NANP-repeat region of CSP, epitopes associated with protective antibodies. However, the frequency of NANP-repeat-specific MBCs declines, while longer-term memory specific for the C-terminus of CSP persists. Despite being class-switched and affinity matured, C- term monoclonal antibodies derived from these MBCs failed to protect mice against a transgenic parasite challenge. Taken together, these findings suggest that RTS,S/AS01 induces a transient, protective NANP repeat-specific B cell response that is subsequently replaced by memory B cells with non-protective reactivities, potentially explaining its limited long-term efficacy and limited response to subsequent challenges or boosters.

immunology↗

bioRxiv2025

Humanized mice enable in vivo evaluation of engineered plasma cell biology and therapeutic function

Engineered plasma cells (ePCs) offer a durable strategy for in vivo delivery of therapeutic antibodies, but standard immunodeficient mouse models lack human immune factors critical for plasma cell survival and function. We utilized a humanized mouse model in which NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were engrafted with human CD34+ stem cells as recipients for infusions with autologous ePCs. In this setting, ePCs localized to plasma cell niches and stably secreted antibodies for over three months. To improve the selection of antibodies for secretion, we developed a B cell receptor surface display screen that identified candidate antibody sequences with high secretion potential. An anti-SARS-CoV-2 antibody (clone 297) selected by this method showed robust secretion both in vitro and vivo, and serum from ePC-engrafted mice potently neutralized SARS-CoV-2 pseudovirus. Together, these findings establish a physiologically relevant model for testing human ePCs, and offer a generalizable strategy for optimizing antibody selection to support long-term therapeutic delivery.

molecular biology↗

bioRxiv2024

Reduced function of the adaptor SH2B3 promotes T1D via altered γc cytokine-regulated, T cell intrinsic immune tolerance

Genome-wide association studies have identified SH2B3 as an important non-MHC gene for islet autoimmunity and type 1 diabetes (T1D). In this study, we found a single SH2B3 haplotype significantly associated with increased risk for human T1D, and this haplotype carries the single nucleotide variant rs3184504*T in SH2B3. To better characterize the role of SH2B3 in T1D, we used mouse modeling and found a T cell-intrinsic role for SH2B3 regulating peripheral tolerance. SH2B3 deficiency had minimal effect on TCR signaling or proliferation across antigen doses, yet enhanced cell survival and cytokine signaling including common gamma chain-dependent and interferon-gamma receptor signaling. SH2B3 deficient CD8+T cells showed augmented STAT5-MYC and effector-related gene expression partially reversed with blocking autocrine IL-2 in culture. Using the RIP-mOVA model, we found CD8+ T cells lacking SH2B3 promoted early islet destruction and diabetes without requiring CD4+ T cell help. SH2B3-deficient cells demonstrated increased survival post-transfer compared to control cells despite a similar proliferation profile in the same host. Next, we created a spontaneous NOD.Sh2b3-/-mouse model and found markedly increased incidence and accelerated T1D across sexes. Collectively, these studies identify SH2B3 as a critical mediator of peripheral T cell tolerance limiting the T cell response to self-antigens. Article HighlightsO_LIThe rs3184504 polymorphism, encoding a hypomorphic variant of the negative regulator SH2B3, strongly associates with T1D. C_LIO_LISH2B3 deficiency results in hypersensitivity to cytokines, including IL-2, in murine CD4+ and CD8+ T cells. C_LIO_LISH2B3 deficient CD8+ T cells exhibit a comparable transcriptome to wild-type CD8+ T cells at baseline, but upon antigen stimulation SH2B3 deficient cells upregulate genes characteristic of enhanced JAK/STAT signaling and effector functions. C_LIO_LIWe found a T-cell intrinsic role of SH2B3 leading to severe islet destruction in an adoptive transfer murine T1D model, while global SH2B3 deficiency accelerated spontaneous NOD diabetes across sexes. C_LI

immunology↗

bioRxiv2024

Monoclonal antibodies derived from B cells in subjects with cystic fibrosis reduce Pseudomonas aeruginosa burden in mice

Pseudomonas aeruginosa (PA) is an opportunistic, frequently multidrug-resistant pathogen that can cause severe infections in hospitalized patients. Antibodies against the PA virulence factor, PcrV, protect from death and disease in a variety of animal models. However, clinical trials of PcrV-binding antibody-based products have thus far failed to demonstrate benefit. Prior candidates were derivations of antibodies identified using protein-immunized animal systems and required extensive engineering to optimize binding and/or reduce immunogenicity. Of note, PA infections are common in people with cystic fibrosis (pwCF), who are generally believed to mount normal adaptive immune responses. Here we utilized a tetramer reagent to detect and isolate PcrV-specific B cells in pwCF and, via single-cell sorting and paired-chain sequencing, identified the B cell receptor (BCR) variable region sequences that confer PcrV-specificity. We derived multiple high affinity anti-PcrV monoclonal antibodies (mAbs) from PcrV-specific B cells across 3 donors, including mAbs that exhibit potent anti-PA activity in a murine pneumonia model. This robust strategy for mAb discovery expands what is known about PA-specific B cells in pwCF and yields novel mAbs with potential for future clinical use.

immunology↗

bioRxiv2024

The IFIH1-A946T risk variant promotes diabetes in a sex-dependent manner

Type 1 diabetes (T1D) is an autoimmune disease in which pancreatic islet {beta}-cells are attacked by the immune system, resulting in insulin deficiency and hyperglycemia. One of the top non-synonymous single-nucleotide polymorphisms (SNP) associated with T1D is in the interferon-induced helicase C domain-containing protein 1 (IFIH1), which encodes an anti-viral cytosolic RNA sensor. This SNP results in an alanine to threonine substitution at amino acid 946 (IFIH1A946T) and confers an increased risk for several autoimmune diseases, including T1D. We hypothesized that the IFIH1A946T risk variant, (IFIH1R) would promote T1D pathogenesis by stimulating type I interferon (IFN I) signaling leading to immune cell alterations. To test this, we developed Ifih1R knock-in mice on the non-obese diabetic (NOD) mouse background, a spontaneous T1D model. Our results revealed a modest increase in diabetes incidence and insulitis in Ifih1R compared to non-risk Ifih1 (Ifih1NR) mice and a significant acceleration of diabetes onset in Ifih1R females. Ifih1R mice exhibited a significantly enhanced interferon stimulated gene (ISG) signature compared to Ifih1NR, indicative of increased IFN I signaling. Ifih1R mice exhibited an increased frequency of plasma cells as well as tissue-dependent changes in the frequency and activation of CD8+ T cells. Our results indicate that IFIH1R may contribute to T1D pathogenesis by altering the frequency and activation of immune cells. These findings advance our knowledge on the connection between the rs1990760 variant and T1D. Further, these data are the first to demonstrate effects of Ifih1R in NOD mice, which will be important to consider for the development of therapeutics for T1D.

immunology↗

bioRxiv2023

Activated PI3Kδ specifically perturbs mouse Treg homeostasis and function leading to immune dysregulation

Foxp3+ regulatory T cells (Treg) are required for maintaining immune tolerance and preventing systemic autoimmunity. PI3K{delta} is required for normal Treg development and function. However, the impacts of dysregulated PI3K{delta} signaling on Treg function remain incompletely understood. Here, we used a conditional mouse model of activated PI3K{delta} syndrome (APDS) to investigate the role of altered PI3K{delta} signaling specifically within the Treg compartment. Aged mice expressing a PIK3CD gain-of-function mutation (aPIK3CD) specifically within the Treg compartment exhibited weight loss and evidence for chronic inflammation as demonstrated by increased memory/effector CD4+ and CD8+ T cells with enhanced IFN-{gamma} secretion, spontaneous germinal center responses and production of broad-spectrum autoantibodies. Intriguingly, aPIK3CD facilitated Treg precursor development within the thymus and an increase in peripheral Treg numbers. Peripheral Treg, however, exhibited an altered phenotype including increased PD1 expression and reduced competitive fitness. Consistent with these findings, Treg specific-aPIK3CD mice mounted an elevated humoral response following immunization with a T-cell dependent antigen, that correlated with a decrease in follicular Treg. Taken together, these findings demonstrate that an optimal threshold of PI3K{delta} activity is critical for Treg homeostasis and function, suggesting that PI3K{delta} signaling in Treg might be therapeutically targeted to either augment or inhibit immune responses.

immunology↗

bioRxiv2023

Advancing Cell Therapies: Single-Cell Profiling, Generation, Expansion, and Gene Delivery in Rhesus Macaque Plasma B Cells

Engineered long lived plasma cells have the potential to be a new area of cell therapy. A key step in developing this cell therapy is testing in a model with an intact immune system similar to humans. To that end, we have developed methods to purify, expand, and differentiate non-human primate (NHP; rhesus macaque) B cells ex vivo. We consistently achieved 10-fold expansion of NHP B cells using a readily available commercial supplement. After only seven days in culture, large percentages of cells in NHP B cell cultures were differentiated. These cells expressed surface markers found in human antibody secreting cells (CD38 and CD138) and secreted immunoglobulin G. From single cell transcriptome analysis of NHP, we verified the presence of plasma cell markers commonly shared with humans, and have unearthed less recognized markers such as CD59 and CD79A. In addition, we identified unique NHP plasma cell markers that are absent in humans including the immune checkpoint molecule CD274 (PD-L1, Programmed Death-Ligand 1). Furthermore, we found that MHC class I molecules were upregulated in NHP plasma cells, in contrast to the pattern observed in humans. Lastly, we also identified the serotypes (AAVD-J) and established the conditions for efficient transduction of NHP B cells with AAV vectors, achieving an editing rate of approximately 60%. We envision that this work will accelerate proof-of-concept in vivo studies using engineered protein-secreting B cells in the NHP model.

bioengineering↗

bioRxiv2023

Human plasma cells engineered to secrete bispecifics drive effective in vivo leukemia killing

Bispecific antibodies are an important tool for the management and treatment of acute leukemias. Advances in genome-engineering have enabled the generation of human plasma cells that secrete therapeutic proteins and are capable of long-term in vivo engraftment in humanized mouse models. As a next step towards clinical translation of engineered plasma cells (ePCs) towards cancer therapy, here we describe approaches for the expression and secretion of bispecific antibodies by human plasma cells. We show that human ePCs expressing either fragment crystallizable domain deficient anti-CD19 x anti-CD3 (blinatumomab) or anti-CD33 x anti-CD3 bispecific antibodies mediate T cell activation and direct T cell killing of specific primary human cell subsets and B-acute lymphoblastic leukemia or acute myeloid leukemia cell lines in vitro. We demonstrate that knockout of the self-expressed antigen, CD19, boosts anti-CD19 bispecific secretion by ePCs and prevents self-targeting. Further, anti-CD19 bispecific-ePCs elicited tumor eradication in vivo following local delivery in flank-implanted Raji lymphoma cells. Finally, immunodeficient mice engrafted with anti-CD19 bispecific-ePCs and autologous T cells potently prevented in vivo growth of CD19+ acute lymphoblastic leukemia in patient-derived xenografts. Collectively, these findings support further development of ePCs for use as a durable, local delivery system for the treatment of acute leukemias, and potentially other cancers. Key pointsO_LIUsing gene editing, we engineered human plasma cells that secrete functional bispecifics to target leukemia cells expressing CD19 or CD33 C_LIO_LIEngineered plasma cells secreting bispecifics suppress patient-derived leukemia in immunodeficient mice C_LI

bioengineering↗

bioRxiv2023

IL-4 downregulates BCL6 to promote memory B cell selection in germinal centers

Germinal center (GC)-derived memory B cells (MBCs) are critical for humoral immunity as they differentiate into protective antibody-secreting cells during re-infection. GC formation and cellular interactions within the GC have been studied in detail, yet the exact signals that allow for the selection and exit of MBCs are not understood. Here, we show that IL-4 signaling in GC B cells directly downregulates BCL6 via negative autoregulation to release cells from the GC program and promote MBC formation. This selection event requires additional survival cues and can therefore result in either GC exit or death. We demonstrate that both increasing IL-4 bioavailability or limiting IL-4 signaling disrupt MBC selection stringency. In this way, IL-4 control of BCL6 expression serves as a tunable switch within the GC to tightly regulate MBC selection and affinity maturation.

immunology↗

bioRxiv2022

SEC-seq: Association of molecular signatures with antibody secretion in thousands of single human plasma cells

Protein secretion drives many functions in vivo; however, methods to link secretions with surface markers and transcriptomes have been lacking. By accumulating secretions close to secreting cells held within cavity-containing hydrogel nanovials, we demonstrate workflows to analyze the amount of IgG secreted from single human antibody-secreting cells and link this information to surface marker expression and transcriptional profiles from the same cells. Measurements using flow cytometry and imaging flow cytometry corroborated an association between levels of IgG secretion and CD138 expression. Using oligonucleotide-labeled antibodies and droplet-based sequencing, we show that pathways encoding protein localization to the endoplasmic reticulum, NADH complex assembly, and mitochondrial respiration were most associated with high IgG secretion. Altogether, this method links secretion information to cell surface and single-cell sequencing information (SEC-seq) and enables exploration of links between genome and secretory function, laying the foundation for numerous discoveries in immunology, stem cell biology, and beyond.

bioengineering↗

bioRxiv2022

PTPN22 R620W gene editing in T cells enhances low avidity TCR responses

A genetic variant in the gene PTPN22 (R620W, rs2476601) is strongly associated with increased risk for multiple autoimmune diseases and has been linked to altered TCR regulation. Here, we utilize Crispr/Cas9 gene editing with donor DNA repair templates in human cord blood-derived, naive T cells to generate PTPN22 risk edited, non-risk edited (silent modification), or knock out T cells from the same donor. PTPN22 risk edited cells exhibited increased activation marker expression following non-specific TCR engagement, findings that mimicked PTPN22 KO cells. Next, using lentiviral delivery of T1D patient-derived islet-antigen specific TCRs, we demonstrate that loss of PTPN22 function led to enhanced signaling in T cells expressing a lower avidity self-reactive TCR. In this setting, loss of PTPN22 mediated enhanced proliferation and Th1 skewing. Importantly, expression of the risk variant in association with a lower avidity TCR also increased proliferation relative to PTPN22 non-risk T cells. Together, these findings suggest that, in primary human T cells, PTPN22 rs2476601 contributes to autoimmunity risk by permitting increased TCR signaling in mildly self-reactive T cells, thereby potentially expanding the self-reactive T cell pool and skewing this population toward an inflammatory phenotype.

immunology↗

bioRxiv2021

Efficient methods for generation and expansion of, and gene delivery to Rhesus Macaque plasma B cells

immunology↗

bioRxiv2021

Evaluation of persistence and fate of ex vivo edited-HSC modified with donor template and Its role in correcting Sickle Cell Disease

Sickle cell disease (SCD) is caused by a single nucleotide transversion in exon 1 of the HBB gene that changes the hydrophobicity of adult globin ({beta}A), leading to substantial morbidity and reduced lifespan. Ex vivo autologous gene editing utilizing co-delivery of a designer nuclease along with a DNA donor template allows for precise homology-directed repair (HDR). These gene corrected cells when engrafted into the bone marrow (BM) can prove to be therapeutic and serves as an alternative to HLA-matched BM transplantation. In the current study, we extensively explored the role of single stranded oligonucleotide (ssODN) and recombinant adeno-associated 6 (rAAV6) donor template delivery to introduce a codon-optimized change (E6optE) or a sickle mutation (E6V) change following Crispr/Cas9-mediated cleavage of HBB in healthy human mobilized peripheral blood stem cells (mPBSCs). We achieved efficient HDR in vitro in edited cells and observed robust human CD45+ engraftment in the BM of NBSGW mice at 16-17 weeks. Notably, recipients of ssODN-modified HSC exhibited a significantly higher proportion of HDR-modified cells within individual BM, CD34+ and CD235+ compartments of both E6optE and E6V cohorts. We further assessed key functional outcomes including RNA transcripts analysis and globin sub-type expression. Our combined findings demonstrate the capacity to achieve clinically relevant HDR in vitro and in vivo using both donor template delivery method. The use of ssODN donor template-delivery is consistently associated with higher levels of gene correction in vivo as demonstrated by sustained engraftment of HDR-modified HSC and erythroid progeny. Finally, the HDR-based globin protein expression was significantly higher in the E6V ssODN-modified animals compared to the rAAV6-modified animals confirming that the ssODN donor template delivery outperforms rAAV6-donor template delivery.

genomics↗