Biology subjects

Arneja, A.

Publications and source records attributed to Arneja, A..

10 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

Sex differences in human IgG1-mediated angiogenesis inhibition depend on Y chromosome-encoded DDX3Y

Prevalent diseases of angiogenesis such as age-related macular degeneration (AMD) exhibit sex-specific prevalence, with female sex being an independent risk factor for the advanced neovascular form of AMD. The basis for these sex differences is poorly understood. Here, we quantify the impact of sex on suppression of aberrant choroidal angiogenesis by human immunoglobulin 1 (hIgG1), which possesses class-wide, antigen-independent anti-angiogenic activity. Males exhibited significantly more robust hIgG1-induced chemotaxis inhibition in primary human and mouse macrophages and anti-angiogenic activity in mouse laser-induced choroidal neovascularization (CNV). Four core genotypes and XY* Turner Syndrome mouse models revealed the Y chromosome as a sex-biasing factor contributing to hIgG1 responses. Transcriptomic and RNAi screening identified DEAD-Box Helicase 3 Y-Linked (DDX3Y) as necessary for the robust inhibitory effects of hIgG1 in males. Male mice as well as human and mouse macrophages lacking DDX3Y exhibited blunted hIgG1 responses in CNV and macrophage chemotaxis, resembling those of females. These results unveil a novel mechanism through which sex chromosome complement differences can impact key processes involved in AMD, carrying implications for various sex-related disorders and conditions involving angiogenesis.

pathology↗

bioRxiv2025

Splenic MARCO+ marginal zone macrophages regulate rapid production of MCP-1 and KC but are dispensable for alloantibody generation in response to stored RBCs in a murine model.

BACKGROUNDAlloimmunization to transfused red blood cells (RBCs) remains a significant clinical problem. However, the cells that initiate immune responses to transfused RBCs remain incompletely characterized. Recently published work has identified splenic marginal zone B (MZB) cells as being critically required for the production of anti-RBC alloantibodies in response to RBCs. In infectious models, MZB cell activation has been shown to depend on a unique population of marginal zone macrophages (MZMs). We hypothesized that MZMs would capture stored RBCs and present them to MZBs, and ultimately MZMs would be required for generation of anti-RBC alloantibodies in response to stored RBC transfusion. STUDY DESIGN AND METHODSStored GFP+ murine RBCs were utilized to determine the splenic localization and erythrophagocytosis by splenic macrophage populations. To determine the functional impact of MZMs, we compared LXR-KO mice, which have been reported to lack MZMs, with wild type mice. Both innate and adaptive immune responses to stored HOD allogenic RBC transfusion were measured in LXR-KO and wild type mice. RESULTSRBC storage leads to a significant increase in the phagocytosis of transfused RBCs by splenic MZMs. LXR-KO mice demonstrated a lack of MZMs and had significantly decreased rapid phase production of cytokines MCP-1 and KC, but similar levels of IL-6. Surprisingly, anti-RBC alloantibody levels were unaffected by the absence of splenic MZMs. CONCLUSIONSSplenic MZMs are involved in the innate response to transfused stored HOD RBCs, contributing to both MCP-1 and KC cytokine production. However, MZMs are dispensable for anti-RBC alloantibody production.

immunology↗

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↗

bioRxiv2025

Initiation of primary T cell--B cell interactions and extrafollicular antibody responses in an organized microphysiological model of the human lymph node

In vitro microphysiological systems (MPS) are needed to replicate events in the lymph node (LN) leading to humoral immunity against new immune threats, but current lymphoid MPS focus largely on recall responses from memory lymphocytes. Here, an LN MPS was developed from primary, naive human lymphocytes in microfluidic 3D culture to model interactions and antibody production at the LN T cell--B cell border. Naive CD4+ T cells exhibited CCL21-dependent chemotaxis, chemokinesis, and activation in the MPS, and were skewed to a T follicular helper (pre-Tfh) phenotype. IgM secretion was induced in co-culture with activated B cells in the presence of a superantigen, staphylococcal enterotoxin B (SEB); micropatterning confirmed that the interaction required physical proximity. SEB-dependence of IgM secretion was greatest at a 1:5 T:B ratio, while seeding more pre-Tfh cells accelerated plasmablast differentiation and clustering. On-chip co-cultures at a 1:5 T:B ratio developed large lymphoid clusters containing CD38+ plasmablasts and CD138+ plasma cells after 15 days, with response varying between donors. Significant plasmablast induction in T-B co-cultures did not require the pre-Tfh phenotype, but pre-Tfh cells were required for inducing IgM secretion. We envision that this LN MPS will enable predictions and mechanistic analyses of human humoral immunity in vitro.

bioengineering↗

bioRxiv2024

Engineered human lymph node stroma model for examining interstitial fluid flow and T cell egress

The lymph node (LN) performs essential roles in immunosurveillance throughout the body. Developing in vitro models of this key tissue is of great importance to enhancing physiological relevance in immunoengineering. The LN consists of stromal populations and immune cells, which are highly organized and bathed in constant interstitial flow. The stroma, notably the fibroblastic reticular cells (FRCs) and the lymphatic endothelial cells (LECs), play crucial roles in guiding T cell migration and are known to be sensitive to fluid flow. During inflammation, interstitial fluid flow rates drastically increase in the LN. It is unknown how these altered flow rates impact crosstalk and cell behavior in the LN, and most existing in vitro models focus on the interactions between T cells, B cells, and dendritic cells rather than with the stroma. To address this gap, we developed a human engineered model of the LN stroma consisting of FRC-laden hydrogel above a monolayer of LECs in a tissue culture insert with gravity-driven interstitial flow. We found that FRCs had enhanced coverage and proliferation in response to high flow rates, while LECs experienced decreased barrier integrity. We added CD4+ and CD8+ T cells and found that their egress was significantly decreased in the presence of interstitial flow, regardless of magnitude. Interestingly, 3.0 {micro}m/s flow, but not 0.8 {micro}m/s flow, correlated with enhanced inflammatory cytokine secretion in the LN stroma. Overall, we demonstrate that interstitial flow is an essential consideration in the lymph node for modulating LN stroma morphology, T cell migration, and inflammation.

bioengineering↗

bioRxiv2024

Ex vivo model of breast cancer cell invasion in live lymph node tissue

Lymph nodes (LNs) are common sites of metastatic invasion in breast cancer, often preceding spread to distant organs and serving as key indicators of clinical disease progression. However, the mechanisms of cancer cell invasion into LNs are not well understood. Existing in vivo models struggle to isolate the specific impacts of the tumor-draining lymph node (TDLN) milieu on cancer cell invasion due to the co-evolving relationship between TDLNs and the upstream tumor. To address these limitations, we used live ex vivo LN tissue slices with intact chemotactic function to model cancer cell spread within a spatially organized microenvironment. After showing that BRPKp110 breast cancer cells were chemoattracted to factors secreted by naive LN tissue in a 3D migration assay, we demonstrated that ex vivo LN slices could support cancer cell seeding, invasion, and spread. This novel approach revealed dynamic, preferential cancer cell invasion within specific anatomical regions of LNs, particularly the subcapsular sinus (SCS) and cortex, as well as chemokine-rich domains of immobilized CXCL13 and CCL1. While CXCR5 was necessary for a portion of BRPKp110 invasion into naive LNs, disruption of CXCR5/CXCL13 signaling alone was insufficient to prevent invasion towards CXCL13-rich domains. Finally, we extended this system to pre-metastatic TDLNs, where the ex vivo model predicted a lower invasion of cancer cells. The reduced invasion was not due to diminished chemokine secretion, but it correlated with elevated intranodal IL-21. In summary, this innovative ex vivo model of cancer cell spread in live LN slices provides a platform to investigate cancer invasion within the intricate tissue microenvironment, supporting time-course analysis and parallel read-outs. We anticipate that this system will enable further research into cancer-immune interactions and allow isolation of specific factors that make TDLNs resistant to cancer cell invasion, which are challenging to dissect in vivo.

bioengineering↗

bioRxiv2024

Mitigating reactive oxygen species production and increasing gel porosity improves lymphocyte motility and fibroblast spreading in photocrosslinked gelatin-thiol hydrogels

On-chip 3D culture systems that incorporate immune cells such as lymphocytes and stromal cells are needed to model immune organs in engineered systems such as organs-on-chip. Photocrosslinking is a useful tool for creating such immune-competent hydrogel cultures with spatial cell organization. However, loss of viability and motility in photocrosslinked gels can limit its utility, especially when working with fragile primary cells. We hypothesized that optimizing photoexposure-induced ROS production, hydrogel porosity or a combination of both factors was necessary to sustain cell viability and motility during culture in photocrosslinked gelatin-thiol (GelSH) hydrogels. Jurkat T cells, primary human CD4+ T cells and human lymphatic fibroblasts were selected as representative lymphoid immune cells to test this hypothesis. Direct exposure of these cells to 385 nm light and LAP photoinitiator dramatically increased ROS levels. Pretreatment with an antioxidant, ascorbic acid (AA), protected the cells from light + LAP-induced ROS and was non-toxic at optimized doses. Furthermore, scanning electron microscopy showed that native GelSH hydrogels had limited porosity, and that adding collagen to GelSH precursor before crosslinking markedly increased gel porosity. Next, we tested the impact of AA pretreatment and increasing gel porosity, alone or in combination, on cell viability and function in 3D GelSH hydrogel cultures. Increasing gel porosity, rather than AA pretreatment, was more critical for rescuing viability of Jurkat T cells and spreading of human lymphatic fibroblasts in GelSH-based gels, but both factors improved the motility of primary human CD4+ T cells. Increased porosity enabled formation of spatially organized co-cultures of primary human CD4+ T cells and human lymphatic fibroblasts in photo-crosslinked gels in a multi-lane microfluidic chip, towards modeling the lymphoid organ microenvironment. Some optimization is still needed to improve homogeneity between regions on the chip. These findings will enable researchers utilizing photocrosslinking methods to develop immunocompetent 3D culture models that support viability and function of sensitive lymphoid cells.

bioengineering↗

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↗

bioRxiv2020

In situ photopatterning of cell laden biomaterials for spatially organized 3D cell cultures in a microfluidic chip

Micropatterning techniques for 3D cell cultures enable the recreation of tissue-level structures, but their combination with well-defined, microscale fluidic systems for perfusion remains challenging. To address this technological gap, we developed a user-friendly in-situ micropatterning protocol that integrates photolithography of crosslinkable, cell-laden hydrogels with a simple microfluidic housing, and tested the impact of crosslinking chemistry on stability and spatial resolution. Working with gelatin functionalized with photo-crosslinkable moieties, we found that inclusion of cells at high densities ([≥] 107/mL) during crosslinking did not impede thiol-norbornene gelation, but decreased the storage moduli of methacryloyl hydrogels. Hydrogel composition and light dose were selected to match the storage moduli of soft tissues. The cell-laden precursor solution was flowed into a microfluidic chamber and exposed to 405 nm light through a photomask to generate the desired pattern. The on-chip 3D cultures were self-standing, and the designs were interchangeable by simply swapping out the photomask. Thiol-ene hydrogels yielded highly accurate feature sizes from 100 - 900 m in diameter, whereas methacryloyl hydrogels yielded slightly enlarged features. Furthermore, only thiol-ene hydrogels were mechanically stable under perfusion overnight. Repeated patterning readily generated multi-region cultures, either separately or adjacent, including non-linear boundaries that are challenging to obtain on-chip. As a proof-of-principle, primary human T cells, were patterned on-chip with high regional specificity. Viability remained high (> 85%) after overnight culture with constant perfusion. We envision that this technology will enable researchers to pattern 3D cultures under fluidic control in biomimetic geometries that were previously difficult to obtain.

bioengineering↗