Immunology

Permanent URI for this collectionhttps://digital.lib.washington.edu/handle/1773/4930

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Monocytic niches enable Mycobacterium Tuberculosis Persistence in Lymph Nodes
(2026-04-20) Shamskhou, Elya; Gerner, Michael Y.; Urdahl, Kevin B.
Tuberculosis remains a leading cause of infectious mortality worldwide, in part due to the ability of Mycobacterium tuberculosis (Mtb) to persist within host tissues despite robust immune activation. While pulmonary infection has historically dominated studies of tuberculosis pathogenesis, emerging evidence suggests that lymphoid tissues play a critical role in shaping disease outcome. This dissertation investigates the lung-draining mediastinal lymph node (medLN) as a central and underappreciated site of Mtb persistence, addressing the paradox that the medLN serves as both the primary site of adaptive immune priming and a long-term bacterial reservoir.Using complementary approaches including mouse infection models, multiparameter confocal microscopy, flow cytometry, single-cell transcriptomics, and mathematical modeling, this work defines the cellular and spatial mechanisms that enable Mtb survival within the medLN. We identify conventional dendritic cells and monocytes as early carriers of Mtb from the lung to the medLN and show that, by three weeks post-infection, Mtb becomes concentrated within monocytic aggregates that support bacterial persistence. Conventional dendritic cells—particularly cDC1 and inflammatory cDC2 subsets—are required for robust TH1-skewed CD4 T cell priming due to sequential waves of IL-12 production in the medLN during the first 3 weeks of infection. However, the monocytic niche in the medLN provides a site of bacterial persistence, where overwhelming numbers of Mtb-specific TH1 effector T cells fail to reduce Mtb burden within the medLN, despite effective control in the lung. These findings reveal a state of functional immunological blindness within the medLN, in which immune activation is uncoupled from immune efficacy. Together, this work redefines lymphoid tissue involvement in tuberculosis pathogenesis, establishes the medLN as a privileged niche for Mtb persistence, and highlights the need for therapeutic and vaccine strategies that target tissue-specific immune constraints rather than immune activation alone.
KIT Supports Small Intestinal Tuft Cell Hyperplasia
(2026-04-20) Lara, Heber Isai; von Moltke, Jakob
A fundamental characteristic of multicellular biology is the continual turnover of specialized cells.These cells are characterized by their unique combination of transcribed RNA, proteins—such as membrane- bound signals and transcription factors—and spatial localization. Together, these properties generate unique cell types with specialized functions that interdependently maintain the multicellular organism. Historically, immunology focused on a diverse group of cells specialized for pathogen elimination. Early studies primarily investigated white blood cells such as T cells or macrophages1; however, there is now a broad appreciation that immune function depends on communication not only among these cells but also with non-hematopoietic cells. Central to this dialogue are secreted proteins, such as cytokines, chemokines, and growth factors, which collectively create an environment that supports the generation and activation of cells critical to an immune response. This coordinated signaling is exemplified in the immune response against a parasitic worm (helminth), where epithelial and lamina propria cells act in concert to initiate and sustain anti-helminth immunity. Soil-transmitted helminths (STHs) mature and reproduce in the small intestine, where the primary immune response is enacted. Helminths are detected by tuft cells, a rare specialized epithelial cell that produces interleukin (IL)-25 and cysteinyl leukotrienes (cysLTs). These mediators act synergistically to activate group 2 innate lymphoid cells (ILC2s) which in turn drive helminth clearance. Activated ILC2s secrete IL-4, -5, and - 13, characteristic cytokines of type 2 immunity. These interleukins coordinate hallmarks of the type 2 immune response such as increased muscle contraction, mucus secretion, and tuft cell hyperplasia. Among these, tuft cell hyperplasia is critical to the worm clearance, not just for the amplification of pathogen detection, but also for increased production of tuft cell-derived effector molecules. This expansion depends on IL-4/13 signaling within the small intestinal epithelium (SIE) and can increase tuft cell frequency up to 10-fold. Yet, despite the magnitude of this response, how IL-4/13 supports the expansion of tuft cells remains unclear. We show that tuft cells across all tissues express the receptor tyrosine kinase KIT, a growth factor known to support cell division, differentiation and survival in multiple cell types. We find that IL-4/13 is necessary and sufficient to upregulate KIT on small intestinal (SI) tuft cells. While epithelial KIT is dispensable for homeostatic turnover, KIT deletion from tuft cells during helminth infection reduces tuft cell hyperplasia and delays helminth clearance. Mechanistically, KIT signaling supports the generation of new tuft cells in SIE crypts. These findings identify a novel tuft cell-specific function for KIT in type 2 immune responses and highlight the coordinated efforts of cytokines and growth factors in establishing and maintaining immunity.
Divergent effects of a Treg-selective IL-2 mutein on Influenza specific T cell responses
(2026-02-05) Albe, Joseph; Campbel, Daniel J
Enhancing regulatory T cell (Treg) function offers a compelling therapeutic strategy for autoimmune disease. Engineered IL-2 muteins more selectively expand functional Tregs with minimal impact on other immune cells compared to wild type IL-2, but their potential to compromise antiviral immunity remains largely unexplored. Here, we used a murine model of Influenza A virus (Flu) infection to determine how IL-2 mutein shapes T cell responses to respiratory virus infection. IL-2 is a pleiotropic cytokine and may have divergent effects depending on the time of administration relative to infection. IL-2 mutein administration prior to infection suppressed Flu-specific (Flu-sp) CD8 T cell responses and altered their localization and phenotype within the lungs, without affecting bystander CD8 T cells. This suppression correlated with reduced antigen presentation molecule expression on conventional dendritic cells (cDCs) early after infection but did not impact Flu-sp CD8 T cell priming. In contrast, administering IL-2 mutein during infection exacerbated disease and drove CD25-dependent expansion of Flu-sp CD8 T cells. Despite these opposing effects on effector responses, regardless of when Fc.Mut24 was given relative to infection, Fc.Mut24-treated mice generated robust antibody responses and protective T cell memory which were maintained for at least 170 days. These findings reveal that Fc.Mut24 has temporally distinct effects on antiviral immunity, dampening early effector responses when given before infection, but enhancing effector expansion and disease severity when delivered during infection. Our results provide critical context for the therapeutic application of IL-2 muteins and highlight the importance of treatment timing in balancing immune modulation with protective immunity.
Generation of immune responses within lymph nodes during type-I inflammation
(2025-10-02) Huang, Jessica; Gerner, Michael
Immune responses must be rapid, tightly orchestrated, and tailored to the encountered stimulus. Lymphatic vessels facilitate this process by continuously collecting immunological information (i.e., antigens, immune cells, and soluble mediators) about the current state of peripheral tissues, and transporting these via the lymph across the lymphatic system. Lymph nodes (LNs), which are critical meeting points for innate and adaptive immune cells, are strategically located along the lymphatic network to intercept this information. Within LNs, immune cells are spatially organized, allowing them to efficiently respond to information delivered by the lymph, and to either promote immune homeostasis or mount protective immune responses. These responses involve the activation and functional cooperation of multiple distinct cell types, and are tailored to the specific inflammatory conditions. The natural patterns of lymph flow can also generate spatial gradients of antigens and agonists within draining LNs, which can in turn further regulate innate cell function and localization, as well as the downstream generation of adaptive immunity. In this dissertation, we explore how information transmitted by the lymph shapes the spatiotemporal organization of innate and adaptive immune responses in LNs, with particular focus on type-I inflammation.
Lymphatic chain gradients regulate the magnitude and heterogeneity of T cell responses to vaccination
(2025-08-01) Conlon, Michael Thomas; Gerner, Michael
Upon activation, T cells proliferate and differentiate into diverse populations, including highly differentiated effector and memory precursor subsets. Initial diversification is influenced by signals sensed during T cell priming within lymphoid tissues. However, the rules governing how cellular heterogeneity is spatially encoded in vivo remain unclear. Here, we show that immunization establishes concentration gradients of antigens and inflammation across interconnected chains of draining lymph nodes (IC-LNs). While T cells are activated at all sites, individual IC-LNs elicit divergent responses: proximal IC-LNs favor the generation of effector cells, whereas distal IC-LNs promote formation of central memory precursor cells. Although both proximal and distal sites contribute to anamnestic responses, T cells from proximal IC-LNs preferentially provide early effector responses at inflamed tissues. Conversely, T cells from distal IC-LNs demonstrate an enhanced capacity to generate long-lasting responses to chronic antigens in cancer settings, including after checkpoint blockade therapy. Therefore, formation of spatial gradients across lymphatic chains following vaccination regulates the magnitude, heterogeneity, and longevity of T cell responses.
Notch-activated basophils support intestinal CD4 T cell fate and function during helminth infection
(2025-08-01) Warner, Lindsey; Tait Wojno, Elia
Parasitic helminth infections affect over 1 billion people worldwide, underscoring the need to study host-parasite interactions for therapeutic intervention. Helminth infection provokes a Type 2 inflammatory response orchestrated by CD4+ T helper 2 (Th2) cells. In the intestine, Th2s elicit an interleukin-13 (IL-13)-dependent “weep and sweep” response from the epithelium to drive parasite clearance. Tissue-specific cues critically optimize intestinal CD4+ T cell responses, but the exact mechanism the regulate intestinal Th2 responses remain unclear. Basophils, a rare granulocyte, are associated with Th2 function. However, the basophil-dependent signals that support intestinal Th2s are incompletely defined. Previously, we located the Notch signaling pathway in basophil activation during Trichuris muris infection, a mouse model of human whipworm. Here, we show that loss of Notch-activation in basophils results in defective parasite clearance and a blunted Th2 response. We found that basophil-intrinsic Notch was not only required for infection-elicited Th2 cytokine responses, but also for maintaining a broader IL-4 production program across a larger population of diverse intestinal CD4+ T cells. Intestinal CD4+ T cell cytokine production was basophil-dependent in vitro and in vivo, but independent of basophil-secreted factors. Our findings highlight an IL-4 autocrine signaling module that mediates intestinal CD4+ T cell fate and function via direct cell-cell interaction with basophils during helminth infection. These data improve our understanding of the tissue-specific mechanisms required for robust Type 2 immune responses and may inform the development of new therapeutic interventions for helminth infection.
Leveraging TCR signaling for the design of sensitive bispecific receptors for cancer therapy
(2025-01-23) Bugos, Grace Alice; Riddell, Stanley R
Immune-based therapies have revolutionized the treatment of cancer by improving the immune system’s capacity to target and kill cancer cells. These advances were built on foundational research in immunology, underscoring that continued immunology research is critical to the improvement of human health. Adoptive T cell therapies, particularly chimeric antigen receptors (CAR) T cell therapies exemplify the direct impact of fundamental immunology on treatment innovations. Chimeric Antigen Receptors (CARs) are engineered by combining and extracellular binding domain with signaling molecules essential T cell activation into one molecule, enabling specific activation of T cells to target tumor cells expressing the ligand for the extracellular binder. Research in T cell biology, particularly the T cell receptor (TCR) enabled the design of CARs by identifying essential TCR activation signals that were incorporated in the CAR architecture. Thousands of patients have been treated with CAR T cell therapies, with remarkable success, particularly in therapy of B cell malignancies and multiple myeloma. However, further advances are required to overcome the escape of tumor cells that express low levels or lack the target antigen improve the durability of responses and expand the application T cell therapies. This thesis presents the development a novel class of receptors for adoptive T cell therapy, termed chimeric T cell receptors (ChTCRs)that provide improved sensitivity and specificity over traditional CAR T cell therapies. Our receptor design leverages an understanding of TCR signaling to more closely mimic native TCR signaling, and improves receptor sensitivity. This approach led to the development of two novel receptors: the monospecific ‘Full ChTCR,’ which has superior antigen sensitivity and tumor control in pre-clinical mouse models relative to existing CARs and other ChTCR variants, and the dual-antigen targeting ‘Bi-ChTCR,’ which has enhanced sensitivity and efficacy in to targeting heterogenous tumors compared to monospecific and bispecific CARs. Additionally, I developed a mass cytometry tool to determine similarities and differences in proximal receptor signaling in engineered T cells and understand how improved signaling may impact cell differentiation and function. Collectively this work demonstrates the value of TCR signaling research in guiding novel receptor design for adoptive T cell therapies. It introduces a tool to study signaling dynamics and reports an adaptable receptor architecture for sensitive targeting of tumor antigen.
Apoptotic cells promote circulating tumor cell survival and metastasis
(2025-01-23) Hagan, Cassidy; Oberst, Andrew
During tumor progression and especially following cytotoxic therapy, cell death of both tumor and stromal cells is widespread. Despite clinical observations that high levels of apoptotic cells correlate with poorer patient outcomes, the physiological effects of dying cells on tumor progression remain incompletely understood. Here, we report that circulating apoptotic cells robustly enhance tumor cell metastasis to the lungs. Using intravenous metastasis models, we observed that the presence of apoptotic cells, but not cells dying by other mechanisms, supports circulating tumor cell (CTC) survival following arrest in the lung vasculature. Apoptotic cells promote CTC survival by recruiting platelets to the forming metastatic niche. Apoptotic cells externalize the phospholipid phosphatidylserine to the outer leaflet of the plasma membrane, which we found increased the activity of the coagulation initiator Tissue factor, thereby triggering the formation of platelet clots that protect proximal CTCs. Inhibiting the ability of apoptotic cells to induce coagulation by knocking out Tissue factor, blocking phosphatidylserine, or administering the anticoagulant heparin abrogated the pro-metastatic effect of apoptotic cells. This work demonstrates a previously unappreciated role for apoptotic cells in facilitating metastasis by establishing CTC-supportive emboli, and suggests points of intervention that may reduce the pro-metastatic effect of apoptotic cells.
Defining mRNA-generated T cell memory in respiratory viral infection
(2025-01-23) Pruner, Kurt B.; Pepper, Marion
T cell immune memory provides protective effector mechanisms to restrict pathogen growth upon secondary infection, although the ability of T cells generated by novel mRNA-LNP vaccines to restrict viral infection in currently unknown. To address this, we studied antigen specific T cell memory generated by mRNA-LNP vaccines in both humans and mice. We report that functional and durable T cell memory is induced by mRNA vaccines in humans, and we define functional T cell correlates of so-called ‘hybrid immunity’ through the study of T cell cytokine production in human blood. Further, we report that mRNA-LNP immunization of mice provides incomplete benefits when compared to previous infection and unexposed naÃ¯ve mice challenged with influenza virus in the absence of neutralizing antibodies. This intermediate state of protection was associated with a lack of resident memory cells following initial vaccination but expeditated T cell recruitment and adaptation to the lung when compared to unexposed mice. Together, our data imply that mRNA-LNP vaccine-generated memory T cells can provide limited protective capacity in the absence of neutralizing antibodies, but this protective benefit could be substantially improved by adapting aspects of previous infection in future vaccine regimens.
Synthetic RIG-I-agonist RNA induces death of hepatocellular carcinoma cells
(2025-01-23) Ulloa, Brittany S.; Gale, Michael
Retinoic acid-inducible gene I (RIG-I) is a critical sensor of viral RNA and is activated in response to binding to RNA containing exposed 5’-triphosphate (5′ppp) and poly-uridine to trigger innate immune activation and response including induction of type I and III interferons (IFNs). RIG-I signaling plays a key role in not only restricting RNA virus infection but also suppressing tumor progression via oncolytic signaling. We evaluated the actions of a specific RIG-I agonist RNA (RAR) as a potential therapeutic against model tumor cell lines representing hepatocellular carcinoma (HCC). RAR constitutes a synthetic-modified RNA motif derived from the hepatitis C virus genome that is specifically recognized by RIG-I and induces innate immune activation when delivered to cells. We found that RAR directs RIG-I-dependent signaling to drive HCC cell death. Analysis of knockout cell lines lacking RIG-I, MAVS, or IRF3 confirmed that RAR-induced cell death signaling propagates through the RIG-I-like receptor (RLR) pathway to mediate caspase activation and HCC cell death. RAR-induced cell death is potentiated by type I IFN. Thus, RAR actions trigger HCC cell death through RIG-I linkage of RLR, caspase, and IFN signaling programs. RAR offers a potent application in antitumor therapeutic strategies leveraging innate immunity against liver cancer.
Defining the necroptotic transcriptional signaling pathway
(2024-10-16) Kofman, Sigal Barrientos; Oberst, Andrew A
Neurons are post-mitotic, non-regenerative cells that have evolved fine-tuned immunological responses to maintain life-long cellular integrity; this includes resistance to common programmed cell death (PCD) pathways, including apoptosis and necroptosis. We have previously demonstrated a necroptosis-independent role for the key necroptotic kinase RIPK3 in host defense against neurotropic flavivirus infection. While this work showed that neuronal RIPK3 expression is essential for chemokine production and recruitment of peripheral immune cells to the infected CNS, the full RIPK3-dependent transcriptional signature and molecular mechanism underlying RIPK3-dependent transcription in neurons are incompletely understood. It also remains unclear what factors govern differential RIPK3 effector functions in different cell types. Here, we show that RIPK3 activation has distinct outcomes in primary cortical neurons and mouse embryonic fibroblasts (MEFs) during Zika virus (ZIKV) infection and sterile activation. We found that RIPK3 activation does not induce death in neurons; in these cells, RIPK3 is the dominant driver of antiviral gene transcription following ZIKV infection. While RIPK3 activation in MEF cells induces cell death, ablation of downstream cell death effectors unveils a RIPK3-dependent transcriptional program which largely overlaps with that observed in ZIKV-infected neurons. Using death resistant MEFs as a model to study RIPK3 signaling revealed that RIPK3 transcription relied on interactions with the RHIM domain-containing proteins RIPK1 and TRIF, effects mirrored in the RIPK3-dependent antiviral transcriptional signature observed in ZIKV-infected neurons. These findings suggest the pleotropic functions of RIPK3 are largely context dependent and that in cells that are resistant to cell death, RIPK3 acts as a mediator of inflammatory transcription.
Interleukin-2 mediated NF-κB -dependent mRNA splicing induces interferon gamma protein production
(2024-10-16) Van Gelder, Rachel Dintzis; Savan, Ram
Interferon-gamma (IFNγ) is a pleiotropic cytokine produced by natural killer (NK) cells during the early response to infection. IFNγ expression is tightly regulated to mount sterilizing immunity while preventing tissue pathology. Post-transcriptional effectors resolve expression of inflammatory cytokines, and several dampen IFNγ expression through IFNG mRNA degradation. NK cells' acute induction of IFNγ defies transcription-translation kinetics, suggesting that cells may be poised for rapid IFNγ production. In this study, we identify mRNA splicing as a major regulator of IFNγ protein production. While treatment with the combination of IL-12 and IL-2 causes synergistic production of IFNG mRNA and protein, we observe that NK cells treated with IL-12 alone transcribe IFNG with its introns intact. When NK cells are treated with both IL-2 and IL-12, IFNG transcript is spliced to form mature mRNA with a concomitant increase in IFNγ protein. We found that IL-2-dependent intron splicing occurs independently of nascent transcription and translation but is NF-κB signaling dependent. We propose that while IL-12 transcriptionally induces IFNG mRNA expression, IL-2 signaling stabilizes IFNG mRNA in an NF-κB dependent manner by inducing swift splicing of detained introns, ensuring that NK cells are poised for robust IFNγ protein expression. This study uncovers a novel role for splicing in regulating IFNγ protein production through a mechanism potentially applicable to immune control of other inflammatory mediators.
Modulating antigen-specific humoral immunity with non-differentiating B cells
(2024-09-09) Pitner, Ragan; Rawlings, David J.
Antibody-derived inhibitors pose an ongoing challenge to the treatment of patients with inherited protein deficiency disorders, limiting the efficacy of both protein replacement therapy and corrective gene therapy. Beyond their central role as producers of serum antibody, B cells also exhibit many unique properties that could be exploited in cell therapy applications, notably including antigen-specific recognition and the linked capacity for antigen presentation. Here we employed CRISPR/Cas9 to demonstrate that ex vivo antigen-primed Blimp1-knockout “decoy” B cells, incapable of differentiation into plasma cells, participated in and downregulated host antigen-specific humoral responses after adoptive transfer. Following ex vivo antigen pulse, adoptively transferred high affinity antigen-specific decoy B cells were diverted into germinal centers en masse, thereby reducing participation by endogenous antigen-specific B cells in T-dependent humoral responses and suppressing both cognate and linked antigen-specific IgG following immunization with conjugated antigen. This effect was dose-dependent and, importantly, did not impact concurrent unrelated antibody responses. We demonstrated the therapeutic potential of this approach by treating factor VIII (FVIII)-knockout mice with antigen-pulsed decoy B cells prior to immunization with a FVIII conjugate protein, thereby blunting the production of serum FVIII-specific IgG by an order of magnitude as well as reducing the proportion of animals exhibiting functional FVIII inhibition by 6-fold. Finally, we demonstrated that these Blimp1-deficient decoy B cells can be simultaneously gene-edited to enable constitutive secretion of the regulatory cytokine IL 10. Together, these results suggest that it will be possible to generate engineered regulatory B cells capable of modulating the activation and differentiation of antigen-specific CD4 T cells.
The role of IL-33 cytokine in allergic sensitization and inflammation
(2024-02-12) Varela, Stephanie; Ziegler, Steve
In this study, we sought to understand the role of interleukin-33 (IL-33) in the context of allergic sensitization and inflammation. To achieve this, we developed two novel models of IL-33 driven allergic sensitization and inflammation. Genetically modified mouse strains with cell specific IL-33 receptor knockouts, hyperactivating receptor variants, and cytokine knock outs were used to characterize these models and determine important responding cell types. Using an intradermal sensitization model with IL-33 and ovalbumin on WT C57BL/6 mice, we observed an increase in draining lymph node cellularity, antigen-specific IgE titers, and Th2 cell generation within skin-draining lymph nodes demonstrating evidence of classic allergic sensitization. Using CD4 T and dendritic cell specific ST2 deficient mouse lines we found that direct IL-33/ST2 signaling on CD4 T cell is necessary for a complete type 2 response. Without this signaling, we found an overall dampened type 2 sensitization response with a significant reduction in total effector Th2 cells and IgE titer. Our findings also suggest a possible role of CD11c+ dendritic cells in responding to IL-33 as we found an increased overall frequency of type 2-skewed CD4 T cells. To expand on our sensitization protocol we included several multi-day oral challenges with ovalbumin to induce antigen dependent eosinophil-rich allergic inflammation within the esophagus of mice. This established an IL-33 driven model of eosinophilic esophagitis (EoE). In characterizing the model we measured esophageal eosinophilia levels over a 7 day time course with a peak occurring after 7x challenges. We also determined that the model was antigen specific and IL-5 dependent. We also found that mice carrying a hyperactive ST2 SNP, rs10204137, showed no difference in eosinophilia levels. Collectively, these studies underscore the significance of IL-33 in promoting allergic sensitization, with CD4 T cells identified as a critical responding cell type in skin-draining lymph nodes, alongside the establishment of an IL-33 driven model for EoE.
IL-4 downregulates BCL6 to promote memory B cell selection in germinal centers
(2024-02-12) Shehata, Laila I; Pepper, Marion
Germinal center (GC)-derived memory B cells (MBCs) play a pivotal role in humoral immunity by differentiating into protective antibody-secreting cells upon re-infection. Despite extensive research focused on GC formation and the cellular interactions occurring within the GC microenvironment, the precise signals which regulate MBC selection and exit remain incompletely understood. In these studies, we focus on the role of interleukin-4 (IL-4) in the GC using murine blood-stage Plasmodium infections as a model of for a type 1 immune response. Our findings demonstrate that IL-4 signaling can trigger the selection and exit of GC B cells by modulating the expression of BCL6, the primary transcription factor within the GC. Specifically, we show that IL-4 induces negative autoregulation of BCL6, leading to a loss in expression of this anti-apoptotic factor within GC B cells. Consequently, in the presence of excess IL-4, there is increased GC B cell death and a loss of selection stringency. Furthermore, we demonstrate that in the absence of IL-4-mediated downregulation of BCL6, B cells with lower affinity can persist within the GC and contribute to the MBC pool. This observation highlights the critical role of IL-4-mediated downregulation of BCL6 in maintaining selection stringency and affinity maturation within the MBC population. By elucidating the role of IL-4 in modulating the fate of GC B cells, we have demonstrated the significance of IL-4 signaling in shaping the dynamics of the GC selection and subsequent memory formation. The importance of both GCs and memory formation are further underscored in chapter 3, in which we analyze the B cell response generated by both protein nanoparticle vaccines and by natural infection with SARS-CoV-2. As the formation of class-switched MBCs and LLPCs are the primary correlate of protection for vaccination, our finding that a nanoparticle displaying the receptor binding domain of SARS-CoV-2 can elicit the formation of a robust GC with class-switched B cells was very promising for the future of nanoparticle vaccines. Collectively, these findings offer novel insights into the mechanisms underlying MBC selection and affinity maturation within the GC. Through the identification of IL-4 as a negative regulator of BCL6 expression and its impact of GC B cell survival, our study advances the current understanding of how the immune system maintains an optimal balance between selection stringency and the generation of diverse MBC populations. Further research in this area will likely explore additional signaling pathways and molecular players involved in the complex network of interactions governing GC-derived MBC selection, and ultimately will contribute to the development of strategies aimed at enhancing immune responses and vaccine efficacy.
Prolonged T – DC macro-clustering within lymph node microenvironments initiates Th2 differentiation in a site-specific manner
(2024-02-12) Lyons-Cohen, Miranda R.; Gerner, Michael Y
T helper 2 (Th2) responses protect against pathogens while also driving allergic inflammation, yet how large-scale Th2 responses are generated in tissue context remains unclear. Here, we used quantitative imaging to investigate early Th2 differentiation within lymph nodes (LNs) following cutaneous allergen administration. Contrary to current models, we observed extensive activation and ‘macro-clustering’ of early Th2 cells with migratory type-2 dendritic cells (cDC2s) generating specialized Th2-promoting microenvironments. Macro-clustering was integrin mediated and promoted localized cytokine exchange among T cells to reinforce differentiation, which contrasted behavior during Th1 responses. Unexpectedly, formation of Th2 macro-clusters was dependent on the site of skin sensitization. Differences between sites were driven by divergent activation states of migratory cDC2 across dermal tissues, with enhanced costimulatory molecule expression by cDC2 in Th2-generating LNs promoting prolonged T cell activation, macro-clustering, and cytokine sensing. Thus, generation of dedicated Th2 priming micro-environments through enhanced costimulatory molecule signaling initiates Th2 responses in vivo and occurs in a skin site-specific manner.
Tuft cell-derived acetylcholine regulates epithelial fluid secretion and helminth clearance
(2023-09-27) Billipp, Tyler; von Moltke, Jakob
Helminth, or parasitic worm, infection afflicts nearly one third of humans worldwide, resulting in massive suffering and comorbidity. The arm of the immune system responsible for combatting helminth infection, called “Type 2” immunity, is also responsible for causing a variety of allergic diseases. Understanding how Type 2 immunity is regulated has the potential to inform both better treatment for helminth infection as well as allergic disease.Mucosal barrier tissues are the site of exposure to Type 2 stimuli and the locus of the ensuing immune response. Both the airways and the gastrointestinal tract are protected by a gradient of mucus, antimicrobial peptides, and fluid secreted by epithelial cells that contribute to host defense and enable key physiological functions of the tissues. The Type 2 immune response triggers epithelial remodeling that increases fluid and mucus secretion as well as contractility of underlying smooth muscle in order to flush away helminths and allergens. Acetylcholine is a key regulator of both epithelial secretion and muscle contraction. Collectively this response is known as “weep and sweep” and familiar to anyone who experiences seasonal allergies. While the effects of Type 2 immunity have been well understood for decades, the initiation of the response remained a mystery until recently, when tuft cells were discovered as the key sensors of intestinal helminth infection. Tuft cells are epithelial cells that possess chemosensory machinery linking sensing of lumenal stimuli via a suite of receptors to basolateral secretion of immune- and neuro-modulating factors including IL-25, leukotrienes, and acetylcholine (ACh). In response to small intestinal (SI) helminth infection or succinate, a metabolite produced by protist and bacterial colonization, tuft cells secrete IL-25 and leukotrienes to activate Group 2 innate lymphoid cells (ILC2s) that produce the hallmark Type 2 cytokines IL-5 and IL-13. IL-13 signals on the intestinal stem cell compartment to drive preferential differentiation of mucus-producing goblet cells and, intriguingly, tuft cells, which rapidly remodels the epithelium in a matter of days. This remodeling, and thus tuft cells, are required for helminth clearance. Additional functions of SI tuft cells or tuft-derived ACh are not known. We show that in response to sensing of succinate or direct activation of the chemosensory ion channel TRPM5, SI tuft cells secrete ACh to induce epithelial fluid secretion in the intestine and airways, independently of neurons. Unlike other tissues where nearly 100% of tuft cells express the enzyme Chat required for ACh synthesis, the frequency of Chat+ SI tuft cells occurred in a gradient from the proximal to distal SI, increasing from approximately 40% to 80% of tuft cells. Succinate-induced fluid secretion was restricted to the distal SI where Chat+ frequency and SUCNR1 expression was highest. Consistent with their high expression of Sucnr1, tuft cells in the trachea also responded to succinate by inducing ACh-dependent fluid secretion. In the proximal SI and colon tuft cell activation via the TRPM5 agonist Class 8 induced fluid secretion, but the exact ligands sensed by tuft cells in these tissues remains to be determined. Oral administration of Class 8 induced fluid secretion in vivo as measured by fecal water content. During Type 2 tissue remodeling, Chat+ tuft cells increase in number, enhancing the fluid secretion response. Upon helminth infection, mice with Chat-deficient tuft cells experience delayed helminth clearance despite normal tuft-ILC2 circuit activation. We conclude that tuft cell-derived ACh regulates epithelial fluid secretion, and that this effector function can contribute to Type 2 immune responses during helminth infection. By coupling chemosensing to rapid epithelial fluid secretion, tuft cells coordinate an epithelium-intrinsic effector unit that can flush offending agents away from the tissue.
Uncovering novel suppressors of endogenous nucleic acid ligands
(2023-09-27) Smith, Julian Robert; Savan, Ram
Type I interferons (IFNs) are important for controlling viral infections, but aberrant IFN expression can result in tissue damage. IFNs are induced following activation of pattern recognition receptors that sense pathogen associated DNA and RNA. RIG-I-like receptors (RLRs), RIG-I and MDA5, sense viral RNA and signal through the adaptor protein MAVS, while cGAS and DDX41 sense viral DNA signal though the adaptor protein STING. Activation of MAVS or STING leads to the phosphorylation and nuclear translocation of the transcription factor IRF3, in turn inducing IFNs. While activation of these pathways is often driven by viral nucleic acids, both pathways can be activated by host endogenous nucleic acids. Therefore, several mechanisms exist to prevent recognition of host nucleic acids by these receptors in order to prevent aberrant expression of IFN and IFN-mediated disease. Identifying factors important for suppressing endogenous DNA and RNA ligands is critical for preventing IFN-induced autoimmunity. In this dissertation, we identify two novel regulators that suppresses endogenous nucleic acids that activate the RLR pathway or the cGAS/DDX41/STING pathway. We discovered that the transcription factor MEF2A is required to suppresses the accumulation of RNA:DNA hybrids and DNA-damage which leads to the unscheduled activation of STING and production of IFN. Interestingly, STING activation required cGAS and DDX41 demonstrating a previously undescribed role for DDX41 driven activation of STING through genomic RNA:DNA hybrids. We also uncovered a novel role for the splicing factor CELF2 as a suppressor of endogenous RNA ligands. Depletion of CELF2 in monocytes leads to a spontaneous IFN and IFN-stimulated gene signature, dependent on the RIG-I-MAVS pathway. These data are the first to demonstrate a role for MEF2A and CELF2 in suppressing endogenous nucleic acid ligands, which can activate host pattern recognition receptors to induce an IFN signature. Overall, these findings suggest that MEF2A and CELF2 are important for preventing IFN-induced inflammation.
Integrin αvβ3 limits Toll-like receptor type I interferon production by plasmacytoid dendritic cells and restricts autoimmunity
(2023-09-27) Lorant, Alina Kathryn; Lacy-Hulbert, Adam
Plasmacytoid dendritic cells (pDCs) are strongly implicated as a major source of type I interferon (IFN-I) in systemic lupus erythematosus (SLE), triggered through Toll-like receptor (TLR)-mediated recognition of nucleic acids released from dying cells. However, relatively little is known about how TLR signaling and IFN-I production are regulated in pDCs. Here I describe a role for integrin αvβ3 in regulating TLR responses and IFN-I production by pDCs in mouse models. I show that αv and β3-/- pDCs produce more IFN-I and inflammatory cytokines than controls when stimulated though TLR7 and TLR9 in vitro and in vivo. This dysregulated TLR signaling results in activation of B cells and promotes germinal center B cell and plasma cell expansion. Furthermore, in a mouse model of TLR7-driven lupus-like disease, deletion of αvβ3 from pDCs causes accelerated autoantibody production and pathology. I therefore identify a pDC-intrinsic role for αvβ3 in regulating TLR signaling and preventing activation of autoreactive B cells. As αvβ3 serves as a receptor for apoptotic cells and cell debris, I hypothesize that this regulatory mechanism provides important contextual cues to pDCs and functions to limit responses to self-derived nucleic acids.
Influenza-specific CD4+ Th1 memory cells in the lung can be functionally altered by allergen exposure
(2023-08-14) Ruterbusch, Mikel Joseph; Pepper, Marion
CD4+ lung-resident memory T cells (Trm) generated in response to influenza infection confer effective protection against subsequent viral exposures. Whether these cells can be altered by environmental antigens and cytokines released during heterologous, antigen-independent immune responses is currently unclear. We therefore investigated how influenza-specific CD4+ Th1 Trm in the lung are impacted by a subsequent Th2-inducing respiratory house dust mite (HDM) exposure. Although naÃ¯ve influenza-specific CD4+ T cells in the secondary lymphoid organs do not respond to HDM, influenza-specific CD4+ Trm in the lungs do respond to a subsequent allergen exposure by altering their phenotype and functional capacity. Changes in transcription factor expression in this population persisted upon heterosubtypic influenza challenge and was associated with decreased morbidity, viral load, and pro-inflammatory cytokine expression in the lung. Further investigation revealed that respiratory cysteine protease or rIL-33 administration was sufficient to induce these changes in the lung-resident influenza-specific CD4+ Trm population. Thus, heterologous antigen exposure or IL-33 release can drive persistent alterations in CD4+ Th1 Trm populations.

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