bluuebunny/crossref_metadata_2025 huggingface dataset:
Discovery of Antigen Specific CD4+ T cells in anti-3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) Immune Mediated Necrotizing Myopathy (IMNM)
Authors: Tiniakou Eleni , Mammen Andrew , Darrah Erika
Published: January 2023

Anti-HMGCR+ IMNM is characterized by IgG antibodies against HMGCR and a strong association with HLA-DRB1* 11:01. Although this implicates HMGCR-specific CD4+ T cells in disease pathogenesis, no such cells have been identified thus far.

Monocyte derived dendritic cells from 6 anti-HMGCR+ IMNM patients were incubated with HMGCR protein and presented peptides were identified using a natural antigen processing assay (NAPA). Briefly, HLA-DR/peptide complexes were isolated by immunoprecipitation and bound peptides were sequenced by mass spectrometry. HMGCR peptides corresponding to putative T cell epitopes were synthesized and used to stimulate peripheral blood mononuclear cells from: 10 anti-HMGCR+ IMNM patients, 10 dermatomyositis patients (DM), 5 HLA-DRB1* 11:01+ healthy controls (HC). HMGCR-reactive CD4+ T cells were identified by flow cytometry based on CD154 upregulation.

A total of 7 different naturally processed HMGCR peptides were identified. The number of distinct peptides presented per patient ranged from 1 to 5, with 5 being presented by at least 2 patients. All HMGCR peptides elicited robust CD4+ T cell responses in 9/10 anti-HMGCR+ IMNM patients, compared to 4/10 DM patients (p=0.017), 1/5 of HC (p=0.0119). The anti-HMGCR+ IMNM patients responded to more epitopes (p=0.0163) and had higher total number of positive CD4+ T cell cells (p= 0.0001).

Our findings represent the first report of antigen-specific CD4+ T cells in anti-HMGCR+ IMNM. Leveraging NAPA, we defined a core set of naturally presented HMGCR peptides defining precise immunologically relevant CD4+ T cell epitopes. Definition of these epitopes is key in understanding disease pathogenesis and will aid in the development of antigen-specific therapeutic tools.

Supported by grants from Rheumatology Research Foundation and the Buck Foundation.
Comparison of salt-soluble wheat allergens in a mouse model vs. known human wheat allergens: evidence from durum and ancient wheats
Authors: Jorgensen Rick Dean , Gao Haoran , Raghunath Rajsri , Chandra Shivam , Noble Amanda , Olson Eric , Ng Perry K W , Gangur Venugopal
Published: January 2023

Wheat allergies are among the major types of food allergies that are potentially life-threatening because of anaphylaxis. Monitoring changes to wheat allergens in novel wheat lines/genotypes is critical to prevent inadvertent introduction of potentially hyper-allergenic varieties from genetic modification. Nonetheless, validated methods for this purpose are unavailable at present. We have previously described a mouse model of wheat allergy using salt-soluble protein extract (SSPE) from durum wheat. As a proof-of-concept study, here we tested the hypothesis that wheat allergens in this mouse model will be identical to those reported for human wheat allergy. We created a mini plasma bank using hyper-IgE immune plasma obtained from Balb/cJ female mice (n=20) that had been sensitized with durum-SSPE along with alum adjuvant followed by repeated booster intraperitoneal injections with SSPE alone. Using hyper-IgE plasma we optimized an IgE-Western Blot (IgEWB) method to identify allergens present in SSPEs from durum wheat and an ancient tauschii wheat. The IgE-binding allergenic bands present in raw and boiled/reduced SSPEs from durum and tauschii wheats were sequenced using LC-MS/MS method. The most abundant allergens were identified and compared with a human wheat allergen database. There were 13 allergens present in durum-SSPE, of which 6 were present only in raw extract, 2 were present only in boiled/reduced extract, and 5 were present in both. There were 10 allergens present in tauschii-SSPE, of which 7 were present only in raw extract, and 3 were present in both raw and boiled/reduced extract. Between durum and tauschii wheats, 14 allergens were present in this mouse model, of which 10 are human wheat allergens.

Supported by USDA/NIFA, MSU
TFR-derived IL-4 is required for antigen-specific IgE but not antigen-specific IgG1 production in a peanut allergy model
Authors: Chen Qiang , Dent Alexander
Published: January 2023

T follicular regulatory cells (TFR) have been shown to regulate antibody production in germinal centers. Cytokine IL-4 controls the class-switching of IgG1 and IgE and is associated with the type II immune response. Though T follicular help cells (TFH) have been identified as a source of IL-4, whether and how IL-4 from TFR cells regulate antibodies including IgE remains unknown. Utilizing a food allergy model in which mice are sensitized using peanut extract and cholera toxin through oral gavage, we found that peanut-specific IgE but not IgG1 is absent with in vivo anti-IL-4Rα antibody treatment, while total IgE is also affected. This differential requirement for IL-4 is further validated using Il4-heterzygous mice, suggesting that IL-4 haploinsufficiency only impairs IgE production. Additionally, the number of germinal center B cells were not reduced in both models. To determine the role of IL-4 from TFR cells specifically, bone marrow chimera mice were constructed in which IL-4 is deficient in TFH and/or TFR cells. Both peanut-specific IgG1 and IgE were impaired when IL-4 is deficient in both TFH and TFR cells, while only peanut-specific IgE is almost absent when IL-4 is deficient in TFR cells. In another mouse model, where mice are immunized with OVA plus alum, the differential roles of IL-4 in IgE and IgG1 production are recapitulated. In conclusion, switching to IgE requires a stronger IL-4 signal compared to IgG1, and TFR cell-derived IL-4 plays a critical role in switching to IgE but not to IgG1.

Supported by grants from NIH (R01AI132771)
Brain mast cell accumulation and activation in an asymptomatic mouse model of cow’s milk allergy
Authors: La Mae Germundson Danielle , Nagamoto-Combs Kumi
Published: January 2023

Mast cells (MCs) are best known as the effector cells of allergy. Due to their presence in the brain and production of potent inflammatory mediators, MCs have been implicated in the pathogenesis of neuroinflammatory disorders. Previously, we showed that acutely challenging allergically sensitized mice increased the numbers of brain MCs. However, the consequences and mechanism of MC recruitment and activation within the brain are still unclear. We hypothesized that MCs increase in the brain due to allergen infiltration into the central nervous system, which subsequently activates MCs that release histamine, increasing blood-brain barrier (BBB) permeability and neuroinflammation. C57BL6/J mice were sensitized to a bovine whey allergen β-lactoglobulin (BLG) and placed on a whey-containing diet for two weeks to simulate prolonged allergen consumption. Although no overt allergic symptoms were observed, BLG-sensitized mice exhibited depression-like behavior after the allergen consumption period. Serum BLG-specific IgE and histamine were also significantly elevated, validating their hypersensitivity status. In the brains of sensitized mice, MC numbers and histamine levels were increased. IgG extravasation and perivascular astrogliosis were also detected, indicating BBB impairment and neuroinflammation, respectively. Furthermore, myelin staining revealed demyelination in select cortical regions of BLG-sensitized mice, suggesting a potential action of MC-derived proteases. Together, our findings support the involvement of MCs in peripheral-to-central communication during a chronic allergic response. MC stabilization or allergen avoidance may be useful to treat neuroinflammation in susceptible individuals.

Supported by grants from the NIH/NIGMS (P20GM103442), UND Host-Pathogen CoBRE Pilot Grant (P20GM113123) and funds from the University of North Dakota SMHS Department of Biomedical Sciences
Psychological Stress of aged mice has long-term impact on Mycobacterium tuberculosis infection by altering composition of lung T cell populations
Authors: Lafuse William P , Sunkum Shrayes , Kumar Naresh , Ahumada Omar Santiagonunez , Turner Joanne , Rajaram Murugesan V S
Published: January 2023

Aging is a major risk factor for chronic infections, including tuberculosis (TB). Elderly TB patients also suffer from elevated levels of psychological stress. It is not known how psychological stress impacts TB pathogenesis. To study the effects of stress on Mycobacterium tuberculosis (M.tb) we used a social disruption stress (SDR) model which involves repeated social defeat in subordinate mice. Young (3 month) and Old (18 month) C57BL/6 mice were subjected to SDR and infected with M.tb. Mice were sacrificed at 30 and 60 days post infection and M.tb burden analyzed by CFU assay and lung pathology by HE staining of lung sections. At day 30, M.tb burden was lower in control old mice compared to young mice and SDR did not significantly alter M.tb burden. However, at day 60, SDR in old mice significantly increased M.tb burden compared to SDR in young mice, indicating that SDR in old mice has a detrimental effect on the ability to control the infection. Our gene expression analysis revealed that T cell genes (JAK3 and LCP2) in the lungs of old SDR infected mice were increased but not in young SDR mice, suggesting that SDR in old mice increased T cell recruitment in the lung. Additionally, we found that Tbet (Th1) and GATA3 (Th2) gene expression were unchanged by SDR, in contrast the expression of RORγT (Th17) was increased by SDR in old mice at day 60. Also, mRNA levels of IL-10 at day 60 were increased by SDR in old mice, but not in young mice, while SDR decreased IFN-γ in both young and old mice. To further define T cell populations, we are analyzing lung sections by confocal microscopy. In conclusion, our studies suggest that psychological stress prior to M.tb prior to infection has long-term impact by altering composition of T cell populations in the lung.

Supported by NIH grants (PO1-AG051428, AI146252 and AI146690)
Bacteria load and hypoxia contribute to glucose uptake by macrophages and T cells in cynomolgus macaque granulomas
Authors: Mattila Joshua T , Gould Victoria A , Junecko Beth A , Bellavia Michael C , Borish H Jacob , White Alexander G , Maiello Pauline , Nyiranshuti Lea , Lin Philana Ling , Scanga Charles A , Anderson Carolyn J , Flynn JoAnne L
Published: January 2023

Granulomas form during tuberculosis (TB) and restrain bacterial dissemination but are also sites of mycobacterial replication and persistence. The immunometabolic state of cells in granulomas has immunologic and diagnostic relevance and PET-CT with the glucose analog FDG demonstrates that granuloma glucose uptake is dynamic and heterogenous within a host. Basic details on glucose uptake, including the cells responsible for FDG PET signal, have not been resolved and filling these gaps will improve interpretation of PET data in TB. Our objective was to identify relationships between glucose (FDG) uptake and granuloma composition and to identify factors that drive this process in M. tuberculosis-infected cynomolgus macaques. We used glucose transporter 1 (GLUT1) to identity cells that may be using glucose as an energy source in granulomas, and compared these data with the cell’s microenvironment, and the granuloma’s bacteria load and FDG PET data to determine how these factors influence GLUT1 expression. We found that GLUT1 was strongly expressed by myeloid cell subsets in specific granuloma microenvironments and this pattern was conserved in granulomas from different organs. We also identified macrophage subsets and T cells that may be important contributors to a granuloma’s potential glucose (FDG) uptake when their GLUT1 expression and population sizes were considered. We also correlated granuloma bacteria loads and hypoxia with GLUT1 expression, suggesting that bacterial antigens and hypoxic conditions drive a granuloma’s glucose uptake. Taken together, our data suggest that granuloma glycolysis and FDG uptake are driven, in part, by cell subset-specific responses to a granuloma’s microbial and microenvironmental milieu.

This work was supported by NIH grants AI134183 and AI118195.
C. elegans host defense pathways protect against microbiota cryptic virulence
Authors: Gonzalez Xavier , Wollenberg Amanda , Irazoqui Javier
Published: January 2023

Animals are colonized by diverse communities that provide essential functions to the superorganism. At homeostasis, such microbiota communities are thought to benefit the healthy host. In contrast, dysbiosis can be both pathogenic and a consequence of disease. Establishing causal relationships among microbiota composition, host immune status, and disease is essential for fundamental understanding of many diseases and for their therapeutic targeting. Here we took a reductionist approach to address the potential for normal microbiota bacteria to cause disease in the immunodeficient host (i.e., the cryptic virulence of pathobionts) using C. elegans. C. elegans are known to rely on several host defense pathways to counteract intestinal infections. Although these pathways are known to play pivotal roles in defense against various human pathogenic bacteria, it is not clear what roles these pathways play for interactions with the native microbiota. To address this gap in knowledge, we determined the functional significance of p38, Wnt, and TFEB pathways in terms of gene expression and host survival of monocolonization by individual members of the C. elegans microbiota. We find that distinct host defense pathways are necessary for normal survival of animals colonized with distinct microbiota members. Our results suggest that specific microbiota members trigger specific host defense pathways, which are necessary for the host to defend against their cryptic virulence. The new insights provide the necessary foundation to characterize the molecular mechanisms by which microbiota members trigger host defense pathways specifically, including potentially novel bacterial metabolites with useful therapeutic properties in mammals.

Supported by gran from NIH (R01GM101056-10) Supported by grant from NIH (Innate Immunity Training Grant (T32 AI095213))
Dynamics of antimicrobial protein secretion in mouse milk
Authors: Abokor Ahmed , Saha Piu , Yeoh Beng San , Golonka Rachel , Vijay-Kumar Matam
Published: January 2023

Breast milk is the preferred food for the newborns and considered as complete ‘edible immune system’. In addition, breast milk is a source of innate and adaptive proteins which not only protects the naïve infant gastrointestinal tract from enteropathogens but also aid in the appropriate initial colonization of gut microbiota. In this study, we analyzed antimicrobial immune proteins in the milk from C57BL/6 dams at several time points, and observed distinctive patterns in the immune protein levels throughout the lactation period. The presence of innate immune proteins serum amyloid A (SAA), CD14, and notably lipocalin-2 (Lcn2) were observed in high quantities with Lcn2 and SAA present at microgram levels suggesting a potential niche for these proteins during neonatal immune development. Moreover, low dose LPS administration to dams significantly increased some of these proteins in the milk. In addition, adaptive immune proteins immunoglobulins (Ig) A and G with IgG were present in milk at higher quantities compared to IgA at day 5 post-delivery. Interestingly, class-switching occurs by day 15 indicating a selective adaptive immune preference towards IgA, which could be due to gut colonization of newborns with more complex gut microbiota with aging and IgA being a major gut homeostatic factor. Additionally, milk from IgA-deficient dams allowed for robust proliferation of E. coli compared to wild-type milk further signifying the potential involvement of IgA during the early colonization of microbes in the neonatal gut. Collectively, our findings provide insight in the various immune proteins with antimicrobial activity may play a major role in the initial microbial colonization in the gut, which has long-lasting consequences on the host.

Supported by NIH R01-CA219144, NCI Diversity Supplement (CA219144-05S1)
E-Syt1 ubiquitination by Nedd4 limits the caspase-11-mediated non-canonical inflammasome and endotoxemia
Authors: Ma Yilei , Lin Guoxin , Guo Hui , Yu Jianyu , Zhang Jian
Published: January 2023

Sepsis caused by gram-negative bacteria is thought to be largely due to the host’s response to LPS or endotoxin. Upon sensing LPS by its cytosolic receptor, caspase-11 (Casp-11) assembles a higher order structure that enables the activation of Casp-11 protease function, leading to gasdermin D cleavage and pyroptosis. However, the mechanisms for negatively regulating the activation of the Casp-11-dependent non-canonical inflammasome during sepsis are largely unknown. Nedd4 is a HECT-type E3 ubiquitin ligase that positively regulate T cell activation. However, its role in innate immunity is largely unknown. In this study, we found that although macrophages lacking Nedd4 produce comparable amounts of TNF-α and IL-6 in response to different TLR ligands, they produce more IL-1β upon LPS priming and stimulation with cholera toxin B (CTB) and EHEC but not ATP, nigericin, or other inflammasome stimuli. These data suggest that Nedd4 specifically regulates non-canonical inflammasomes. In support of this notion, mice lacking Nedd4, or lacking Nedd4 in myeloid cells, succumb to endotoxemia, which is rescued by deficiency for Casp-11 but not NLRP3. Mechanistically, Nedd4 appears to specifically bind to extended synaptotagmin 1 (E-Syt1), and targets E-Syt1 for ubiquitination, and loss of Nedd4 stabilize E-Syt1 protein which potentiates Casp-11 oligomerization and activation. Therefore, our data unveil a novel mechanism by which Nedd4 puts a brake on the Casp-11-dependent non-canonical inflammasome, thus preventing over-activation of innate immune responses during sepsis. The molecular insights gained in this study provide novel targets for modulating Casp-11-dependent inflammasome-mediated sepsis in patients.
Examining the relationship between epidermal gamma delta T cells and neuropeptide Y
Authors: Almaguer Berenice , Jameson Julie
Published: January 2023

Neuropeptide Y (NPY) is one of the most abundant peptides present in the central nervous system and is responsible for regulating food intake, energy metabolism, and emotional expression. Emerging evidence indicates that NPY could potentially play a role in serving as a transmitter between the nervous system and immune system and even within the immune system itself. Epidermal gamma delta (γδ) T cells play a role in governing wound repair and homeostasis within the skin by producing growth factors, cytokines, and performing cytolysis. Wound repair involves the transmission of pain signals and immune functions simultaneously, suggesting interaction and collaboration between the nervous and immune system. We isolated and cultured epidermal γδ T cells from wild type mice and used flow cytometry to determine the expression of NPY1R on epidermal γδ T cells prior to and after activation. The data indicates that NPY1R is present on epidermal γδ T cells and that expression increases when upon activation. In addition, we performed immunofluorescence microscopy to acquire images of skin sections and epidermal sheets to determine expression of NPY1R on epidermal γδ T cells during skin homeostasis. This data shows that NPY1R is present on select populations of epidermal γδ T cells in the skin and future directions will examine how this impacts the role of epidermal γδ T cells in wound repair and homeostasis in the skin.

Supported by the Arnold & Mabel Beckman Foundation (Beckman Scholars Program)
The neurotrophic factor Neuritin regulates T cell anergy and T regulatory cell function
Authors: Yu Hong , Nishio Hiroshi , Barbi Joseph , Mitchell-Flack Marisa , Vignali Paolo , Zheng Ying , Lebid Andriana , Chang Kwang-Yu , Fu Juan , Blosser Lee , Tam Ada , Pardoll Drew
Published: January 2023

T cell activation and tolerance are tightly regulated to ensure effective elimination of foreign antigen while maintaining immune tolerance to self-antigens. Development of T cell anergy and regulatory T cell (Treg) mediated suppression both contribute to the establishment of immune tolerance. Here, we show that neuritin (Nrn1), a conserved GPI-anchored surface molecule important for the development, survival and function of neurons, is highly expressed in anergic and Treg cells. Nrn1 deficient CD4 cells are resistant to Treg cell mediated suppression, display defective anergy induction, and have reduced peripheral Treg generation. Nrn1 deficient Foxp3+ Treg cells exhibit reduced control of inflammatory colitis. Moreover, upon induction of experimental autoimmune encephalomyelitis (EAE), Nrn1 deficient mice develop non-remitting disease and have increased spinal cord inflammatory infiltrates. These in vivo findings underscore the importance of Nrn1 in immune tolerance. Recently, Nrn1 was identified as an accessory component of the ionotropic AMPA receptor (AMPAR) complex in neurons. AMPARs mediate glutamate dependent cation flux and regulate cell membrane potential. Cell membrane potential can impact nutrient uptake, calcium influx, cell size, proliferation and survival. In vitro analysis reveals that Nrn1 deficient Treg cells exhibit reduced proliferation and survival, associated with higher membrane potential, reduced nutrient sensitivity, reduced glycolysis and mTOR activation. AMPAR blockade can correct proliferation defect in Nrn1 deficient Treg cells. These findings reveal Nrn1 as an important regulator of immune tolerance functioning through the modulation of glutamate activated AMPAR.
Transcription coactivator OCA-B/Pou2af1 is necessary and sufficient to promote T cell-intrinsic CD4 memory
Authors: Tantin Dean R , Sun Wenxiang , Kim Heejoo , Perovanovic Jelena , Ibarra Andrea , Hale J Scott , Williams Matthew A
Published: January 2023

The establishment of durable immunological memory remains a poorly understood aspect of lymphocyte biology and a limiting factor in vaccine development. Using a T cell-conditional OCA-B (Pou2af1) knockout model and a mouse viral pathogen, we show that expression of the transcription cofactor OCA-B within T cells is necessary for proper CD4+ memory T cell formation. We also show that ectopic OCA-B expression is sufficient to drive T cells towards a memory fate while having no effect on primary antiviral effector response. Bulk and single-cell gene expression profiling comparing cells transduced with OCA-B and empty vector at peak primary viral response identifies changes in gene expression consistent with later memory formation. Intersecting differentially expressed genes in bulk RNA-seq with prior ChIP-seq identifies direct targets increased (Tbx21, Zeb2, Gadd45b, Socs2) and decreased (Zbtb16, Ccr1) by ectopic OCA-B expression. Single-cell RNA-seq reveals expansion of short-lived effector T cell compartments with increased expression of Gadd45b and Socs2, as well as increased expression of memory-associated genes such as Slamf6, Il7r and Tcf1 in smaller clusters of effector cells with memory potential. We also describe a new OCA-B-mCherry reporter mouse that efficiently labels B and T lymphocytes and that shows high expression in CD4+ TCM cells. Cumulatively, the results demonstrate that OCA-B expression in T cells is necessary and sufficient to promote CD4 T cell memory in vivo.

Supported by grants from NIH (R01 AI100873)
The extracellular ATP receptor P2RX7 imprints a pro-memory transcriptional signature in effector CD8+ T cells
Authors: Van Dijk Sarah , Vardam-Kaur Trupti , da Silva Henrique Borges
Published: January 2023

Development of central memory (Tcm) and resident memory (Trm) CD8+ T cells, which respectively promote immunity in the circulation and in barrier tissues, is not completely understood. Tcm and Trm cells may arise from common precursors, however their fate-inducing signals are elusive. We found that virus-specific effector CD8+ T cells display heterogeneous expression of the extracellular ATP sensor P2RX7. P2RX7-high expression is confined, at peak effector phase, to CD62L+ memory precursors which preferentially form Tcm cells. Among early effector CD8+ T cells, asymmetrical P2RX7 distribution correlated with distinct transcriptional signatures, with P2RX7-high cells enriched for memory and tissue-residency sets. P2RX7-high early effectors preferentially form both Tcm and Trm cells. Defective Tcm and Trm formation in P2RX7 deficiency is significantly reverted when the transcriptional repressor Zeb2 is ablated. Mechanistically, P2RX7 negatively regulates Zeb2 expression, at least partially through TGF-β sensing in early effector CD8+ T cells. Our study indicates that unequal P2RX7 upregulation in effector CD8+ T cells is a foundational element of the early Tcm/Trm fate.

Supported by grants from NIH (R00 AI139381, NIAID).
Maternally derived epidermal growth factor mediates protection in neonates from opportunistic intestinal pathogens.
Authors: Greenfield Kara G , Lothert Petra , Harlow Olivia , Knoop Kathryn
Published: January 2023

The intestinal epithelial barrier is vital in preventing translocating bacteria and subsequent systemic infections. This protection is provided by host factors, by the intestinal microbiota, and, during early life, by breast milk. While the beneficial functions of breast milk are numerous to the nursing infant, it has been observed that proteins such as antibodies and growth factors are secreted in higher concentrations early during lactation and decrease as the milk matures. One such growth factor, epidermal growth factor (EGF), regulates the development of colonic goblet cell-associated antigen passages (GAPs). Decrease of EGF through disruption of synchronous breastfeeding or deletion of the epidermal growth factor receptor from goblet cells alters the kinetics of GAP formation, which can serve as a passage for pathogenic bacteria. In a murine model of early GAP formation utilizing an inhibitor of EGF receptor, we observed a variety of gut-resident pathogens were able to utilize GAPs to disseminate systemically and cause disease, including Escherichia coli, Enterococcus faecalis, Enterobacter cloacae, and Pseudomonas aeruginosa. In human studies, we found a strong correlation between concentrations of EGF in the diet and stool of very low birth weight infants. Notably, formula-fed infants contained significantly less EGF in both diet and stool. Future work will assess EGF in donor milk, which we find has highly variable EGF concentration. This work has strong implications for how diet influences risk of developing infections from gut-resident pathogens and how maternal EGF present in breastmilk is synchronized to the newborn infant to provide appropriate protection in early life.

Supported by grants from NIH (AI144721, DK122187)
The cellular dynamics of early and transitional human breast milk
Authors: LeMaster Cas , Pierce Stephen , Geanes Eric S , Khanal Santosh , Elliott Staci , Scott Allison , Louiselle Daniel , McLennan Rebecca , Truog William , Maulik Devika , Lewis Tamorah , Pastinen Tomi , Bradley Todd
Published: January 2023

Breast milk (BM) is a complex fluid containing factors essential for infant nutrition and immunity. Breastfeeding has been shown to be protective against infections and other immune-mediated diseases during the lactation period and beyond in later childhood. This suggests that BM also imprints the neonatal immune system and influences long-term health. BM also contains populations of maternal-derived cells. Which factors in BM that are important for neonatal health and how they change during lactation have not been well-defined. In this study, we used a single-cell transcriptomic approach to identify and define cell types of early and transitional milk. We collected BM samples from mothers of infants 2–5 days (early milk) and 8–12 days (transitional milk) after delivery. We applied single-cell RNA sequencing on over 154,000 BM-derived cells. We identified 25 transcriptionally distinct populations of cells in the BM. As expected, the most abundant cells in BM were mammary epithelial cells and macrophages. Monocytes, T cells, dendritic cells, and neutrophils were also present and had a higher frequency in week 2, suggesting that some immune cells may remain abundant in the early days of lactation and slowly decline as milk matures. We also detected a small number of stem and progenitor, natural killer and B cells in the BM at a higher frequency in week 1. This work provides an atlas of the cellular component in human milk at two timepoints of lactation. In addition to cell identity and frequencies, we have also uncovered unique molecular pathways that are activated in BM cells. This work will lay the foundation for future studies of how these cells influence neonatal health.

Supported by funding from Children’s Mercy Kansas City
Systemic and mucosal IgA responses are variably induced in response to SARS-CoV-2 mRNA vaccination and are associated with protection against subsequent infection
Authors: Sheikh-Mohamed Salma , Chao Gary , Isho Baweleta , Zuo Michelle , Cohen Carmit , Lustig Yaniv , Nahass Georgie , Salomon Rachel E , Blacker Grace , Fazel-Zarandi Mahya , Rathod Bhavisha , Colwill Karen , Jamal Alainna J , Li Zhijie , deLaunay Keelia Quinn , Takaoka Alyson , Garnham-Takaoka Julia , Patel Anjali , Fahim Christine , Patterson Aimee , Liu Angel , Haq Nazrana , Barati Shiva , Gilbert Lois , Green Karen , Mozafarihashjin Mohammad , Samaan Phillip , Budylowski Patrick , Siqueira Walter , Mubareka Samira , Ostrowski Mario , Rini James , Rojas Olga , Weissman Irving L , Tal Michal Caspi , McGeer Alison , Regev Gili , Straus Sharon , Gingras Anne-Claude , Gommerman Jennifer L
Published: January 2023

SARS-CoV-2 is a novel respiratory virus that has quickly spread across the globe. The virus uses a protein called Spike and its associated receptor binding domain (RBD) to interact with angiotensin converting enzyme-2 (ACE-2) on the surface of epithelial cells in the respiratory tract. Although a definite correlate of protection against COVID-19 has yet to emerge, many studies have quantified anti-Spike and anti-RBD IgG antibody (Ab) levels, as well as neutralizing Ab in the blood to ascertain immunity. This approach misses out on Ab that are produced in the upper respiratory tract (URT) mucosa – the site of viral encounter. Whether intramuscularly (i.m.) administered COVID-19 vaccines can promote immunity in the mucosa is not well understood. We recently completed a study where we showed that anti-Spike/RBD IgG could be detected in the saliva following i.m. vaccination with either two doses of mRNA vaccines (Pfizer or Moderna) or with a heterologous dosing of Astra Zeneca followed by an mRNA vaccine. Administration of a second dose of mRNA boosted the IgG but not IgA response, with only 30% of participants remaining positive for IgA at this timepoint. At 6 months post-dose 2, these participants had diminished anti-Spike/RBD IgG levels, although secretory component associated anti-Spike Ab were more stable. Examining two prospective cohorts we found that participants who experienced breakthrough infections with SARS-CoV-2 had lower levels of vaccine-induced serum anti-Spike/RBD IgA at 2–4 weeks post-dose 2 compared to participants who did not experience an infection, whereas IgG levels were comparable between groups. These data suggest that COVID-19 vaccines that elicit a durable IgA response may have utility in preventing infection.

We received funding support from CIHR (Fund #15992), a COVID-19 Immunity Task force grant, an “Ontario Together” province of Ontario grant, a CIHR team grant to CoVARR-Net, a Donation from the Royal Bank of Canada (RBC) and a donation from the Krembil Foundation to the Sinai Health System Foundation.
Accelerated tumorigenesis in a colorectal cancer model in Siglec-E knockout mice
Authors: Maniaci Brandon Lee , Friedman David , Crotts Sydney , Rajcula Matthew , Theodore Keith , Lee Hyun Se Kim , Shapiro Virginia
Published: January 2023

The lifetime risk for colorectal cancer in the United States is approximately 4%. Individuals with Inflammatory Bowel Disease, including Ulcerative Colitis and Crohn’s Disease, have a substantially increased risk of developing colorectal cancer. The mechanisms for accelerated tumorigenesis due to enhanced inflammation are not fully characterized. Siglecs (sialic acid immunoglobulin lectin-like proteins) are a family of inhibitory receptors that are negative regulators of the immune response. Siglec-E is an inhibitory receptor that is expressed by innate immune cells, including monocytes, macrophages, neutrophils and dendritic cells. Interestingly, mice deficient in Siglec-E have accelerated development of tumors and reduced survival in a spontaneous mouse model of colorectal cancer (TS4-cre LSL-KRasG12D APClox468/wt). While tumors develop at approximately 6 months in mice with Siglec-E, tumors develop at approximately two months in Siglec-E knockout mice. Initial results indicate that Siglec-E knockout mice also have accelerated gut inflammation using the DSS colitis model as compared to WT mice. Current studies are examining inflammation that develops during tumorigenesis in Siglec-E knockout mice.
Ovarian cancer immunogenicity is governed by a narrow subset of progenitor tissue-resident memory T-cells
Authors: Galindo Carmen Maria Anadon , Yu Xiaoqing , Hanggi Kay , Biswas Subir , Chaurio Ricardo , Mandal Gunjan , Martin Alexandra , Payne Kyle K , Innamarato Pasquale P , Harro Carly M , Mine Jessica , Sprenger Kimberly , Cortina Carla , Powers John J , Perez Bradford A , Gatenbee Chandler D , Prabhakaran Sandhya , Marchion Douglas , Heemskerk Mirjam H , Curiel Tyler J , Anderson Alexander R A , Wenham Robert M , Rodriguez Paulo C , Conejo-Garcia Jose R
Published: January 2023

Despite repeated associations between T-cell infiltration and patient outcome, human ovarian cancer remains poorly responsive to immunotherapy. We report that hallmarks of tumor recognition in ovarian cancer-infiltrating T-cells are primarily restricted to tissue-resident memory (TRM) cells. In mouse models we found that TRM T-cells were better than the re-circulating counterpart at controlling tumor growth. Single-cell RNA/TCR/ATAC sequencing of 83,454 CD3+CD8+CD103+CD69+ TRM cells and 24,175 CD3+CD8+CD103− re-circulating TILs showed that progenitor (TCF1low) tissue-resident memory T-cells (TRMstem cells) arise from transitional recirculating T-cells, which depends on antigen affinity/persistence, resulting in oligoclonal, trogocytic, effector lymphocytes. This effector population develops into proliferative lymphocytes that eventually become exhausted TRMs. Immunohistochemistry of 122 high-grade serous ovarian cancer tissues showed that only TRMstem cells, but not re-circulating TCF1+ T-cells, predict ovarian cancer outcome. Therefore, ovarian cancer is indeed an immunogenic disease that depends on ~13% of CD8+ tumor-infiltrating T-cells (~3% of CD8+ clonotypes), which are primed against high-affinity antigens and maintain waves of effector TRM cells.

Support for Shared Resources was provided by Cancer Center Support Grant (CCSG) CA076292 to H. Lee Moffitt Cancer Center and by CCSG CA010815 to The Wistar Institute. This study was supported by grants from NIH (R01CA157664, R01CA124515, R01CA178687, R01CA211913 and U01CA232758 to JRCG; R01CA184185 and RO1CA262121 to PCR.)
Sexual dimorphism and the effect of therapeutic low-dose estrogen in head and neck squamous cell carcinoma
Authors: Knitz Michael W , Bickett Thomas E , Darragh Laurel B , Bhatia Shilpa , Van Court Benjamin , Piper Miles , Douglas Sarah , Karam Sana D
Published: January 2023

Differences between males and females have been identified in a variety of immune-related diseases. Generally, females show stronger immune responses, but also present a higher incidence of auto immune disease. Reasons for this have not been concretely identified but estrogen is believed to be a main driver of sex-dependent differences in biological behavior of disease and response to therapy. Using castration, oophorectomy, and estrogen rescue experiments in various preclinical head and neck squamous cell carcinoma (HNSCC) models, we identify estrogen as a regulator of immune response to treatment. The effect of estrogen is dose-dependent, as higher doses fail to demonstrate the beneficial effects of low dose estrogen. This response is dependent on regulatory T cells and dendritic cells, as knockout of these cell types eliminates the disparity between male and female responses. We also identified estrogen receptor beta agonists as a potential therapeutic strategy providing similar treatment benefit as low dose estrogen, reversing the effects of oophorectomy and stimulating cytotoxic T cell function. Survival analysis from HNSCC patients provided correlational validation of the potential benefit of estrogen as it demonstrates increased overall survival in younger women. Our data implicate low dose estrogen as a potential therapeutic strategy to enhance response to immunotherapy.

Sana D. Karam is funded by the NIDCR (R01 DE028529-01, R01 DE028282-01) and Colorado Head and Neck Cancer SPORE and receives funding from Astra Zeneca, Genentech, and Ionis. This study was partly supported by the National Institutes of Health P30CA046934 Cancer Center Support Grant, including the Bioinformatics and Biostatistics Shared Resource Core, the Cancer Center Flow Cytometry Shared Resource, and the Genomics and Microarray Shared Resource at the University of Colorado. This study was also partly supported by the Cancer League of Colorado.
Sex differences in B cell frequencies and antibody responses following influenza vaccination in healthcare workers during the 2019–2020 season
Authors: Park Han-Sol , Kuo Helen , Liu Hsuan , Fenstermacher Katherine , Shapiro Janna , Jedlicka Anne , Dhakal Santosh , Ursin Rebecca , Wilson Jo , Shea Patrick , Morgan Rosemary , Gearhart Patricia J , Rothman Richard , Pekosz Andrew , Klein Sabra L
Published: January 2023

Annual influenza vaccination for healthcare workers (HCWs) is required at Johns Hopkins to reduce influenza spread in healthcare facilities. We conducted a clinical study to evaluate sex differences in virus-specific antibody-producing B cells (i.e., plasmablasts) following receipt of the seasonal influenza vaccine in a highly vaccinated population of healthcare workers. To understand sex differences in immune responses to the influenza vaccine in the HCWs, 83 participants consented to blood draws at baseline and 75 participants returned for the 7- and 28-days post-vaccination survey and blood draw. Participants received their annual quadrivalent influenza vaccine (QIV). Influenza A specific plasmablasts were quantified at baseline and day 7 post-vaccination. Males had a significantly greater percentage of influenza-specific plasmablasts than females after vaccination (p<0.0003) Plasma collected at baseline and at day 28 post-vaccination was used to measure neutralizing antibody (nAb) titers against H1N1 and H3N2 vaccine viruses. Neutralizing antibody responses against either the H1N1 and H3N2 viruses increase after vaccination to the same degree in both males and females. The proportion of plasmablasts at day 7 was significantly associated with the vaccine-induced fold rise in nAb. This relationship differed by sex, whereby for a given proportion of plasmablasts, females tended to mount a greater fold-rise in nAb than males. These data suggest that despite having potentially fewer plasmablasts after vaccination, females mount an antiviral antibody response to the influenza vaccine viruses equivalent to males. Identification of transcriptional differences in plasmablasts from male and female HCWs is ongoing.
Estradiol replacement improves vaccine-induced antibody responses and protection against influenza in aged female mice
Authors: Mueller Alice R L , Dhakal Santosh , Caputo Christopher , Seddu Kumba , Klein Sabra L
Published: January 2023

Adult female mice generate greater vaccine-induced immunity and protection than their male counterparts, but this female advantage is lost with age. We hypothesized that treatment of aged female mice with exogenous estradiol would improve vaccine-induced antibody responses and protection from both pulmonary infection and disease after live influenza virus challenge. To investigate this, adult (2–3 months old) and aged (17–18 months old) female C57BL/6 mice were implanted with either empty (placebo) capsules or capsules loaded with 17β-estradiol (E2) hormone, and then vaccinated with mouse-adapted A/California/04/09 (ma2009 H1N1) vaccine. Total IgG and neutralizing antibody titers (nAb) were quantified using blood samples collected post vaccination. >1 month post vaccination, mice were challenged intranasally with ma2009 H1N1 drift variant virus. A subset of mice was euthanized at 3 days post challenge to collect lungs and measure infectious virus titer. Another subset was followed for 14 days to measure changes in body mass as a measure of disease severity. We found that aged female mice treated with E2 produced significantly greater IgG and nAb responses compared to placebo-treated aged females, and the responses of E2-treated aged females were comparable to the antibody responses generated in adult females. Treatment with E2 also reduced morbidity, but not pulmonary virus titers, following live virus challenge. Our data suggest that estrogen replacement in aged female mice improves their humoral immune response to influenza vaccination and protects them from severe disease.

Supported by U54AG062333
Preventing neonatal herpes: Protection after maternal mRNA-lipid nanoparticle vaccination equals or exceeds that from prior maternal genital infection in murine models
Authors: Desmond Angela , LaTourette Philip C , Awasthi Sita , Egan Kevin P , Hook Lauren M , Brice Angela K , Lubinski John M , Naughton Alexis M , Fowler Bernard , Beattie Mitchell , Pardi Norbert , Cohen Gary H , Weissman Drew , Friedman Harvey M
Published: January 2023

Neonates are vulnerable to poor outcomes from herpes simplex virus (HSV), but maternal infection eliciting non-sterilizing immunity prior to delivery provides some protection. We are therefore pursuing birthing parent vaccination with nucleoside-modified mRNA-lipid nanoparticles (mRNA-LNPs) to prevent neonatal herpes. Our trivalent vaccine encodes HSV type 2 (HSV-2) proteins involved in attachment and immune evasion (gC2, gD2, and gE2). We have shown that this vaccine protects against genital HSV and neonatal HSV-2 in mice. Here, we tested how well murine dam vaccination prior to pregnancy protects pups against HSV type 1 (HSV-1) challenge compared to prior maternal intravaginal HSV-1 infection. Dams were immunized intramuscularly with the HSV-2 trivalent mRNA-LNP vaccine or a control immunogen (PolyC RNA-LNP), or were infected intravaginally with HSV-1 (HSV-1 ivag). Pups were challenged intranasally with HSV-1 on the 3rd day of life. Pups born to dams previously immunized or infected survived through 28 days (mRNA 95% vs. HSV-1 ivag 96%, N.S.; vs. PolyC 13%, p<0.0001). Likewise, they were protected from HSV-1 dissemination to the brain, lung, liver, spleen, and kidney by plaque assay (mRNA 2/45 organs affected vs. HSV-1 ivag 6/20, p=0.0083; vs. PolyC 69/80, p<0.0001). Additionally, fewer organs showed necrosis by histopathology of brain, lung, and liver, though not statistically significant, and fewer mRNA and HSV-1 ivag pups had detectable HSV-1 DNA in the trigeminal ganglia by qPCR (mRNA 6/30 vs. HSV-1 ivag 7/26, N.S.; vs. PolyC 17/18, p<0.0001). These studies demonstrate that the mRNA vaccine provides comparable to improved protection against neonatal HSV-1 challenge versus prior maternal HSV-1 intravaginal infection.

AD was supported by NIH NIAID T32 AI118684. HMF, SA, GHC, and DW were supported by NIH NIAID R01 AI139618 and an unrestricted grant from BioNTech SE. KPE was supported by NIH T32 NS007180. The funders had no input or influence on the content of this abstract.
The immunologic effects of different adjuvants on enhancing homologous and cross-protection by influenza virus vaccination in young and aged mice
Authors: Bhatnagar Noopur , Kim Ki-Hye , Subbiah Jeeva , Park Bo Ryoung , Ko Eun-Ju , Wang Pengfei , Kang Sang-Moo
Published: January 2023

Seasonal vaccination is ineffective in the elderly population and in conferring cross-protection against antigenically different influenza viruses. Therefore, the cross-protective efficacy of influenza vaccines needs to be improved. Here, we compared the effects of different adjuvants (QS-21+MPL, CpG+MPL, BCG CWS, Saponin VSA-1, Quil-A, and Alum) on enhancing the immunogenicity and homologous and cross-protection by influenza vaccination in mice. QS-21+MPL adjuvant was most effective in inducing Th1 T cell, cross-reactive IgG, and hemagglutination inhibiting antibody responses to vaccination. QS-21+MPL and CpG+MPL adjuvants exhibited high potency by preventing weight loss and reducing viral loads and conferring enhanced homologous and cross-protection in young and aged mice. BCG cell-wall skeleton (CWS) displayed lower adjuvant efficacy in inducing Th1 IgG responses and conferring cross-protection. VSA-1 (an analog of licensed adjuvant QS-21) exhibited adjuvant effects on enhancing the protective efficacy against homologous and heterosubtypic viruses comparable to QS-21. Saponin Quil-A exhibited adjuvant effects in CD4-deficient mice suggesting a CD4-independent adjuvant mechanism, in contrast to CpG that requires intact CD4 T cells. The findings suggest that QS-21+MPL, CpG+MPL, and VSA-1 exhibit desirable adjuvant properties on enhancing cross-protective humoral and cellular immunity to vaccination, with a unique pattern of innate immune responses, contributing to improved homologous and heterosubtypic protection. This study has significance in better understanding the effects of potent adjuvants on enhancing homologous and cross-protection to influenza vaccination in young and older adults.

This study was supported by NIH/NIAID grants AI093772 (S.M.K.), AI154656 (S.M.K), and AI147042 (S.M.K).
Robust efficacy and long-lasting humoral immunity induced by a respiratory syncytial virus prefusion F-based nanoparticle vaccine in genetically diverse animal models
Authors: Stephens Laura , Ross Kathleen A , McLellan Jason S , Narasimhan Balaji , Varga Steven M
Published: January 2023

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in children, accounting for 7% of deaths in those less than one year of age. Despite multiple exposures to RSV throughout life, natural infection affords only partial protection and there is currently no licensed vaccine against RSV. We developed a vaccine candidate, RSVNanoVax, composed of polyanhydride nanoparticles encapsulating the RSV prefusion F protein and a CpG1668 adjuvant. To assess the efficacy of RSVNanoVax we utilized two model systems with increased genetic variability, Swiss Webster mice and the cotton rat. Following an intranasal prime-boost vaccination with RSVNanoVax, Swiss Webster mice exhibited robust titers of RSV F-directed IgG antibodies in both the serum and lung tissue that were sustained out to at least 1-year post-vaccination. RSV F-directed IgA was also induced within the lung tissue and nasal passages, suggesting that RSVNanoVax stimulates long-lasting systemic and mucosal antibody responses in outbred mice. Serum antibodies maintained robust neutralizing activity against both RSV A and B virus strains. Following RSV challenge, vaccinated mice exhibited complete viral clearance in the lungs as early as day 2 post-infection. Further studies performed in cotton rats demonstrated that RSVNanoVax conferred protection from both viral replication in the lungs as well as RSV-induced pulmonary immunopathology following viral challenge. Vaccinated cotton rats also exhibited robust titers of RSV F-directed serum antibodies with neutralizing capacity. Overall, RSVNanoVax represents a promising RSV vaccine candidate capable of providing long-term protection and immunity in genetically diverse populations.

Supported by grants from NIH (R01 AI124093, T32 CA078586, T32 AI007485)