bluuebunny/crossref_metadata_2025 huggingface dataset:
Depletion of Polymorphonuclear Myeloid-Derived Suppressor Cells Increases Efficacy of Listeria monocytogenes Cancer Vaccine
Authors: Williams Jacob E , Castillo Jesse Garcia , Fernandez Sebastian , Portnoy Daniel A , DuPage Michel
Published: August 2023

The tumor microenvironment is highly immunosuppressive, allowing cancer to evade the immune system by inducing the dysfunction, exhaustion, or subversion of CD8+ T-cell functions. Cancer immunotherapies using attenuated strains of Listeria monocytogenes (Listeria) engineered to express tumor antigens have been demonstrated to promote cancer antigen-specific CD8+ T-cells that infiltrate the tumor microenvironment and control mouse models of cancer. However, in human clinical trials, initial IV therapies utilizing Listeria did not demonstrate strong clinical efficacy, suggesting that the generation of tumor-specific CD8+ T-cells recognizing tumor antigens alone is insufficient to provide robust tumor control. Here, we demonstrate that intratumoral (IT) or intravenous (IV) inoculation of an attenuated form of Listeria(ΔActA) demonstrated no therapeutic benefit in the context of establish MC38 tumor-bearing mice while predominantly recruiting and infecting CD11b+Ly6G+CD14+ cells in the tumor microenvironment, markers associated with polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs). In vitro analysis of PMN-MDSC infection by Listeria revealed increased immunosuppressive function, indicating a possible adverse outcome of Listeria treatment in vivo. Further, IV or IT injection of Listeria in the context of PMN-MDSC depletion increased the therapeutic efficacy of Listeria therapy and promoted a robust CD8+ T-cell mediated anti-tumor response. Collectively, our results demonstrate that depletion of PMN-MDSCs may be required to mediate the therapeutic efficacy of Listeria inoculation in the context of cancer immunotherapy and the production of an effective anti-tumor immune response.

Supported by grants from NIH (DP2CA247830) & Cancer Research Coordinating Committee (CRCC) (C23CR5612)
Systemic Cancer Immunostimulatory Therapies and Acute Viral Infection Induce Corticosteroid-Mediated Thymic Involution and Naïve T cell Output in Mice and Humans
Authors: Yoon Daniel J , Khuat Lam Tan , Collins Craig , Vick Logan , Sckisel Gail , Minnar Christine Mall , Dunai Cordelia , Curti Brendan , Chao Nelson J , Blazar Bruce , Longo Dan L , Canter Robert , Monjazeb Arta , Murphy William J
Published: August 2023

Thymic involution (TI) occurs with aging and during stress responses, decreasing naïve T cell output. Thymic progenitor apoptosis can occur directly by corticosteroids (CS) leading to transient involution followed by recovery. The effects of systemic immunotherapies (IT) used in cancer or immune stimulation via acute viral infection on the thymus has not been well-characterized and was the primary goal of this study. IT treatment of mice with high dose IL-2 (HD IL-2) or models of acute viral infection (mouse cytomegalovirus, MCMV) all resulted in rapid TI due to apoptosis of CD4/CD8 double-positive progenitors followed with a reduction in naïve T cell content. Interestingly, cessation of therapy or resolution of viral infection resulted in greater thymic size compared to untreated recipients, indicating a rebound effect. In both models, increased CS levels in the serum of mice preceded TI. Clinically, patients receiving HD IL-2 for cancer had significantly reduced T cell receptor excision circles, a marker for naïve T cell output, also correlating with increased cortisol levels. Using a model of sub-lethal MCMV infection that did not increase systemic pro-inflammatory cytokines, we observed a similar correlation of TI and increased CS levels. To further analyze the effects of CS on TI, mice were adrenalectomized and then given HD IL-2, where significantly reduced CS levels correlated with partial protection from TI. These results indicate that strong systemic immunostimulation by either IT or acute viral infections markedly induce transient CS-mediated TI, leading to a decrease in naïve T cells. Therefore, targeting CS responses in preventing TI and, especially with the aged, allow for naïve T cell populations to be maintained.
Mucosal vaccination with an adenoviral vector vaccine protects against pulmonary breast cancer metastasis by robust induction of tissue-resident memory T-cells in the lung
Authors: Oltmanns Friederike Elise , Antão Ana Vieira , Irrgang Pascal , Schmidt Anna , Viherlehto Vera , Jörg Leticia , Tenbusch Matthias , Lapuente Dennis
Published: August 2023

The presence of tissue-resident memory T-cells (TRM) in solid tumors positively correlates with an improved overall prognosis and responsiveness to checkpoint inhibitor therapy. Rationally designed vaccine approaches capable of inducing antitumor TRM at cancer sites are needed to harness the full potential of localized immunity in tumor therapy. Our project aims to establish tumor antigen-specific lung TRM by mucosal vaccination with an adenoviral (Ad) vaccine to prevent and treat lung metastasis in the murine 4T1 breast cancer model.

We employ Ad serotype 5 vectors encoding for 4T1 tumor-associated antigens from Mage-b and MuLV gp70 for intranasal vaccination, supplemented with an Interleukin-1β-encoding vector to specifically boost lung TRM induction. Indeed, particularly if Ad-IL-1β was co-delivered, mucosal vaccination established high numbers of tumor antigen-specific CD69+CD103+ lung TRM as shown by phenotypic and functional analyses in combination with intravascular staining.

Vaccine efficacy was evaluated in a 4T1 pseudometastasis model mimicking a prophylactic vaccination against lung metastasis in breast cancer patients. Local immunization led to a significant protection against pulmonary metastasis and improved survival compared to non-vaccinated and, notably, intramuscularly immunized mice. By using FTY720 to inhibit T-cell circulation upon tumor challenge, we confirmed that TRM are crucial contributors to the vaccine efficacy.

In conclusion, induction of tumor-antigen specific lung TRM are a key point in local protection against pulmonary metastasis. Ongoing studies analyze the therapeutic use of this vaccination strategy as well as its potential in combination with immune checkpoint inhibition.

Supported by grants from ‘Anschubfinanzierung (ELAN) im IZKF’ and ‘Doktor Robert Pfleger-Stiftung’.
Targeting dendritic cell inhibitory receptors to down regulate immune response in inflammatory disease
Authors: Wei Hsi-Ju , Chen Liang , Patil Suresh , Barbon Jeffrey , Waire James , McCarthy Donna , Deora Arun , Laroux Stephen , Jin Liang , Tian Yu , Veldman Trudi , Ghayur Tariq , Hegde Subramanya
Published: August 2023

Immune regulator receptors play a crucial role in immune homeostasis by balancing activating vs inhibitory immune responses and are distributed across cells of myeloid and lymphoid lineages. Inhibitory receptors contain Immune Receptor Tyrosine-based Inhibitory Motifs (ITIM) and suppress immune response via SH2 domain-containing protein tyrosine phosphatases (SHP2). Dendritic Cell Inhibitory Receptor (DCIR) is a C-type lectin receptor expressed on monocytes, dendritic cells, and neutrophils. It has been implicated in controlling autoimmunity in preclinical models and polymorphisms linked to patients’ susceptibility to autoimmune diseases such as RA, Lupus, and Sjogren’s. Hence ligation of DCIR using monoclonal antibodies may provide inhibitory signals and therapeutic benefit. Here we show that agonistic anti-human DCIR antibodies (mAb) induce DCIR ITIM phosphorylation and SHP2 association. The DCIR/SHP2 complex diminished the SHP2 association of Syk with FcgR to inhibit TNFa, IL-1b, and IL-6 in human myeloid cells by attenuating the phosphorylation of SHP2, Syk, p38 and p65. Proteomic and RNAseq analyses also suggest that MAPK pathways are regulated by the agonistic mAbs. In human DCIR knock-in mice: (i) DCIR+ cells were enriched in inflamed crypts in DSS-induced colitis model; (ii) Agonistic mAbs reversed the weight loss and reduced the accumulation of neutrophils in the inflamed colon; (iii) Agonistic mAbs significantly reduced the recruitment of neutrophils and leukocytes in zymosanD-induced peritonitis model. Moreover, the increase of DCIR in activated neutrophils can be targeted by mAbs for their clearance. In summary, anti-DCIR agonistic mAbs modulate inhibitory functions and dampen inflammatory responses.
Class switched memory B cell development and transcriptional programming is regulated by EZH2
Authors: Wiggins Keenan J , Rose James , Boss Jeremy M , Scharer Christopher D
Published: August 2023

The most effective vaccines involve the development of antigen specific memory B cells (MBCs). MBCs make up a heterogenous population derived from germinal center (GC) dependent or independent processes, resulting in primarily immunoglobulin class switched and non-class switched MBCs, respectively. Class switched IgG MBCs are programmed to become plasma cells following a reinfection and non-class switched MBCs are programmed to seed secondary germinal centers. It is not clearly defined what epigenetic factors influence MBC differentiation and programming. EZH2 is a histone methyltransferase that catalyzes H3K27me3, resulting in gene repression. EZH2 has been shown to regulate multiple aspects of B cell differentiation, including development, germinal centers, and plasma cells; however, it is unknown if EZH2 regulates MBC development. To study EZH2, multiple knockout models have been established where EZH2 is conditionally deleted (cEZH2-KO). Here we used the influenza (PR8) model to determine the kinetics of antigen-specific MBC differentiation following a live infection in cEZH2-KO mice. We show that antigen positive MBCs are altered when EZH2 is deleted at days 14 and 39 post infection. Of the MBC subsets, IgG+, CD73+, and DP (PDL2+CD80+) MBCs were reliant on EZH2 for development but not IgM MBCs. These data indicate that extrafollicular/non-GC derived MBC differentiate in an EZH2-independent manner which reveals a distinct gene profile for extrafollicular/non-GC derived MBCs. EZH2 is necessary for repression of key gene regulators which determines the fate of class switched and non-switched MBCs. Overall, this model highlights the role of EZH2-dependent programming regarding MBC development and function.

Supported by R01 AI148471 from NIH/NIAID to CDS
The critical role of copper homeostasis during the immune response
Authors: Mandarano Alexandra H , McGargill Maureen A
Published: August 2023

Copper homeostasis is tightly regulated in living organisms, as copper is essential to numerous cellular processes but toxic in excess. Dysregulated copper homeostasis is associated with numerous human diseases, including genetic diseases of copper transport, cancer, autoimmune disease, and infection. Although patients with mutations in copper transporters have an increased susceptibility to infection, little is known about the changes in immune function in these diseases. Further, altering copper availability has shown promise in treating either autoimmunity or infection, but the mechanisms underlying this effect are unresolved. We examined serum copper levels in human samples during acute influenza or SARS-CoV-2 virus infections, revealing unique virus-dependent copper dynamics. As copper is required for important immune cell processes such as metabolism, signaling and maintenance of reactive oxygen species, we next investigated the effect of modulating copper levels on T cell activation. We found that copper chelation impaired T cell proliferation, reduced expression of T cell activation markers, and altered gene expression following stimulation. To determine the impact of copper levels during the immune response in vivo, we then treated mice with copper chelation prior to and during an influenza A virus infection. Notably, mice treated with copper chelation prior to infection had altered serum copper dynamics, greater weight loss, and more flu-specific T cells compared to untreated mice. Overall, our results demonstrate the importance of copper homeostasis during the immune response. Ongoing experiments will investigate the mechanisms underlying this role in immune function during disease.

Supported by ALSAC and NIH (5T32AI106700).
The interferon-rich skin environment regulates Langerhans cell ADAM17 to promote photosensitivity in lupus
Authors: Zyulina Victoria , Li Thomas M , Seltzer Ethan S , Dacic Marija , Chinenov Yurii , Daamen Andrea , Veiga Keila R , Schwartz Noa , Oliver David J , Lora Jose , Jabbari Ali , Liu Yong , Munir Hafsa , Shipman William D , Sandoval Marvin J , Sollohub Isabel F , Ambler William G , Mishra Bikash , Taber Sarah F , Onel Karen B , Zippin Jonathan , Rashighi Mehdi , Krueger James G , Anandasabapathy Niroshana , Rogatsky Inez , Blobel Carl P , Lipsky Peter E , Lu Theresa
Published: August 2023

Photosensitivity, a sensitivity to ultraviolet radiation, is a hallmark symptom of systemic and cutaneous lupus erythematosus (SLE and CLE). We have previously shown that photosensitivity in SLE models is caused by reduced function of Langerhans cells (LC) disintegrin and metalloprotease 17 (ADAM17), a sheddase that normally limits UVR-induced skin inflammation by activating epidermal growth factor receptor (EGFR) ligands. We also showed that LC ADAM17 activity is reduced in murine models and human SLE and that this LC ADAM17 dysfunction contributed to photosensitivity. The aim of this study is to understand why lupus LCs are dysfunctional and how type I interferon (IFN-I) in lupus skin contributes to LC ADAM17 dysregulation.

In this study we utilize multiple photosensitive SLE models and skin obtained from healthy human volunteers. LC ADAM17 activity and expression was measured by flow cytometry.

We found IFN-I signatures in non-lesional skin of MRL/lpr and B6.Sle1yaa photosensitive SLE models in addition to that of CLE patients, suggesting the similarities in gene expression patterns between human and mouse lupus skin. We also showed that IFN-I inhibits murine and human LC ADAM17 activity, and anti-IFNAR1 in SLE models restores LC ADAM17 function and reduces photosensitivity in EGFR− and LC ADAM17-dependent manners.

Together our findings suggest a model whereby the IFN-I-rich microenvironment in non-lesional lupus skin inhibits UVR-induced ADAM17 activity, leading to photosensitivity. These results suggest that the beneficial effects of the recently FDA-approved anifrolumab on human lupus skin may be attributable to effects on improving LC ADAM17 activity.
Foxp3 exon 2 regulates keratinocyte-specific IgE and basophil extracellular traps in cutaneous autoimmune photosensitivity
Authors: Domeier Phillip P , Stefani Caroline , Chen Shuting , Snyder Jessica M , Lacy-Hulbert Adam , Eisenbarth Stephanie C , Zhou Baohua , Ziegler Steven F
Published: August 2023

Ultraviolet B (UVB) light is a common environmental trigger that can induce flares in several autoimmune diseases, but the mechanisms that mediate UVB-induced photosensitivity remain poorly understood. We used mice which express an autoimmune-driving isoform of Foxp3, called FoxP3ΔE2 mice, to develop a model autoimmune cutaneous photosensitivity. In the steady-state, FoxP3ΔE2 mice have high serum titers of IgE that target skin-associated autoantigens, and increased numbers of IL4-producing T follicular helper cells that target skin-associated proteins (Keratin 14) in the absence of overt immune challenge. After skin exposure to UVB light, FoxP3ΔE2 mice had further elevated IgE, worsened skin inflammation, and increased IgE complex deposition that localized to the challenge site. IgE production in UVB-challenged mice correlated with increased frequencies of germinal center (GC) B cells, elevated percentages of IL-4+ T follicular helper cells, and numbers of basophils with high surface expression of IgE in the draining lymph nodes of UVB-challenged skin. We also observed a significant increase in IgE accumulation, increased numbers of Tug8+ basophil extracellular traps in UVB-challenged skin and increased frequencies of activated plasmacytoid dendritic cells (CD80+CD86+) in the skin and skin-draining lymph nodes of FoxP3ΔE2 mice when compared to wild-type littermates. Collectively, we found that T follicular regulatory cells in FoxP3ΔE2 mice fail to maintain self-tolerance in the GC, resulting in increased production of skin reactive IgE and worsened skin inflammation driven by basophil extracellular trap accumulation in UVB-damaged skin.

Supported by grants from the NIH (F32 AI154787, R01 AI136475, R21 AI169418, and R21 AI152444)
Paneth cells regulates innate lymphoid cells and B1 cells throughout the body
Authors: Joldrichsen Marisa R , Yoo Sungjun , Rayner Rachael , Cormet-Boyaka Estelle , Boyaka Prosper N
Published: August 2023

Paneth cells are a subset of small intestinal epithelial cells specialized to maintain gastrointestinal homeostasis through production of antimicrobial products and cytokines. The fact that dysbiosis has been associated with several noninfectious pathologies, has solidified the notion that Paneth cells contribute to the general health. However, little is known about Paneth cells beyond their role as regulator of the gut microbiota. Using mice lacking Paneth cells (Sox9ΔIEC mice), we now report that Paneth cells also regulate the composition of immune cell subsets in Gut-Associated Lymphoid Tissues (GALT) and but also in sites distant from the gut. Thus, loss of Paneth cells significantly decreased the number of ILC3s and increased the number of ILC2s in the intestinal lamina propria. Paneth cell deficiency also affected Peyer’s patches. In this regard, Paneth cell deficient mice displayed an increased numbers of Peyer’s patches which were of larger size than Peyer’s patches in control wild-type mice. Furthermore, Peyer’s patches of Paneth cell deficient mice contained increase numbers of IgA+CD19+B cells and B1a (CD19+CD5+) cells. Interestingly, when compared to control wild-type mice, the numbers of B1a cells were found to be also increased in mesenteric lymph nodes of Paneth cell deficient mice, but also at sites distant from the gut including the lungs, and bone marrow. Also, in the lungs there was an increase in ILC2s. Taken together, these findings indicate that Paneth cells play a key role as regulators of innate immune responses throughout the body via regulation of ILC subsets and B1 cells.

NIH Grants
Expansion of the intraepithelial lymphocyte (IEL) compartment results in an increased bioenergetic profile and reduced IFNγ production in γδ IELs
Authors: Alonso Sara , Jia Luo , Laguerta Alyssa , Edelblum Karen
Published: August 2023

Intestinal γδ intraepithelial lymphocytes (γδ IEL) serve as the first line of defense against pathogen invasion. We recently identified a transmissible hyperproliferative γδ IEL (γδHYP) phenotype that protects against enteric infection, and these mice have no signs of overt intestinal pathology. Given that IELs are metabolically constrained to prevent aberrant activation, we hypothesized that the bioenergetics of γδHYP IELs may regulate their effector function. Electron microscopy revealed a 70% increase in mitochondrial number (p=0.005) and 24% increase in mitochondrial area (p=0.04) in γδHYP IELs relative to WT. Seahorse mitochondrial stress assays on γδ IELs showed a 50% increase in spare respiratory capacity in γδHYP IELs compared to WT (p=0.014). Mitochondrial metabolism was shown to influence γδ T cell cytokine production, and similarly, we found that stimulation with PMA and ionomycin reduced the frequency of IFNγ+ γδ IELs by 59% in γδHYP relative to WT (p<0.0001). We also observed that γδHYP IELs exhibit higher CD39 expression compared to WT (p<0.0001), and a CD39hi subset produces less IFNγ compared to CD39neg and CD39int cells (p<0.0001) The addition of the ATP-synthase inhibitor oligomycin during IEL stimulation increased IFNγ production by both WT and γδHYP IELs by 66% and 44%, respectively (p<0.0001). Together, our data demonstrate that the shift towards high CD39 expression and elevated mitochondrial metabolism contribute to decreased IFNγ production in γδHYP IELs. Further understanding of the mechanisms regulating γδ IEL homeostasis and effector function may ultimately allow fine tuning of mucosal surveillance to confer protection against intestinal injury or infection while limiting aberrant cytotoxicity.

Supported by NJCCR (COCR23PRF024)
Myeloid cells and sphingosine-1-phosphate are required for TCRαβ+ intraepithelial lymphocyte recruitment to the colon epithelium
Authors: Danielson Sarah Mann , Lefferts Adam , Kuhn Kristine A
Published: August 2023

Intraepithelial lymphocytes (IELs) of the colon are essential for barrier maintenance and repair after injury. As bacterial depletion or global MyD88 deficiency results in a significant reduction of colon IELs and impaired barrier integrity, we aimed to connect the signals of how bacteria recruit colon IELs to the epithelium using conditional knockouts of MyD88 to target T cells (Lck-Cre), IECs (Villin-Cre), and myeloid cells (LysM-Cre). Only in the colons of LysM-Cre+ mice were TCRαβ+ IELs reduced, at 3.3-fold (p<0.005 compared to Cre− littermate controls) without effect on lamina propria T cells or myeloid cells. TCRγδ+ IELs were unaffected in all knockouts, suggesting they are recruited via different mechanisms. We then examined upregulated cell surface gene expression of IELs during recruitment, identifying sphingosine-1-phosphate receptor 1 (S1PR1) as a candidate for further study. Indeed, S1PR1 expression is significantly increased at 3.7-fold in IELs (p<0.05 compared to lamina propria T cells). In accordance with these findings, we demonstrated that TCRαβ+ IEL recruitment is blocked by administration of the S1PR1 receptor agonist FTY720 (p<0.05 compared to vehicle treatment). Furthermore, myeloid cell production of sphingosine-1-phosphate (S1P) was required for colon TCRαβ+ IEL recruitment in a MyD88-dependent manner. In the TNFΔARE/+ model of spontaneous colitis, loss of myeloid MyD88 resulted in protection from disease (p<0.05 compared to Cre-littermate controls). Overall, these data demonstrate the requirement for myeloid-produced S1P for TCRαβ+ IEL recruitment to the colon and suggest that TCRαβ+ IELs can contribute to intestinal inflammation under a specific genetic predisposition.
Weight cycling may induce lipid handling in mast cells
Authors: Caslin Heather L , Cottam Matthew A , Betjemann Alexa , Mashayekhi Mona , Silver Heidi J , Hasty Alyssa
Published: August 2023

Weight cycling worsens diabetes risk in both humans and mice. We recently found that weight loss and weight cycling induce many immunological changes in the adipose tissue of mice by single cell RNA-sequencing. We also found a substantial increase in adipose mast cells with weight cycling, but not weight loss. The purpose of this study was to confirm and characterize the changes in mast cells with weight cycling. However, using flow cytometry (cKit+FcER1+) and Toluidine blue staining, we found that adipose mast cells are greatest in the weight loss group, in contrast to our prior study using single cell RNA-sequencing. Upon further analysis, we found two unique mast cell populations in our single cell sequencing dataset: one that we consider traditional mast cells and one with lower expression of mast cell surface and protease genes and elevated expression of lipid handling and antigen presentation genes. The second fraction appeared similar to lipid-associated macrophages and accounted for most of the mast cells in the weight cycled group. By flow cytometry, we confirmed the %CD9+, MHCII (I-A/I-E)+, and CD74+ mast cells were highest in weight cycled mice. We also found two mast cell populations in human subcutaneous adipose tissue that appear similar to the murine populations in our dataset, with one population significantly correlating with weight variability. Together, these data suggest that weight cycling may induce a population of lipid-associated mast cells. We next aim to to induce lipid handling in bone marrow derived mast cells by repeated exposure to adipose conditioned media in order to understand the mechanisms which induce lipid handling.

This project was funded by a Veterans Affairs Merit Award 5I01BX002195 to AHH. AHH is also supported by a Career Scientist Award from the Veterans Affairs (IK6 BX005649). HLC was funded by an American Heart Association Postdoctoral Fellowship (20POST35120547), MAC was funded by an NIH F31 Predoctoral Fellowship (1F31DK123881), and both were previously supported by the Molecular Endocrinology Training Program (T32 DK07563). Additionally, the human mast cell work was funded by VICTR grants to HLC and MM. The Vanderbilt Institute for Clinical and Translational Research (VICTR) is funded by the National Center for Advancing Translational Sciences (NCATS) Clinical Translational Science Award (CTSA) Program, Award Number 5UL1TR002243-03.
Dietary whey protein increases brain leukocytes in mice regardless of their hypersensitivity status
Authors: Ekanayake Dilini Vimarshani , Nookala Suba , Nagamoto-Combs Kumi
Published: August 2023

Cow’s milk allergy (CMA) often manifests as milder reactions and may be linked to neurological problems. Previously, we demonstrated that C57BL/6J mice sensitized to a bovine whey allergen, β-lactoglobulin (BLG, Bos d 5), moderately increased BLG-specific IgE levels and exhibited behavioral changes without severe allergic reactions. When these non-anaphylactic CMA mice were placed on a whey-protein (WP)-containing diet for 2 weeks to simulate continuous dairy consumption, we found neuropathology indicative of neuroinflammation and cortical demyelination. Since immune cells migrate to the central nervous system (CNS) and promote neuroinflammation in demyelinating conditions such as multiple sclerosis, we hypothesized that the number of leukocytes would increase in BLG-sensitized mouse brains to orchestrate neuropathology. To test this hypothesis, we used flow cytometry to determine the number and phenotypes of leukocytes in the brains of naïve, sham, and BLG-sensitized mice after the 2 weeks of the WP diet. The frequencies of cells expressing common leukocyte marker CD45, pan T cell marker CD3, cytotoxic T cell marker CD8, integrin CD11b, myeloid cell marker CD14, and co-stimulatory marker CD86 significantly increased, regardless of the sensitization status. The percentages of these cells were low in mice that never received WP. This result indicated that WP diet consumption alone increased CNS leukocyte populations. Additional immunophenotyping is needed to determine whether the identified cells can be differentiated among the experimental groups. Detailed characterization of CNS leukocyte phenotypes and dynamics will help elucidate the mechanism of CMA-induced neuroinflammation and cortical demyelination.
Continuous consumption of whey protein affects motor and cognitive functions in a mouse model of cow’s milk hypersensitivity
Authors: Verma Geetika , Nagamoto-Combs Kumi
Published: August 2023

Hypersensitization to a food allergen can occur in the absence of immediate allergic reactions, a condition referred to as asymptomatic sensitization. Using mice asymptomatically sensitized to a bovine whey allergen, β-lactoglobulin (BLG, Bos d 5), we have previously shown that chronic exposure to the allergen results in behavior changes with increased neuroinflammation and cortical demyelination. However, whether allergen avoidance can resolve these pathologies is unknown. Thus, we aimed to compare immunological and behavioral changes in sham- or BLG-sensitized mice with or without the removal of dietary whey after the mice had consumed either a control diet (CTL) or a whey-protein-containing diet (WP) for 2 weeks. As previously reported, BLG-sensitized mice did not display overt physical reactions during the 2-week allergen exposure period, while their plasma levels of BLG-specific IgE and IgG1 were elevated compared to the respective sham groups. Following an additional 2 weeks on either the CTL or WP diet, these antibodies remained elevated in some of the sensitized mice in both diet groups. The grip-strength test indicated that sham mice that switched to the CTL diet after the 2-week WP diet increased limb strengths. In contrast, BLG-sensitized mice on either diet, as well as sham mice that stayed on the WP diet, showed a decline in limb strengths, suggesting that WP consumption, whether during sensitization or as part of the daily diet, affected their motor function. Furthermore, removing dietary WP improved spatial memory performance with the cross-maze test in both sham and BLG-sensitized mice. These results suggested that continuous exposure to a dietary allergen may influence motor and cognitive functions.

Supported by grants from NIH (R01 AI168563)
B cell intrinsic Toll-like receptor 9 signaling regulates class switching to IgE
Authors: Chang Karen , Nelson Alexander J , Wu Yee Ling
Published: August 2023

Chronic allergic diseases such as allergic asthma are driven by excessive production of allergen specific IgE. B cells produce IgE by class switch recombination (CSR) in response to T cell help and Th2 cytokines. However, development of allergic diseases can also be influenced by factors such as early-life exposure to microbial products. In vivo, stimulation of Toll-like receptor 9 (TLR9) with microbial DNA reduces IgE levels, largely thought to be a response to Th1 cytokines. However, the direct effects of TLR9 signaling on B cell class switching to IgE remain unclear. To delineate the B cell intrinsic effects of TLR9 stimulation, we treated mouse splenic B cells in vitro with IL-4 and anti-CD40 antibody with or without a synthetic TLR9 agonist, CpG oligodeoxynucleotides (CpG). Compared to stimulation with IL-4 and anti-CD40 alone, the addition of CpG decreased IgE production. Using B cells from reporter mice that track germline transcription, a prerequisite for CSR, we found that CpG inhibited Iɛ but increased Iγ1 germline transcription. By Western blot analyses, we probed the expression of transcription factors known to negatively regulate Iɛ and positively regulate Iγ1 transcription. These transcription factors were synergistically upregulated in the presence of CpG with IL-4 and anti-CD40. Together, these data illustrate a molecular mechanism by which microbial products directly perturb the production of IgE and influence the development of allergic disease.

R01 HL165120
Nutritional factors govern gut homeostasis via oral tolerance
Authors: Nagai Motoyoshi , Okawa Takuma , Nakata Kazuaki , Kawamura Yuki I , Hase Koji
Published: August 2023

Oral tolerance is one of the most critical systems to regulate allergic disorders. However, how dietary factors regulate the induction and maintenance of oral tolerance remains to be elucidated. To address this, we temporarily omitted the food supply by fasting before oral ovalbumin (OVA) administration. We observed that fasting mitigated the expansion of OVA-specific Treg cells. This abnormality mainly resulted from functional defects in CX3CR1high macrophages responsible for the uptake of luminal OVA and the reduction of tolerogenic CD103+dendritic cells (DCs). Eventually, fasting impaired the preventive effect of orally administrated OVA in the development of asthma and allergic rhinitis. We further explored the food ingredients essential for the induction of oral tolerance and found that the intake of dextrin and a basic amino acid is indispensable for oral tolerance induction. These observations illustrate that nutritional signals are critical in maintaining immune homeostasis via induction of tolerance to ingested food antigens.
Maternal-neonatal model of immune protection to type 2 pulmonary inflammation
Authors: Krishnamoorthy Nandini , Smith Ethan , Bruggemann Thayse , Walker Katherine , Chiang Nan , Serhan Charles N , Levy Bruce D
Published: August 2023

Asthma tends to “run in families” is a concept supported by several research studies of asthma genetics, but this only explains a relatively small fraction of asthma inheritance. We are now standing this paradigm on its head by asking the opposite question: Can asthma protection be passed on from one generation to the next? Epidemiological and experimental studies have determined that the early postnatal period offers a critical window for the establishment of lung protection. Maternal milk represents a potentially important transgenerational mechanism by which offspring may acquire early-life immunity from their mothers. To study this transfer of protection we have developed a new preclinical animal model of maternal transference of immune protection. In this model, maternal exposure to endotoxin postpartum resulted in infant protection from allergic inflammation to the common indoor allergen house dust mite and to a clinical strain of Respiratory Syncytial Virus (RSV). The overall downregulation of type 2 immune responses to an allergen and a virus indicated that the mechanism of protection was common for different antigens. Metabololipidomics analysis of maternal milk from endotoxin-exposed mothers revealed elevated levels of select specialized pro-resolving mediators (SPMs) from essential fatty acids. Young animals from endotoxin-exposed mothers had overall lower allergic immune responses, which was related to altered pulmonary dendritic cell (DC) subsets. Importantly, these protective effects of asthmatic inflammation reduction were also recapitulated with the direct administration of SPMs. This study provides a novel transgenerational immunological conduit of protection in a heretofore-unexamined process
The role of NFAT in CD8+Foxp3+ Tregs
Authors: Patino Liliana C , Ortega-Recalde Oscar , Gauthier Thierry , Liu Na , Jin Wenwen , Chen WanJun
Published: August 2023

CD8+Foxp3+ regulatory T cells (CD8 Treg) correspond to a very low frequency population which can be observed during GVHD at early stage or in some cancer types. However, in vitro, around 30–60% of CD8 can express Foxp3 after induction culture with TCR stimulation and TGF-β (iCD8Treg). Why in healthy mice and humans this cell population has very low or non-frequency been still an unresolved question. In vitro, iCD8Tregs can suppress effector cells in a similar way as iCD4Treg. However, in vivo, in the IBD model, we observed that in contrast to iCD4Treg, iCD8Treg lose the Foxp3 expression and consequently their suppressive function. Also, iCD8Treg increase the IFN-γ production. These results suggest that iCD8Treg are not able to maintain the Foxp3 expression in comparison with iCD4Treg cells. What signaling pathway is responsible for the Foxp3 losing in iCD8Treg? We found a positive correlation between Foxp3 maintenance and cyclosporin A (CSA) concentration, and a reduction of IFN-γ in culture in iCD8Treg, but not for iCD4Treg. We evaluated the effect of CSA, an NFAT inhibitor, on the Tregs induction culture (with TGF-β) in the CD8 cells and found that there was no increase of Foxp3 expression. These results suggest that NFAT signaling plays a role in the Foxp3 maintenance but not in the induction of the iCD8Treg and this role is different from iCD4 Tregs.
A STAT5 activator protects CD8+ T cells in immunosuppressive environments
Authors: Shudde Claire E , Gu Zehui , Piper Taylor L , Zheng Yating , Courtney Adam H
Published: August 2023

CD8+ T cells are central mediators of an anti-tumor immune response because they can recognize and kill cancerous cells. However, cancer can evade this attack by establishing a tumor microenvironment (TME) that suppresses CD8+ T cells by depriving them of pro-survival cytokines, such as IL-2. IL-2 has been directly administered to cancer patients as an immunotherapy, but was found to cause broad immune activation that could result in multi-organ failure and possessed poor pharmacologic properties. To address these limitations, we have devised a strategy to activate the intracellular mediator of IL-2 signaling – STAT5. The activator of endogenous STAT5 that we developed was evaluated for its capacity to maintain T cell survival and function. To recapitulate a niche devoid of pro-survival cytokines like the TME, we activated CD8+ T cells in the absence of IL-2 and evaluated their viability in culture. We determined that direct activation of STAT5 could maintain CD8+ T cell viability up to 10 days post-activation, whereas control cells died off by day 3. To investigate whether STAT5 activation could be used to retain CD8+ T cell function, we assessed the potential of these cells to become activated by antigen and kill cancerous cells. We found that T cells that persisted in the absence of IL-2 due to STAT5 activation could upregulate a reporter of T cell activation (Nur77-GFP) and kill cancer cells ex vivo. Moreover, STAT5 activation enabled CD8+ T cells to produce IFNγ upon restimulation by antigen presenting cells. Our data demonstrate that a STAT5 activator can be used to maintain T cell survival, activation, and cytotoxic capacity in cytokine-depleted environments, suggesting its potential to counteract an immunosuppressive TME.

Supported by grants from NIH (T32GM140223), V Foundation for Cancer Research, and the Concern Foundation
The role of the ubiquitin ligase Cul4b in B lymphocytes
Authors: Davis Destiny K , Elliott Gabrielle , Renshaw Lindsay , Moser Emily
Published: August 2023

Vaccination or infection induces antibody secretion by long-lived plasma cells, creating durable immune protection against secondary infections. These plasma cells develop from B cells that have undergone affinity maturation, which requires somatic hypermutation in proliferating B cells, forming single strand DNA breaks that are repaired by mismatch repair machinery. Cullin 4b (Cul4b) is an E3 ubiquitin ligase found in most cells, including B cells, but its function in B cells is unknown. Cul4b is one of two docking proteins forming the backbone of the Cullin RING Ligase 4 (CRL4) complex, which ubiquitinates substrate proteins for degradation. Previous studies have shown that Cul4b promotes DNA repair with cell proliferation, and high Cul4b is linked to aggressive colon cancer. Because proliferation and DNA repair are essential for humoral immunity, we hypothesized that Cul4b also functions in B cells. To test this, we generated mice that lack Cul4b in B cells. We immunized these mice with an mRNA vaccine against an H1N1 Influenza A virus, and measured virus-specific serum antibody. We found that Influenza-specific antibodies were dramatically reduced in mice serum that lacked Cul4b in B cells. To determine if Cul4b promotes DNA damage repair in B cells, we induced single-strand DNA breaks with UV irradiation and then stimulated the cells to proliferate in vitro. We found that Cul4b promoted B lymphocyte proliferation after UV exposure. Understanding the Cul4b-DNA damage repair pathway in B cells will identify new drug targets to transiently amplify this pathway to improve vaccines and encourage stronger antibody responses, or to inhibit this pathway to prevent tumor progression in cancer.
Phenotypic Evaluation of TREX1 Null Mice as a Model for Lupus-Like Autoimmune Disease
Authors: Li Zhiying , Liang Juan , Chen Zhong , Xu Shuxin , Chen Yingjiao , Tong Huan , Ku Cunxiang , Zhao Jing , Xiang Gao
Published: August 2023

TREX1 (DNase III) is the major mammalian 3’-5’ DNA exonuclease and thought to play a major role in cell death and genomic DNA degradation. Dysfunction of Trex1 has been suggested to activate immune response by self DNA, as TREX1 null mice develop inflammatory myocarditis similar to autoimmune cardiomyopathy and produce type 1 IFN. TREX1 D18N causes a monogenic cutaneous form of lupus called familial chilblain lupus and exhibits dysfunctioned dsDNA-degrading activity, providing a link between dsDNA degradation and nucleic acid-mediated autoimmune diseases. Using CRISPR-Cas9, we generated a Trex1−/− mouse by knocking out exon 2 and performed comprehensive phenotypic analyses to evaluate the loss of function of TREX1 and its potential application in SLE research.

Both female and male TREX1 null mice showed dramatically increased post-wean morbidity and significantly increased serum ALT, AST and LDH levels, compared to wildtype and heterozygotes from 6 to 24 weeks of age, indicating liver and heart injuries. Pathohistological analysis showed inflammatory immune cell infiltration in liver, heart, spleen, kidney, lung and skin among TREX1 null mice. Serum anti-dsDNA antibody levels were significantly increased and remained high from 7 to 25 weeks of age, consistent with Trex1’s being the major DNA exonuclease. Urine protein to creatinine ratio was dramatically increased at 28 weeks of age among TREX1 null mice.

In conclusion, the Trex1−/− mice we generated in this work showed lupus-like inflammatory autoimmune responses in multiple organs, providing an urgently needed tool to study Trex1-mediated autoimmunity and to evaluate potential therapeutic treatments for lupus.
Dysregulation of complement regulator CD55 in the initiation of autoimmunity
Authors: Lee Iris , Ling Ivana , Pham Christine T , Kendall Peggy L
Published: August 2023

CD21 is a complement receptor involved in B cell tolerance. CD21 low age-associated B cells (ABCs) are an autoimmune-prone population of B cells that are expanded in autoimmunity. B cell receptor (BCR) and toll-like receptor 9 (TLR9) co-stimulation poise B cells for this autoimmunity-associated cell fate. We and others have discovered that BCR and TLR9 co-stimulation also causes downregulation of CD55, a complement regulatory protein that dampens complement activation by interfering with the production of complement fragments. We posit that CD55, by controlling the generation of complement fragments, regulates BCR-TLR9-triggered B cell responses in the context of autoimmunity. Congruent with this hypothesis, CD55 deficiency has been shown to cause exaggerated experimental encephalitis and disease in the MRL/lpr mouse model of lupus. Our overarching hypothesis is that CD55 regulates B cell responses in the context of autoimmune disease. For this study, we examined CD86 upregulation, proliferation, and cytokine production in B cells after knocking out CD55. We found that CD55 did not impact the CD86 upregulation or proliferation of B cells, but did induce a proinflammatory cytokine profile with increased IL-6 production. In addition, crossing CD55 deficiency onto anti-insulin B cells, a well-studied anergic model, showed that CD55 deficiency partially abrogated anergy-mediated tolerance, increasing the ability of these B cells to proliferate when stimulated through TLR9. These findings may have implications for the future use of complement inhibitors in the treatment of autoimmune disease.

Iris Lee holds a Dean’s Scholars Award from the Washington University Division of Physician-Scientists, which is funded by a Burroughs Wellcome Fund Physician-Scientist Institutional Award. The Kendall lab is supported by VA Merit I01BX002882 and R01 DK084246.
Interferon regulatory factor 3 disrupt the normal differentiation of epidermal keratinocyte by promoting the nuclear localization of interleukin-33
Authors: Jeong Na-Hee , Kim Sang-Hyun
Published: August 2023

Atopic dermatitis (AD) is a complex and multifactorial skin disease characterized by skin inflammation and intense pruritus. AD is often diagnosed based on symptoms, but a precise diagnosis and treatment are difficult due to various symptoms. This study aimed to develop a safe and effective therapeutic agent and identify a novel therapeutic target for AD. We verified the functions and mechanism of interferon regulatory factor3 (IRF3) as a novel therapeutic target of AD. IRF3, a transcription factor regulating various pro-inflammatory cytokines, including IL-33, was significantly increased in the skin lesions of AD mice and patients with AD. However, deficiency of IRF3 relieved AD symptoms and pathophysiological changes, including skin thickening (the dermis and epidermis thickening), immune cell infiltration, serum immunoglobulins level, and pro-inflammatory cytokines. In damaged AD tissues, IRF3 regulated the expression of cytokeratin 1 (KRT1) and 10 (KRT10). IRF3 also altered the expression of nuclear IL-33 in the epidermis and the extracellular IL-33 in the dermis of AD mouse tissues. In vitro experiments using human keratinocytes, knockdown or overexpression of IRF3 regulated the nuclear localization and the extracellular release of IL-33. Thus, IRF3 is an essential factor in the onset and exacerbation of AD by inducing skin barrier abnormalities as well as activation of the immune system. Collectively, our studies identify IRF3 as a positive regulator for IL-33, highlighting a novel therapeutic target for AD and suggesting a novel solution for treating intractable dermatitis.
Inflammatory conditions shape phenotypic and functional characteristics of lung-resident memory T cells
Authors: Schmidt Anna , Fuchs Jana , Lapuente Dennis , Hübner Julian , Antão Ana Isabel Vieira , Irrgang Pascal , Oltmanns Friederike , Paudyal Basudev , McNee Adam , Vatzia Eleni , Carr Veronica , Villanueva Sonia , Tchilian Elma , Tenbusch Matthias
Published: August 2023

Lung tissue-resident memory T cells (TRM) are critical for the local control of viral respiratory tract infections. However, the exact mechanisms of TRM stimulation and maintenance are not fully understood, rendering the induction of protective TRM responses by vaccination an ambitious goal.

Here, TRM populations induced by natural influenza A virus (IAV) infection or by mucosal immunization using adenoviral vector vaccines encoding the IAV antigens HA and NP as well as IL-1β as adjuvant were analyzed. Phenotypic and functional TRM responses were assessed in mice over 200 days via flow cytometry. Interestingly, long-lasting TRM responses with a less cytotoxic character were found scattered within the lung tissue after mucosal immunization. By contrast, TRM triggered by prior IAV infection surrounded bronchus-associated B cell clusters and their quantity waned over time more rapidly. Furthermore, single cell RNA sequencing revealed different gene expression profiles of NP-specific TRM depending on the priming condition. However, both TRM populations provided protection from heterologous challenge, resulting in an expansion of cognate antigen-specific TRM. Substantial differences in IAV-specific T-cell immunity after adenoviral vector immunization or prior infections could be confirmed in the Babraham pig model. Noticeably, increased TRM responses did not correlate with protection against an infection with a heterologous IAV strain, indicating potentially different correlates of protection in pig and mice.

In conclusion, mucosal immunization was associated with a prolonged tissue retention of functional TRM, opening up a promising line of enquiry for the design of future vaccines against respiratory viruses.

Main parts of this work were supported by grants from IZKF (advanced project A90). The translational part into the pig model was carried out at the Pirbright Institute, supported by grants from VetBioNet (grant agreement No 731014, Ref: VBN_20_49) and BBSRC.