bluuebunny/crossref_metadata_2025 huggingface dataset:
Changes in Myeloid-Derived Suppressor Cell function during neonatal sepsis
Authors: Vance Jordan , Rawson Travis , Witt Michelle R. , Seman Brittany G. , Bradford Shelby , Robinson Cory M.
Published: January 2023

Infants are more susceptible to infectious disease due to immaturity of the immune system. Our laboratory has shown that the immune suppressive cytokine, interleukin (IL)-27, is elevated in neonates. We and others have also shown that myeloid-derived suppressor cells (MDSCs) are more abundant in neonates, and these cells are a source of IL-27 in neonates. MDSCs suppress immunity with additional effector functions such as nitric oxide synthase (NOS2), arginase-1 (ARG1), and production of oxidative radicals to. We hypothesized that MDSCs gain enhanced immune suppressive function during neonatal sepsis. We utilized a murine model of neonatal Escherichia coli-induced sepsis that that demonstrates reduced weight gain in infected animals compared with controls, and high levels of bacteria in the blood and peripheral tissues. MDSCs were isolated from control or infected pups and the expression of effector function genes evaluated. Gene expression analysis revealed that NOS2, ARG1, and IL-27 gene expression was increased in MDSCs from infected mice. To understand whether or not these changes manifest at the protein/functional level and to further understand if they could be replicated in vitro, MDSCs isolated from control mice were cultured with E. coli. A similar pattern of changes in expression was observed with NOS2, ARG1, and IL-27 genes in vitro when the MDSCs were cultured with bacteria. This was followed by increased arginase activity, elevated nitrite levels, and secreted IL-27. These results suggest that gram-negative bacteria can directly act on MDSCs to enhance effector function associated with immune suppression. They also support further study of MDSC activity in regulating host control of infection early in life.
Retinoic acid-induced expression of CD103 in human DCs is dependent on p38 MAPK signaling and NFAT1c
Authors: Roe Mandi M , Swain Steve , Bimczok Diane
Published: January 2023

Mucosal CD103+ (αE integrin+) DCs are critically involved in driving a T regulatory response and enabling mucosal homeostasis. However, the regulation of CD103 expression in human DCs is not fully understood. We previously found that retinoic acid (RA) induces CD103 expression in human monocyte-derived DCs (MoDCs). Here, we investigated the molecular mechanisms leading to RA-induced CD103 expression on human DCs. We used a phospho-protein array to screen RA-treated MoDCs and found multiple potential targets. We focused on p38 MAPK based on previous studies that showed reduced CD103+ DCs in p38 MAPK KO mice. Western blots confirmed increased phosphorylation of p38 MAPK in RA-treated MoDCs. To determine whether p38 MAPK signaling influences CD103 expression, we next used an inhibitor of p38 MAPK and evaluated CD103 expression on MoDCs. The p38 MAPK inhibitor SB202190 significantly decreased expression of CD103 protein and mRNA in RA treated, but not in control MoDCs (p=0.037 and p=0.047 respectively). The transcription factor NFAT1c is a downstream target of p38 MAPK that has been shown to bind to the promotor region of CD103 in T cells. To determine whether NFAT1c regulates RA-induced CD103 expression, we next exposed RA-treated MoDCs to an NFAT1c peptide inhibitor. We demonstrated a significant dose-dependent decrease in CD103 protein expression of RA-treated MoDCs with increasing concentrations of the NFAT1c inhibitor (p=0.03). These data indicate that RA-induced expression of CD103 is dependent on p38 MAPK signaling and NFAT1c in human DCs and give insight into molecular mechanisms of DC signaling.
HMGB1-induced ILC2s activate dendritic cells by producing IL-9 in mice asthma
Authors: Xu Huaxi , Wan Jie , Su Zhaoliang , Wang Shengjun
Published: January 2023

High-mobility group box-1 (HMGB1), a unique and important pathogenic protein, has been implicated as a pro-inflammatory cytokine involved in the pathogenesis of various inflammatory and autoimmune diseases, and CD4 T-cell differentiation and modulation. It has been reported that HMGB1 can promote Th2 response, aggravating the severity of asthma in mice, thus ILC2s contribute to Th2 polarization and maintain this immune imbalance. However, little is known about possible role of HMGB1 on regulation of ILC2s and on the activity of dendritic cells (DCs) in asthma. In this study, we detected the IL-9 expression level in HMGB1-stimulated ILC2s in vitro or asthmatic mice, analyzed the effect of IL-9 derived from ILC2s on DCs function and their role in the development of asthma. Our results showed that HMGB1 could activate ILC2s and promoted IL-9 secretion, and A-box of HMGB1 eliminated this phenomenon. In asthma mice model, A-box of HMGB1 relieved asthma severity. Bone marrow-derived dendritic cells (BMDCs ) could be activated in ILC2s and BMDCs co-culture system, but this effect was inhibited when anti-IL-9 was added in vitro. In addition, IL-9-activated BMDCs co-cultured with naïve T cells promoted the Th2 cells differentiation. The Jak1/stat3 and NF-κB pathway contributed to BMDCs activation involved by IL-9. In asthma mice model, injection of anti-CD90.2 and anti-IL-9 could relieve asthma severity, but transferred exogenous IL-9 made asthma worse. It indicated that increased HMGB1 in the lung of asthmatic mice activated ILC2s to secrete IL-9, which regulated the activity of DC involved in promoting Th2 cells differentiation and finally aggravated asthma.
STEM knowledge translational outreach efforts – connecting with Immunology in the classroom for Pacific Intermediate students
Authors: Sika-Paotonu Dianne
Published: January 2023

The appropriate contextualisation of complex scientific content for young students can be a challenge. Supporting improved interest and engagement with STEM related content by all students in the classroom at the same time, can also be difficult. The purpose of this work was to use an Immunological in-class teaching focus to improve interest in STEM related teaching content for underrepresented student groups at Intermediate school level. A STEM knowledge translational programme organised by an external organisation, included as part of their programme an outreach activity contribution made by a female Pacific Biomedical Scientist for Pacific students aged 10–12 years (Year 7 & 8). Science related learning that was context specific was a primary focus for the Pacific students. Clinical research work components relating to the autoimmune condition Acute Rheumatic Fever (ARF) were presented to the Pacific College students as high rates of ARF and RHD are found in Māori and Pacific Peoples in NZ and the Pacific Region. Enthusiastic in-class interactions between the Biomedical Scientist and the Pacific students were observed. It was noted that the students present were highly motivated to ask questions relating to the Immunologically based content that had been presented. Verbal and written feedback indicated the students (and teachers) enjoyed this particular session and were challenged to consider undertaking STEM related courses in future. This work demonstrated the importance of promoting STEM related teaching content that is of clinical relevance and presented appropriately for Pacific students. The value of using of role modelling in this context was also recognised.
An intervention to increase accuracy and understanding in undergraduate micropipette use
Authors: Avery Lyndsay , Dukes April , Warner Marcie
Published: January 2023

Micropipetting is an essential skill for the budding immunologist and is widely used across biological and physical science disciplines, yet few undergraduates are adequately introduced to this skill. The mentors of undergraduates seeking research experience point to micropipette inexperience as a common barrier to success. As part of our SEA-PHAGES authentic research laboratory, we designed a hands-on intervention to give thorough training in micropipetting to first-year undergraduates. The intervention included assignment of a pre-class video on micropipetting, an activity to orient students to the micropipette parts and functions, and a TA-led demonstration of proper micropipette use and volume adjustment. Success of the intervention was analyzed using data collected from a practical test following the lab activity. In addition, we compared the students’ end-of-semester experimental success to that of students from previous semesters lacking the micropipetting activity, using archived electronic laboratory notebooks. We found that students progressed through experiments faster and with more success after experiencing the micropipette intervention. Our analysis showed that some aspects of proper micropipette use need to be emphasized, such as proper depth of tip entry and the correct tactile discrimination of the first/second stop on the micropipette. In addition, we found that students have misconceptions and gaps in knowledge about the metric system that are an additional barrier to proper micropipette use. This project presents a successful platform for teaching micropipette technique, and demonstrates that this leads to increased likelihood of success for the students in this real-world laboratory setting.
A host cell-intrinsic innate regulatory circuit limits inflammasome activity and promotes immune escape of Salmonella inside macrophages
Authors: Akhade Ajay Suresh , Atif Shaikh M , Lakshmi Bhavana S , Dikshit Neha , Qadri Ayub , Subramanian Naeha
Published: January 2023

Inflammasomes have been implicated in the detection and clearance of a variety of bacterial pathogens, but little is known about whether there is active cross-talk between the host sensing mechanism and the expression of stimulatory ligands by the pathogen. Here we show that inflammasome activation regulates expression of the NLRC4 and TLR5 ligand, flagellin, by Salmonella. Host lysophospholipids released upon NLRC4-mediated pyroptosis increase flagellin expression by extracellular bacteria that enhances pyroptosis upon internalization, establishing a positive feedback loop that potentiates Salmonella detection and clearance. A TLR-dependent host negative feedback response later inhibits inflammasome activation and lysophospholipid biosynthesis within cells, prompting the pathogen to switch to a flagellin-low phenotype and establish an intracellular survival niche inside macrophages. Ablation of this host regulatory circuit prevents downregulation of flagellin by intracellular Salmonella and promotes bacterial clearance in vivo. Our data identify modulation of expression of a bacterium-derived inflammasome ligand by the very process of inflammasome activation as a novel mode of host-pathogen cross-talk and reveal a host mechanism that is adapted by Salmonella for flagellin downregulation and immune escape within macrophages.
PtdIns4P on dispersed trans-Golgi network mediates NLRP3 inflammasome activation
Authors: Chen Jueqi , Chen Zhijian J
Published: January 2023

The NLRP3 inflammasome, which has been linked to human inflammatory diseases, is activated by diverse stimuli. How these stimuli activate NLRP3 is unknown. Here we show that different NLRP3 stimuli lead to disassembly of the trans-Golgi network (TGN). NLRP3 is recruited to the dispersed TGN (dTGN) through ionic bonding between its conserved polybasic region and negatively charged phosphatidylinositol-4-phosphate (PtdIns4P) on the dTGN. The dTGN then serves as a scaffold for NLRP3 aggregation into multiple puncta, leading to polymerization of the adaptor protein ASC, thereby activating the downstream signalling cascade. Disruption of the interaction between NLRP3 and PtdIns4P on the dTGN blocked NLRP3 aggregation and downstream signalling. These results indicate that recruitment of NLRP3 to dTGN is an early and common cellular event that leads to NLRP3 aggregation and activation in response to diverse stimuli.
Nucleic acid sensing in a 3D hydrogel model of B16F10 mouse melanoma
Authors: Heller Loree , Bulysheva Anna , Arpag-Mcintosh Sezgi , Semenova Nina , Cemazar Maja
Published: January 2023

Pattern recognition receptors (PRRs) are nearly ubiquitous in immune and non-immune mammalian cells. PRRs detect molecular pattern molecules associated with pathogen invasion as well as internal damage markers. A subset of pattern-recognition receptors, found endosomally or cytosolically, detect nucleic acids including DNA. Recently, a number of cytosolic DNA-specific PRRs expressed in non-immune cells have been described. Binding and activation of these sensors induces the production of Type I interferons and pro-inflammatory cytokines and chemokines.

Many cell types, including B16F10 mouse melanoma cells, respond to DNA electroporation with the upregulation of specific PRRs on the mRNA and protein levels. We developed B16F10 melanoma cell hydrogels to test whether cells in these constructs (“3D cells”) responded similarly to cells in suspension. The cells formed small spheroids within the hydrogels, similar to in vivo tumor morphology, cell-cell, and cell-matrix microenvironment. 3D cells maintained a nearly 73% proliferation rate compared to a 94% rate for cells cultured in 2D monolayers. The relative metabolism kinetics of 3D cells were similar to monolayers in culture, as was transgene expression after delivery by electroporation. However, while similar PRR mRNAs were upregulated, including DAI/ZBP1, DDX60, and p204, the relative levels of upregulation differed significantly between suspended B16F10 and 3D cells. In addition, p204 was upregulated in control constructs but not in suspension cells receiving pulses without DNA. Supporting these observations, the induced cytokine profile also differed. These data emphasize the importance of testing multiple models before generalizing to tissue responses.
Outcomes of ERK Signaling Differ in Macrophages and Dendritic Cells
Authors: Groft Sarah Grace , Nagy Nancy , Shukla Supriya , Sweet David , Boom W Henry , Harding Clifford V
Published: January 2023

Stimulation of dendritic cells and macrophages by pathogen-related products activates a variety of immune responses. Pathogen-induced cell signaling and associated outcome research is abundant in macrophages, but minimal in dendritic cells. We investigated the role of ERK signaling in the induction of cytokines such as IL-12p40 and IL-10 following activation by Pam3Cys or Mycobacterium tuberculosis (Mtb) H37Ra. ERK ablation was accomplished pharmacologically with MERK1/2 inhibitor U0126 or genetically by using cells from Tpl2−/− mice (TPL2 connects TLR to ERK). We utilized western blotting to examine ERK activation, ELISA to assess cytokine production, and qRT-PCR to investigate mRNA levels. In macrophages, blockade of ERK signaling inhibited IL-10 production and increased IL-12p40 production (6–10 fold increase in IL-12p40 mRNA and 3–4 fold increase in protein). In dendritic cells, ERK blockade similarly inhibited IL-10 production, but produced a very different change in IL-12p40 expression: ERK blockade decreased IL-12p40 production (2-fold decrease in IL-12p40 mRNA and protein). This result suggests that the impact of ERK signaling in response to these stimuli differs between macrophages and dendritic cells for a subset of ERK-regulated genes. ERK-mediated suppression of IL-12p40 in macrophages may prevent excess inflammation and associated tissue damage, while ERK-mediated induction of IL-12p40 in dendritic cells may promote priming of Th1 responses. Future studies will examine other genes and proteins involved in infection responses. Greater understanding of the role that ERK signaling plays in different cell types may inform the development of host-directed therapy for infections such as tuberculosis.
Analysis of health and lung injury markers in mice treated with TLR4-interacting SPA4 peptide
Authors: Awasthi Shanjana , Rahman Negar , Hedrick Andria , Awasthi Vibhudutta , Breshears Melanie , Kosanke Stanley
Published: January 2023

The SPA4 peptide (GDFRYSDGTPVNYTNWYRGE) interacts with TLR4, affects intracellular signaling, improves host defense, and alleviates tissue injury in mouse models of bacterial lung infection and lipopolysaccharide-induced lung inflammation. In this study, we investigated the effect of different doses of SPA4 peptide on health, and systemic and lung injury parameters in outbred CD-1 mice. Mice were intratracheally administered with 0.625, 1.25, 2.5, 5, and 10 μg SPA4 peptide per g body weight every day for three days. Mice left untreated or those treated with equivalent volume of vehicle were included as controls. Signs of sickness and body weights were monitored every 24 h; food and water were provided ad libitum. After 24 h of last intratracheal instillation, mice were euthanized, and blood and major organs were collected. Blood gas and electrolytes (pCO2, total CO2, pO2, pH, anion gap, base excess, total hemoglobin, Na+, K+, Cl−, HCO3−) were analyzed, and markers of lung injury (lung wet/dry weight ratio, and levels of albumin, lactate, and total protein) were determined in lung tissue homogenates. Major organs (stomach, intestines, lung, liver, spleen, kidney, heart, and thymus) were fixed, sectioned, stained with hematoxylin and eosin, and examined for tissue damage or inflammatory response. No signs of sickness or changes in body weight were noted. Blood gas and electrolyte parameters were not significantly altered in SPA4 peptide-treated mice. The lung injury parameters were also not significantly different in SPA4 peptide-treated mice. Histological examination revealed no sign of tissue injury or inflammation in any of the major organs. Together, these results provide an initial safety assessment of the SPA4 peptide.
NHR-49/PPARα regulates HLH-30/TFEB-mediated innate immune response via a flavincontaining monooxygenase in C. elegans
Authors: Wani Khursheed , Taubert Stefan , Irazoqui Javier
Published: January 2023

The nematode C. elegans mounts an innate immune response against Gram-positive bacteria S. aureus by the transcriptional induction of multiple host defense genes. The expression of a majority of these genes is under the control of HLH-30, C. elegans homolog of TFEB (transcription factor EB) that also functions in the innate immune response in mammals. In addition to its role in host defense against S. aureus, HLH-30 acts as a nutritionally-controlled stress response factor. We wanted to deconvolve the response of C. elegans to nutritional challenge from the antibacterial response against S. aureus. To this end, we performed RNAseq profiling in wildtype and hlh-30 mutants that were either infected with S. aureus or were starved. Our RNAseq analysis showed that multiple HLH-30-dependent or-independent genes are specifically induced by S. aureus infection, and not by starvation. One of the genes that is highly induced by infection and not starvation is fmo-2, which encodes a member of the flavin-containing monooxygenase (FMO) family in C. elegans. We decided to characterize the role of FMO-2 in host defense in C. elegans by employing genetic, transgenic, and molecular approaches. We found that FMO-2 is important for host defense against S. aureus infection, and its induction during S. aureus infection is controlled by two transcription factors, HLH-30 and NHR-49 (C. elegans ortholog of the mammalian nuclear-hormone receptor, PPARα). Our results propose a model in which NHR-49 functions upstream of HLH-30 to bind to fmo-2 promoter and to induce its upregulation by functioning specifically in C. elegans intestine (the site of innate immune response), thereby providing host defense against S. aureus infection.
Induction of COX-2 gene transcription and prostaglandin E2 (PGE2) production in primary human monocytes by MDP adjuvant: role of MAPK and p300 HAT
Authors: Liu Fengjie , Romantseva Tatiana , Golding Hana , Zaitseva Marina
Published: January 2023

Muramyl dipeptide (MDP) is the minimal structure responsible for adjuvant activity of peptidoglycans. Early clinical trials of vaccines formulated with MDP revealed adjuvant-induced pyrogenic response. Here we studied signaling pathway leading to production of mediator of fever, PGE2, in human monocytes activated with MDP.

MDP alone induced nuclear translocation of NF-kB but only minimal levels of COX-2 mRNA expression and PGE2 production in monocytes. T cell conditioned medium (TCM) prepared from CD3 bead-purified T cells dramatically upregulated COX-2 mRNA and PGE2 in MDP-activated monocytes but did not increase nuclear NF-kB. COX-2 mRNA expression and PGE2 production in monocytes activated with MDP and TCM were sensitive to the inhibitors of ERK1/2, p38, and JNK MAPK. Western blotting confirmed that MDP alone and TCM alone induced phosphorylation of p38 and SAPK/JNK and of ERK1/2, respectively. In addition to MAPK, the role of p300 histone acetyltransferase (HAT) was studied by ChIP assay. TCM induced binding of p300 to a COX-2 promoter. Inhibition of p300 blocked binding of p300 to COX-2 promoter, acetylation of H3 and H4 histones, upregulation of COX-2 mRNA and of PGE2 in monocytes activated with MDP/TCM. Importantly, inhibitors of either p38 or ERK1/2 prevented recruitment of p300 to COX-2 promoter. Taken together these results suggest cooperation between two signaling pathways in monocytes: nuclear translocation of NF-kB and activation of SAPK/JNK and p38 MAPK are induced by MDP, while activation of ERK1/2 and recruitment of p300 to COX-2 promoter are induced by TCM. These two pathways are equally important for p300-facilitated acetylation of H3 and H4 histones at the COX-2 promoter and for up-regulated COX-2/PGE2 production.
Lung-resident memory CD4 T cells do not divide extensively during protective responses against Influenza virus
Authors: McKinstry Karl Kai , Finn Caroline M
Published: January 2023

Tissue-resident memory (TRM) CD4 T cells are emerging as critical orchestrators of immunity but little is known about their response kinetics during secondary challenges. We recently developed an adoptive transfer model in which virus-specific CD4 TRM isolated from lungs of Influenza A virus (IAV)-primed mice are transferred intranasally to new congenic hosts. Here, we track these donor TRM cells after IAV challenge of the adoptive hosts to gain insight into unique aspects of CD4 TRM responses to infection. Our findings demonstrate that the TRM rapidly activate elements of innate defense in the lung, including dendritic cells and NK cells. The TRM response is limited to the lung as no donor cells are detected in the spleen or mediastinal draining lymph nodes through the first week of infection. Although the CD4 TRM are highly protective and can clear even lethal IAV challenge in unprimed hosts, they undergo only limited division. To ask if the TRM-mediated protection requires influx of systemic T cells to the lung, we treated adoptive hosts with FTY720 to block the migration of host T cells during infection. TRM-mediated protection was not impacted by FTY720 treatment, indicating that the local memory CD4 cells can protect independently of T cell responses initiated in secondary lymphoid organs. Finally, we could detect the donor TRM in lungs for at least 21 days after infection and they were rapidly activated by antigen given intranasally. Our results thus show that lung CD4 TRM initiate highly protective responses in the lungs of otherwise naïve mice and have the capacity to respond against multiple rounds of infection. As such their generation through vaccination is a powerful way to improve immunity against seasonal and pandemic IAV.
An Intranasally Administered Antibody Cocktail Prevents Ricin Toxin-Induced Lung Inflammation and Alveolar Macrophage Death
Authors: Mantis Nicholas J , Rong Yinghui , Torres-Velez Fernando J
Published: January 2023

Ricin toxin, a plant-derived, mannosylated glycoprotein, elicits an incapacitating and potentially lethal, pulmonary inflammatory response in the airways following inhalation. Uptake by alveolar macrophages (AM) and other pulmonary cell types occurs via ricin’s B subunit (RTB), a galactose-specific lectin, and the mannose receptor (MR; CD206). Ricin’s A subunit (RTA) is a ribosome-inactivating protein capable of inducing apoptosis in all mammalian cell types. It was recently reported that a single monoclonal antibody (MAb), PB10, directed against an immunodominant epitope on RTA, is able rescue Rhesus macaques against lethal dose of ricin administered by aerosol. In this study, we now demonstrate in mice that the effectiveness PB10 is significantly improved when combined with a second MAb, SylH3, against RTB. Mice treated with PB10 alone survived lethal-dose intranasal ricin challenge, but experienced significant weight loss, moderate pulmonary inflammation (e.g., elevated IL-1 and IL-6 levels, PMN influx), and apoptosis of alveolar and tissue macrophages. In contrast, mice treated with the PB10/SylH3 cocktail were essentially impervious to pulmonary ricin toxin exposure, as evidenced by no weight loss, no change in local IL-1 and IL-6 levels, retention of alveolar and tissue macrophage numbers, and a significant dampening of PMN recruitment to the bronchoalveolar lavage (BAL) fluids. The PB10/SylH3 cocktail only marginally reduced ricin binding to target cells in the BAL, suggesting that the antibody mixture neutralizes ricin by interfering with one or more steps in the RTB- and MR-dependent uptake pathways.
Inducible thymic epithelial cell-based rejuvenation in thymic aging-associated inflammaging (Supported by NIH/NIAID R01AI121147)
Authors: Oh Jiyoung , Thomas Rachel , Wang Weikan , Su Dongming
Published: January 2023

Age-related thymic involution primarily results from a decline in the transcription factor forkhead box N1 (FOXN1), while input of exogenous FOXN1 is able to significantly rescue thymic aging. Given that FOXN1 over-expressing fibroblasts (inducible thymic epithelial cells, iTECs) can generate an ectopic de novo thymus and engrafted young TECs can lead to middle-aged thymus regrowth, we asked whether engrafting iTECs directly into the aged thymus and perithymus can be a strategy to rescue aged thymic function. The re-establishment of central tolerance is crucial to attenuate age-associated chronic inflammation in the elderly (termed inflammaging). This will reduce increased self(auto)-reactivity, an important contributor to inflammaging. Intrathymic transplantation of iTECs into the aged murine thymus revealed that iTECs drove partial regrowth of the aged thymus, equally well in both male and female mice. The iTECs also rejuvenated aged thymic architecture, including regeneration of well-organized thymic medulla, and improvement of aged thymic function, including re-enforcement of thymocyte negative selection-related Aire gene expression. This led to reduction of self-reactivity-resulted inflammaging, including pro-inflammatory cytokines (IL-6 and IL-1b) and lymphoinfiltration to non-lymphoid organs, in old mice. Our findings support this potential novel strategy for enforcing the declined thymocyte negative selection to partially restore central tolerance in the elderly and reduce inflammaging.
T regulatory antigen-specific multi-peptide immunotherapy induce clinical responses in autoimmune diseases
Authors: Marquez-Manriquez Juan P , MATUTE-BRISENO JOSE ANTONIO , Alejandro Lucero-Diaz PEDRO ALEJANDRO , CAMACHO-HERNANDEZ ALEJANDRO
Published: January 2023

Background

The current treatment for several fatal autoimmune diseases is mainly palliative with minimal clinical benefit. By other hand the biological and clinically relevant potential targets to stop the auto immune response are mainly unknown, including the innate and adaptive auto-immune micro-environment. We performed a pilot trial with (n=5) progressive patients with myasthenia gravis (MG), multiple sclerosis (MS) and amyotrophic sclerosis lateral (ASL) using a multi-peptide T regulatory immunotherapy.

Methods

We performed a systematic review to found potential proteins involved in the auto-immune response in these three autoimmune diseases using the terms “misfolded proteins and autoimmune disease and prognosis and multivariate analysis”. initially N=25 repository samples from autoimmune disease patients were analyzed to test the predicted T regulatory peptides immunogenicity for ELISA, IL-10 ELISPOT and T –cell expansion.

Results

We found that predicted T regulatory peptides from Bcl-2, peptide C (p=0.001), survivi, peptide A (0.001) and VCP, peptide 8 = (P=0.005) were able to expand antigen-specific T regulatory cells and there was a clinical correlation in symptoms improvement in all the three diseases (mOS=28 months).

Discussion

The multi-peptide T regulatory peptide immunotherapy is feasible therapeutic approach to treat incurable autoimmune diseases and this data could be used as proof of principle to prepare a large clinical study in order to potentially improve clinical outcomes in combination with other approaches that may impact the clinical scenario in autoimmune diseases with limited or nor therapeutic options. No clinical toxicity was observed.
Lipid Nanoparticle-Mediated Delivery of Enhanced Costimulation Blockade to Prevent Type 1 Diabetes
Authors: ZHANG YICHUAN , Wang Jialu , Calderon-Colon Xiomara , Tiburzi Olivia N. , Lozano Marcos Iglesias , Patrone Julia B. , Raimondi Giorgio
Published: January 2023

Type 1 diabetes (T1D) remains an untreatable autoimmune disease caused by the destruction of pancreatic beta cells by autoreactive T cells. Costimulation blockade, blocking the CD28 pathway via CTLA4-Ig, is one promising immunotherapeutic, but recent observations suggest its efficacy is antagonized by inflammatory factors. As antigen presenting cells (APCs) malfunctions and aberrant accumulation of type 1 interferons are associated with T1D, we pose that inhibiting the signaling of inflammatory cytokines via Tofacitinib (Tofa), a JAK inhibitor, would enhance the efficacy of CTLA4-Ig in preventing T1D development. The objective of this study was to design a controlled and localized delivery of Tofa via biocompatible lipid nanoparticles, Nanostructured Lipid Carrier (NLC), and assess the immunomodulatory impact of this strategy.

We identified a specific composition of NLC that had negligible toxicity, could be readily taken up by multiple immune cells, and had a favorable Tofa encapsulation efficiency. Live animal imaging confirmed these particles have unique property of accumulating in lymphoid tissues, particularly in spleen, pancreatic and mesenteric lymph nodes when administrated via oral gavage. Ex-vivo, Tofa-NLC rapidly delivered Tofa to mouse APCs preventing their maturation and the release of inflammatory cytokines. Ongoing experiments show that short-term administration of Tofa-NLC via oral gavage at early (3-week-old) or late (10-week-old) stage in disease prone NOD mice promotes a significant reduction of T1D onset.

Overall, Tofa-NLC represent a promising strategy to complement CTLA4-Ig and we envision this combined strategy could enhance the efficacy of antigen specific immunotherapy.
Foxp3 cooperates with Ikaros to control the suppressive function of regulatory T cells
Authors: Thomas Rajan M , Wang Liqing , Chen Chunxia , Hancock Wayne W , Wells Andrew D
Published: January 2023

Ikaros (encoded by Ikzf1) is a lymphocyte-specific transcription factor that controls a wide spectrum of T cell functions, including T helper differentiation, cytokine production, and tolerance in response to antigenic stimuli. Using heterozygous and dominant-negative mouse models, we have shown previously that Ikaros controls IL-2 production by conventional T cells, T helper differentiation, CD8+ effector differentiation, and anergy induction. While the role of Ikaros in many T cell subsets has been established, its role in Treg-mediated peripheral tolerance has not been examined. To examine the Treg-specific role of Ikaros, we generated Ikzf1-fl/fl-Foxp3-YFP-Cre+ mouse with conditional deletion of Ikaros in Tregs. Ikzf1-fl/fl-Foxp3-YFP-Cre+ mouse exhibited enlarged secondary lymphoid tissues, with increased frequencies of effector memory Tconv and Treg. We show that Ikaros expression by Tregs is required for immune tolerance in an allogenic cardiac transplant model. While CD28- and CD40L-blockade induced long-term cardiac allograft tolerance in control recipients, Ikzf1-fl/fl-Foxp3-YFP-Cre+ recipients rejected the allografts in a short duration (p<0.01). We show that Ikaros forms a complex with Foxp3, which is required for Foxp3 to bind to and repress inflammatory cytokine genes. Ikaros is also required for normal iTreg development in vitro, as TGF-B/IL-2-induced iTreg development is impaired in conventional CD4+ T cells with reduced Ikaros activity. These studies suggest that Ikaros interacts with Foxp3 and directs Foxp3 to exert its suppressive function in Tregs.
Graft plasmacytoid dendritic cells improve thymic function in allogeneic hematopoietic cell transplantation
Authors: Zhu Jingru , Wang Yitong , Antonova Alina Ulezko , Li Yiwen , Giver Cynthia R , Hassan Mojibade , Waller Edmund K
Published: January 2023

Allogeneic hematopoietic stem cell transplantation(allo-HSCT) is a well established cure for a variety of hematologic diseases. However, graft-versus-host disease(GVHD) remain the primary obstacles to successful transplant. GVHD results from donor T-cell recognition of a genetically disparate recipient. Thymus has been recognized as a recipient tissue that can be severely damaged by GVHD. Plasmacytoid dendritic cells(pDCs) is a distinct subset of DC, which have both immunogenic and tolerogenic effect in allo-HSCT, licensed by the microenvironment to respond to the milieu in which they migrate. Impaired thymic DC has been the major causation of dysfunctional thymic negative selection, thus generating allo-reactive T cells and GVHD. To further demonstrate pDCs from the graft are migrating to thymus to protecting host from GVHD. We transplanted B10.BR mouse with pDCs from MHC mismatched GFP-labeled C57/B6 mouse, along with hematopoietic stem cells and T-cells from normal C57/B6 mouse. The results from the confocal imaging suggest that pDCs are migrating to thymus 14 days after allo-HSCT. CCR9 expression is important for pDCs migrating to thymus. We transplated mice with either wild type pDCs or CCR9 knock out pDCs. Survival rate is significantly lower in group transplanted with CCR9-knockout pDCs suggesting thymus is a critical place for pDCs to induce tolerogenic response. We also tranplanted wild tipe pDCs with thymectomized host and normal host. Thymectomy group have better survival rate compared to non-thymectomized group, further confirming the association between pDCs and thymus. Improved thymic function post transplant with pDCs from the graft may have a potential to decrease GVHD.
Carbomer-based adjuvant potentiates CTL immunity by enhancing cross-presentation of antigens by NOX2 and TAP-dependent mechanisms
Authors: Lee Woojong , Bakke Brock , Sonsalla Shelly , Sanchez Darren , Reyes Leticia , Iyer Gopal , Suresh M.
Published: January 2023

We have previously showed that a carbomer-based adjuvant (CBA), Adjuplex® (ADJ) enhanced systemic and mucosal CD8 T cell responses to non-replicating antigens. Here, we investigated the molecular mechanism(s) of how the CBA enhance cross-presentation of antigens by dendritic cells (DCs). We found that ADJ enhanced cross-presentation of antigens in DCs by several mechanisms, including augmenting antigen degradation, reducing endosomal acidity, promoting antigen localization to early endosomes and increasing ROS production leading to escape of antigen from endosomes to the cytosol. Additionally, we find that ADJ-induced cross-presentation of antigens by DCs required NOX2 and TAP1. Taken together, we propose that ADJ enhances cross-presentation of antigens by promoting NOX2-dependent ROS production leading to endosomal antigen leakage, proteolysis of antigen by proteasomes and TAP-dependent peptide transport into MHC-containing cellular compartment. Thus, our studies have revealed a novel mechanism for ADJ-induced cross-presentation of antigen, highlighting the significance of incorporating CBA as a vaccine adjuvant to elicit strong cytotoxic T cell responses.
Determinants of TCRα-β chain pairing in generating TCR diversity
Authors: Tanno Hidetaka , Gould Timothy M , Durrett Russell E , McDaniel Jonathan R , Cao Wenqiang , Gollihar Jimmy , Steven Cate , Tanno Yuri , Ellington Andrew D , Ippolito Gregory C , Hildebrand William , Georgiou George , Goronzy Jorg J
Published: January 2023

Advances in next-generation sequencing have allowed insights into the enormous diversity of the human T cell receptor (TCR). Earlier studies have suggested that ~108 unique TRB genes exist at the periphery. In contrast, the factors that determine the pairing of TCRα and TCRβ chains have remained elusive due to the lack of high-throughput single-cell sequencing technology. We have developed a high-throughput single-cell sequencing technology that enables identification of >3×104 paired TRA:TRB sequences per sample and have conducted sequencing of naïve and memory CD4+ and of total T cells from 15 healthy individuals, including six pairs of monozygotic twins. We found that the frequencies of TRAV:TRBV pairing are essentially determined by the frequencies of the individual gene segments, suggesting the absence of structural constraints in the pairing of the α and β chains. Twins are more similar in the frequencies of TRAV and TRBV combinations than are unrelated individuals, but this difference was explained by a genetic influence on AV and BV gene segment frequencies. Twins and unrelated individuals were compared for the sharing of identical TCR sequences. TRA sequence sharing among twins was comparable to that among unrelated individuals, but TRB sharing was significantly higher in twins. Sharing of the entire TCR was regularly found in twins but not detected for naïve CD4+ T cells from unrelated individuals. Results were similar for naïve and memory T cells, with the genetic influence more prominent in memory T cells, suggesting the additional influence of peripheral selection. In support of this interpretation, the sharing of identical TCR sequences in unrelated individuals correlated with the number of shared MHC class II alleles.
HIV-1-specific B cell immune responses in Rhesus Macaques induced by early region deleted-Ad5 vectors
Authors: sangare Kotou , Hogge Christopher , Hait Sabrina Helmold , Hoang Tanya , Robert-Guroff Marjorie , Thomas Michael A
Published: January 2023

As a vaccine delivery vector, Adenovirus (Ad) is recognized for its ability to prime immune responses against HIV and other infectious agents. We reported that early region 1B55k (E1B55k) deleted vector induced cellular immunity, transgene-specific antibodies and innate immune signals including chemokines, cytokines and NKG2D ligands MIC A/B in immunized mice. However, the immune priming ability of E3-E1B55k-E4orf1 and E3-E1B55k- E4orf1-4deleted Ad vectors is unknown. Here we compared HIV-specific immune responses generated by E3deleted, E3-E1B55k, E3-E1B55k-E4orf1and E3-E1B55k-E4orf1-4deleted Ad5 vectors encoding full-length single-chain HIVBalgp120 linked to the D1 and D2 domains of rhesus macaque (RM) CD4 (rhFLSC). RMs were immunized twice mucosally with each vector. Blood and rectal tissue were collected at pre and 2-week post-immunization time points. The frequency of plasmablasts (PB)/plasma cells (PC), memory B cells, and antibody secreting cells and antibody titers were assessed by flow cytometry, ELISpot and ELISA, respectively. All vectors induced increased frequencies of rectal PB, PC and rhFLSC specific memory B cells. rhFLSC specific IgA and IgG secreting cells were detected in all immunized macaques; the IgA secreting cells were significantly increased compared to pre values. This was accompanied by elevated levels of IgG, IgG1 and IgA serum antibodies. In summary all four vectors induced B cells responses while retaining replicability. Moreover, the largest deletion, E3-E1B55k-E4orf1-4, improved the transgene carrying capacity of the Ad vector while maintaining the ability to induce B cell immunity important for protective efficacy.
Rainbow trout as a model for the study of neuroimmune interactions in the nasal mucosa
Authors: Salinas Irene , Sepahi Ali , Kraus Aurora , Das Pankoj
Published: January 2023

Many pathogens exploit the olfactory route to infect the lower respiratory tract or the central nervous system. Understanding the mechanisms by which the nasal mucosa halts pathogen progression is vital for the control of many human diseases. We have recently developed a model for the study of neuroimmune interactions in the olfactory organ of rainbow trout based on our initial discovery of a nasopharynx-associated lymphoid tissue in bony fish. Our studies indicate that viruses activate neurons in the olfactory epithelium and olfactory bulb of rainbow trout and that neuronal-viral interactions are required for the onset of ultra-rapid innate immune responses. These responses, that occur within minutes, have been overlooked thus far, and are characterized by the influx of non-conventional CD8 T cells that express TCRa/b. Further studies indicate that these response occur in response to a wide-range of microbial stimuli, albeit at lesser magnitude than in the case of live viruses. Current research efforts in our laboratory involve the validation of this model using human-relevant pathogens and vaccines such as the Flu vaccine. These results underscore the importance of understanding neuronal immune responses to pathogens and vaccines in order to develop better immunotherapies that exploit the nasal route.
Segregation of chicken MHC-Y haplotypes in high and low antibody selected lines provides evidence that MHC-Y contributes to the genetics of immune responses
Authors: Zhang Jibin , Goto Ronald M , Honaker Christa F , Siegel Paul B , Miller Marcia M
Published: January 2023

MHC-Y is a second genomic region in chickens containing genes encoding highly polymorphic MHC class I molecules. MHC-Y class I molecules have a distinctive surface distribution of polymorphic residues. The function of MHC-Y class I molecules is unknown. To begin to determine whether genetic differences at MHC-Y influence immune responses, we have defined the MHC-Y haplotypes segregating in the Virginia Tech high antibody (HAS) and low antibody (LAS) lines. HAS and LAS have been selected for 45 generations for high and low antibody responses to sheep red blood cells, respectively. The HAS and LAS lines are known from previous studies to differ at a number of loci that influence immune responses, including the classical MHC-B region. We MHC-Y typed the HAS and LAS lines at the 44th and 45th generation using a microsatellite sequence found immediately upstream of the MHC-Y class I gene start sites. Typing revealed the presence of five MHC-Y haplotypes overall. Two haplotypes were found only in the HAS line. The frequency of one haplotype was 60%. In LAS, three other haplotypes were common. The separation of different MHC-Y haplotypes in the HAS and LAS lines suggests that MHC-Y locus is another polymorphic genomic region changing in response to selection for high and low antibody phenotypes. This finding is among the first linking MHC-Y genetics to immune responses in chickens.

Supported in part by USDA NIFA Grant 2016-10247.
Sex-specific DLA Class II haplotypes are associated with primary hypoadrenocorticism in Standard Poodle dogs
Authors: Treeful Amy , Rendahl Aaron , Friedenberg Steven
Published: January 2023

Autoimmune Addison’s Disease (AAD) is a life-threatening endocrine disorder that occurs spontaneously in humans and dogs and whose clinical features are highly similar between the species. Associations between MHC class II genes and AAD have been shown in several human studies. Our goal was to identify MHC class II associations with AAD in a large population of Standard Poodles, a breed highly predisposed to AAD. We sequenced exon 2 of the class II genes DRB1, DQA1, and DQB1 in 110 affected and 101 unaffected Standard Poodles and tested for association with AAD. Two haplotypes were found to confer risk of developing AAD in a gender-specific manner: 015:01-006:01-023:01 in males (□2 p = 0.03, OR 2.1) and 009:01-001:01-008:011 in females (□2 p = 0.02, OR 8.43). Sex-specific associations have been previously described in human populations, but this is the first report of this kind in dogs. Consistent with findings in other studies, we also found the DQA1*006:01 allele (□2 p = 0.04) to be associated with AAD in males independent of haplotype. In females, the haplotype 009:01-001:01-008:011 appears to confer a very high risk for developing AAD, though its frequency was rare (9 of 124 females) in our study population. Further studies are warranted to assess the usefulness of this haplotype as a genetic risk marker for AAD in female Standard Poodles. Our findings highlight the importance of evaluating MHC class II-disease associations in large populations, and accounting for both gender and population structure in order to uncover clinically relevant associations.
Autoreactive potential of universal influenza vaccines
Authors: McGargill Maureen Ann , Pillai Meenu R , Chang Ti-Cheng , Crawford Jeremy , Keating Rachael , Labombarde Jocelyn , Lewis Carlessia , Guthmiller Jenna , Li Quan_Zhen , Wilson Patrick C , Thomas Paul
Published: January 2023

A universal influenza vaccine could save millions of lives in the event of a deadly pandemic. It is not clear why antibodies specific for conserved regions of influenza viruses are so rare. One possibility is that these antibodies have a higher potential to cross-react to self-proteins, and therefore B cells that generate these antibodies are deleted through tolerance mechanisms. Therefore, we examined whether cross-reactive influenza antibodies had a higher potential to be autoreactive than antibodies specific for one subtype of influenza. We previously demonstrated that H3N2-vaccinated mice treated with a low dose of rapamycin had more cross-reactive influenza antibodies and were better protected against subsequent lethal infections of multiple subtypes. Thus, we utilized rapamycin to increase the frequency of influenza cross-reactive antibodies, and tested whether these antibodies were more reactive to self-proteins than strain-specific antibodies. We found that mice with increased levels of cross-reactive influenza antibodies also had more IgM antibodies specific for self-antigens. Although the increase in autoreactive IgM antibodies was transient, it correlated with increased susceptibility to disease in mouse models of multiple sclerosis and Guillian-Barre Syndrome. Together, our results suggest that influenza cross-reactive antibodies have the potential to be autoreactive. These data have important implications for developing universal influenza vaccines designed to generate durable influenza cross-reactive antibodies.