bluuebunny/crossref_metadata_2025 huggingface dataset:
Neonatal dendritic cells alter the immunodominance hierarchy of the CD8 T cell response during influenza infection
Authors: Heil Luke , Oliphant Samantha , Lines Louise , Hollifield Melissa , Garvy Beth A
Published: January 2023

Neonates are more susceptible to influenza virus infection than adults, resulting in increased morbidity and mortality as well as delayed clearance of the virus. Multiple differences between the adult and neonatal immune response to influenza help explain this vulnerability. Dendritic cells are of particular interest in this process as their decreased function in neonates results in the poor T cell activation observed during neonatal influenza infections. We sought to understand how differences in neonatal dendritic cells shape CD8 T cell specificity and immunodominance during influenza infection as well as how this may affect memory formation and viral clearance. We found that neonatal C57BL/6 mice display an altered CD8 T cell immunodominance hierarchy, preferentially responding to the influenza protein PA rather than the dominant adult epitope in the NP protein. Additionally, upon secondary infection, mice first infected as pups suffered increased morbidity compared to mice infected previously as adults. Finally, transfer of influenza infected adult dendritic cells to pups resulted in increased T cell activation and enhanced viral clearance. Taken together, these data suggest that neonatal dendritic cells alter CD8 immunodominance, and this may compromise viral clearance and memory formation.
Investigation of CD8+ T Cell Immunity in Zika Virus Infection Using Mouse Models
Authors: Ngono Annie Elong , Vizcarra Edward A , Tang William W , Sheets Nicholas , Joo Yunichel , Kim Kenneth , Gorman Matthew J , Diamond Michael S , Shresta Sujan
Published: January 2023

CD8+ T cells may play a dual role in both protection against and pathogenesis of Flaviviruses, including Zika virus (ZIKV). However, virtually nothing is currently known about the role CD8+ T cells play during ZIKV infection in humans and animal models. The goal of our study is to investigate the role of CD8+ T cells during primary ZIKV infection in mice. Using LysMCre+IFNARfl/fl C57BL/6 (H-2b) mice that lack type I IFN receptor in a subset of myeloid cells, we characterized a new mouse model susceptible to infection with both East African lineage (MR766) and Asian lineage (FSS13025) ZIKV strains. A screening of 244 ZIKV epitopes predicted to bind H-2b class I molecules, defined a specific map of 26 and 15 peptides for MR766 and FSS13025 ZIKV strains, respectively. Intracellular cytokine staining confirmed the identity of these epitopes and demonstrated induction of polyfunctional ZIKV-specific CD8+ T cells. Furthermore, adoptive transfer of ZIKV-immune CD8+ T cells reduced viral burdens, whereas depletion of CD8+ T cells led to higher tissue burdens and mortality was increased in ZIKV-infected CD8−/− mice compared to Wild-type. These results demonstrate that CD8+ T cells are important mediators of protection during primary ZIKV infection and provide a H-2b mouse model for investigating ZIKV-specific CD8+ T cell responses. Based on these findings, studies are in progress towards defining the role of resident memory CD8+ T cells in protection against ZIKV infection using our recently developed mouse models of ZIKV systemic infection and sexual transmission.
Clinical and Immunologic Biomarkers for Regression of High Grade Cervical Dysplasia and Clearance of HPV16 and HPV18 Infection after Immunotherapy with VGX-3100 in a Phase IIb Clinical Trial
Authors: Morrow Matthew P , Kraynyak Kimberly A , Sylvester Albert J , Dallas Michael , Knoblock Dawson , Boyer Jean D , Yan Jian , Vang Russell , Khan Amir , Humeau Laurent , Sardesai Niranjan , Kim J Joseph , Plotkin Stanley , Weiner David , Trimble Cornelia Liu , Bagarazzi Mark
Published: January 2023

We have previously demonstrated proof-of-principle of VGX-3100, an immunotherapy which targets HPV16/18 E6 and E7, in a Phase IIb study in women with premalignant cervical lesions caused by HPV16 or HPV18. After completion of the treatment regimen, cervical lesions underwent complete regression (CR) concomitant with elimination of underlying HPV16/18 infection in 40.0% of VGX-3100-treated patients and in only 14.3% of patients receiving placebo (p=0.002). Here we detail characteristics present that either correlated with or predicted therapeutic benefit with VGX-3100. Combining HPV typing and Pap smear data collected following the third dose (Week 14) was predictive for both CR and the elimination of HPV16/18 at the efficacy assessment twenty two weeks later (predictive value 94%, sensitivity 96%). Patients who had a significant elevation of peripheral blood CD137+ Perforin+ CD8+ T cells specific for the HPV type causing disease (p=0.001) were more likely to have lesion regression and HPV elimination than women without a detectable peripheral blood response. In comparisons of the intensity of cervical mucosal CD103+ and Perforin+ cell infiltrates pre- and post-vaccination, significant increases were observed in patients displaying exhibiting CR with elimination of HPV infection (p=0.027 and p=0.043, respectively). In sum, we found that quantitative measures associated with an effector response to VGX-3100 antigens were associated with subsequent lesion regression. These analyses indicate that certain immunologic responses are associated with successful resolution of HPV 16/18-induced pre-malignancy, with particular emphasis on the upregulation of Perforin in the immunotherapy induced immune response.
Controlled release of mTOR inhibitors to enhance CD8+ T cell memory
Authors: Zeng Qin , Jewell Christopher M
Published: January 2023

Recent studies demonstrate, they frequent, low-dose administration of rapamycin (Rapa) during vaccination can help maintain the plasticity of antigen-specific CD8+ T cells, polarizing the phenotypes of these cells toward central memory T cells (TCM). However, these effects required multiple, systemic doses of Rapa. Here we hypothesized that local, intra-LN controlled release of Rapa from these depots during vaccination would polarize T cells toward highly proliferative TCM during T cell expansion. to enhance T cell immunity. To test this idea, vaccine were formulated by mixing free ovalbumin (OVA) and PLGA microparticles loaded with a TLR9 agonist (CpG). Mice were first immunized with the vaccine, and then received daily i.p injection of Rapa at low doses (75 μg/kg) for 28 days. This regimen confirmed i.p injection of Rapa increased the percentage of OVA-specific CD8+ T cells in PBMCs by 2.4 times on day 14 and specific TCM levels by 4.3 times on day 49 compared to vaccine without Rapa. We next assess the effects of controlled release by using the vaccine above but treating mice with a single dose of controlled release depots loaded with defined doses of Rapa. In mice, this approach showed functional as similar as daily i.p injection of Rapa. While high dose of Rapa (20.9 μg to 36.4 μg per dose) suppressed the immunity, Rapa at a low dose (0.07 μg to 4.17 μg per dose) in MP significantly promote the CD8+ T cell immunity over 7 weeks, where specific CD8+ T cells was promoted by 2.9 times on day 14 and specific TCM on day 49 by 2.4 times of vaccine without Rapa. These result suggested that sustained release of immunomodulator in biomaterials depots directly in the LN microenvironment can significantly enhance the antigen specific CD8+ T cells immunity.
The QuickSwitch Quant HLA-A*02:01 Tetramer Kit can be used for determining the biological activity of a cancer vaccine.
Authors: Delcommenne Marc C , Hrytsenko Olga , Tram Cynthia , Weir Genevieve , Stanford Marianne M.
Published: January 2023

We have devised a fast and user-friendly assay (QuickSwitch™ Quant) that can both help determine binding of novel peptides to MHC class I molecules and generate new specificity MHC class I tetramers for peptide specific T cell detection. This study aimed to determine whether the QuickSwitch™ Quant HLA-A*02:01 Tetramer Kit can also be used for evaluating the biological activity of a vaccine. The tested vaccine was DPX-Survivac, an ovarian cancer vaccine candidate which consists of several survivin peptide antigens that are each restricted to a different human class I allele. We sought to evaluate the specificity and sensitivity of the QuickSwitch™ Quant HLA-A* 02:01 Tetramer Kit-PE for assessing the biological activity of SurA2.M, an HLA-A2-restricted peptide, in DPX-Survivac. The complete vaccine also contains non-HLA-A2 restricted peptides, lipids and a polynucleotide adjuvant. We tested the detection of the peptide prepared individually in a buffered solution or in the DPX-Survivac vaccine prepared in an aqueous formulation. Results indicate that peptide exchange rate of SurA2.M is similar whether it is dissolved individually in a buffered solution or mixed with other components of the vaccine. Results also may be dependent on the affinity of the peptide for HLA-A2. Thus, by optimizing a concentration curve using individual peptides, the QuickSwitch™ Quant HLA-A*02:01 Tetramer Kit can be used to quantify the concentration of HLA-A2 restricted peptides in simple solutions or more complex formulations.
Genetic linkage analysis of cancer vaccine response in Diversity Outbred mice
Authors: Gibson Heather , Reyes Joyce , Jones Richard , Dyson Greg , Wei Wei-Zen
Published: January 2023

Recent advances in cancer immunotherapy demonstrate the power of anti-tumor immunity, however only a fraction of patients maintain durable responses. To test the hypothesis that genetic variation determines cancer immunotherapy response, we conducted genetic linkage analysis of HER2 vaccine response in Diversity Outbred (DO) mice. Our HER2 DNA vaccine has shown varying anti-tumor activity in mice, cats and human patients. Differential vaccine response was found in HER2 transgenic (Tg) mice of BALB/c versus C57BL/6 backgrounds. DO mice, produced by non-sibling crossing of 8 mouse stains (A/J, C57BL/6, 129S1/Svlm, NOD, NZO, CAST/EiJ, PWK/PhJ and WSB/EiJ) incorporate >90% of all genetic variation in mice. The fully sequenced founder strain genomes enable genetic linkage analysis using Mouse Universal Genotyping Array (GigaMUGA), which consists of >150,000 genetic markers, mostly single nucleotide variants. DO mice were bred with C57BL/6 HER2 Tg mice, generating HER2 DO mice that express human HER2 as a self-antigen. A wide range of IgG (1.8 – 50 μg/mL) and IFNg producing T cells (0 – 348/106 splenocytes) were detected after 2× vaccination with an adenoviral vaccine encoding human HER2. Genetic loci associated with positive response (p<10−5) were sought by linear regression analysis. Immune-related genes within identified loci were examined. Preliminary analysis links IgG response to NFkB, IL6, and IFNg-related gene clusters, and IFNg producing T cell response to NFkB, IL6, IL10, Akt and p38 MAPK-related gene clusters. Further analysis will define and rank genetic variants of candidate immune regulators to help identify vaccine responsive individuals.
Foxp3+ regulatory T cell expression of IL-10 is required for IL-33-mediated expansion of regulatory B cells.
Authors: Roessing Anna , Cherukuri Aravind , Rothstein David , Turnquist Heth Roderick
Published: January 2023

BACKGROUND

FoxP3+ regulatory T cells (Tregs) are crucial to self- and antigen-specific tolerance. IL-33 drives expansion of Tregs expressing the IL-33 receptor, ST2, and secreting high levels of IL-10. Regulatory B cells (Bregs) are recently identified negative regulators of the immune system and depend on IL-10 to suppress effector T cell responses. In new data, we find that IL-33 also expands Bregs, however, the relationship between ST2+ Treg and Bregs cells remains unknown. Herein we elucidated if a directional relationship exists between IL-33-expanded Bregs and Tregs.

METHODS

B6 eGFP-FoxP3-Diptheria Toxin (DT) receptor mice were used to establish whether IL-33-expanded Treg were needed for IL-33-aided increases of TIM-1+ IL-10+ Bregs. B6 Foxp3-Cre X IL-10fl/fl mice were used to determine the need for IL-10 expression by Tregs for IL-33-mediated expansion of Bregs.

RESULTS

Assessment of splenocytes for Tregs and Bregs by flow cytometry showed that IL-33 administration led to a 3–5-fold increase in ST2+ Tregs. IL-33 also led to a 3-fold increase in the frequency of IL-10+ Breg, most of which expressed ST2. A lack of Tregs hindered the ability of IL-33 to increase Bregs. While Treg expression IL-10 was not required for ST2+ Treg expansion by IL-33, IL-10 expression by Tregs is crucial for Breg expansion by IL-33.

CONCLUSIONS

We have uncovered a relationship between Tregs and Bregs in response to IL-33. Specifically, while we show that Bregs express ST2, our data suggest that IL-33 expansion of Bregs is indirectly mediated by Treg IL-10 production.
A new model to investigate cell death-independent mechanisms of direct Treg suppression of B cells
Authors: Golding Amit , Weingartner Elizabeth
Published: January 2023

Tregs are well-known for their role in maintaining peripheral self-tolerance and immune homeostasis. One such role is regulation of the humoral response, which is most clearly demonstrated by abundant auto-antibody production in Scurfin mice and IPEX patients, both of which lack functional Tregs. Indirect Treg regulation of the humoral response via their influence on helper T cells, in particular in germinal centers, is well established. Prior demonstration of direct Treg inhibition of B cells has also been demonstrated, but is mostly attributable to Treg killing of B cells. We have re-addressed the role of Tregs in directly suppressing Ig secretion specifically, and we have begun to elucidate the mechanism behind this suppression. Because it is likely that regulation is dependent on the developmental stage of the B cell, we used a homogeneous, immature, antibody secreting cell line, Ramos B cells. We demonstrate that human Tregs can directly suppress basal and stimulated IgM secretion by Ramos through a death- and contact-independent mechanism. This suppression is enacted by a soluble, anti-inflammatory factor and does not appear to alter the rate of IgM transcription but instead specifically affects the amount of secreted IgM mRNA by altering RNA processing. The finding that Tregs suppress IgM secretion by immature B cells via a soluble factor is novel. We plan to further characterize this suppression and determine if it is altered in disease states that present with high immunoglobulin titers.
Restoration of Immune Tolerance in Type 1 Diabetes by Modulating Interleukin-2 Receptor Signaling
Authors: Yuan Xiaomei , Dong Yi , Tso J. Yun , Fu Wenxian
Published: January 2023

Type 1 diabetes (T1D) is an organ-specific autoimmune disease, characterized by the destruction of insulin-producing beta cells in pancreatic islets. A variety of lymphocytes, including T cells and natural killer (NK) cells infiltrate into the islets. Their roles in T1D are rather different--effector T cells and NK cells accelerate, while regulatory T cells, dampen the development of T1D. Signaling through interleukin-2 (IL-2) receptor beta (IL-2Rβ, also known as CD122) is critical for the differentiation and function of both T cells and NK cells. However, it remains poorly understood how these cells respond to IL-2Rβ signaling during the development of T1D. Here we show that in non-obese diabetic (NOD) mice, the most commonly used animal model for human T1D, administration of a non-immunogenic monoclonal antibody (mAb) selectively ablates pathogenic cells, including memory CD4+ T cells, CD8+ T cells and NK cells. In contrast, CD4+ Foxp3+ regulatory T (Treg) cells are only mildly affected. This leads to significantly elevated ratios between Treg cells and pathogenic cells. This reset balance preferentially occurs in pancreatic islets, without systemic immunosuppression. Importantly, IL-2Rβ blockade suppresses both insulitis and diabetes in NOD mice. Moreover, IL-2Rβ blockade significantly suppresses interferon gamma (IFN-γ) production in T cells and NK cells, but promotes interleukin-17 (IL-17) production in CD4+ T cells. Therefore, the modulation of IL-2Rβ signaling represents a novel and efficient approach to restore immune tolerance in T1D.
Modulation of T cell function by CD271
Authors: Lebert-Ghali Charles-Etienne , Bonkoungou Carole , Fournier Marilaine , Sood Aditi , Melichar Heather J
Published: January 2023

Co-signaling molecules are essential modulators of the immune system. These molecules belong to the immunoglobulin and tumor necrosis factor receptor superfamilies (IgSF and TNFRSF, respectively). Despite intensive study of these protein families, the functions and binding partners for many IgSF and TNFRSF members remain largely unknown. In particular, CD271 (NGFR and TNFRSF16) is expressed on neural crest-derived cells, several immunomodulatory cell types, as well as on diverse cancers cells, and our data attribute new immunoregulatory properties to CD271 that may play a role in both cancer evasion and autoimmune disease. Indeed, in vitro stimulation of mouse and human T cells in the presence of recombinant CD271-Fc protein inhibits T cell activation, proliferation and cytokine production. Additionally, we co-cultured antigen-specific mouse T cells with artificial antigen presenting cells overexpressing CD271 and observed a partial inhibition of T cell proliferation in the presence of CD271. Further, although the proportion and activation state of immune cells are similar in adult CD271 knockout (KO) and control mice at steady-state, our preliminary data suggest that CD271 KO mice have a more severe phenotype in autoimmune models as compared to control animals. Together, these results demonstrate that CD271 modulates T cell function and may inform therapeutic approaches in autoimmunity and cancer.
Human gingiva-derived mesenchymal stem cells inhibit xeno-graft-versus-host disease through CD39 and IDO signals
Authors: Wang Julie , Huang Feng , Olsen Nancy J , Zheng Song Guo
Published: January 2023

Mesenchymal stem cells have the capacity to maintain immune homeostasis and prevent autoimmunity. We recently reported that human-derived gingival mesenchymal stem cells (GMSCs) have strong capacity to suppress immune responses and T cell-mediated collagen-induced arthritis in animals. However, it is unclear whether these cells can suppress human T cell-mediated diseases. Here, we used a xeno-GVHD model in the NOD/SCID mouse, which is a useful preclinical construct for evaluating the therapeutic and translational potential of this approach for applications in human disease. We found that GMSCs potently suppressed the proliferation of PBMC and T cells in vitro. Co-transfer of GMSC with human PBMC significantly suppressed human cell engraftment and markedly prolonged the mouse survival. Moreover, we demonstrated that GMSCs inhibited human PBMC-initiated xenogenic responses via CD39 and IDO signals. These findings suggest the potential for GMSCs to suppress human immune responses in immune system-mediated diseases, offering a potential clinical option to be used for modulating GVHD and autoimmune diseases.
Exosomes from B lymphocytes are a major source of Fas ligand
Authors: Lundy Steven K , Taitano Sophina H , van der Vlugt Lucien EPM
Published: January 2023

Exosomes are nanovesicles released by a variety of cell types, including antigen-presenting cells. The ability of exosomes to induce immunity has prompted great interest in their clinical use. Murine studies have also indicated that circulating FasL+MHCII+ exosomes can be tolerogenic in allergic and delayed-type hypersensitivity responses. B cells are capable of expressing FasL and are a major in vivo source of exosomes, but their role in producing tolerogenic exosomes is incompletely explored. In the current study, we evaluated B cells as a source of FasL+ exosomes in vivo and signals that induced B cells to release FasL+ exosomes in vitro.

In B-cell deficient mice, a major loss in total splenic exosomes and exosome-associated FasL was observed, suggesting B cells are the major source of FasL+ exosomes in vivo. After sorting B cells into CD107a+, CD5+ or CD9+ cells, all subsets showed some intracellular storage of FasL, yet FasL was predominately found in the CD9+ subset. Searching for signals that induced FasL+ exosomes in vitro, we stimulated B cell signaling through Toll-like receptors (TLRs), the B-cell receptor (BCR), and/or CD40. In response to BCR ligation, B cells produced 20% more FasL compared to medium controls, which increased to 50% in the presence of any TLR ligand. Interestingly, B cells increased their intracellular FasL storage, but failed to increase their FasL+ exosome release when stimulated through CD40 alone. TLR-4, TLR-7(/8) or TLR-9 ligation with CD40 activation induced the FasL+ exosome release, which was further enhanced by BCR ligation. These data suggest that signals known to induce the differentiation of IL-10-producing B cells, also enhance FasL+ exosome production as another potential suppressive mechanism.
Fast and easy immunomagnetic positive selection of PE- or biotin-conjugated antibody-labelled cells with Releasable Rapidspheres results in highly purified, functional and particle-free cells
Authors: Ewen Catherine , Kokaji Andy I , Clarke Samuel J , Lambertz Ulrike , De Jong Susan , Woodside Steven M
Published: January 2023

Immunomagnetic cell isolation is a cost-effective and gentle method of obtaining purified cell populations. However, isolating novel or rare cell populations often relies on relatively time consuming and expensive cell sorting techniques due to the lack of off-the-shelf immunomagnetic cell isolation kits. To bridge this gap, we have developed the EasySep™ Release isolation kits for PE- or biotin-conjugated antibodies. EasySep™ Release uses magnetic particles with low non-specific binding characteristics that can be rapidly removed from isolated cells after positive selection.

The system is highly customizable, targeting cells through the use of any biotin or PE-conjugated primary antibody or ligand. Starting with a variety of sample materials, including single cell suspensions from mouse spleen, lymph nodes, lungs, rat spleen, or human PBMCs, the EasySep™ Release kits have demonstrated purities consistently above 90%.

The EasySep™ Release technology can also be used to isolate subsets of cells when used in combination with our other cell separation products. For example, functional and particle-free mouse CD25+ or CD304+ CD4+ regulatory T cells were isolated by using our EasySep™ PE Positive Releasable Rapidspheres™ kit and our EasySep™ Mouse CD4+ T Cell Isolation kit. We have used EasySep™ Release with EasySep™ Dextran Rapidspheres™ to isolate CD4/CD8 double positive mouse thymocytes (double positive selection), and to isolate mouse lung epithelial cells by EpCAM positive selection followed by the removal of contaminating leukocytes via CD45 depletion.

These kits offer a customizable, and cost-effective magnetic isolation approach for the isolation of virtually any cell population.
A novel real-time imaging technique to quantify mechanisms of cell death in neutrophils
Authors: Chan Diana Wen , Zaal Kristien J , Ralston Evelyn , Gupta Sarthak , Kaplan Mariana J
Published: January 2023

Neutrophils have distinct mechanisms of cell death, including the formation of neutrophil extracellular traps (NETs- a process called NETosis). NETosis is characterized by reactive oxygen species generation, chromatin decondensation, membrane rupture and extrusion of a meshwork of chromatin bound to granule peptides. Objective, efficient techniques to quantify NETosis are required. We developed a new method to automatically quantify the percentage of neutrophils undergoing NETosis using the IncuCyte ZOOM™ (Essen BioScience, Inc.) platform to image neutrophils at 20× in a 96-well plate and a dual-dye system dependent on membrane integrity to stain DNA, collecting both phase and fluorescence images. DNA in healthy control neutrophils were stained with a cell membrane permeable NUCLEAR-ID Red DNA dye. Neutrophils were then incubated with Sytox, a membrane-impermeable DNA dye, used to assess cell death. Conditions were run in triplicate and 3 images per well were captured at 10-minute intervals. A processing definition was set and optimized to count all neutrophils (NUCLEAR-ID stained) and neutrophils undergoing cell death (Sytox stained) using fluorescence intensity and stained area size to identify cells undergoing NETosis. Changes in nuclear morphology, quantified by the processing definition, distinguished PMA, Ionomycin and SLE sera induced NETosis from staurosporine induced apoptosis. Findings were confirmed and correlated with the gold-standard of immunofluorescence microscopy. This novel real time assay distinguishes types of neutrophil cell death and quantifies NETosis in a rapid, accurate and reproducible way, significantly improving the study of neutrophils.
Cyanine TruStain™, an effective blocking buffer to eliminate non-specific cyanine-like dye-mediated monocyte binding
Authors: Ransom John T , Elia Jeanne , Godfrey William , Wang Jing , Jiang Nan , Yang Xifeng , Monell Craig
Published: January 2023

Cyanine dyes and their analogs are commonly used as acceptors in commercially available antibody-tandem fluorophore conjugates. However, it has been confirmed that the dyes have a tendency to bind monocytes or macrophages nonspecifically, which limits the ability to do multi-color flow cytometric analysis for lower density antigens. We are introducing Cyanine TruStain™ Buffer which eliminates this nonspecific binding. This reagent blocks nonspecific binding of monocytes and macrophages to Cyanine dyes, (PE/Cy7, PE/Cy5, PercpCy5.5, APC/Cy7, APC/Fire™ 750, PE/Dazzle™ 594). It does not affect binding of, e.g., CD64, CD14 and other tested antibodies. Multiple Cyanine dyes can be blocked at once and stability data shows no decrease in signal intensity compared to the conjugate alone. In addition the reagent has no impact on cell viability.

Mock sorting experiments were performed with PBMC to determine if the Cyanine TruStain™ Buffer stimulates cells to produce cytokines and chemokines. Our data indicates minimal to no stimulation above conjugated antibody alone for IL-6, IL-8, IL-10 and MCP-1 production. We also tested Cyanine TruStain™ Buffer for its ability to negatively affect PBMC proliferation in response to anti-CD3/anti-CD28 stimulation. We observed minimal to no effect on proliferation by measuring BrdU incorporation. In summary, we are introducing a blocking reagent to eliminate nonspecific binding to Cyanine dyes which shows minimal perturbation of cell functions.
Self-assembly of immune signals to induce and control tolerance
Authors: Jewell Christopher M , Tostanoski Lisa H , Royal Walter
Published: January 2023

Multiple sclerosis (MS) is an autoimmune disease in which myelin insulating neurons is attacked. New research in humans shows excess inflammation through toll like receptors (TLR) contribute to disease. In animals, co-administration of self-antigens and regulatory cues leads to more specific tolerance. Polymers and other biomaterials can enable this co-delivery, along with attractive features such as tunable cargo loading, targeting, and cargo protection. However, these materials often exhibit intrinsic properties that can cause inflammation; features that might exacerbate disease. Thus, we designed immune polyelectrolyte multilayers (iPEMs) mimicking attractive features of biomaterial, but constructed entirely from immune signals. iPEMs were built by electrostatic interactions between myelin-derived, cationic peptides with an anionic, regulatory TLR9 antagonist (GpG). We hypothesized iPEMs would reduce TLR-driven inflammation during T cell differentiation, biasing expanding myelin-reactive T cells away from effector cells (TH1, TH17) and toward regulatory T cells (TREG) that control disease specifically. In culture, iPEMs co-deliver each signal to dendritic cells, reduce TLR9 levels, deactivate DCs, and promote myelin-specific TREG. These results depend on the presence of both GpG and myelin. In mice, iPEMs reduce TH17 and TH1 cells, expand TREG, and reverse MS-like disease (EAE). In samples from human MS patients, iPEMs also activate T cells, but polarize cytokine profiles toward tolerance and away from inflammation. This approach represents a simple, modular platform for combatting autoimmunity by programming the combinations and relative concentrations of self-antigens and regulatory TLR ligands.
Mass mining: a crowdsourcing approach for meta-analyzing gene expression signatures of autoimmunity using large-scale public data sets
Authors: Sparks Rachel , Lau William W ,   OMiCC Jamboree Working Group, Tsang John S
Published: January 2023

The volume and diversity of large-scale biological data available in the public domain continues to grow. This data has the potential to be reused to answer questions beyond those envisioned when the data was generated; however, few immunologists have sufficient bioinformatics expertise to do so. We used OMiCC, a free online platform that enables programming-free meta-analysis of public gene expression data and facilitates “crowdsharing” the work of annotating and constructing data compendia. We organized an “OMiCC Jamboree” to evaluate if biologists without bioinformatics training could use OMiCC to identify and annotate public gene expression datasets and design proper disease versus control comparisons for meta-analysis. Twenty-nine volunteer NIH biologists gathered to search and annotate public microarray data of human autoimmune conditions and the corresponding mouse models. Meta-analyses across studies explored 1) gene expression signatures for each disease, 2) pan-disease signatures, and 3) cross-species signatures. A large number of differentially expressed genes and enriched pathways were identified for each disease, with substantial overlap among diseases both within and between species, including pan-disease and pan-species signatures such as those associated with interferon.

Supported by the Intramural Research Programs of NIAID and CIT, NIH.
Identification of novel immune biomarkers in lymphoma
Authors: Derakhshandeh Roshanak , Webb Tonya J
Published: January 2023

The diagnosis and treatment options for lymphoma patients have changed significantly over the past few decades as new immunophenotypic markers and novel biomarkers have emerged. Natural killer T (NKT) cells play a critical role in anti-tumor immunity. However, NKT cells are approximately 50% lower in cancer patients compared to healthy donors of the same age and gender. Thus, prior to initiating immunotherapy, a specific and highly sensitive assessment of NKT cell function should occur; yet, no such assay is currently available. Thus, we have developed a qPCR-based diagnostic assay that can be used to rapidly assess natural killer T (NKT) and total T cell function. In this study we assessed NKT cell number and function in healthy donors (n=30) and lymphoma patients (n=50) by flow cytometry, ELISA, and qPCR. We detected NKT cell function in 82% of healthy donors, compared to 44% of lymphoma patients. To investigate the mechanisms by which NKT cells were functionally impaired in lymphoma patients, we examined circulating levels of cytokines, such as Semaphorin 4D, IL-10, and IL-6. It was found that lymphoma patients have significantly higher levels of both pro- and anti-inflammatory cytokines, compared to healthy donors. These data implicate an immunoregulatory role for NKT cells in lymphoma.
Deficiency of MyD88 signaling in CD4 Tconvs increases Tregs suppression through loss of ST2 signaling
Authors: Griesenauer Brad , Zhang Jilu , Ramadan Abdulraouf M , Paczesny Sophie
Published: January 2023

Acute graft-versus-host disease (aGVHD) hinders the efficacy of allogeneic hematopoietic cell transplantation (allo-HCT). We found that plasma soluble suppression of tumorigenicity 2 (sST2) predicted GVHD-related mortality in allo-HCT patients. ST2 signals through the adapter protein MyD88. Lack of MyD88 in CD4 Tconvs has been shown to decrease ovalbumin or a peptide to the haplotype H2b stimulated Th1/Th17 cells via the IL-1R. Thus, we hypothesized that absence of MyD88 signaling would protect against aGVHD through IL-1R, ST2, or both.

We found that knocking out MyD88 in the donor T cells protected against aGVHD while recipients of IL-1R−/− donor T cells showed no difference. We found that the aGVHD protection was entirely driven by MyD88−/− CD4 T cells. There were no differences in proliferation, apoptosis, or migration in WT or MyD88−/− CD4 T cells. We next explored the cell-intrinsic role of MyD88−/− Tconvs and Tregs. We did not find a difference in survival in aGVHD models with MyD88−/− Tconvs or MyD88−/− Tregs. However, use of MyD88−/− Tconvs with WT or MyD88−/− Tregs ameliorated aGVHD. We hypothesized that this was mediated by lack of ST2 on donor Tconvs. Recipients of ST2−/− Tconvs with WT or ST2−/− Tregs lowered aGVHD mortality, mirroring MyD88−/− Tconvs. Transcriptome analysis comparing 10 days post-HCT CD4 T cells from MyD88−/− versus WT recipients showed lower levels of Irak1, Il1rl1 (gene of ST2), Ifng, Csf2, Stat5, and Jak2 as well as decreased systemic plasma sST2 and IFN-γ levels. Our data suggests that Tregs suppression from lack of MyD88 signaling in Tconvs during alloreactivity uses the ST2 but not the IL-1R pathway, possibly by different antigen stimulation. MyD88 represents an aGVHD therapeutic target sparing Treg function.
TIM-1 blockade of the donor graft provides protection against lethal GVHD
Authors: Iliopoulou Bettina , Hsu Katie , Pierini Antonio , Freeman Gordon J. , Dekruyff Rosemarie H , Meyer Everett
Published: January 2023

Graft-versus-host disease (GVHD) continues to be a common cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). The T cell immunoglobulin and mucin (TIM) proteins represent a family of molecules that act in concert with T–cell receptor and costimulatory signals to regulate expansion, differentiation and effector function of T and NKT cells. TIM-1 binds to phosphytidylserine, exposed on the surface of apoptotic cells. Given the immunoregulatory role of TIM-1, we explored how TIM-1 influences GVHD in a murine model of allogeneic HSCT which causes massive apoptosis. Here we show that mice treated with an antagonistic anti-TIM-1 mAb that blocks phosphatidylserine recognition are protected from lethal GVHD. Protection against GVHD appears to be mediated by TIM-1 expression on the donor graft as TIM-1 knockout mice showed minimal survival advantage after allogeneic HSCT. Mice treated with anti-TIM-1 mAb cleared the A20 tumor cells suggesting that GVL effects are preserved. By bioluminescent imaging, it was determined that TIM-1 blockade does not alter the homing and expansion of donor T cells in vivo, nor does it affect T or NKT cells proliferation in vitro. Moreover, using microarray analysis, we found that anti-TIM-1 treatment protects from lethal GVHD by promoting an anti-inflammatory environment in the gut tissue. Antagonistic anti-TIM-1 mAb also protects from lethal GVHD in a xenogeneic model of GVHD. Overall, these findings can form the basis for the development of novel therapeutic strategies to prevent GVHD and improve HSCT.
Maximizing the potential of Treg-based therapies for transplant rejection via computational immune-modeling: effect of dose, timing, and distribution
Authors: Raimondi Giorgio , Arun Anirudh , Dorabiala Olga , Arciero Julia
Published: January 2023

Regulatory T cells (Treg) are a promising target to treat transplant rejection but the optimal conditions for their effective clinical use still remain to be identified. Exploring all possible Treg conditions is unrealistic using conventional in vivo experimentation. Computational modeling provides an efficient and inexpensive solution to guide experimentation and the design of therapeutic protocols. Here, strategies for the adoptive transfer of Tregs are theorized using an experimentally-based mathematical model of the immune response to murine heart transplants. The model tracks populations of innate and adaptive immunity and proxies for pro- and anti-inflammatory factors within the graft and a representative lymph node. The model is used to predict the impact of different Treg adoptive transfer strategies on graft survival. In particular: timing of dosing, dosing rate, activation status of Tregs, and site of accumulation post-injection. Model results show that activated Tregs accumulating directly to the graft are most protective. The administration day of a single dose of Tregs has an unexpected impact on graft survival: delaying administration to POD2 or POD3 is more effective than peri-transplant transfer. The model also suggests that distributing a total dose over multiple days exerts a more protective effect than administering the same dose amount on a single day. Finally, the model predicts that a delayed dose at a lower rate can have a greater impact on graft survival than multiple doses at the same rate, suggesting that the timing of doses is most important. Thus, theoretical simulations are an invaluable tool that reveals unexpected treatment strategies for transplant patients that can be validated experimentally.
Acute graft-versus-host disease is regulated by an IL-17-sensitive microbiome.
Authors: Varelias Antiopi , Ormerod Kate L , Bunting Mark D , Koyama Motoko , Gartlan Kate H , Kuns Rachel D , Lachner Nancy , Locke Kelly R , Lim Chun Y , Henden Andrea S , Zhang Ping , Clouston Andrew D , Hasnain Sumaira Z , McGuckin Michael A , Blazar Bruce R , MacDonald Kelli PA , Hugenholtz Philip , Hill Geoff R
Published: January 2023

Donor T cell-derived IL-17A can mediate late immunopathology in graft-versus-host disease (GVHD), however protective roles remain unclear. Using multiple cytokine and cytokine receptor subunit knockout mice we demonstrate that stem cell transplant (SCT) recipients lacking the ability to generate or signal IL-17 develop intestinal hyper-acute GVHD. This protective effect is restricted to the molecular interaction of IL-17A and/or IL-17F with the IL-17RA/C receptor. The protection from GVHD afforded by IL-17A required secretion from, and signaling in, both hematopoietic and non-hematopoietic host tissue. Given the intestinal-specificity of the disease in these animals, we hypothesized a microbiome contribution. Cohousing of WT with IL-17RA and IL-17RC deficient mice, dramatically enhanced the susceptibility of WT mice to acute GVHD. Furthermore, the gut microbiome of WT mice shifted towards that of the IL-17RA/C mice during cohousing prior to transplant, confirming that IL-17-sensitive gut microbiota controls susceptibility to acute GVHD. Finally, induced IL-17A deletion peri-transplant also enhanced acute GVHD, consistent with an additional protective role for this cytokine independent of effects on dysbiosis. Importantly, this implies that blocking IL-17 in a clinical trial could have adverse effects via dysbiosis, particularly in the early post-transplant setting and raises caution about their potential effects if used long term.
On Hyperleucocytosis and Its Bearing on Specific Therapy
Authors: Zinsser Hans , Tsen Edgar
Published: January 2023

The literature of the last ten years contains a great many reports of the successful treatment of various acute infectious diseases with suspensions of the dead bacteria causing the disease, i.e., homologous “vaccines.” However, although this method has attained universal acceptance as a prophylactic measure in typhoid fever, cholera and paratyphoid infections, and promises similar prospects in some other conditions, it can hardly be said that it has been more than hopefully suggestive as a therapeutic procedure used during the course of actual disease. Indeed there has been an increasing suspicion among bacteriologists that the effects produced, when bacterial protein was injected into patients, were not in any sense specific, and depended rather upon the general character of the injected substances as bacterial proteins, than upon their specifically immunizing properties.
The Selection of Horses for the Production of Diphtheria Antitoxin by an Intra-Palpebral Toxin Test
Authors: Hitchens A. Parker , Tingley E. K.
Published: January 2023

An interesting result of the work of Park and Zingher1 upon the immunization of persons with mixtures of diphtheria toxin and antitoxin has been with regard to the production of antitoxin by various individuals. It has been shown that those persons who already possess immunity to diphtheria toxin more readily react to the injections with the production of antitoxin than do those individuals who possess little or no natural immunity to diphtheria toxin. This being the case, it occurred to us that if the same thing held true with horses, we might have, in a modification of the Schick test, some indication of the productive possibilities of horses with regard to diphtheria antitoxin. The experiments that we are about to report indicate that, to a considerable degree, the antitoxin productiveness of horses can be anticipated with the use of the Schick test.
Studies in Anaphylaxis
Authors: Weil Richard , Eggleston Cary
Published: January 2023

This study, which was being conducted in connection with the work described in the preceding paper, has been interrupted by the war. In view of the indefinite postponement of its complete publication, it seems advisable at the present time to give a brief preliminary statement of the results, in so far as they have been clearly established. The following protocol of one of the experiments is illustrative of the essential findings.

The liver was removed from a dog actively sensitized to horse serum, and placed in a warm perfusion chamber. By means of a cannula tied in the portal vein the liver was washed out with a liter of salt solution. The defibrinated blood of the same animal was divided into equal portions of 200 cc. each. The organ was then perfused with one of these portions, the blood being passed through a number of times successively.
Studies on Pollen and Pollen Extracts
Authors: Cromwell Hobart W. , Moore Marjorie B.
Published: January 2023

Skin sensitivity to pollen extracts has been demonstrated in actively immunized rabbits using trypan blue as an aid, after the method of Ramsdell. The reaction is specific, and is more delicate than the precipitin test as indication of an immune response of the animal. The localization of the trypan blue also occurs in the abdominal fluid of the immune animal following intraperitoneal injection of the pollen antigen. Passive transfer of the skin sensitivity from immune to normal rabbit has been accomplished by the use of large quantities of immune serum.
The Production of Group Specific Human Hemagglutinins with a Note on the Separation of the Isoagglutinogens from the Red Corpuscles
Authors: Davidsohn I.
Published: January 2023

From the foregoing there is evidence to the effect: That it was possible in a relatively small percentage of cases to remove partly or completely the group agglutinogens from the human red corpuscles by the mechanical procedure of repeated washing and shaking with physiological saline, thus confirming the results of Schuetz and Wohlisch, and of Hallauer.That it is possible to produce group specific anti-A and anti-B immune sera by adsorbing the heterologous agglutinins in rabbit immune sera with the corpuscles A or B, respectively.That by using unwashed human red bloo corpuscles a relatively high percentage of group specific agglutinating sera is obtained.
Exposure to silver nanoparticles primes mast cells for enhanced IgE/FcɛR1-mediated activation
Authors: Alsaleh Nasser B. , Brown Jared M.
Published: January 2023

Nanotechnology has been greatly expanding over the past few years and engineered nanomaterials (ENM) are being employed into many consumer products; yet, a basic understanding about their potential associated adverse responses is largely lacking. Silver nanoparticles (AgNP) are one of the most utilized ENMs primarily for their antimicrobial properties. We recently demonstrated a non-IgE activation of signal transduction pathways including PI3K, PLCγ, PKC and calcium signaling leading to robust mast cell degranulation (early-phase response) following AgNP exposure. Here, we examined late-phase activation, reactive oxygen species (ROS) formation, stress response and IgE-induced activation following exposure to AgNPs. Our data revealed that, unlike IgE, exposure to 20 nm AgNP resulted in a delayed (24 hours) activation of the MAPK pathway without release of inflammatory cytokines. Although AgNPs induced transient ROS formation (within the first hour), long-term exposure (6, 24 hours) was neither associated with cellular stress response (e.g. glutathione levels) nor it involved apoptotic or necrotic cell death. Importantly, despite the lack of major AgNP toxicity, our results demonstrated that exposure to AgNPs primed mast cells for exacerbated IgE/FcɛR1 activation. Specifically, exposure to AgNPs for 24–72 hours resulted in enhanced IgE-mediated phosphorylation of FcɛR1-linked protein tyrosine kinases, degranulation, and cytokine release. Together, albeit exposure to AgNPs appeared non-toxic to mast cells, our findings suggest that AgNPs might prime mast cell for exacerbated allergic type responses and further investigation is warranted to understand the underlying mechanism.
Lipoxin A4 restrains mast cell function and inhibits type 2 mediated cutaneous inflammation
Authors: Baxendell Haylee , Haduch Alexandra , Alwine Jeremy , Naumov Nicholas , Falo Louis D. , Sumpter Tina L.
Published: January 2023

Atopic dermatitis (AD) is a frequently occurring allergic inflammatory disease. In AD, IgE-activated dermal mast cells amplify type 2 immune responses. Current MC-therapies have limited success, necessitating exploration of MC-targeting agents for AD treatment. Lipoxin A4 (LXA4) is an immunoresolvin family member derived from arachadonic acid that signals primarily through the formyl peptide receptor 2 (FPR2). Limited reports investigate the relationship between LXA4, MCs and cutaneous inflammation. Here, we tested the hypotheses that LXA4 downregulates MC function and cutaneous inflammation utilizing bone marrow derived (BM)MCs and the murine type 2 skewed, MC-dependent, FITC-contact hypersensitivity model (CHS), respectively. In resting BMMCs, antagonism of FPR2 and pharmacological inhibition of LXA4 synthesis increased IL-6 and IL-13 release, suggesting that resting BMMCs generate LXA4. Exogenous LXA4 inhibited IgE-initiated MC activation, blocking degranulation and IL-13, TNF, and IL-6 release. Mechanistically, exogenous LXA4 blocked STAT5B and NFkB nuclear translocation and reduced mRNA for STAT5 and NFkB transcribed genes. FPR2 antagonists failed to reverse the effects of exogenous LXA4, suggesting that a second inhibitory receptor may be activated by LXA4 in BMMCs. In vivo, LXA4 reduced ear swelling in the MC-dependent FITC-CHS model. Collectively, these data suggest that LXA4 restrains MC function and may be therapeutically beneficial in AD.