bluuebunny/crossref_metadata_2025 huggingface dataset:
Over-expression of BDNF in Primary Mouse B cells
Authors: Torres Vanessa , Matsui Takeshi K. , Stowe Ann Marie
Published: January 2023

Background

Neuronal networks require significant neurotrophic support for recovery after stroke, but delivery of neurotrophins (NTs) failed in clinical trials due to short half-lives and limited brain permeability. B cells, however, infiltrate the post-stroke brain and possess the ability to produce NTs (e.g. brain-derived neurotrophic factor (BDNF)). Thus, we hypothesized that neurotrophic B cells could enhance neuroplasticity after stroke.

Methods

A CMV-BDNF-mCherry plasmid was transfected into HEK-293T cells or LPS-stimulated splenic B cells isolated from 6–10 week old C57Bl/6J mice. Transfected B cells were co-cultured with supporting splenocytes for 24, 48, or 72h. Flow cytometry assessed mCherry+ B cells and BDNF secretion was measured via ELISA. Graphpad Prism analyzed statistical differences. Significance was p<0.05.

Results

The CMV-BDNF-mCherry plasmid was successfully transfected into HEK-293T cells compared to non-transfected controls, and led to long-term BDNF secretion (48h-3,162.9pg/mL, 72h-5,106.7pg/mL; both p<0.05 vs. 24h-720pg/mL). For splenic B cells, the percentage of transfected mCherry+ B cells was highest at 24h after transfection (12.6% of CD45+CD19+ cells) and reduced by 48h (8.77%; p<0.01 vs. 24h) and 72h (6.03%; p<0.01 vs. 24h). This trend was also reflected in absolute mCherry+ B cell numbers. Despite this reduction in mCherry+ B cells, BDNF secretion was consistently detected over 72h (24h-303.3pg/mL, 48h-453pg/mL, 72h-317.6pg/mL).

Conclusions

Our studies show successful overexpression of BDNF in primary B cells. We are currently assessing the magnitude of B cell-derived neurotrophic support on neuroplasticity under homeostatic conditions and in vitro stroke injury models.
Humoral Immune Response Modulation by Aryl hydrocarbon Receptor Activation (AhR) in CD11c+ Cells: Exploring Gender-specific and Ligand-dependent Effects on Antibody Responses in Mice.
Authors: Kreitinger Joanna , Shepherd Celine Ann Beamer , Shepherd David M.
Published: January 2023

Modulation of antibody responses is often a summation of effects on B cells, helper T cells, and dendritic cells (DCs). All three of these immune cells express AhR, a ligand-activated transcription factor with endogenous and exogenous ligands known to modulate immune responses. Importantly, antibody responses are keenly sensitive to AhR activation by TCDD, its prototypical ligand and common environmental pollutant. However, the specific contribution of AhR activation in DCs has not yet been defined, particularly in the context of gender-specific and ligand-dependent effects. We aimed to determine the effects of AhR activation in DCs by endogenous and exogenous ligands on OVA-specific antibody responses in mice. Specifically, we hypothesized that AhR activation in DCs alone suppresses antigen-specific serum antibody production, selectively affecting different isotypes. To test this hypothesis, we used mice conditionally deficient of AhR in CD11c+ DCs (AHRCD11c). Control (AhRd) and AHRCD11c mice were treated with vehicle, ITE or TCDD via oral gavage, and subsequently immunized i.p. with whole OVA/adjuvant to induce a systemic, antigen-specific humoral response. One week later, AhRd mice showed significantly reduced OVA-specific IgM and IgG2a titers while IgA was significantly increased; effects that were ameliorated in AhRCD11c mice. Interestingly, TCDD-induced IgA increases were both AhR- and gender-dependent with female mice showing increased IgA production. Collectively, these results demonstrate that DCs contribute to, but are not wholly responsible for, AhR-mediated effects on antigen-specific serum antibody generation. Research supported by ES013784 (DMS) and AAI Careers in Immunology Fellowship (JMK).
Changes in metabolic phenotype and cellular ATP production during CD4+ T cell activation.
Authors: Romero Natalia , Swain Pamela , Kam Yoonseok , Dranka Brian P.
Published: January 2023

Activation of CD4+ T cells is followed by rapid proliferation and differentiation into specific subsets (Treg or Teff). These transitions are accompanied by tight regulated changes in energetic demand and cellular metabolic reprogramming. Using Agilent Seahorse extracellular flux analysis, we have developed a cell-based assay for simultaneous measurement of the two-main cellular ATP-producing pathways, i.e. glycolysis and oxidative phosphorylation. The assay allows for quantification of real time changes in total ATP production rate, and the fractional contribution of the individual pathways to support bioenergetic demands. We found that naïve CD4+ T cells obtain most of its ATP from mitochondrial oxidative phosphorylation. After activation with anti CD3/CD28 conjugated beads, total ATP production rate significantly increases, and that rise is sustained by an increase in glycolytic ATP production, but also in mitochondrial ATP production. When glucose is replaced by galactose in the assay medium, T cell activation is inhibited despite partial compensation of total ATP production through mitochondrial respiration. Time-course of bioenergetic phenotype during T-cell expansion shows a deep increase in ATP production rate up to day 6–7 with glycolysis contributing up to 70% of total ATP production. When glucose is replaced by galactose 72 hr after activation, total ATP production rate was fully compensated by mitochondrial ATP production but still the inhibition of glycolytic activity by galactose reduces cell proliferation. The results obtained highlight the role of glycolytic activity beyond energetic supply and its importance for biosynthetic and redox requirements or cell activation and proliferation.
Physiological microbial exposure has the capacity to substantially influence inflammatory responses in vivo
Authors: Sjaastad Frances V. , Pierson Mark , Huggins Matthew A. , Danahy Derek B. , Badovinac Vladimir P. , Griffith Thomas S. , Hamilton Sara E.
Published: January 2023

Physiological exposure to microbes profoundly effects the basal immune state, based on data from ‘dirty’ mice with a diverse microbial experience obtained after co-housing with pet store mice. Most T cells in dirty mice and adult humans have an effector/memory phenotype, but specific pathogen free (SPF) mice or newborn humans have few Ag-experienced memory T cells. These differences prompted us to define the acute cytokine/chemokine response in SPF or co-housed inbred C57Bl/6 (B6) mice. Co-housing for 60 d increased the frequency of CD44hi CD8 T cells and led to higher basal serum cytokine/chemokine levels (vs. SPF mice). We then examined to what extent these basal immune differences affected the response to different inflammatory stimuli. Infection of SPF mice with virulent Listeria significantly increased serum cytokines/chemokines (e.g., IFNg, TNF, CXCL1, CXCL10, CCL7) 24–72 h after infection. The same infection produced little-to-no change in the serum of dirty mice, but they had significantly lower bacterial burden. After LPS challenge, SPF and dirty mice had similar increases in serum cytokines/chemokines, but mortality was increased in dirty mice. Lastly, while an increase in serum cytokines/chemokines occurred 6–12 h after sepsis induction (CLP model) in both SPF and dirty mice, they were 2–8-fold higher in dirty mice. Acute lymphopenia was observed in both sets of CLP-treated mice; yet, mortality was higher in CLP-treated dirty mice. Thus, physiological microbial exposure influences the cytokine ‘storm’ and survival suggesting the immune status of the host controls the magnitude of the response and outcome. The data also point out the need to further develop the ‘dirty’ mouse model to complement current studies in SPF mice.
CD28 co-stimulation drives memory phenotype (MP) Treg cell and MP CD4+Foxp3− effector T cell homeostatic proliferation.
Authors: Panda Abir Kumar , Shevach Ethan M.
Published: January 2023

Foxp3+Tregs, CD4+Foxp3− effector T cells and CD8+ T cells are composed of naïve (NP, CD44loCD62Lhi) and memory (MP, CD44hiCD62Llo) subsets. About 30% of MP T cells are cycling (Ki-67+) in-vivo. To determine the factors that drive T cell homeostatic proliferation in-vivo, we investigated the roles of cytokines (TNFa, IL-2, IL-7, and IL-33), TCR-MHC-II interactions, co-stimulatory (CD28, ICOS, CD40, GITR, OX40), and co-inhibitory (CTLA-4, PD1, BTLA, TIGIT) receptors. Blockade of CD28-CD80/86 signaling completely inhibited MP Treg and MP CD4+Foxp3− effector T cell proliferation but did not affect MP CD8+ T cell proliferation. Inhibition of cytokine activity did not affect the proliferation of any of the T cell populations. Marked enhancement of homeostatic proliferation of MP Treg and MP CD4+Foxp3−T cells was observed by blocking TCR-MHCII interactions, CTLA4-CD80/CD86 interactions, or by agonistic mAbs to CD40, GITR, and OX40. Blocking PD1-PDL1 interactions modestly enhanced proliferation, while blocking BTLA, TIM3, ICOS and TIGIT did not affect MP T cell proliferation. Importantly, blocking of CD28-CD80/86 signaling completely abrogated the augmentation of proliferation observed after blocking TCR and CTLA4, PD1, or by agonistic mAbs to GITR, OX40, and CD40. Our results support a complex pathway in which CD28-CD80/CD86 interactions play a critical role in Treg and MP CD4+Foxp3−T cells proliferation in-vivo. CTLA4-CD80/CD86 interactions act as a brake, while MHCII-TCR interactions negatively regulate the inhibitory functions of CTLA-4. These findings have important implications for the use of biologic agents to enhance Treg numbers/functions, as well as for the use of checkpoint inhibitors.

Supported by DIR, NIAID.
Translating knowledge and raising awareness of Immunology related human disease mechanisms via engagement with the general public and lay audience
Authors: Sika-Paotonu Dianne
Published: January 2023

Acute rheumatic fever (ARF) is an autoimmune condition caused by untreated Group A Streptococcal (GAS) infection of the upper respiratory tract (and possibly skin). Multiple or severe attacks of ARF can cause cardiac damage known as rheumatic heart disease (RHD). Effective management for ARF requires painful monthly intramuscular injections of Benzathine Penicillin G (BPG) known as secondary prophylaxis. The goal of secondary prophylaxis is to prevent GAS infections that may lead to the recurrence of ARF. Rates of adherence to secondary prophylaxis are usually low due to the frequency and duration of injection, pain and access to proper and timely healthcare. A less painful and longer acting BPG formulation is urgently needed. Research being undertaken in New Zealand seeks to support global BPG reformulation efforts. The purpose of this work was to support knowledge translation of BPG reformulation research efforts and the associated fundamental Immunology concepts within a public setting. A general public and lay audience outreach event was held through the local university, inviting members of the public to attend. A presentation by a female Pacific Biomedical scientist was offered to support increased knowledge and awareness of emergent rheumatic fever related issues locally and internationally. This event was well attended by the general public. Questions from the audience relating to ARF/RHD dominated, with strong interest expressed in the global efforts to reformulate Penicillin. Additional positive verbal and written feedback was received from those in attendance. This public outreach engagement event supported communication of research findings that was culturally appropriate, clear and meaningful.
Inflammasomes confer protection via IL-18 and pyroptosis, and are negatively regulated by IFN-γ-dependent nitric oxide during Brucella infection
Authors: Lacey Carolyn , Mitchell William J. , Skyberg Jerod A.
Published: January 2023

Brucellosis, caused by the intracellular bacterial pathogen Brucella, is a zoonotic disease for which arthritis is the most common focal complication in humans. Here we show caspase-1 (casp1) and caspase-11 (casp11) initiate Brucella-induced joint inflammation, but later in infection also restrict Brucella replication. In contrast, AIM2 and NLRP3 were dispensable for control of joint infection by Brucella. Both IL-1 and IL-18 promoted joint inflammation, however only IL-18 contributed to protection against Brucella infection of the joint. In vitro studies demonstrated that casp1 and casp11 both induce pyroptosis which limited Brucella infection in macrophages. While IFN-γ production in part required casp1/11 and IL-18, casp1/11-dependent clearance of Brucella was only partially IFN-γ dependent. However, IFN-γ deficiency resulted in severe inflammation that was entirely inflammasome dependent, and in particular reliant on NLRP3. IFN-γ was vital for induction of the nitric oxide (NO) producing enzyme, iNOS, in infected joints, and NO inhibited casp1 activation in Brucella-infected macrophages in vitro. During Brucella infection in vivo, iNOS inhibition exacerbated arthritis, and administration of a NO donor reduced joint inflammation in IFN-γ-deficient mice. Collectively these data demonstrate inflammasomes induce early inflammation in an IL-18 and IL-1 dependent manner, and inflammasome-dependent IL-18 and pyroptosis restrict Brucella burdens in the joint. Moreover, IFN-γ reduces inflammation by inhibiting excessive casp1 activation through production of NO. Thus NO donors, in combination with antibiotics, may be an effective therapy for treating Brucella-induced inflammation.
TRAF3 regulates Pim2 and c-Myc-mediated B cell survival
Authors: Whillock Amy Louise , Mambetsariev Nurbek , Lin Wai W. , Stunz Laura L. , Bishop Gail A.
Published: January 2023

Tumor necrosis factor receptor associated factor 3 (TRAF3) is an adaptor protein that plays an important role in B cell homeostasis. TRAF3 deletions or inactivating mutations are common in human B cell malignancies. In human and mouse B cells, TRAF3 negatively regulates signaling of several receptors important for B cell homeostasis (including BAFFR, CD40, and IL-6R). B cell specific TRAF3-deficient (B-Traf3−/−) mice have enlarged spleens and lymph nodes and an increased frequency of B cell tumors as they age. B cells from B-Traf3−/− mice have markedly increased survival and autoantibody production. The mechanisms by which TRAF3 regulates B cell survival are not yet well defined. We found that TRAF3-deficient mouse B cells and human multiple myeloma cell lines with low expression of TRAF3 had increased RNA and protein expression of the pro-survival kinase Pim2. Additionally, TRAF3-deficient mouse B cells had increased phosphorylation of Pim2 targets BAD, p70S6K, and 4E-BP1. TRAF3-deficient B cells showed increased survival relative to TRAF3-sufficient B cells after treatment with the Pim inhibitors SGI-1776 and TP-3654. Pim2 can promote survival by phosphorylating and stabilizing the oncogenic transcription factor c-Myc. Loss of TRAF3 led to transcription-independent c-Myc protein elevation that was dependent on Pim2. K48 polyubiquitination of c-Myc was decreased in the absence of TRAF3, consistent with increased c-myc protein stability. TRAF3 deficiency correlated with B cell resistance to apoptosis mediated by the c-Myc transcription inhibitor JQ1. Our results show that TRAF3 promotes B cell survival by regulating expression of and interaction between Pim2 and c-Myc.
The pro-bacterial effect of Type 1 Interferon signaling requires its own negative regulator USP18
Authors: Shaabani Namir , Honke Nadine , Prinz Marco , Knobeloch Klaus-peter , Zhang Dong-Er , Teijaro John R.
Published: January 2023

Elevated Type 1 interferon (IFN-I) production following bacterial and viral infections has been causally linked with promoting host susceptibility to primary and secondary bacterial infections, some which can result in severe morbidity and Mortality. Using a Listeria monocytogenes (L.m.) model system, we found that among the many Interferon-induced genes (ISGs), USP18 is a central ISG that increases susceptibility to primary and secondary L.m. infection downstream of IFN-I signaling. Knocking out Usp18 specifically in DCs enhanced bacterial control, despite an elevated IFN-I gene signature compared to WT mice. Moreover, Usp18 pro-bacterial effect hinges on the ability of Usp18 to inhibit TNF-α signaling. We extended our findings to demonstrate that Usp18 expression also regulates IFN-I sensitive Staphylococcus aureus infection, suggesting that Usp18 may regulate many IFN-I-sensitive bacterial infections. In summary, we identify a single downstream ISG responsible for promoting primary and secondary bacterial infection – a significant conceptual advance. Targeting host USP18 will have the distinct advantage of exerting less selective pressure on bacterial populations and thus likely attenuate the rate at which resistance develops.
Circulating bacterial DNA is associated with disease severity in patients with S. aureus bacteremia
Authors: Gutierrez Johnny , Guimaraes Alessander , Peck Melicent C. , Barhanu Aklile , Xu Min , Cao Yi , Kim Janice , Yan Donghong , Chang Joanna K. , Dinoso Jason , Koss Catherine , Clemenzi-Allen Angelo , Chambers Henry F. , Rosenberger Carrie , Baruch Amos
Published: January 2023

Staphylococcus aureus bacteremia is a leading cause of morbidity and mortality and yet tools to assess severity of infection and response to treatment are still lacking. This study aimed to establish a link between bacterial DNA quantified in blood and clinical severity in bacteremic patients. We developed a workflow coupled with a highly sensitive quantitative PCR method to quantify S. aureus bacterial DNA. This assay was used to measure whole blood S. aureus DNA (SA-DNA) and plasma-derived S. aureus cell-free DNA (SA-cfDNA) in a set of longitudinal samples from 45 patients with confirmed S. aureus bacteremia. SA-DNA was detectable at baseline and declined following treatment with antibiotics. Notably, baseline SA-DNA correlated with disease severity and levels were sustained in cases of complicated infections versus with non-complicated infections. In addition, plasma-derived S. aureus cell-free DNA could be readily detected in bacteremic patients and showed concordance with whole blood SA-DNA. High levels of SA-DNA and SA-cfDNA were associated with metrics of clinical severity, including longer durations of treatment, elevated white blood cell count, and positive blood cultures. Taken together, these data suggest that circulating bacterial DNA is more reflective of whole body bacterial load than blood culture and may aid in monitoring clearance of tissue reservoirs of infection during antibiotic therapy.
Comparative genomic analysis of Escherichia coli strains resistant to grazing by the social amoeba Dictyostelium discoideum
Authors: Snyder Michelle , Hazen Tracy , Michalski Jane , Rasko David
Published: January 2023

Diarrheagenic Escherichia coli remain a significant health challenge, particularly in developing countries. Challenges in the use of small animal models for enteric infections highlight the need for development of alternative model systems. The social amoeba, Dictyostelium discoideum, which phagocytizes bacteria for nutritional purposes, serves as a host model for several pathogenic bacteria, including E. coli. Previous studies characterized interactions of D. discoideum with commensal and extraintestinal E. coli. In the current study, we extend this characterization to analyze the grazing interactions of D. discoideum with 35 representative diarrheagenic, extraintestinal and commensal E. coli strains. We found that E. coli strains sensitive and resistant to D. discoideum grazing are present in each pathotype. Whole genome phylogenomic analysis revealed that grazing resistance to D. discoideum is distributed among the phylotypes, although resistant strains are enriched in the B2 phylogenomic group. Comparative genomic analysis identified genes that were more prevalent among strains resistant to D. discoideum grazing. While not providing direct insight into conserved mechanisms of resistance, these identified genes do provide avenues for further investigation. Phenotypic analysis revealed that E. coli strains resistant to D. discoideum grazing also exhibited increased resistance to the reducing agent, β-ME. These data extend the characterization of interactions between D. discoideum and E. coli, and through comparative genomics identify candidate E. coli genes that may be associated with resistance to grazing by the model phagocyte D. discoideum, and by extension with resistance to phagocytic immune cells.
NK cells inhibit anti-M.bovis BCG T cell responses by lysing BCG-infected macrophages and aggravating pulmonary inflammation
Authors: Fang Min , Wang Dongfang , Gu Xiuling , Liu Xiaoman , Wei Songtao , Wang Bin
Published: January 2023

Tuberculosis remains a threat to public health. The major problem for curing this disease is latent infection, of which the underlying mechanisms are still not fully understood. Previous studies indicate that NK cells do not play a role in inhibiting the growth of Mycobacterium tuberculosis in the lung, and recent studies have revealed that NK cells regulate the adaptive immunity during mycobacterial infection. By using a novel mouse model of direct lung infection with M.bovis BCG, we found that the presence of NK cells postponed the priming and activation of T cells after BCG infection. In addition, depletion of NK cells before infection alleviated pulmonary pathology. Further studies showed that NK cells lysed BCG-infected macrophages in an NKG2D dependent manner. Thus, NK cells did not play a direct role in control BCG, but by lysing BCG-infected macrophages, NK cells aggravated the pulmonary inflammation and impaired anti-BCG T cell immunity. Our results may have important implications for the design of immune therapy to treat tuberculosis.
TGF-β1 regulates epidermal LCs in a Smad-independent pathway
Authors: HUANG LINTING , Li Gui-Hua , Zhang Xilin , Yin Congcong , Zhou Li , Mi Qing-sheng
Published: January 2023

Transforming growth factor-β1 (TGF-β1) is a key factor for Langerhans cell (LC) homeostasis and maintaining in the epidermis, but the underlying mechanisms in TGF-β signaling pathways remain elusive. Canonically, binding of TGF-β to TGF-βR activates Smad2 or Smad3, which subsequently heterodimerizes with Smad4 and then translocates into the nucleus acting as transcriptional regulators of TGF-β1 target genes. In order to investigate if Smad-dependent pathways are required for epidermal LC maintenance, we first crossed CSF1R-Cre and hLangerin-Cre transgenic mice with Smad2fl/fl and Smad4fl/fl, mice to generate the mutant mice with conditional Smad2 and Smad4 deletion at birth (CSF1Rcre) or 3 days after birth (hlangerinCre). Surprisingly, deletion of individual Smad2 or Smad4 in LCs did not significantly affect their ratios in skin and their expression of MHC-II, CD80 and CD86 in both models. The phagocytic capacity of LCs from KO mice was unaltered in vivo and in vitro compared to WT LCs. Furthermore, upon in vitro stimulation, MHC-II, CD80 and CD86 were comparably unregulated on LCs between KO and WT mice. We further tested LC number and maturation in conventional Smad3KO mice as well as Smad2/4 double KO mice induced by CSF1Rcre, the number and maturation of LCs are comparable between mutant and WT mice. Overall, our data suggest that conventional TGF-β/Smad2/3/4 signaling pathways may not be required for maintaining LC homeostasis and immature stage in the epidermis and LC maturation upon stimulation. The underlying mechanisms that Smad2/3/4-independent TGF-β pathways involved in LC maintenance and function remain to be further determined.
Oropharyngeal (OPG) vaccination activates head and neck lymphoid tissues required for immune protection against mucosal Brucella infection
Authors: Clapp Beata , Yang Xinghong , Hoffman Carol , Pascual David W.
Published: January 2023

Human brucellosis remains a global health problem with no human vaccines existing. Most human infections occur mucosally with eventual systemic brucellae dissemination. Current interventions primarily focus on the systemic disease, rather than considering neutralizing the infection at its source. A number of studies show that the OPG mucosa is the principal site of brucellae uptake following oral or inhalational exposures. We have devised an OPG infection method using an attenuated Brucella mutant by allowing the mice to drink (ad bibitum) from a pipette tip. BALB/c mice were fed once daily for 3 days with 108 CFUs. Individual head and neck lymph nodes (HNLNs), and spleens were examined at 2 weeks post-infection for bacterial colonization and proinflamatory cytokine responses. Elevated colonization was observed in submandibular LNs (SmLNs) and deep cervical LNs (CLNs). Modest colonization was detected in the parotid LNs (PrLNs) and spleens. Splenic and individual HNLN lymphocytes were analyzed for IFN-g, TNF-a, granzyme B, and perforin production by flow cytometry. SmLN, CLN, and PrLN IFN-g-producing CD8+ T cells significantly exceeded IFN-g-producing CD4+ T cells by 2.5-, 2.5-, and 1.5-fold, respectively. Increased numbers of CD4+ and CD8+ T cells expressing TNF-a, granzyme B, and perforin were also observed. All activated lymphocytes were predominantly of effector-memory phenotype, CD44+CD62LlowCCR7low. These studies demonstrate that ad bibitum vaccination against brucellosis results in colonization and activation of the HNLNs, mimicking natural human infection, enabling future inquiry into the immune mechanisms responsible for protection to mucosal Brucella infections. Work supported by NIH AI-125546.
Large-scale use of knockout validation to confirm antibody specificity
Authors: Zhong Zhen , Sassi Monica , Heaton Sam , Koch Sofia , De Block Gert , Conlon Daniel , Lochead Julia , Dreja Hanna , Munro Melanie , Solache Alejandra , Hamilton Bruce
Published: January 2023

Antibodies are among the most common tools in basic science and clinical research. However, an increasing number of studies have shown that not all antibodies are specific, leading to a growing issue of experimental irreproducibility. This is in part due to the cross-reactivity between antibodies and off-target proteins, as well as the variability between different antibody batches. These unspecific antibodies result in wasted time and resources and compromise the advancement of science.

In response to this, Abcam launched a knockout (KO) validation program in 2015 as one of its antibody quality control protocols. This initiative uses KO cells lines made possible through a partnership with Horizon Discovery. Target genes are mutated via CRISPR-Cas9 within a haploid cell line, which results in a frameshift and a complete loss of gene expression. These KO cell lines function as true negative controls that can be used at a large scale to confirm antibody specificity to the protein of interest.

We have KO-validated hundreds of antibodies, including several that are relevant to immunology and immuno-oncology research. Here we present the data related to key targets validated in western blot, flow cytometry and immunocytochemistry. The antibodies have been tested in KO and wild-type cells to confirm their specificity. By providing researchers with reliable and specific antibodies that work first time, we hope to minimize wasted resources and improve antibody reproducibility.
Magnetic enrichment of tumor infiltrating T cells reduces experimental time and prevents biased functional characterization
Authors: Evaristo Cesar , Siemer Ramona , Agorku David , Brauner Janina , Hardt Olaf , Dose Christian , Richter Anne
Published: January 2023

Tumor-Infiltrating (TI) T cells play an important role in anti-tumor immunity, highlighted by multiple immunotherapy strategies approved by the FDA. However, the benefit of current immunotherapies is still limited, such that additional research is needed to improve efficacy. Preclinical tumor models are essential to advance immunotherapy research. However, tumor composition is highly variable and T cell numbers can be very limited. Even flow cytometry can be very time consuming. Importantly, TI T cells are embedded in a cellular environment where antigen is abundant and surrounding cells express immunomodulatory molecules, such that unbiased cell-intrinsic functional characterization is impossible. Pre-enrichment of TI T cells is highly desirable to reduce hands-on time, and generate high quality and reliable data.

We developed new CD4, CD8 and Pan T cell specific enrichment reagents for magnetic cell sorting from mouse tumors. Single cell suspension was obtained by automated dissociation. T cell isolation was done either manually, or using semi or full automation. TI T cell frequencies ranged from 0.2–9%, were isolated to purities above 80% with yields ranging from 60–95%. Pan T cell isolation maintained the original CD4:CD8 ratio. CD69 was not upregulated after 18h culture, demonstrating that isolation did not result in T cell activation. Importantly, functional analysis of bulk vs. purified cells revealed significant influence of tumor and stromal environment in cytokine expression by TI T cells, which was not due to the isolation protocol.

Our data, demonstrates that magnetic enrichment reduces time of analysis, increases quality of analysis and is essential for the functional characterization of TI T cells.
Cross platform validation of a unique method for deep genomic and proteomic analysis of rare immune cell populations
Authors: Lopez Peter , Gregory Michael , Miller Kelley , Fuhrman Kit , Ray Karina , Hinerfeld Douglas
Published: January 2023

Advances in flow and mass cytometry expand the number of cell parameters that can be interrogated improving our understanding of the myriad immune cell types. These technologies, however, are limited in the types of analytes profiled in a single sample. In addition to efforts to understand the immune system for the treatment of autoimmune diseases, the immune system is increasingly a target for cancer therapeutics. These efforts require deep characterization to understand rare cell populations, requiring next-generation methods that expand on flow cytometry.

3D Flow™ Analysis integrates flow cytometry cell sorting and the nCounter® Vantage 3D™ RNA:Protein Immune Cell Profiling Assay* to characterize sorted immune cell populations with direct digital counting of 770 RNA and 30 proteins. To determine if the method is agnostic to the cell sorting technology used, 3D Flow analysis was run with the Becton Dickenson FACSARIA IIu, Sony SH800z, and Beckman Coulter MoFloXDP. Stimulated and unstimulated PBMC were co-stained with flow antibodies for CD3, CD8, and CD4 plus 30 DNA-labeled antibodies for NanoString readout. 5,000 CD3+/CD4+ and CD3+/CD8+ cells were sorted on each platform and immediately lysed to release cellular RNA and DNA tags from NanoString antibodies. RNA and DNA tags were directly hybridized to NanoString barcodes and run on the nCounter system.

Expression of 30 proteins and 770 RNA across cell populations correlated between the three sorting platforms, showing 3D Flow is agnostic to the sorting technology used. Importantly, high-plex RNA data was obtained without additional molecular biology methods, such as RNA purification or library construction, simplifying incorporation into a flow cytometry laboratory.
Rapid Specificity Determination and FACS Selection of Plasma Cells for Single Cell Antibody Cloning
Authors: McBride Kevin M. , Mu Yunxiang , Zelazowska Monika Anna , Dong Qiwen , Krug Laurie , Plummer Joshua
Published: January 2023

Plasma cells (PC) are terminally differentiated antibody secreting cells that are the major source of serum immunoglobulins (Ig). Identifying antigen specific PCs is an important goal for both immunologic studies and monoclonal antibody production. However, the lack of Ig surface expression has impeded direct identification of PCs expressing antigen specific antibodies. We devised a simple method termed antigen specificity of plasma cell determination (ASPCeD), to isolate live PCs producing antigen specific antibodies from total spleen or bone marrow populations using one-step flow cytometry. To demonstrate the utility of this technique we immunized mice against a viral protein immunogen, and combined ASPCeD with established single cell immunoglobulin cloning. We rapidly produced sequence diverse, recombinant monoclonal antibodies, highly specific for the viral protein target and infected cells. Furthermore, we have produced post-translational modification specific antibodies using this technique. Thus, ASPCeD is a novel method to identify antigen specific PCs and an improved means for high throughput recombinant antibody production over existing methodology.
The Benzathine Penicillin G (BPG) reformulation preferences study – the importance of cultural awareness and appropriate governance concerning Rheumatic Fever related research in New Zealand
Authors: Sika-Paotonu Dianne , Tiatia Ramona , Sung Yun K. , Thornley Craig , Betty Bryan , Wineera-Parai Ranei , Eddie Barbara , Baker Michael , Maloney Margaret , Sanchez-Felix Manuel , Spector Jonathan , Carapetis Jonathan
Published: January 2023

Acute Rheumatic Fever (ARF) is the autoimmune condition caused by untreated group A streptococcal (GAS) infection of the throat and possibly skin. Repeated or severe untreated attacks of ARF can cause permanent heart damage known as rheumatic heart disease (RHD). ARF/RHD rates in the Pacific (Samoa, Tonga and Fiji) have been reported as being 5 times higher than that found in NZ. Pacific Peoples living in NZ currently comprise ~ 7% of the NZ population so prioritization of ARF/RHD prevention, treatment and management efforts within the Pacific region and NZ is warranted due to high incidence rates and migration between countries. As a GAS vaccine is not yet available, the most effective recommended preventative measure for ARF requires painful monthly injections of Benzathine Penicillin G (BPG) be given intramuscularly for 10 years. Known as secondary prophylaxis, the purpose is to prevent GAS infections that can lead to ARF and cause RHD. A reformulated BPG is desperately needed. To support the generation of a more appropriate BPG reformulation that encourages treatment adherence, this work explored the BPG reformulation preferences of predominantly Māori and Pacific children/teens in New Zealand currently receiving monthly BPG intramuscular injections, their families and healthcare providers. The appropriate governance, cultural leadership and guidance essential for this project work to progress safely by the research team was provided by Pasifika and Māori health researchers, leaders, representatives, scientists and academics with careful considerations integrated into the development of the software applications and into the research work.
A bead-based multiplex immunoassay for cancer autoantibody biomarker discovery
Authors: Pepin D , Godeny Michael , Lie Wen-Rong
Published: January 2023

Accurate, reliable and affordable blood tests are needed for early detection of cancer. Here we show data for the development and validation of a sensitive Luminex® bead-based multiplex immunoassay for detecting cancer autoantibodies in multiple cancer types. The cancer autoantibody biomarkers in this new multiplex panel include: CTAG1B/NY-ESO-1, Cyclin B/CCNB1, ENO1, Galectin-1, Galectin-3, Her2, HIF1α, HSP60, IMP2/IGF2BP2/p62, IMP3/IGF2BP3/KOC, MUC1, p16-INK4a, p53, SOX2, and Survivin/BIRC5. This multiplex immunoassay is sensitive and robust. In a 96-well plate, a mixture of recombinant protein-immobilized magnetic bead sets was incubated with serum samples for 2 hours with gentle shaking; after washing, the beads were incubated with detection antibody phycoerythrin (PE)-labeled anti-human IgG for 1.5 hours. After the final wash, the beads were resuspended in sheath fluid and the median fluorescent intensity (MFI) data of 50 beads per bead set were analyzed on a Luminex® reader. The performance of this panel was demonstrated using a set of serum samples from 78 cancer cases and 18 healthy controls. Elevated cancer autoantibodies were detected in serum from patients with various cancer types, as compared to healthy controls. In conclusion, this cancer autoantibody multiplex immunoassay has potential application as a non-invasive tool for studying early detection, diagnosis or monitoring of cancer.
Aptamer-based screening of cerebrospinal fluid for protein biomarkers of multiple sclerosis
Authors: Soomro Sanam , Hughes Samuel , Greenberg Benjamin , Hanly John , Mohan Chandra
Published: January 2023

Background

Multiple sclerosis is a degenerative autoimmune disease marked by the presence of physical, mental, and psychiatric symptoms. Diagnosis is often made very late in the disease. Hence, there is a need for better diagnostic and prognostic biomarkers as well as new therapeutic targets. In this study, MS patients’ CSF was interrogated for potential diagnostic biomarkers using an aptamer based screen.

Methods

The CSF of 6 MS, 10 neurological disease controls (NC), and 8 healthy controls (HC) were subjected to an aptamer based screen of 1128 unique human proteins. Dysregulated proteins were selected using group wise fold change (FC) and Mann Whitney tests. A subset of the elevated proteins was chosen for validation by ELISA in a set of pilot CSF samples (11 MS and 6 HC).

Results

The aptamer-based screen showed 12 proteins to be elevated in MS vs HC CSF and 22 proteins to be elevated in MS vs NC CSF (FC > 1.3 P < 0.05). Among these, IP10, ITAC, BAFF, and LGMN were elevated in MS CSF but not NC, compared to healthy controls (FC > 1.25 P < 0.1). Of the ELISA-validated proteins, BCMA, IP-10, CD48, and MMP9 showed elevation in MS CSF in comparison to healthy CSF, while LGMN did not. I-TAC and MMP-7 could not be detected by ELISA.

Conclusion

An ultra-sensitive and comprehensive aptamer-based biomarker screening platform has uncovered several elevated proteins in MS CSF, including chemokines, cytokines, and B-cell drivers. Although validation is still in progress, the identified proteins show promise as diagnostic markers or therapeutic targets for MS.
Type 2 innate lymphoid cells impair IL-33-mediated tumor suppression
Authors: Long Alan W. , Dominguez Donye , Qin Lei , Zhang Minghui , Fan Jie , Zhang Bin
Published: January 2023

Although a number of studies have recently explored the contribution of the adaptive immunity in interleukin 33 (IL-33)-mediated antitumor effects, the involvement of innate immunity has been weakly characterized. Using Rag1−/− mice (lacking T and B lymphocytes), we show here that either systemic administration of recombinant IL-33 or ectopic expression of IL-33 in melanoma cells is sufficient to significantly delay tumor growth independent of adaptive antitumor immunity. We have demonstrated that IL-33-mediated antitumor effects rely on expansion and activation of NK cells. Concurrently, IL-33 also promoted the expansion of active type 2 innate lymphoid cells (ILC2s), via its receptor, ST2. Elevated ILC2 presence coincided with decreased NK activation and cytotoxicity, and resulted in increased tumor growth, indicating a protumor role for ILC2s. IL-33-induced ILC2 activity elicited greater expression of the immunosuppressive ecto-enzyme CD73. Removal of CD73 from ILC2s in culture with NK cells resulted in markedly increased activation levels in NK cells, providing a potential mechanism by which ILC2s may suppress NK cell-mediated tumor killing. Thus, our data reveal an important contribution of IL-33-induced ILC2 to tumor growth by weakening NK cell activation and tumor killing, independent of adaptive immunity.
The immune enhancement activity of exopolysacharide from marine bacteria on the IBDV immune response induced by B87 vaccine
Authors: He Xiumiao , Wei Ping
Published: January 2023

Infectious bursal disease (IBD), which is caused by infectious bursal disease virus (IBDV), is a fatal or immunosuppressive disease of young chickens. Immunization is still an important way to prevent the outbreak of the disease. The purpose of this study was aimed to investigate the immune enhancement activity of two kinds of exopolysacharide (EPS), on the IBDV immune response induced by an IBD vaccine (B87).

B87 vaccine plus EPS or B87 only were performed twice (at 7 and 14-day old) on commercial Three-Yellow-Chickens. The antibodies and IL-1 level were evaluated after immunization. The vvIBDV challenging was performed at 28-day old. The protection were assessed at 3 and 7 days post infection (dpi).

The results showed that IBDV antibodies and IL-1 level in 04 EPS+B87 and 09 EPS+B87 groups were significantly higher than B87 group. Further, the IL-1 level in groups received EPS only were significantly higher than the group received PBS only. High levels of IBDV antibody and IL-1 lasted at least for 5 weeks. After challenged with vvIBDV, the morbidity in 04 EPS+B87 and 09 EPS+B87 groups was 20%, lower than any other. The BBIX of bursa and thymus in 04 EPS+B87 and 09 EPS+B87 groups were significantly higher than the B87 group but lower than negative control, suggested that either the 04 and 09 EPS might have the ability to repair the immune organ damages induced by B87 vaccine. The trend of T cells (both CD4+ and CD8+) and B cells number, and the viral load, at 3 and 7 dpi, respectively, were consistent with the changing trend in BBIX value. We concluded that either 04 or 09 EPS showed the activity of immune enhancement in Three-Yellow-Chickens, the combination of EPS and B87 could induce higher titers of anti-IBDV antibody and protect chickens from vvIBDV challenge.
Analysis of dendritic cell activation by differential filovirus GP concentration in virus-like particle vaccines
Authors: Charley Krystal R. , Clarke Elizbeth , Bradfute Steven
Published: January 2023

Filoviruses, specifically ebola- and marburgviruses, can cause severe hemorrhagic fever in humans. Currently, there are no US licensed vaccines available against filoviruses. An important filovirus protein required for cellular entry is glycoprotein (GP). GPs are also used as vaccine antigens that can trigger antibody production, which makes them an important factor in vaccine development. Distinct vaccine platforms are made in different cell types, such as mammalian and insect cells. Different glycosylation patterns and GP distribution in virus-like particles (VLPs) were observed by our lab in GPs expressed in mammalian cell lines, but comparative data is needed to determine how differential filovirus GP concentration in the VLP platform affects the immune response in vaccination. We hypothesize that differential filovirus GP concentration will yield diverse results in immunogenicity and will provide insight into specifically how we can utilize GP concentration to improve vaccine development. We are using ebolavirus GPs to develop VLPs by transfecting 293T (mammalian) cells with VP40, a structural protein, and GP DNA from the different filoviruses. Preliminary results show that changing the amount of GP in transfection appears to affect the amount of VLPs generated and the relative amount of GP compared to VP40. We are currently adding the VLPs to dendritic cells (DCs), a key subset of innate immune cells, to measure activation by flow cytometry. We expect to measure how differential filovirus GP concentration in VLPs affects DC activation. This will result in broad significance in development of filovirus vaccine platforms.
An interferon-inducible mitochondrial circuit implicated in cell death, regulated by miRNAs, and conserved in vertebrates uncovered by evolution-guided studies
Authors: Hancks Dustin , Elde Nels
Published: January 2023

Genomes are shaped by selective pressure imposed by pathogens. For instance, over evolutionary time direct-binding of immune factors, such as pattern-recognition receptors, by pathogen-encoded inhibitors can leave a historical record of this pressure as elevated rates of amino acid replacement across closely related species - a hallmark of positive selection. We hypothesized that positive selection could be leveraged to ascribe novel immune functions to poorly characterized genes. Using signatures of positive selection similar to those observed in critical antiviral genes (e.g. APOBEC3G, PKR), we carried out an evolution-guided screen to identify novel immune factors. Our top-candidate is a small mitochondrial protein strongly induced by interferon in human and mouse cells, conserved to zebrafish, and encoded by a poxvirus. Loss-of-function analysis uncovered a defect in staurosporine-induced cell death in the presence of interferon in the absence of this factor. Unexpectedly, this defect appears due to a miRNA, which is perfectly conserved (22/22nt) in certain fish species, encoded by the ISG mRNA. This miRNA targets a highly-conserved sequence in its paralog, a factor also rapidly evolving; a constitutively expressed mitochondrial protein that interacts with the OXPHOS complexes and conserved to yeast. Strikingly, another paralog and a related miRNA (that targets the same microRNA response element in the other paralog) are upregulated in response to hypoxic stress. These data allude to a vertebrate-specific circuit implicated in cell death that is induced by distinct types of cellular stress (e.g. infection, hypoxia), regulated by related miRNAs, implemented by mitochondrial paralogs and antagonized by viruses.
Development of a computational model to quantify antigenic similarity between Dengue viruses
Authors: Smith Brittany K. , Fremont Daved H.
Published: January 2023

Dengue virus (DENV) causes dengue fever, the most prevalent arthropod-borne viral illness in humans. Globally, four DENV serotypes cause an estimated 100 million new cases of dengue fever and 250,000 cases of dengue hemorrhagic fever per year. DENV serotypes differ by 25 to 40 percent in the envelope (E) protein. Although primary DENV infection results in life-long immunity to the infecting serotype, subsequent infection with a different serotype increases risk of severe disease. It has been argued that this is due to weakly neutralizing, cross-reactive antibodies that mediate viral uptake by Fc receptor-bearing cells. The unique role of DENV antibody cross-reactivity in disease outcome necessitates characterization of viral antigenic similarity and resulting disease pathogenesis.

Inspired by influenza models, we are developing a model that incorporates sequence data, neutralization titers and structural information to quantify antigenic similarity between circulating DENV strains. We first identify important E protein antigenic positions by considering sequence conservation and correlation with cross-reactivity. Amino acid similarity at antigenic positions is then combined with structural data to calculate antigenic variance. The objectives of this study are to (1) identify E protein sequence positions and structural properties (epitopes) associated with antigenic variation and (2) introduce a model to quickly predict antigenic distance between DENV strains that can be assessed by neutralization experiments. We expect many antigenic positions to be located in epitopes described in the Immune Epitope Database, although some distant positions may exert allosteric affects by altering E protein structure or dynamics.