bluuebunny/crossref_metadata_2025 huggingface dataset:
Commensal-dendritic-cell interaction specifies a unique protective skin immune signature.
Authors: Bouladoux Nicolas , Naik Shruti , Linehan Jonathan L , Han Seong-Ji , Harrison Oliver J , Tussiwand Roxanne , Murphy Kenneth M , Merad Miriam , Segre Julia A , Belkaid Yasmine
Published: January 2023

The skin represents the body’s primary interface with the environment, acting as the first line of physical and immunological defense. This organ is also home to trillions of microbes (bacteria, fungi and viruses) that play an important role in tissue homeostasis and local immunity. Skin microbial communities are highly diverse and can be remodeled over time or in response to environmental challenges. How in the context of this complexity, individual commensals may differentially modulate skin immunity and what the consequences of these responses for tissue physiology are remain unclear. Here, we demonstrate that defined commensals dominantly impact skin immunity and identify the cellular mediators involved in this specification. In particular, colonization with Staphylococcus epidermidis induces IL-17A+CD8+T cells that home to the epidermis, enhance innate barrier immunity and limit pathogen invasion. These commensal-specific T cell responses result from the coordinated action of skin resident dendritic cell subsets and are not associated with inflammation, revealing that tissue resident cells are poised to sense and respond to alterations in microbial communities. This dialogue may represent an evolutionary means by which the skin immune system uses fluctuating commensal clues to calibrate barrier immunity and provide heterologous protection against invasive pathogens. Altogether these findings reveal that the skin immune landscape is a highly dynamic environment that can be rapidly and specifically remodeled by encounters with defined commensals, findings that have profound implications for our understanding of tissue specific immunity and pathologies.
Imaging mass cytometry - elemental immunohistochemistry for multiparametric imaging and quantitation
Authors: Ornatsky Olga , Chag Qing , Swanson Eric , Closson Taunia , Bouzekri Alexandre , Loboda Alexander , Baranov Vladimir
Published: January 2023

Pathology assessment of tissue sections provides prognostic evaluation and helps select optimum treatment regimens. Digital pathology has made significant strides in the analysis of several markers. There is a growing understanding of cell heterogeneity within tumor tissue, role of microenvironment, and the impact of immune cells on cancer development. Sophisticated tools are needed to provide quantitative information for a large number of biomarkers, low-abundance small molecules and chemotherapeutic drugs, while retaining spatial resolution of cells and tissue architecture. Imaging mass cytometry (IMC) is a novel technology that can simultaneously detect and quantitatively measure more than 50 metal-containing reagents in tissue sections at 1 μm resolution. IMC combines laser ablation with the Helios CyTOF®. We will describe in detail technology, workflow, multiplexing protocols, image analysis and show representative data for human and mouse sections. We will demonstrate the use of metal-containing histological stains for tissue architecture, and endogenous element identification [iodine, platinum]. Combined detection of protein targets and transcripts within cells will be presented. Validation will be demonstrated on sequential tissue sections prepared by conventional immunohistochemistry.

Precision medicine is based on access to high-density data (proteomics and genomics) which provides accurate diagnosis and information on the best therapeutic approach. IMC is a highly multiparametric, quantitative method for phenotypic, signaling pathway, and cell state protein identification together with spatial information within tissue sections.
Identifying interactions between cytokine levels and clinical outcomes in influenza infection using a novel data-driven method for defining cytokine modules
Authors: Hertz Tomer , Fiore-Gartland Andrew , Panoskaltsis-Mortari Angela , Thomas Paul Glyndwr , Randolph Adrienne
Published: January 2023

Cytokines and chemokines are key signaling molecules of the immune system. Recent technological advances enable measurement of multiplexed cytokine profiles in blood as well as sites of infection such as the nasal and alveolar mucosa. These profiles can then be used to identify potential biomarkers of clinical outcomes following influenza infection, by comparing groups of individuals with different outcomes. Recently, several associations between single cytokines and mortality have been reported. However, single analyte association testing ignores context-dependent covariation in cytokine secretion and decreases statistical power due to multiple testing. We have developed a novel data-driven approach to analyze cytokine and chemokine profiles that uses unsupervised clustering to form functional modules. We applied this method to three cohorts of severely ill children infected with influenza. Innate profiles were assayed using a 42-plex Luminex panel at enrollment. Clinical outcomes were collected including hospitalization, septic shock, ALI/ARDS and death. Though many modules were consistent with the immunological literature, some of the modules included cytokines that have not previously been associated, generating novel hypotheses. Modules generated from blood were different than those generated at the site of infection. Furthermore, we found that the modules were significantly associated with clinical outcomes in all three cohorts, while individual cytokines were not statistically significant. Overall, our approach provides a novel method to characterize cytokine and chemokine profiles that both increases statistical power and provides novel hypotheses about context-specific innate signaling.
An optimized IFN-γ ELISpot assay using T-activated
® proteins for determination of CMV-specific cell-mediated immunity
Authors: Boeckl Katharina , Körber Nina , Barabas Sascha , Deml Ludwig , Bauer Tanja
Published: January 2023

In healthy individuals, Cytomegalovirus (CMV) infections are controlled by CMV-specific cell-mediated immunity (CMI). However, functional impairment of the CMI in immunocompromized individuals can lead to uncontrolled CMV-replication. Thus, monitoring CMV-specific CMI is highly relevant for the prognosis of CMV-associated diseases. Here we show the optimization and technical validation of a monitoring tool for the determination of CMV-specific CMI.

Phenotype, quantity and cytokine expression profile of T cells from 19 CMV-seropositive healthy donors reactivated by CMV (IE-1, pp65) -specific pools of overlapping 15mer peptides and the corresponding T-activated proteins® were compared using a 10-parameter flow cytometry-based intracellular cytokine staining assay. Subsequently, an IFN-g ELISpot protocol using T-activated proteins® was established and validated.

Pp65 peptide pool stimulation revealed significant higher CD8+ T-cell responses as the corresponding T-activated proteins® (p=0.006). CD4+ T-cell responses however were comparable. Testing of T-activated proteins® in seronegative donors revealed a specificity of 100%. Use of T-activated proteins® with the optimized IFN-g ELISpot resulted in coefficients of variation for inter-assay, inter-operator and inter-site variations of less than 22 %. A linear correlation between the amount of CMV protein-reactive cells and total PBMC counts was observed.

Given the natural antigen processing of T-activated proteins® along the cross-presentation pathway, reflecting the in vivo situation, these proteins in combination with the optimized IFN-g ELISpot serve as a valuable tool to monitor the CMV-specific CMI.
Unbiased High-Dimensional Identification of Lymphocytes in Mouse Uterus
Authors: Li Yan , Vazquez Jessica , Stanic-Kostic Aleksandar
Published: January 2023

Introduction

Uterine lymphocytes play an essential role at the maternal-fetal interface, contributing to vascular remodeling, fetal tolerance and protection against infection. Despite intense research interest, simultaneous population assessment of diverse immune cells has been limited by the use of few concurrent markers and difficulty in assigning unambiguous cell identity. We present the first comprehensive, unbiased identification of mouse uterine immune subsets by application of dimensionality reduction of highly-polychromatic flow cytometry.

Methods

Uterine specimens were dissected from C57BL/6 mice and mononuclear cells (MCs) were isolated by mechanical and enzymatic disruption. MCs labeled by flurochrome-conjugate antibodies. Data acquisition and analysis were performed using BD Fortessa flow cytometer in an 18 parameter, 5-laser configuration and Flowjo, respectively. Dimensionality reduction by Barnes-Hut modification of t-distributed Stochastic Neighbor Embedding (t-SNE) and density-based k-means clustering was performed using the open-source ACCENSE package.

Results

Analysis revealed uterine B-, T-, Dendritic, and Innate Lymphoid cell subsets. Analysis with ACCENSE allowed unbiased mapping of these subsets onto 2-dimensional scaffolds, without the necessity for expert manual gating.

Conclusion

We present the first comprehensive analysis of uterine immune cells, demonstrating a complex mixture of innate and adaptive immune mediators. The use of dimensionality reduction and population clustering allows allocation of uterine immune cell phenotype in an unambiguous fashion, enhancing reproducibility and functional analysis across studies.
LEARNING ABOUT HUMAN KIDNEY IMMUNOLOGY FROM THERAPEUTIC MONOCLONAL ANTIBODY-INDUCED KIDNEY INJURY: EFFECT OF PD1 BLOCKADE
Authors: Muczynski Kimberly , Anderson Susan , Torres Mark-Joseph
Published: January 2023

Human renal microvascular endothelial cells (RMEC) express high levels of MHC class II proteins under normal physiologic conditions. They are positioned to interact with circulating leukocytes. Yet, under normal conditions kidney inflammation does not occur.

Isolated RMEC can peptide presentation to influenza specific T cells. T cell proliferation and cytokine secretion are blocked by antibodies to CD40, CD58 and HLA-DR; and enhanced by antibodies to PDL1 (B7-H1,CD274). Using cytometry we find high levels of CD58 and CD274 on RMEC in normal human kidneys and CD40 on some RMEC of transplanted kidneys. We hypothesized that RMEC PDL1 interaction with T cell PD-1 (CD279) prevents T cell activation in human kidneys.

New PD-1 blocking monoclonal antibodies, such as nivolumab, are being used to treat advanced cancers by selectively blocking PD-1 interactions with ligands PD-L1 (CD274, B7H1) and PD-L2 (CD273, B7H2) present on tumor cells. We have identified four cases of acute kidney injury in patients receiving PD-1 pathway blockade. Biopsies revealed an intense interstitial nephritis in the area of the class II expressing RMEC.

Our interpretation of RMEC’s ability to activate T cells in a class II-peptide dependent manner, their high levels of PD-L1 (CD274) and now the development of interstitial inflammation in patients receiving anti-PD1 monoclonal therapy is that RMEC are poised to present peptide from circulating antigen as an immune surveillance system, with the PD1 pathway functioning to restrain T cell activation. Other human organs also express high levels of HLA class II on microvascular endothelial cells. Hence our hypothesis of a kidney capillary endothelium forming an immune surveillance system may also pertain to other organs.
Stem cell-derived tissue-associated regulatory T cells suppress autoimmune diabetes
Authors: Haque Mohammad Shamsul
Published: January 2023

CD4+ regulatory T cells (Tregs) are essential for normal immune surveillance, and their dysfunction can lead the development of autoimmune diseases, such as type-1 diabetes. Pluripotent stem cells (PSCs) can be utilized to obtain a renewable source of healthy Tregs to treat autoimmune diabetes as they have the ability to produce almost all cell types in the body, including Tregs. However, the right conditions for the development of antigen (Ag)-specific Tregs from PSCs (i.e., PSC-Tregs) have not been fully defined. In this study, we developed a new approach to generate functional Ag-specific Tregs from induced PSC (iPSCs), i.e., iPSC-Tregs, which have the ability to suppress autoimmunity in a murine model of autoimmune diabetes in Ag-associated fashion. We retrovirally transduced murine iPSCs with a DsRed reporter construct containing genes of major histocompatibility complex (MHC) II (I-Ab)-restricted ovalbumin (OVA)-specific T cell receptor (TCR) and the transcriptional factor FoxP3. We in vitro differentiated the DsRed+ iPSC into OVA-specific iPSC-Tregs with an OP9 stromal cell line expressing Notch ligands DL1, DL4 and I-Ab in the presence of recombinant cytokines of rIL-7 and rFlt3L. We visualized the expression of CD3, TCR, CD4, CD25, and CTLA-4 on OVA-specific iPSC-Tregs. Moreover, adoptive transfer of such Tregs dramatically suppressed autoimmunity in a well-established OVA-induced model of autoimmune diabetes (RIP-OVA × OT-I double transgenic mice), including the inflammation and prevents the insulin secreting pancreatic beta cells from destruction. Our results indicate that PSCs can be used to develop Ag-specific Tregs, which have a therapeutic potential for autoimmune diabetes.
PD-L1/PD-1 Immunotherapy Modulates Effector T Cells Homeostasis and Function in Murine Pancreatic Cancer
Authors: Rivera Ivannie Ortiz , Iclozan Cristina , McGill Andrew , Ghansah Tomar
Published: January 2023

Pancreatic Cancer (PC) is one of the most aggressive and deadliest types of cancer, and it is projected to be the second leading cause of cancer-related deaths in the U.S. by the year 2030.

PC evades immune surveillance by disrupting the immune homeostasis of effector T cells (Teff). T cells homeostasis is critical for proper anti-tumor immune responses. Preliminary flow cytometry data revealed that murine pancreatic (Panc02) cancer cells produce inflammatory soluble factors and in addition, express Major Histocompatibility Complex class I (MHC-I) and Programmed Death Ligand 1 (PD-L1) and 2 (PD-L2). PD-L1/2 can bind to PD-1 receptors on Teff and induce apoptosis or anergy, dampening the anti-tumor immunity. Cytokine bead array and flow cytometry analysis of serum from peripheral blood from C57BL/6 mice injected with Panc02 cells (TB mice) showed a significant increase in inflammatory factors compared to Control (CTRL) mice. Additionally, Teff from splenocytes from TB mice showed significant reduction in Teff percentages compared to CTRL mice, in vitro. In this study, CD3+ cells from CTRL mice were co-incubated with Panc02 cells pre-treated with anti-PD-L1 antibody (ab) to evaluate the modulation of Teff and cytokines production, in vitro. Mice were inoculated with the Panc02 pre-treated cells with anti-PD-L1 ab, to evaluate the modulation of Teff and cytokines production, in vivo. The results from this project may lead to the identification of new immunotherapeutic strategies to stabilize Teff homeostasis and function that could increase anti-tumor immune responses and combat PC progression.
Control of tumor growth by TSLP during colorectal cancer
Authors: de Jesus-Carrion Steven , Ziegler Steven F
Published: January 2023

Thymic stromal lymphopoietin (TSLP) is a cytokine involved in promoting tumor growth during breast and pancreatic cancer by promoting Th2 cell-mediated inflammation. Despite being essential to maintain Treg cell homeostasis in the mouse and human intestine, a role for TSLP in colorectal cancer has never been shown. To determine if TSLP affects tumor growth during colorectal cancer, we utilized a murine model of colitis-associated colorectal cancer. TSLP deficient mice exhibited decreased tumor numbers when compared to the WT littermate controls. Moreover, we found that tumors in TSLP−/− mice were also significantly smaller in size than WT mouse tumors. TSLP was mainly localized in the tumor tissue, indicating that it might be produced by cancer or cancer-associated cells. Further, TSLP receptor (TSLPR) expression was significantly upregulated after development of cancer. To determine if TSLP signals directly on the tumor cells, we utilized Villincre TSLPRflox mice in which the TSLPR expression is absent only in intestinal epithelial cells (IECs). Consistent with the TSLP−/− mice, lack of TSLP signaling on IECs led to a decrease in tumor number as well as tumor size, suggesting that TSLP signals directly on the IECs to promote tumor growth. Overall, these data show a novel role for TSLP in controlling tumor progression during colorectal cancer and identify it as a potential target for immunotherapy intervention.
Interleukin-10 Attenuate Tumor Growth by Inhibiting Interleukin-6/Signal transducer and activator of transcription 3 Axis on Myeloid-Derived Suppressor Cells
Authors: Ko Hyun-Jeong , Kim Seong-Ryeol , Song Jae-Hyoung , Lee Bo-Ra
Published: January 2023

Interleukin-10 (IL-10) is a well-characterized anti-inflammatory cytokine, but its role in anti-cancer immunity is still controversial. When IL-10 knockout (IL-10−/−) mice were subcutaneously injected with TC-1 cancer cells expressing HPV E6 and E7 proteins, tumor growth was accelerated in IL-10−/− mice than wild type (WT) mice. Interestingly, the production of interleukin-6 (IL-6) was significantly increased in IL-10−/− mice than in WT mice. The blockade of IL-6 by systemic administration of anti-IL-6 receptor (IL-6R) monoclonal antibody (mAb) inhibited the tumor growth, and inhibited the generation of myeloid-derived suppressor cells (MDSCs) which were increased in IL-10 deficiency. MDSCs and tumor cells from IL-10−/− mice had increased phosphorylated-signal transducer and activator of transcription 3 (p-STAT3) level and the treatment of mice with STAT3 inhibitor, S3I reduced the tumor growth and inhibited the expansion of MDSCs. Accordingly, S3I also reduced tumor necrosis factor-a (TNF-a) and IL-6 in tumor. Although S3I treatment relieved T cells from suppression by reducing the number of MDSCs, it seems that the proportion of MDSCs having M2 macrophage phenotype was increased among myeloid cells since the transcription of Arg1, Mgl1, Mgl2, and, Fizz1, were significantly increased and the expression of some suppressive functional mediators of MDSCs, including Arg1 and iNOS was up-regulated. Combined treatment of anti-IL-6 mAb and S3I could further suppress the generation of M2-type MDSC and regulate immunosuppressive function of MDSCs, and finally inhibited tumor growth more efficaciously. These results suggested that combined inhibition of IL-6 and STAT3 could be a potential candidate as an anti-cancer strategy.
Hypoxic tumor microenvironment of head and neck cancers downregulates Kv1.3 channels in tumor infiltrating lymphocytes
Authors: Chimote Ameet A , Hajdu Peter , Sfyris Alexandros M , Casper Keith A , Conforti Laura
Published: January 2023

The clinical outcome of solid malignancies depends on the ability of cytotoxic CD8+ T cells to infiltrate the tumor and destroy cancer cells. Unfortunately, tumor infiltrating lymphocytes (TILs) exhibit attenuated functions. Kv1.3 channels are vital to the T cell effector functions as they regulate the Ca2+ signaling necessary for cytokine and granzyme production. We have previously reported that the expression of functional Kv1.3 channels in healthy T cells is suppressed by hypoxia. Solid tumors like head and neck squamous cell carcinomas (HNSCC) are characterized by a hypoxic tumor microenvironment (TME). Herein we investigated whether the hypoxic TME in HNSCC inhibits Kv1.3 expression and function in TILs. Electrophysiological experiments showed a 70% reduction in functional Kv1.3 channels in freshly isolated HNSCC TILs compared to peripheral blood T cells. Kv1.3 currents in TILs ranged from 0 to 600 pA with a median of 90 pA. Immunofluorescence analysis of Kv1.3 and HIF1α (marker of hypoxia) in HNSCC tumor sections revealed that 88% of the CD8+ TILs expressing high Kv1.3 (Kv1.3high: above the median Kv1.3 fluorescence in all TILs) have a low HIF1α level. Furthermore, CD8+ cells preferentially localize in the stroma (68% of the TILs) than in the tumor epithelia (32%), and only 35% of the CD8+ cells in the tumor epithelia are Kv1.3high compared to 55% in the stroma, with a 20% reduction in the median Kv1.3 fluorescence of CD8+ cells in the tumor epithelia compared to the stroma. Overall, our data indicate that exposure of lymphocytes to the hypoxic HNSCC TME leads to decreased Kv1.3 expression. This reduction in Kv1.3 may contribute to the attenuated cytotoxicity of TILs and improper immune surveillance in solid tumors. (NIH2R01CA095286)
The surface alpha-enolase targeting as a novel immunotherapeutical strategy for pancreatic cancer
Authors: Cappello Paola , Principe Moitza , Chattaragada Michelle Samuel , Curto Roberta , Giordano Daniele , Roux Cecilia , Novelli Francesco
Published: January 2023

Pancreatic Ductal Adenocarcinoma (PDA) is a very aggressive tumor for which effective therapeutical strategies are still lacking. The global five-years survival is of 5% and surgery is the only potentially curative treatment. The PDA-associated antigen α-enolase (ENO1), beside its glycolytic function, acts as a plasminogen receptor, promoting activation into plasmin, involved in extracellular matrix degradation. Antibodies against ENO1 are detected in more than 60% of PDA patients and correlate with a better prognosis. Furthermore, anti-ENO1 antibodies are induced in mice after ENO1-DNA vaccination. We observed an increased ENO1 expression on the cell surface of both PDA and myeloid cells (MDSC), suggesting a role in tumor progression and spreading. The immunotherapy represents a chance to selectively target PDA cells and MDSC. Mouse anti-human ENO1 monoclonal antibody (mAb) inhibits plasminogen-dependent invasion of human PDA cells and metastatic spreading in immunosuppressed mice, as well as MDSC adhesion to pancreatic endothelial cells and in vitro and in vivo migration. Similarly MDSC arginase activity and secretion of IL-6 decrease after ENO1 triggering, while anti-ENO1 treatment does not affect costimulatory molecule expression and MDSC suppression functions. Notably, a single administration of Adeno-Associated Virus (AAV)-expressing an anti-ENO1 mAb reduced the number of lung metastases in immunosuppressed mice injected with PDA cells. In conclusion, the antibodies anti-ENO1 may inhibit PDA cell and myeloid cell invasion and modulate T cell response, making the immunotherapy more effective.
Quantitation of sub-visible particles in therapeutic protein formulations using Imaging Flow Cytometry
Authors: Probst Christine Elizabeth , Zeng Yuanchun , Zhu Rong-Rong
Published: January 2023

Monitoring particles within therapeutic protein formulations is necessary to evaluate drug safety and efficacy. Current regulations require analysis of particles greater than 10 μm, however quantitation of particles 1–10 μm is increasingly expected due to potential immunogenicity. These small particles are difficult to measure with current methods, and new methods must be developed. Amnis Imaging Flow Cytometry (IFC) is an existing technique that is conceptually well-suited for this application because provides: (1) Sensitive detection particles across a wide size range (1–100 μm), (2) Direct particle size/shape measurements from brightfield imagery (3) Particle identification using fluorescent dyes, (4) High sampling efficiency (95%) (5) Low sample volume requirement (20 μL). Here we demonstrate IFC may be used to measure particles common to this application, including protein aggregates, silicone oil droplets, polystyrene bead standards, and newly developed protein surrogate standards. IFC provided sensitive detection of particles as small as 100 nm and could be used to discriminate protein aggregates from silicone oil. Interestingly, IFC analysis of one sample revealed transparent protein aggregates invisible using current gold-standard methods. The results demonstrate that IFC is a useful orthogonal technique to current methods for interrogating small and translucent particles within protein formulations.
ImmTACs: Bi-specific TCR-based reagents for targeted cancer immunotherapy.
Authors: Jakobsen Bent
Published: January 2023

Immune system based therapies constitute a promising class of treatment for many types of cancers. Whilst T cells can mediate tumor destruction, their effectiveness is limited due to negative thymic selection, down-regulation of HLA expression by cancer cells and an immunosuppressive microenvironment. To overcome the poor immunogenicity of tumors, Immunocore has developed ImmTACs (immune mobilizing monoclonal T cell receptors (mTCR) against cancer). A soluble mTCR, engineered to recognise a given tumor associated peptide-HLA complex with exceptionally high sensitivity and specificity, redirects host polyclonal T cells via an anti-CD3 antibody fragment, facilitating targeted T cell recognition of tumors. Antigens that exist on tumor cells but not on normal cells are rare, thus the selection and validation of appropriate target antigens and the testing of ImmTACs for specificity is critical. Antigen candidates are selected based on their levels of expression in cancer vs healthy tissues by RT-PCR and the presence of the targeted peptide on the cell surface is confirmed by mass spectrometry. As ImmTACs are specific for humans, efficacy, specificity and off target effects are determined through a detailed molecular and cellular testing programme, using antigen positive tumor cells and HLA appropriate primary human cell lines representing a range of tissues. IMCgp100, the lead ImmTAC, is currently in a Phase I/II clinical trial for the treatment of advanced melanoma. The maximum tolerated dose has been established and emerging data demonstrate several durable responses. IMCgp100 is well tolerated and there is evidence of T cell mobilisation in the tumor microenvironment, release of cytokines and tumor shrinkage.
Galectin-3 inhibition using novel inhibitor GR-MD-02 improves survival and immune function while reducing tumor vasculature
Authors: Linch Stefanie N , Kasiewicz Melissa J , McNamara Michael J , Hilgart-Martiszus Ian F , Farhad Mohammad , Traber Peter G , Redmond William L
Published: January 2023

Immunosuppression and reduced cytotoxic function of tumor-infiltrating lymphocytes (TIL) are major obstacles to creating effective therapies for patients. It is known that Galectin-3 (Gal3), a lectin family member, is expressed and secreted by numerous cancers and immune cell subsets. Serum Gal3 expression is higher in patients with metastatic versus non-metastatic disease, and is associated with reduced survival in metastatic melanoma. Furthermore, Gal3 has been implicated in disease progression via the promotion of angiogenesis and metastasis, the inhibition of TIL function, and promoting M2 polarization of macrophages and mobilization of myeloid cells from the bone marrow to promote a metastatic niche within the tumor.

We hypothesized that Gal3 inhibition would improve TIL function while inhibiting the growth and metastasis of tumors. Through collaboration with Galectin Therapeutics, we tested Gal3 inhibition using the novel Gal3 inhibitor, GR-MD-02, with agonist anti-OX40 therapy. We observed that Gal3 inhibition/OX40 agonism improved survival in the MCA-205 sarcoma and 4T-1 mammary carcinoma models, and prolonged survival in the TRAMP-C1 prostate cancer model. Importantly, Gal3 inhibition/OX40 agonism reduced lung metastases in the 4T-1 model. Within the tumor, we observed an increase in the number of proliferating and IFNg-producing TIL. Gal3 inhibition/OX40 agonism was also associated with a decrease in tumor vasculature, as determined by CD31 staining by IHC within the tumors. These studies, along with clinical data from a liver fibrosis trial, supported testing GR-MD-02 in combination with additional immunotherapies to augment T cell function in patients with metastatic melanoma.
Chlamydia trachomatis Recombinant MOMP Encapsulated within PLGA 85/15 Nanoparticles Activates Mouse Dendritic Cells by Triggering Enhanced Co-stimulatory Molecules and T Helper 1 Type Cytokines
Authors: Sahu Rajnish , Dixit Saurabh , Duncan Skyla , Singh Shree R , Dennis Vida
Published: January 2023

Chlamydia trachomatis (CT), the leading cause of bacterial sexually transmitted diseases, is of public health significance because it causes considerable morbidity and socioeconomic burden worldwide. The quest for vaccine development against this pathogen has escalated to reduce its occurrence and global economic burden. Recently nanovaccines have emerged as attractive therapeutics due to flexibility in their formulation, biocompatibility and adjuvant properties. We have developed a nanovaccine against CT employing its MOMP (major outer membrane protein) and encapsulating it in PLGA [poly (D, L-lactic-co-glycolic acid) 85/15 nanoparticles. We hypothesized that encapsulated MOMP will be effective in activating dendritic cells (DCs) for induction of Th1 immune responses, which are needed for protection against CT. Exposure of mouse DCs to encapsulated MOMP at concentrations of 0.01 to 1 μg/mL revealed marked IL-6 and IL-12p40 secretions, with the latter being 20-fold more abundant. Encapsulated MOMP induced lower IL-10 and TNF levels with an elevated IL-12p40/IL-10 ratio, suggesting a predominant Th1 response. Time-kinetics studies (4 to 96 hr) showed increasing IL-12p40 concomitant with decreasing IL-10 concentrations. Analyses of CD11c+ DCs by FACS indicated heightened expression of the co-stimulatory molecules, CD80, CD86 and CD40 in response to encapsulated MOMP stimulation. TaqMan qPCR for mRNA transcripts validated the upregulation of TLR-2, CD80, CD86 and particularly CD40, which are important for adaptive immune responses. Our data suggest that encapsulated MOMP is a potential nanovaccine candidate by triggering enhanced co-stimulatory molecules and Th1 responses, which are required to protect against CT.
Enhanced protective efficacy of a PRRSV DNA vaccine by absorption onto biodegradable PLGA/BPEI nanoparticle
Authors: Li Bin , Du Luping , He Kongwang
Published: January 2023

Porcine reproductive and respiratory syndrome (PRRS) is one of economically devastating viral diseases of pigs, which has recently caused catastrophic losses in swine industry worldwide. Developing preventive measures to control PRRS outbreaks has been a challenge to the swine industry worldwide. Current PRRSV vaccination strategies provide only a limited protection. The safer and more effective new vaccines are urgently needed.

Nanoparticles (NPs) as vaccine carriers have a broad application prospect in design of vaccine systems. PLGA nanoparticle-mediated delivery of vaccines has shown to be effective in eliciting protective immune response, administered by either mucosal or systemic routes. PEI, due to its high positive charge density, has been widely used as a cationic polymer for the preparation of the cationic PLGA particles.

In this study, a synthetic ORF5 gene (SynORF5) of PRRSV as a model vaccine (pcDNA3.1-SynORF5), which has been employed and demonstrated that it could significantly induce the humoral and cellular immunity responses, was absorbed onto the PLGA/PEI nanoparticles. Moreover, we prepared PLGA nanoparticle-adsorbed PRRSV DNA vaccine and PEI-DNA complex, respectively. The results showed that all the experimental groups could significantly enhanced GP5-specific ELISA antibody, PRRSV-specific neutralizing antibody, IFN-γ level, as well as lymphocyte proliferation than the responses induced by pcDNA3.1-SynORF5. Among the experimental groups, PLGA-branched PEI nanoparticle induced the most efficient immune responses. The delivery systems and adjuvant in the research provide new path for the development of the new generation of vaccines against PRRSV.
Unprecedented short-term in vivo protection in mice with a single immunization of a DNA GP vaccine against heterologous mouse-adapted Ebola virus challenge
Authors: Patel Ami , Racine Trina , Reuschel Emma L. , Karuppiah Muthumani , Shedlock Devon , Yan Jian , Khan Amir , Tierney Kevin , Qiu Xiangguo , Kobinger Gary P , Sardesai Niranjan , Weiner David B
Published: January 2023

The recent Ebola outbreak in West Africa is a reminder that correlates of protection against filovirus infection are still not well understood. DNA vectors are a serology-independent platform that allow for vector re-administration with minimal side-effects. We designed a new micro-consensus DNA vaccine that expresses a Zaire Ebolavirus (EBOV) glycoprotein (GP) based on 2002–2008 EBOV outbreak strains. This novel glycoprotein is 3% distant from the GP expressed by the 1976 EBOV Mayinga outbreak strain. The optimized GP DNA vaccine was administered in mice by intramuscular injection followed by electroporation (IM-EP) and elicited strong total IgG antibody and T cell responses. To assess protection from challenge, we administered the GP DNA vaccine or pVax1 control (40ug) to BALB/c mice (n=10/group) by IM-EP at days 0, -7, -14, or -28 before lethal challenge (1000LD50) with a mouse-adapted EBOV Mayinga strain. Importantly we observed 100% protection against the lethal heterologous challenge when the DNA vaccine was administered 28 days before challenge illustrating the potency of this vaccine in this model. More importantly, we observed 100% protection and 90% protection when the DNA vaccine was administered at days -14 and day -7, respectively. Most surprising was the observation of 40% survival in the group that received vaccine at Day 0 (2–3 hours) before challenge. Rapid, short-term protection has been previously observed with a VSV-ZEBOVGP viral vector vaccine but never before with a DNA vaccine. The data suggests that protection in mice following administration of a heterologous DNA vaccine can be afforded by mechanisms independent solely of antibody titers. Ongoing studies are underway in NHPs to investigate these findings.
Vitamin A deficient mice are highly susceptible to Streptococcus pneumoniae and exhibit a poor response to vaccination with Prevnar-13
Authors: Penkert Rhiannon , Hrincius Eike , Smith Amanda P , Smith Amber M , Rosch Jason , Hurwitz Julia L
Published: January 2023

Streptococcus pneumoniae (pneumococcus) is frequently found in the human respiratory tract and can cause a wide range of diseases from mild otitis to fatal pneumonia or meningitis. It is estimated that pneumococcus kills 1.6 million people each year, approximately half of whom are children in developing nations. Given that vitamin A deficiency (VAD) occurs in both developed and developing countries, we sought to explore the relationship between VAD and pneumococcal disease. Previously, we have shown that mice with VAD fail to clear respiratory virus infections efficiently, resulting in a prolonged state of inflammation. Here we show that mice with VAD are more likely to develop a severe lung infection following intranasal infection with pneumococcus compared to vitamin A sufficient controls. We also found that VAD increases morbidity and mortality from secondary pneumococcal infections in influenza-infected mice. This increased sensitivity to pneumococcal infections led us to investigate the effectiveness of the human pneumococcal vaccine Prevnar-13 (PCV) in the context of VAD. Vaccinated mice with VAD mounted only minimal IgM and IgG antibody responses toward PCV compared to controls. Upon challenge with an invasive strain (TIGR4) of pneumococcus, vaccinated control animals were nearly 100% protected from disease with no detectable bacteremia at 24 hours post-infection, whereas nearly 1/3 of vaccinated VAD animals developed bacteremia within 24 hours and ultimately succumbed to the infection. Our results suggest that clinical analyses of children in both developed and developing countries should be conducted to ensure that PCV successfully protects against pneumococcal disease in individuals with VAD.
A novel therapeutic vaccination delays cART-resistant HIV-1 reservoir rebound in HIV-1 infected humanized mice
Authors: Cheng Liang , Li Guangming , Li Dan , Zhang Zheng , Li Feng , Zurawski Sandra , Zurawski Gerard , Levy Yves , Su Lishan
Published: January 2023

Highly active anti-retroviral therapy (HAART) prevents disease progression but does not eradiate HIV, and this therapy often does not fully restore HIV patients’ immune status. Thus, there remains an unmet need to develop new stratagies to “cure” (eradication or control of HIV-1 with no ART) individuals of HIV infection. Modulating host’s cellular immune response is a promising way to cure HIV infection. We have developed a prototype vaccine with a string of 5 highly conserved T cell epitope rich regions of HIV-1 Gag, Nef and Pol fused to a monoclonal antibody that bind the antigen presenting cell activating receptor CD40(aCD40.HIV5pep). Here we use the humanized mouse model to evaluate the therapeutic effect of the aCD40.HIV5pep vaccine to chronic HIV infection. We first screened a batch of novel TLR adjuvants which have the potential to enhance the immunogenicity of aCD40.HIV5pep in vivo and showed that when vaccinated with Poly I:C as adjuvants, aCD40.HIV5pep can induce human CD8+ CTL response in humanized mice. Interestingly, we found that Poly I:C treatment can activate cART-resistant HIV-1 reservoir in in the presence of cART in chronically infected humanized mice. Finally, we showed that Poly I:C plus aCD40.HIV5pep therapeutic vaccination significantly delayed HIV virus rebound in HIV-1 infected humanized mice after withdraw of cART treatment. Our proof-of-concept study in humanized mice has significant implication for the development of therapeutic vaccine to cue HIV infection.
IGHV and IGHD encoding antibodies with exceptionally long CDR3H are predominant in the bovine neonatal B cells
Authors: Kaushik Azad Kumar , Pasman Yfke , Merico Daniel
Published: January 2023

We sequentially analyzed 18 RNA-seq libraries from three newborn calves as compared to their dams to understand the development of neonatal humoral immunity. A significant shift in neonatal transcriptome occurs within first week of life post-birth. A set of 110 differentially expressed immune-related genes is noted at birth that include complement, MHCII, chemokine receptors, defensins and cytokines etc. As compared to adults, IGKC is expressed at higher levels in the neonates, despite predominant IGLC expression at birth. The IGHM and IGHD transcripts are expressed at birth and IGHM achieves adult levels by day 7. This is followed by IGHA and IGHG transcripts 14–28 days post birth. Interestingly, most 3′ IGHV1S1(BF4E9) gene, known to encode antibodies with exceptionally long CDR3H, is preferentially expressed in neonatal B cells. In parallel, single longest IGHD2(DH2) gene that encodes such an atypical CDR3H is expressed at high levels at birth. It seems that IGHV1S1(BF4E9) gene has an important functional role in the development and expansion of bovine neonatal B-cell repertoire. To conclude, the bovine neonate acquires humoral immunocompetence by four weeks of neonatal life. [Supported by NSERC Canada Discovery Grant]
Indelible labeling and tracking of influenza virus infected antigen presenting cells
Authors: Langlois Ryan A , Fiege Jessica K
Published: January 2023

Influenza A virus is a seasonal pathogen with the potential to unpredictably cause catastrophic pandemics. The virus primarily replicates in epithelial cells of the upper and lower respiratory tract but can also infect a spectrum of other cell lineages, including cells of the immune system. Classically infected cells have been tracked through detection of virus products (RNA or protein) or through virus-derived reporters. However, these methods cannot define any potentially remaining infected cells after active replication has ceased. Using a novel virus expressing Cre recombinase to indelibly label infected cells in reporter mice, we unexpectedly found that epithelial cells were capable of surviving influenza virus infection long-term (Heaton and Langlois et al J. Ex. Med. 2014). We have subsequently discovered that infected CD45+ immune cells are also capable of surviving acute influenza virus infection in vivo. Importantly this virus-reporter system is able to distinguish between immune cells that are directly infected versus those that acquire virus protein/antigen exogenously. We find that both dendritic cells and macrophages, but not B cells, are directly infected within the lung and that the number of these cells increases during the acute infection phase. However, infected dendritic cells and macrophages do not persist in the lungs nor do they migrate systemically. Together these data demonstrate a new tool to track infected cells and defines the migration, turnover, and survival of infected antigen presenting cells.
Influenza A virus specific and Epstein-Barr Virus cross-reactive CD8 T cells during acute symptomatic flu infection.
Authors: Mishra Rabinarayan , Gil Anna , Aslan Nuray , Luzuriaga Katherine , Selin Liisa K
Published: January 2023

Previously we have shown expansion of cross-reactive CD8 T cells between Epstein-Barr Virus (EBV) and Influenza A virus in HLA-A2+ patients during infectious mononucleosis. During acute flu infection expanded IAV-M1-GIL58–66 specific CD8 T cells and IAV-M1-GIL58–66/EBV-BRLF-1-YVL109–117 and IAV-M1-GIL58–66/EBV-BMLF1280–288-GLC double-tetramer+ CD8 T cells were detected directly ex-vivo in 5 HLA-A2+ patients. IAV-M158–66, EBV-BRLF1119–117 and -BMLF1280–288 TCR repertoires changed over the course of infection and in comparison to healthy donors. After three weeks culture with Flu-M1 and EBV-BMLF1 ad EBV-BRLF1 peptides, CD8 T cells were sorted and analyzed by gene array in order to assess large-scale changes in immune responses in cognate or cross-reactive T cell populations in different patients. Global expression profile indicates cognate M1-GIL58–66 and BRLF1-YVL109–117 specific cells had similar immune-response gene signatures, but the EBV-BMLF1280–288 -GLC specific CD8 cells were more similar to the crossreactive populations. Crossreactive M1-GIL58–66/BRLF1-YVL109–117 cells from the BRLF1-YVL line were different in their activation status than the cognate BRLF1-specific cells, consistent with BRLF1-YVL ligand stimulation of different gene activation profiles in these two populations. These results indicate that during symptomatic flu infection there is an expansion of IAV/EBV crossreactive memory CD8 T cell responses. Ongoing studies are investigating whether EBV-IAV cross-reactive CD8+ T cells may contribute to immunopathology during acute IAV infection (NIH/NIAID PO1 AI 049320).
Identification of the immunological parameters that associate with severe influenza disease or recovery in mice
Authors: Forero Adriana , Nishida Andrew , Steuerman Yael , Gat-Viks Irit , Katze Michael G. , Morrison Juliet
Published: January 2023

The severity of influenza virus infection is largely determined by the interplay between the virus and the host. Understanding the pathological drivers of influenza A viruses serves to rapidly characterize the host response to newly emerging influenza viruses and derive prognostic signatures of disease severity. We implemented a differential network approach to uncover novel distinctions between the global transcriptional host response to highly pathogenic and low pathogenic H1N1, H7N9, H7N7, and H5N1 influenza A infections in BALB/c mice. We identified crucial mediators of inflammatory, coagulation, and tissue repair responses that discriminate between disease outcomes. It has been demonstrated that these responses are largely impacted by the immune cell influx into the site of infection. We examined the contribution of resident and lung-infiltrating immune cell quantity and activation state to disease phenotypes by implementing digital cell quantification (DCQ), which allowed us to model the in vivo dynamics of immune cells across viral strains based on time and strain-dependent infected lung transcriptional profiles. Through linear regression modeling, we revealed that distinct monocyte, granulocyte, DC, and T cell populations predict disease morbidity. Additionally, we demonstrated that both the kinetics of CD8+ T cell infiltration and the extent of their activation state, serve as a key predictor of immunopathological consequences of influenza infection. Overall, we provide a multidimensional analysis that delineates how innate immune responses relate to aberrant tissue repair responses and drive subsequent host adaptive immune response that control the severity of influenza virus disease in the murine model.
Prior administration of staphylococcal enterotoxin B augments the CD8+ T cell response to influenza A virus.
Authors: Meilleur Courtney Erin , Memarnejadian Arash , McCormick John K. , Haeryfar S.M. Mansour
Published: January 2023

Staphylococcal enterotoxin B (SEB) is a bacterial protein capable of binding up to 20% of the entire T cell repertoire. T cells that have come into contact with SEB initially proliferate and secrete inflammatory cytokines; however, this is followed by anergy or death of the affected cells. This population of deleted or functionally inactivated cells is likely to include T cell clones that are important for immunity to other pathogens such as influenza A virus (IAV). For this reason, we hypothesize that SEB exposure will alter the magnitude and breadth of the CD8+ T cell response to IAV. Using mouse model of intraperitoneal vaccination with IAV, we found that prior exposure to SEB actually increased the number of IAV-specific CD8+ T cells for several IAV epitopes during the primary phase of the immune response. The number of cells specific for these epitopes remained elevated even after the peak of the primary CD8+ T cell response had passed. In addition, repetition of the first experiment with several different naturally circulating and lab-adapted strains of IAV gave similar results, showing that the effect of SEB on virus-specific CD8+ T cells is the same for a potentially diverse range of IAV strains. Furthermore, the ability of the CD8+ T cells to kill target cells pulsed with cognate IAV antigen was also enhanced after SEB exposure, for both primary and memory CD8+ T cells. Curiously, we did not observe an effect of SEB when it is given in the context of a replicative IAV infection in the lungs. This work reveals an unexpected role for SEB as an enhancer of CD8+ T cell immunity in this model.
Can the common cold protect you from influenza? A murine model of respiratory viral co-infection.
Authors: Gonzalez Andres , Clary John , Rodgers Jade , Rodriguez Anna , Miura Tanya
Published: January 2023

Clinical cases demonstrate that up to 30% of respiratory infections involve more than one virus with rhinovirus being one of the most common in co-infections. Interestingly, rhinovirus has been found to exacerbate disease when co-infecting with adenovirus, whereas it can modulate disease when co-infecting with influenza A virus. However, there has yet to be a clear connection for how these viruses interact in the host to alter immune responses and disease severity. We are using a murine model of respiratory viral co-infection to determine how minor group rhinovirus (RV1B) affects pathogenesis during influenza A virus (PR8) infection. BALB/c mice were infected with RV1B or mock-infected two days before infecting with a high (100% lethal) or low (40% lethal) dose of PR8. Mice co-infected with RV1B and low dose PR8 had reduced clinical signs, weight loss and mortality compared to mice infected with PR8 alone. However, RV1B did not reduce disease severity upon infection with a high dose of PR8. Groups of mice were infected with RV1B or mock-infected two days prior to infection with low dose PR8 and were sacrificed 4 and 7 days later to quantify PR8 in the lungs. Single- and co-infected mice had similar PR8 titers on day 4; however, the co-infected group completely cleared PR8 on day 7 while the single infected group still had detectable titers. These results suggest that rhinovirus stimulates an immune response that promotes viral clearance and recovery upon infection with influenza A virus. Ongoing work will characterize this response to explain how respiratory viral co-infections can manipulate host immune responses to change disease outcome.