bluuebunny/crossref_metadata_2025 huggingface dataset:
Linking human health effects to water quality using a rapid, noninvasive salivary-IgG antibody multiplex immunoassay
Authors: Augustine S. Jason , Eason Tarsha N , Griffin Shannon M , Simmons Kaneatra J , Sams Elizabeth A , Oshima Kevin H , Dufour Al , Wade Timothy J
Published: January 2023

Understanding and curbing the mounting global increase in waterborne pathogen outbreaks are quickly becoming major priorities for public health professionals and policy makers. According to the World Health Organization (WHO), 60% of global diarrheal deaths are caused by unsafe water and lack of sanitation or hygiene. The WHO estimates that at least 2 billion people use a drinking water source contaminated with feces. Contaminated water -- shown to transmit diseases such as cholera, dysentery, typhoid, hepatitis A, and polio -- is estimated to cause almost half a million diarrheal deaths each year. The United States Environmental Protection Agency has prioritized efforts to understand the links between water quality and human health effects. To that end, we have developed and applied a salivary-IgG antibody multiplex immunoassay to measure human exposures and associated health effects to multiple pathogens simultaneously. Saliva is emerging as a cost-effective, noninvasive biofluid that is well-accepted by children. The multiplex immunoassay has afforded the ability to assess immunopositivity, immunoprevalence, co-infections and incident infections (immunoconversions) to Helicobacter pylori, Campylobacter jejuni, Cryptosporidium parvum, Toxoplasma gondii, hepatitis A virus and noroviruses GI.I and GII.4 at several beaches throughout the US. Further, we’ve found evidence of asymptomatic norovirus and hepatitis A infections in visitors to a fecally contaminated beach. The assay produces results in as little as one hour and when used in conjunction with epidemiologic and water quality studies, provides valuable information that links human health effects more directly to water quality.
Phage display technology and molecular simulation combination to characterize anti-Opisthorchis viverrini antibodies
Authors: Siripanthong Sitthinon , Techasen Anchalee , Nantasenamat Chanin , Malik Aijaz Ahmad , Sithithaworn Paiboon , Leelayuwat Chanvit , Jumnainsong Amonrat
Published: January 2023

KKU505 hybridoma cell was used to produce monoclonal anti-Opisthrochis viverrini (OV) antibody. Unfortunately, hybridoma technology has many problems with instability for antibody production. Many studies have shown the possibility of developing a monoclonal antibody production using the antibody phage display. This paper presents antibody phage display technology for producing the phage display of the KKU505 Fab fragment and characterization using experiments and molecular simulations.

The characterization was successfully displayed on KKU505 Fab fragment phage particle using experimental means integrated with the molecular simulation. For the experiments, indirect ELISA was shown to be specific to the OV antigen in this KKU505 Fab fragment on the phage particle. Immunohistochemistry has been shown to have a localization and reaction pattern of phage presenting KKU505 Fab fragment with biliary mouse tissue infected with O. viverrini. In particular, the significance of the antibody-antigen dock was studied in order to gain a better understanding of the mechanisms underlying the KKU505 mAb and of the crude OV antigen interaction by Western blot.

All results showed that the fragment KKU505 Fab displayed on the phage particle has specific to the OV crude antigens. This study has shown that high potential phage displays technology shows monoclonal antibody production development. We have shown how the interactions of the KKU505 Fab fragment and OV antigen are evaluated in a successful application of experiment and molecular simulation. This fragment of the KKU505 Fab display on the particle is being developed to be used as a tool for diagnosing human opisthorchiasis.
Robust T- and B-cell clonotype detection using simultaneous targeted mRNA and immune repertoire profiling of single cells
Authors: Acob Ricelle A , Truong Tina , Levine Jay , Liu Kai , Jensen Devon , Prosen Dennis , Lazaruk Katherine
Published: January 2023

The key cell types contributing to the diversity of the immune repertoire are the T cells, with their T-cell receptor (TCR) and B cells with their B-cell receptor (BCR). During T- and B-cell development, the cell undergoes recombination of the germline gene segments, variable (V), diversity (D), and joining (J), also called V(D)J recombination. We developed a single cell clonotype detection assay for the BD Rhapsody™ Single-Cell Analysis System capable of processing thousands of single cells at once. We aimed to capture ~20,000 peripheral blood mononuclear cells from each individual donor (total of five) in a BD Rhapsody™ cartridge and took a sub-set of ~3,000 cells for immune repertoire profiling analysis of the complementarity determining region 3 (CDR3) of the variable regions of TCRα/TCRβ and the IgG heavy/light chains. The BD Rhapsody® Human Immune Response Panel for targeted mRNA gene expression analysis was used to classify the lymphocytes and monocyte populations. Results demonstrate that the BD Rhapsody™ VDJ assay can detect clonotype expression differences among different donors with >80% sensitivity and specificity. The abundance of TCR and BCR transcripts was determined by the addition of a unique molecular index to each mRNA molecule. Overall, the current technology demonstrates the ability to evaluate immune populations using single cell next generation sequencing. This assay could be applied not only to study the immune repertoire but to understand the biology of immune-related diseases and immune response to therapies.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.

BD, the BD Logo and Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2019 BD. All rights reserved.
Gentle cell sorting of immune cells for downstream single-cell RNA-seq
Authors: Jagnandan Nicole , Morachis Jose
Published: January 2023

Cell sorters have played an instrumental role in the progression of immunological discoveries. The ability to isolate specific immune cell types from a heterogenous population while maintaining high viability is critical to the discovery and generation of disease therapies. To isolate fragile and rare immune cell types, a gentle low pressure cell sorter is necessary. In this study, regulatory T cells (Tregs) were sorted from peripheral blood mononuclear cells using the WOLF® Cell Sorter developed by NanoCellect® and a traditional jet-in-air cell sorter. The isolated cells were then analyzed for single-cell RNA-seq using the 10x Genomics workflow. We also analyzed how different sorting methods affect the expression of stress markers induced during sorting. This study showed that despite the small percentage of Tregs in peripheral blood mononuclear cells, the WOLF Cell Sorter is able to obtain high purity, high viability, and high quality sequencing results. The WOLF Cell Sorter was also able analyze several subsets within the purified Treg populations in higher definition. Furthermore, fewer stress markers were induced when compared to the traditional jet-in-air cell sorter. This study provides novel insights into a robust and simplified workflow to use when isolating fragile immune cell types.
Label-free 3-D quantitative phase imaging cytometry with deep learning: identifying naive, memory, and senescent T cells
Authors: Ryu DongHun , Kim Kyunghwan , Cho Hayeon , Dao Khoi , Kim Young Seo , Ahn Daewoong , Min Hyun-Seok , Shin Eui-Cheol , Park YongKeun
Published: January 2023

The current prevailing methods for identifying immune cell subsets exploit a group of differentiation markers (CDs) targeted by fluorochrome or metal conjugated antibodies. However, such labeling methods, requiring a staining process and specific reagents, prevent rapid and cost-effective identification of immune cell subsets. Therefore we developed a label-free imaging cytometry platform that synergistically used refractive index (RI) tomography and three-dimensional (3-D) deep learning. We constructed and trained a deep learning classifier that learns unique representations from the 3-D RI map of each cell obtained using RI tomography without labeling. In this study, we were able to classify human naïve, memory, and senescent T cells according to the expression of CD4, CD8, CD45RA, CCR7 and CD57 using the label-free classifier within milliseconds, with high precision (>95%) even though the morphological and biochemical characteristics extracted from the RI tomograms of the T cells are almost homogeneous. This cannot be achieved by conventional machine learning approaches that only exploit the set of manually extracted features. Our label-free cell sorting platform will facilitate rapid and cost-effective immunological and biomedical studies by eliminating the laborious labeling process.
Adarza’s ZIVA multiplex protein detection platform demonstrates flexibility with custom targets for monitoring both inflammation and anti-drug antibody response
Authors: Schmuke Jon J , Pillers Michelle , Mueller Ernie , Cooper Matt , Carter Alun , Kabakibi Ayman
Published: January 2023

We have used Arrayed Imaging Reflectometry (AIR™), a label-free protein microarray technology that relies on the target binding-induced perturbation of an antireflective coating on the surface of a silicon chip, to create a multiplex antibody array. ZIVA is a new multiplex protein detection platform that leverages AIR™ technology. Understanding the immunogenicity of a therapeutic protein is critical to the development of bioactive stability and efficacy. ZIVA technology can be customized to monitor for multiple biomarker types including markers of inflammation as well as specific ADA targets. In this example, biosensors were custom printed to monitor for the presence of an anti-drug antibodies generated against a single chain antibody presented on the surface of engineered CAR-T cells. Samples that are positive for ADA response may also be evaluated for presence of neutralizing antibodies (NAB) by evaluating specific controls also included on the biosensors. With array capabilities from low to hundreds of plex combined with a hands-free, automation-friendly workflow enabled by the ZIVA instrument, the technology has been designed for the ultimate user-experience.
High-throughput multi-dimensional single antigen-specific T cell profiling
Authors: Jiang Ning , Ma Ke-Yue , Schonnesen Alexandra A , Xia Amanda , Fan Yubo
Published: January 2023

Recent advances in single cell analysis has made it possible to interrogate the transcriptome of single cells in high-throughput manner. However, multi-dimensional profiling of antigen-specific T cells at single cell level for hundreds of antigens has not been realized, which becomes a bottleneck to the understanding of these important cells in immunological diseases.

Here we describe a new technology, TetTCR-SeqHD, that extends our previously developed technology, TetTCR-Seq for Tetramer associated TCR sequencing, to additional two dimensions, single cell gene expression and phenotype. In addition, we expand the throughput to tens of thousands of single T cells. We achieved these by using DNA-barcoded pMHC tetramers and antibodies in combination with single cell gene expression analysis, thus, paralleling all read-outs using next-gen sequencing.

To validate the technology, we have used previously generated polyclonal T cells to 6 different antigens with known TCR sequences. This analysis showed that we could achieve less than 5% on false discovery rate. We then used TetTCR-SeqHD on a panel of type 1 diabetes related peptides and showed unexpected features of antigen-specific T cells that separate healthy individuals from type 1 diabetes patients. In addition, TCR transgenics T cells confirmed their antigen specificity.

In summary, TetTCR-SeqHD enables us to analyze antigen-specific T cells with an extraordinary depth and breadth, which enables discoveries directly in humans. TetTCR-SeqHD would be a useful technology for studying many different diseases where screening for T cells antigens and deep profiling of antigen-specific T cells are highly sought after.
Donor deubiquitinase OTUD1 deficiency attenuates the severity of aGVHD mice after allo-HSCT by suppressing T cell activation and expansion
Authors: Cheng Qiao , Liu Yin , Wang Dong , Zhang Wenli , Xu Yang , Wu Depei
Published: January 2023

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an important means to treat malignant diseases currently. However, the occurrence and development of acute graft versus host disease (aGVHD) seriously affect the survival and prognosis of patients. aGVHD is mainly mediated by the allogeneic immune response of activated donor T cells. Ubiquitination plays essential roles in T cell activation and is counteracted by multiple deubiquitinases (DUBs) with poorly understood. Here we show that OTUD1 deficiency negatively regulates the activation and effector function of naïve CD4+ T cells in vitro, and the proliferation of CD4+ T cells was also suppressed. Moreover, we found that OTUD1-deficient donor CD4+ T cells proliferates less in recipients compared to OTUD1+/+CD4+ T cells transferred. Interestingly, we found that OTUD1 expression was up-regulated in allo-HSCT mice when compared with syngeneic hematopoietic stem cell transplantation (syn-HSCT) mice. We further documented donor OTUD1 deficiency significantly prolongs the survival of aGVHD mice, and decreases the aGVHD scores. Moreover, OTUD1-deficient donor CD4+ T cells also attenuate the severity of aGVHD mice. Associated mechanistic studies revealed that the frequency and activation of CD4+ T cells were significantly reduced in recipients receiving OTUD1−/− bone marrow (BM) cells and splenocytes compared to those transferred OTUD1+/+ BM cells and splenocytes. Furthermore, overexpression of OTUD1 promotes NFAT-luciferase activity and up-regulates CD3-TCR complex stability. These results yield that targeting OTUD1 to regulate T cell-mediated allogeneic responses may be an effective therapeutic strategy to prevent and control aGVHD.
The role of TCF-1 in graft-versus-host disease and antitumor effects
Authors: Harris Rebecca , Karimi Mobin
Published: January 2023

Allogeneic hematopoietic stem cell transplants (HSCT) are used as a curative therapy for malignancies such as cancer. Residual tumor cells and recurrence are controlled by the graft-versus-tumor (GVT) effect, mediated by alloreactive T cells. However, these same cells also cause graft-versus-host disease (GVHD), a serious side effect. Naïve mature T cells in the graft recognize foreign HLA molecules on the host’s cells, and are alloactivated. The T cells then proliferate, produce cytokines, and migrate to the GVHD target organs (liver, skin, and small intestine), damaging host tissues. Separation of detrimental GVHD from beneficial GVT remains a serious unaddressed problem. Thus, the goal of this project is to achieve this separation through T cell signaling modulation.

The transcription factors T-bet and Eomes impact GVHD and GVT. They are downstream of T Cell Factor-1 (TCF-1), which is important in thymocytes and T cell activation. To determine the role of TCF-1 in alloactivation, mice with T cell-specific deletion of TCF-1 were phenotyped. These mice had increased expression of Eomes, Tbet, CD8+ central memory T cells, and polarization of CD44 expression. They also showed reduced activating T cells, and reduced expression of CXCR5 (a major chemokine receptor) when alloactivated. Finally, GVHD severity was reduced in mice transplanted with TCF-1-deficient T cells, and survival was improved in these mice. These data suggest that loss of TCF-1 results in a more activated state, yet GVHD is still reduced. Therefore, these cells may still be capable of antitumor immunity, separating GVHD from GVT. This work will help to improve outcomes for HSCT patients and enhance understanding of how mature alloactivated T cells are regulated.
Effect of CD3/CD28 activator on the cytotoxicity of human ex-vivo expanded γδ T cells against osteosarcoma cells
Authors: KO YUNMI , Lee Jun Ah
Published: January 2023

Background

We investigated the effect of CD3/CD28 activator on the ex-vivo expansion of human γδ T cells and their cytotoxic effect against osteosarcoma cells.

Methods

Peripheral blood mononuclear cells from healthy donors were culture in three different conditions for 10 days: basal expansion media (COM media), COM media with zoledronate+hrIL-2 (EX media), and EX media with CD3/CD28 activator (EX28 media). Percentage of expanded γδ T cells was analyzed by flow cytometry. IFN-γ and TNF-α secreted in the culture media was measured using ELISA assay. Cytotoxicity of γδ T cells against KHOS/NP cells was measured by MTT assay.

Results

Compared with COM media, the fold increase of cell expansion were 33.6 in EX media and 368.2 in EX28 media. The population of γδ T cells expanded in COM media represented 4.9% of CD3+ T cells, while γδ T cells expanded in EX media reached 71% of CD3+ T cells. The population of γδ T cells expanded in EX28 media represented 18% of CD25+ T cells, but not CD3+ T cells. The concentration of IFN-γ and TNF-α in the culture media increased both in EX (1000-fold, 340-fold) and EX28 (2340-fold, 1849-fold) media, compared with unstimulated control. Unexpectedly, IFN-γ and TNF-α production per cells were higher in EX media than in the EX28 media. The γδ T cells expanded in EX media showed potent cytotoxicity against KHOS/NP osteosarcoma cells in a ratio-dependent manner, but γδ T cells expanded in EX28 media did not show significant cytotoxic effect.

Conclusion

CD3/CD28 activator were not as potent as the combination of zoledronate and hrIL-2, for expansion γδ T cells nor its cytotoxic activity against KHOS/NP cells, although it increased the total number of T cells.
Combination therapy to enhance NK cell anti-tumor responses
Authors: Wolf Natalie K , Nicolai Chris , Dang Susanna , Synder Gail , Blaj Cristina , McWhirter Sarah , Picton Lora , Garcia K. Christopher , Raulet David H
Published: January 2023

Current cancer immunotherapies are targeted at mobilizing CD8 T cells, although numerous tumors have few or no antigens for CD8 T cells, or have lost MHC I, thereby evading CD8 T cells. There is therefore a need for improved therapies that mobilize other immune cells, such as NK cells, to the tumor microenvironment. A recent therapy candidate, cyclic dinucleotide (CDN), activates the cGAS-STING pathway of the innate immune system. Intratumoral CDN injections induce type I IFNs and other mediators that amplify the CD8 T cell response and induce regressions of primary tumors. MHC I-deficient tumors are not susceptible to CD8 T cell-mediated rejection, but data from our lab demonstrates that STING activation still leads to long-term tumor regressions, mediated by NK cells and in some cases CD4 T cells, in 30–100% of animals in 5/6 transplant models tested. In order to improve upon CDN monotherapy, we are testing combinations of CDN therapy with other therapies to target and activate NK cells in the tumor microenvironment and prevent or delay the onset of desensitization. Our results show that engineered IL-2 family cytokines synergize with CDN therapy to mobilize anti-tumor responses by NK cells and in some cases CD4 T cells. These anti-tumor responses are due to increased recruitment of NK cells to the tumor and sustained activation of these recruited NK cells. Our combination therapy regimen shows enhanced systemic effects, both in vivo in abscopal tumor models, and ex vivo by cytolysis of target cells mediated by splenocytes. Overall, our work demonstrates the impact of a novel combination therapy in mobilizing powerful NK cell-mediated anti-tumor activity, providing evidence for the potential of NK cell-targeted immunotherapeutics for cancer.
CD122-selective IL-2 complexes target γδ T and NK cells to reduce tumor-promoting Th17 effects and synergize with αPD-L1 to treat primary and metastatic bladder cancer
Authors: Reyes Ryan Michael , Deng Yilun , Zhang Deyi , Mukherjee Neelam , Ji Niannian , Wheeler Karen , Gupta Harshita B. , Garcia Myrna , Kornepati Anand , Svatek Robert S , Curiel Tyler J
Published: January 2023

αPD-L1 bladder cancer (BC) immunotherapy is effective in <30% of cases. To address the large αPD-L1-unresponsive subset of patients, we tested αIL-2/IL-2 complexes (IL-2c) that block IL-2 from binding high-affinity IL-2Rα (CD25) for preferential IL-2Rβ (CD122) binding. Regulatory T cells (Tregs) capture IL-2 by CD25 whereas CD8+T, γδ T, and NK cells use CD122. We hypothesized that the tumor microenvironment, including local immune cells in primary versus metastatic BC, differentially affects immunotherapy responses. We used PD-L1+ mouse BC cell lines MB49 and MBT-2, for intravesical ([IVe] in bladder) and intravenous (IV) challenge studies of local versus metastatic BC. αPD-L1 or IL-2c alone reduced tumor burden and extended survival in IVe MB49 and MBT-2. Treg depletion using FOXP3DTR mice further enhanced IVe IL-2c effects, consistent with the known tumor-promoting role of Tregs in human BC. Using in vivo cell depletion approaches, we found that γδ T cells and NK cells, but not CD8+ T cells, were necessary for IL-2c efficacy in bladder. γδ T cells also reduced intratumoral Th17 cells that promote MB49 growth and are elevated in human BC. We confirmed γδ T cell effects in δ TCR KO mice, which abrogated IL-2c efficacy but not αPD-L1 efficacy. Neither αPD-L1 nor IL-2c alone treated metastatic MB49 and MBT-2 BC but the combination improved survival in both. These data are consistent with our recent findings in human BC patients in whom γδ T cell and NK cell cytotoxicity improved BCG immunotherapy. Thus, IL-2c is a promising novel BC immunotherapy that can improve bladder-specific immunity in primary BC. In metastatic BC, combination with αPD-L1 may also be a successful BC treatment strategy due to engagement of innate and adaptive immune responses.
Mitofission impairs the anti-tumor activity of NK cells in tumor microenvironment
Authors: Zheng Xiaohu , Sun Rui , Tian Zhigang , Wei Haiming
Published: January 2023

The survival and functional performance of NK cells is a metabolically demanding process. However, there is largely unknown how inappropriate metabolic reprogramming underlies disease microenvironments leads to NK dysfunction. Here, through analysis of human liver cancer samples (n=102) and benign liver samples (n=22), we found tumor infiltrative NK (TINK) cells present small and fragmented mitochondria in the cytoplasm, while normal liver NK and peripheral NK cells have big and tubular mitochondria. We also observed hypoxic tumor microenvironment is the main factor to induce NK cell excessive mitochondrial fission into fragmentation and that dependent on increasing Drp1 activity. Consequently, the fragmentation causes the metabolic disturbance and losing of TINK cells and that predicts poor survival in liver cancer patients. Revising mitochondrial morphology by targeting Drp1 improves mitochondrial metabolism and anti-tumor capacity of NK cells. Thus, disease-related mitochondrial restructuring drives NK cell exhaust and that is manipulated to the benefit of temper or enhance immunity.
Interactions of natural killer cells and pancreatic cancer cells in simulated microgravity
Authors: Gregg Randal K , Falwell Harrison , Bludorn Grant , Laken Jordan A , Boos William F , Arnold Jennifer P , Bradley Jillian H
Published: January 2023

One of the major concerns of long-duration spaceflight is the exposure of astronauts to space radiation and the potential for cancer development. Natural killer (NK) cells continuously survey the body for such abnormalities and upon detection are activated to produce pro-inflammatory cytokines as well as cytotoxins which induce apoptosis of the cancer cells. This study employed the rotary cell culture system to generate a simulated microgravity (SMG) environment in order to assess its effect upon the growth of human cancer cells and their susceptibility to lysis by NK cells. We found that SMG diminishes the numbers of both pancreatic cancer cells and NK92MI, a human NK cell line, over a 7 d culture period. Additionally, SMG NK92MI cells had a ~50% decrease in cytotoxicity against PANC-1 pancreatic cancer cells. Surprisingly, a long-term SMG culture of up to 14 d resulted in complete restoration of NK92MI cell cytotoxic function. Interestingly, SMG cultured NK92MI cells produced more IFN-gamma than Static NK cells throughout the time periods assessed. In comparison, 7 d culture of pancreatic cancer cells in SMG led to a ~50% reduction in the ability of either Static or SMG NK92MI cells to induce apoptosis in the SMG cultured cancer cells. Therefore, over a two-week period in SMG culture, NK92MI cells were able to adapt to the environment and recover cytotoxic function, however, pancreatic cancer cells cultured in SMG acquired resistance to lysis by the NK cells. Hence, cancer cells developing in astronauts in microgravity might have a greater potential to resist NK cell control than in patients here on Earth which upon elucidation may aid in the identification of relevant new tumor targets for therapeutics.
Interferon associated adaptive immune resistance represented by immune checkpoint expression and immune cell localization in high-grade serous ovarian cancer
Authors: Li Deyang , Vidotto Thiago , Shakfa Noor , Asante Afrakoma-Afriyie , Peterson Nicole , de Ladurantaye Manon , Mes-Masson Anne-Marie , Francis Julie-Ann , Koti Madhuri
Published: January 2023

High-grade serous carcinoma (HGSC) is a deadly malignancy leading to ~70% of the 140,000 ovarian cancer deaths globally each year. Resistance to platinum chemotherapy followed by recurrence and an incurable disease occurs in most HGSC patients. Contemporary immune checkpoint blockade therapies have shown minimal efficacy in this cancer. Our previous investigations established that tumour interferon (IFN) activation status and CD8+ T cell density are predictors of chemotherapy response in HGSC. Furthermore, we also showed that IFN induced chemokine CXCL10 is a key determinant of increased survival via immune cell recruitment in the tumour immune microenvironment (TIME). Given that signal transducer and activator of transcription 1 (STAT1) is central to the feed forward loops of cellular IFN responses, we investigated STAT1 associated transcriptomic alterations and spatial profiles of immune cells in 204 pre-treatment HGSC tumours.

RNA-sequencing based whole transcriptomic profiling revealed that higher STAT1 expression significantly correlated with higher immunomodulatory gene expression, including immune checkpoints and activators, in both chemotherapy sensitive and resistant tumours. Findings were independently validated in a cohort of 379 HGSC tumour RNA-Seq profiles from The Cancer Genome Atlas Network ovarian cancer dataset. Multiplex immunofluorescence based spatial profiling of CD8+ T cells, FoxP3+ T regulatory T cells, CD68+M1, and CD163+ M2 macrophages and expression of PD-L1, PD-1, IDO1 immune checkpoints was performed. Findings from our study provide evidence for IFN mediated adaptive immune resistance in the HGSC TIME and will potentially inform the design of rational chemo-immunotherapy approaches.
Virally delivered cytokine therapy drives immune equilibrium in melanoma
Authors: Walsh Michael , Lenehan Patrick , Ali Lestat , Dougan Michael , Knipe David , Dougan Stephanie K
Published: January 2023

Although a modified, replicating herpes simplex virus (HSV) encoding GM-CSF was FDA-approved for the treatment of melanoma (T-VEC), which was revolutionary for the field of oncolytic virotherapy, there are several areas for improvement. First, the live virus cannot be safely administered to immunocompromised patients. Second, GM-CSF induces immune-suppressive myeloid cells and is likely not the optimal insert. To this end, our lab has used a non-replicating HSV virus, termed d106S, to serve as a safe viral vector for the treatment of cancers. We have repurposed the non-lytic HSV-1 d106S vaccine vector for local delivery of IL-12. Our replication-defective d106S virus releases a large burst of IL-12 locally within the tumor environment, synergizing with a type I IFN response induced by the virus. We have shown that d106S-IL12 induces regression and long-term stable immune equilibrium in murine B16 melanoma. These results are promising, demonstrating that the d106S vector can deliver immunotherapeutic cargo and induce shrinkage of established tumors. Intriguingly, most mice do not fully clear their tumors but establish an equilibrium phase and withdrawal of therapy eventually leads to tumor outgrowth. We have profiled the immune response induced by d106S-IL12 at separate time points, identifying several points of possible intervention, including blockade of innate inflammatory cytokines IL-1β, TNFα and IL-6. Indeed, blockade of each of these inflammatory cytokines provides a boost to the response initiated by d106S-IL12, which we have further profiled with single-cell RNA-sequencing.
A flagellin-adjuvanted cancer vaccine combined with photodynamic therapy enhanced anti-tumor immune response in B16-F10 melanoma cancer model
Authors: Hwang Hye suk , Lee Shee Eun , Rhee Joon Haeng
Published: January 2023

Immune checkpoint inhibitors have now become a standard therapy for malignant melanoma. However, immune checkpoint inhibitor monotherapy has shown effective in only a limited number of patients. Photodynamic (PDT) and immune combined therapy is a promising modality for the treatment of metastatic melanoma. Herein, we report the combination therapy with PDT and flagellin adjuvanted- Tyrp1/Trp2/gp100 peptides based vaccination (Fla-vax) in mouse models of B16-F10 melanoma cancer. We used the bilateral tumor model to determine whether the Fla-vax could induce systemic anti-tumor effects. The combination therapy with PDT and Fla-vax sufficiently induced systemic anti-tumor immune responses for local and abscopal tumor control,, with a significant increase in the systemic IFNγ/TNFα secretion from cytotoxic T lymphocytes (CTLs) and tumor-infiltrated effector memory CD8+ T cells. It was also found that the combined therapy greatly induced the number of migratory CD103+ DCs which is secreting CXCL10. The therapeutic benefit of PDT and Fla-vax proved to be CD8+ T-cell dependent, and the antitumor activity was additive with that provided by programmed death-1 (PD-1)-targeted immunotherapy. These results suggest the combination of active immunotherapy with tumor ablation by PDT as a feasible novel treatment strategy for advanced melanoma cancer.
Innate immune cell modulation using cargo-less nanoparticles to ameliorate severe inflammation
Authors: Lasola Jackline Joy Martin , Kamdem Henry , McDaniel Michael , Pearson Ryan M.
Published: January 2023

1.6 million people are diagnosed with sepsis each year in the US with a mortality rate of 25-30%. Attempts to improve patient outcomes include strategies to abrogate the overexaggerated immune response seen in sepsis but have thus far failed. It has been hypothesized that these have collectively been unsuccessful as they target a single molecular pathway and do not address redundancies in immune activation pathways. We have shown that nanomaterials are inherently potent immunomodulators that can broadly reduce the release of inflammatory cytokines. As such, we aimed to understand the physical and biological mechanisms that underlie the macrophage response to nanomaterials in the setting of LPS stimulation. PLA-based particle formulations were prepared with similar sizes (~500 nm) and different charges depending on surfactant (−50 to −20 mV). Particles prepared with an anionic surfactant (PEMA) are more effective than those with a neutral surfactant (PVA) at inhibiting inflammatory cytokine secretion in a manner independent of NF-κB activation. Additional studies show that PLA-PVA particles colocalize with TLR agonist at the cell surface, while agonist localization was abolished in cells treated with PLA-PEMA particles. LPS-induced endotoxemia studies show that i.p. administration of PLA-PEMA particles prior to challenge with LPS significantly improved survival of mice compared to those challenged with LPS alone. This work contributes to the fundamental understanding of the physicochemical properties of biomaterials and their modulation of immune cell responsiveness towards activating stimuli and suggests that nanomaterials are novel immune modulators in cases of acute and potentially life-threatening inflammation.
In-silico based screening system to develop allosteric modulators of Bruton’s tyrosine kinase
Authors: Berthelette Alyssa , Kolli Nilima , Amatya Neha , Andreotti Amy , Berg Leslie , Kang Joonsoo , Reboldi Andrea
Published: January 2023

Ibrutinib is a covalent inhibitor that binds to the Bruton’s tyrosine kinase (BTK) ATP-dependent active site and it is currently used for treating a variety of B cell tumors. A high rate of drug resistance develops in Ibrutinib-treated patients through mutations at the BTK kinase domain, resulting in more aggressive diseases. Thus, there is significant interest in identifying modulators that inhibit BTK in an active site-independent manner, specifically via allosteric modulation. We used an in-silico system to identify small molecules that bind outside of the BTK active site. Candidate compounds were then tested for direct binding to full length BTK protein using Saturated Transfer Difference Nuclear Magnetic Resonance (STD-NMR) and were subsequently used in primary mouse B cell-based assays to determine their functional relevance. Ex vivo B cells were triggered via the BCR in the presence of compounds and signaling events downstream of the BCR, including Nur77 upregulation and calcium flux, were measured by flow cytometry. A series of candidate allosteric BTK inhibitors were then confirmed to retain suppressive activity in an ABC-DLBC lymphoma line that carries the Ibrutinib resistant mutation. Additionally, primary human CLL, but not healthy B cells, were susceptible to candidate inhibitor-mediated death. These results indicate that a screening approach integrating in silico docking, NMR measurements, and cellular assays with primary B cells can be used to identify BTK inhibitors that act outside of the kinase domain. Allosteric BTK inhibitors will broaden the range of treatment options for B cell cancers, possibly overcoming or preventing the Ibrutinib-induced resistance when used as a combination therapy.
Targeting of ITK-SYK fusion oncogene through in-silico generated and biologically validated ITK-PH domain modulators
Authors: Ferraj Enxhi , Kolli Nilima , Berthelette Alyssa , Frascoli Michela , Andreotti Amy , Berg Leslie J. , Reboldi Andrea , Kang Joonsoo
Published: January 2023

A subset of human Peripheral T cell Lymphomas, Not Otherwise Specified (PTCL-NOS), harbors a chromosomal translocation of interleukin-2 (IL-2) inducible T cell kinase (ITK) and spleen tyrosine kinase (SYK), giving rise to the composite ITK-SYK protein. ITK, a TEC family member kinase, plays an essential role in TCR and CD28 signaling. SYK signaling pathway is more broadly linked to activation of diverse lymphoid and myeloid cell types. ITK-SYK fusion protein consists of the PH domain of ITK and the SYK kinase domain allowing localization to the signalosome complex at the membrane via the PH domain and constitutive activity in T cells. Given the ubiquitous activity of SYK, it is predicted that targeting the ITK-PH domain of the fusion protein with small molecules can be a precise and less toxic approach to suppress ITK-SYK+PTCL-NOS in vivo. To this end, we performed a computational screen of small molecule libraries to identify candidate ITK-PH domain interacting compounds. A thermal shift assay was used to verify the specificity of binding of candidates to the target protein domain. Cellular assays tested small molecule activity in human and mouse T cells constitutively expressing the ITK-SYK fusion protein. We showed that the first-pass small molecule candidates specific to ITK-PH domain inhibit cytokine production driven by ITK-SYK fusion protein in human and mouse T cells. Our results demonstrate the efficiency of the virtual screening to identify drug candidates with biological activity in lymphocytes. Development of drugs that target ITK-PH domain is a rationale strategy towards first-in-class therapeutics for this subset of PTCL-NOS with very poor prognoses.
Host-directed therapy in foals reduces the severity of clinical pneumonia against Rhodococcus equi intrabronchial challenge
Authors: Bordin Angela Ilha , Giguere Steeve , Bray Jocelyne , Berghaus Londa , Hook Magnus , Scott Brenton , Johnson Rena , Cohen Noah
Published: January 2023

Pneumonia caused by Rhodococcus equi, an intracellular bacterium that infects macrophages, is an important cause of disease and death in immunocompromised hosts, especially foals. No vaccine is available, and control is based on antibiotic treatment of subclinical cases, increasing the pressure for emergence of antibiotic resistance and indicating novel control approaches are needed. TLR agonists have immunomodulatory function. Our first objective was to evaluate the ex vivo effects of treatment of neonatal foal alveolar macrophages (AMs) with a combination of TLR2 and TLR9 agonists (PUL-042). Using bronchoalveolar lavage (BAL), we collected AMs from 48 foals and determined the killing of intracellular R. equi using the ratio of CFUs at 48 hours (T48) to post-phagocytosis (T0) (i.e, T48:T0). Concentration of pro- and anti-inflammatory cytokines were measured in AM culture supernatant, BAL fluid, and serum. Compared to control, PUL-042 decreased TNF-a in BAL fluid but increased IL-6, IFN-g, and TNF-a in AMs after ex vivo infection with R. equi, suggesting that nebulization induced an immune response upon infection that is favorable for killing intracellular pathogens. We also evaluated the in vivo the efficacy of PUL-042 in reducing the severity of clinical pneumonia in foals after intrabronchial challenge with R. equi. Infected foals nebulized with PUL-042 had a shorter duration of clinical signs of pneumonia and of presence of lung abscesses when compared to foals that did not receive nebulization, consistent with our ex vivo findings. Our results show potential for enhancing neonatal immune responses against respiratory pathogens and reducing severity of clinical pneumonia following intrabronchial challenge with R. equi.
Rapid characterization of MHC class I diversity in desert bighorn sheep reveals population-specific allele expression
Authors: Dolan Brian P , Beechler Brianna R. , Dugovich Brian S. , Epps Clinton W. , Jolles Anna E.
Published: January 2023

Diversity of MHC class I alleles within animal populations is necessary to prevent pathogen escape from adaptive immune responses. However, demonstrating this phenomenon is difficult especially in wild animal species where the effect of pathogen escape is more profound than in laboratory animal models. Desert bighorn sheep (Ovis canadensis nelsoni) are a charismatic megafauna native to the southwest of North America which are threatened by a number of infectious diseases and, more recently, paranasal tumors. We sought to develop a relatively inexpensive and rapid way to identify the diversity of MHC class I genes and alleles within individual bighorn sheep. Both DNA and RNA were isolated from leukocytes collected from 154 adult bighorn sheep from across different mountain ranges in Southern California. Previously identified primers from domestic sheep were used to amplify exons 2 and 3 from OMHC I genes while appending adapters for PacBio circular consensus sequencing. PCR amplicons from individual animals were then subjected to a second round of PCR to append index sequences to allow assigning of individual sequences to unique animals. We successfully identified over 40 unique MHC class I sequences expressed by bighorn sheep. Between 2 and 5 loci were amplified in each individual animal. Analysis of gDNA revealed several pseudogenes and other DNA sequences which were not transcribed. Finally, we demonstrate that particular DNA sequences are found within particular populations, suggesting that as bighorn populations fragment, there is a loss of MHC class I diversity.
Assessment of immunocontraceptive vaccine for population control: evaluation of a recombinant fusion GnRH vaccine in mice
Authors: Chang Ai-Mei , Hou Ding-Liang , Chen Chen-Chih , Ke Guan-Ming , Lee Jai-Wei
Published: January 2023

Gonadotropin-releasing hormone (GnRH) plays a pivotal role in regulating the reproductive endocrine system. Accumulated lines of evidence indicated that GnRH immunocontraception vaccine is a potential tool for control and management of animal population. However, the poor immunogenicity of GnRH peptide has limited its efficacy and field application. In the present study, a modified antigen, containing 8 GnRH repeats and immunostimulatory fusion protein, was formulated an oil-based MONTANIDE™ ISA 206 VG adjuvant to enhance the immunogenicity of the GnRH vaccine. The vaccine was vaccinated in 4-week-old male ICR mice (n = 10) and boosted twice, 4 and 8 weeks, respectively after the primary immunization. Mice injected with saline served as the unimmunized control (n = 10). Results demonstrated that the serum GnRH-specific antibody in vaccinated mice was significantly (p < 0.05) increased 5 weeks, peaked at 12 weeks, and remained high until 18 weeks after the primary immunization. In comparison with the control, vaccinated mice had significantly (p < 0.05) reduced serum testosterone concentration (0.856 ± 0.411 v.s. 160.77 ± 1. 865 ng/ml) and hampered development of testicles (weight and spermatogenesis) at 24 weeks after the primary immunization. Finally, a mating test indicated that the conception rate of female mice housed with vaccinated and unvaccinated males was 29% and 75%, respectively. The recombinant GnRH vaccine may be a potential approach for controlling animal population through immunocontraception.
Resilience of High Immune Response (HIR) Genetics in the Context of Climate Change: Effects of Heat Stress on Cattle with Diverse Immune Response Genotypes
Authors: Mallard Bonnie A , Husseini Nasrin , Cartwright Shannon , Livernois Ali , Hodgins Doug , Altvater-Hughes Tess , Beard Shannon , Karrow Niel , Canovas Angela , Schmied Julie
Published: January 2023

As the temperature-humidity index (THI) increases, animals are less able to regulate body temperature leading to decreases in production and immune function. The High Immune Response (HIR™) technology classifies animals based on estimated breeding values (EBVs) of their IR capacity as high (H), average (A), or low (L). H-responders with their unique IR genotype have less disease than A or L, but have not been evaluated for adaptation to heat stress. Therefore, rectal temperatures of beef & dairy cows with known IR classification were recorded during THI< 74 (normal) and THI>74 (above normal). Results show that 50% of beef & 65% of dairy cows had body temperature above 39.2 at THI>74. However, cows that ranked H for antibody response were significantly better at regulating temperature and respiration at THI>74, than A or L. In vitro experiments showed variability in PBMC function when cells were heat stressed (42□, 4hrs). PBMC were stimulated with ConA to measure proliferation or LPS to assess nitric oxide (NO) production. HSP70 was measured in control and heat treated cells. Dairy results indicated that H responders had significantly greater cell proliferation across all treatments and greater NO and HSP production with significance depending on the treatment. Beef results indicate that HSP70 from unstimulated PBMC increased after 1 or 2 heat treatments, and the magnitude of the change varied by IR class. Conversely, PBMCs challenged with LPS produced more NO before than after heat treatments. These results indicate that cattle with diverse immune response genotypes vary in their ability to withstand heat stress, and that it should be possible to identify resilient individuals with both greater immunity and adaptation to climate warming.
IDENTIFICATION OF PRIMORDIAL ORGANIZED LYMPHOID STRUCTURE IN THE SPLEEN OF TELEOST FISH
Authors: Sunyer J. Oriol , Shibasali Yasuhiro , Takizawa Fumio , Yang Ding , Boudinot Pierre , Krasnov Aleksei
Published: January 2023

Induction of adaptive immune responses in higher vertebrate species occur within organized lymphoid structures (e.g. lymph nodes, Peyer’s patches). It has been proposed that such structures emerged throughout evolutionary time with the goal to maximize encounters between antigens, antigens-presenting cells and B-T lymphocytes. Fish lack such structures and thus, it remains unknown how and where antigen-specific immunoglobulin responses are induced in these species. To understand how systemic immune responses are induced in teleost lymphoid organs, Rainbow Trout were immunized with several soluble protein antigens. Overall, our results identified the spleen as the major site for CD4+ T and IgM+ B cell proliferation in systemic lymphoid organs upon immunization. The proliferating splenic IgM+ B cells were frequently observed as clusters in the vicinity of melano-macrophage centers. Moreover, in these areas we observed aggregates of B and T lymphocytes with a loose organized structure reminiscent of the cellular architecture frequently associated with tertiary lymphoid organs. Laser dissection microdissection of these areas enabled us to evaluating the immunoglobulin IgM repertoires within these structures upon immunization. Critically, repertoire analysis identified processes of antigen-specific B cell clonal expansion. In conclusion, these data points to the previously unrecognized existence of primordial semi-organized lymphoid tissue in the spleen of teleost fish in which adaptive IgM immune responses are induced. Our findings provide strong evidence that the induction of antigen-specific immune responses in all bony vertebrates requires the formation of organized or semi-organized lymphoid structures.
Phenotypic and functional characterization of bovine mucosal associated invariant T (MAIT) cells
Authors: HONG SUYEON , McGill Jodi L
Published: January 2023

Mucosal associated invariant T (MAIT) cells are innate-like T cells that are prevalent in the blood and tissues of many mammalian species. MAIT cells recognize small metabolites derived from the riboflavin pathway that are presented by the highly conserved MHC-related protein-1 (MR1). MAIT cells have been described to play a proinflammatory role during several viral and bacterial diseases in humans and in rodent models. While MAIT cells have been described in cattle at the transcriptional level, little is known about the phenotype or function of this population in the bovine during steady-state or disease conditions. Here, using tetramers that bind to the MR1-restriced T cell receptors on MAIT cells, we investigated the frequency and phenotype of MAIT cells in the peripheral blood and bronchoalveolar lavage of healthy calves and animals with respiratory disease. We determined that cattle have 2 subsets of MAIT cells, defined by their expression of CD8; that bovine MAIT cells have the capacity to secrete IFNγ and IL-17 in response to mitogen stimulation; and that the frequency of MAIT cells in the bronchoalveolar lavage increases in the context of respiratory infection, suggesting that MAIT cells may play an important role in host defense in the respiratory tract of cattle.