bluuebunny/crossref_metadata_2025 huggingface dataset:
miR-146a modulates IgA class-switch DNA recombination by targeting Smad2, Smad3 and Smad4 genes
Authors: Daw Cassidy C. , Zan Hong , Casali Paolo
Published: January 2023

IgA is the predominant antibody isotype at mucosal surfaces. It plays a critical role in mucosal homeostasis and provides a first line of immune protection. Class-switch DNA recombination (CSR) to IgA is a strictly controlled process that involves cytokine induction of transcription factors and expression induced cytidine deaminase (AID). CSR to IgA is initiated by TGF-β activation of Smad2, Smad3, Smad4, and Runx3, which bind to the Iα promotor region to activate germline Iα-Cα transcription. This critically opens up the Igα locus and is greatly enhanced by retinoic acid (RA). As we have suggested, CSR is modulated by epigenetic modifications, including microRNA-mediated gene regulation. miR-146a has been shown to regulate T cell, dendritic cell and macrophage functions in innate and adaptive immune responses. Its role, however, in B cells and in the antibody response, is unknown. Here we showed that, in B cells, miR-146a expression is downregulated by LPS/CD154, TGF-β, IL-4, anti-BCR plus RA, the stimuli that induce a robust CSR to IgA. Deficiency of miR-146a in miR-146a−/− B cells resulted in upregulated Smad2, Smad3 and Smad4 expression, increased recruitment of Smad2, Smad3 and Smad4 to Ia promoter, as well as elevated germline Iα-Cα transcription and CSR to IgA, without a significant alteration in AID expression and CSR to other isotypes, including IgG1. Indeed, as we also showed, miR-146a targets the 3′UTRs of Smad2, Smad3 and Smad4 mRNAs, thereby inhibiting expression of these genes. Thus, by downregulating the level of Smad2, Smad3 and Smad4 in B cells, miR-146a plays an important role in the modulation of the IgA response.
Erythroid suppressor cells promote feto-maternal tolerance through arginase-2 and PDL-1
Authors: Elahi Shokrollah , Delyea Cole , Bozorgmehr Najmeh , Dunsmore Garett , Shahbaz Shima , Huang Vivian
Published: January 2023

Survival of the allogenic pregnancy depends on the maintenance of immune tolerance to paternal alloantigen at the feto-maternal interface. Multiple localized mechanisms contribute to fetal evasion from the mother’s immune rejection as fetus is exposed to a wide range of stimulatory substances such as maternal allo-antigens, microbes and amniotic fluids. Here, we demonstrate that CD71+ erythroid cells are expanded at the feto-maternal interface and in the periphery during pregnancy. These cells exhibit immunosuppressive property and their abundance is associated with a Th2 skewed immune response, as their depletion results in a pro-inflammatory immune response at the feto-maternal interface. In addition to their function in suppressing pro-inflammatory responses in vitro, maternal CD71+ erythroid cells inhibit an aggressive allogeneic response directed against the fetus such as reduction in TNF-a production through arginase-2 activity and PD-1/PDL-1 interactions. Their depletion leads to the failure of gestation due to immunological rejection of the fetus. Similarly, fetal liver CD71+ erythroid cells exhibit immunosuppressive activity. Therefore, immunosuppression mediated by CD71+ erythroid cells on both sides (mother/fetus) is crucial for feto-maternal tolerance. Thus, our results reveal a previously unappreciated role for CD71+ erythroid cells in pregnancy and indicate that these cells mediate homeostatic immunosuppressive/immune-regulatory responses during pregnancy.
Unique Cytokine Profile of Novel Innate Lymphoid Cells in Human Decidua
Authors: Vazquez Jessica , Li Yan , Stanic-Kostic Aleksandar
Published: January 2023

Immune cells at the maternal-fetal interface play a complex role in regulation of vascular remodeling, fetal tolerance and protection from infection. We have previously presented the identification of two novel CD56highNKs based on Eomes expression (Eomeslo/hi). Activation of these subsets revealed preferential production of IL-8 and IL-17A by decidual innate lymphoid cells (ILCs).

Decidual specimens were dissected from term placentas and mononuclear cells (MCs) were isolated by mechanical (GentleMACS) and enzymatic (Collagenase, DNAse) disruption. MCs were then simulated with PMA/ionomycin or IL-23, then labeled by flurochrome-conjugate antibodies against surface CD3, 14, 16, 19, 34, 45, 49a, 56, 94, 117, 127, 335 and intracellular Granzyme B, IL-8, IL-17A, IL-22, INFγ, TNFα. Data acquisition was performed using BD Fortessa flow cytometer in a 5 laser, 18 detector configuration and data analysis was performed using FlowJo 10.2.

Activation of decidual ILCs revealed a unique milieu of cytokine production. The novel subsets, CD56high Eomeslo/hi, preferentially produced IL-8 and IL-17A, while decreasing production of INFγ, Granzyme B, and TNFα. In addition, as expected, ILC3s and LTi-like cells produced IL-17A and IL-22. Furthermore, differences in response were observed between decidual basalis and decidual parietalis ILCs, with decidual parietalis ILCs being more responsive to stimulation.

Preliminary results suggest that both CD56high Eomeslo/hi subsets demonstrate a similar altered cytokine profile that is conducive to decidual remodeling. Further gene expression analysis will reveal if these novel subsets represent a truly unique population or are members of the expanding tissue-resident NK cell family.
Novel strategies to assess the synaptic interface of primary human T cells from peripheral blood and lymphoid tissue
Authors: Steblyanko Maria A. , Anikeeva Nadia , Buggert Marcus , Betts Michael R. , Sykulev Yuri
Published: January 2023

Our current understanding of the kinetics and structural features of T cell synaptic interfaces has largely been determined through the use of glass supported artificial bilayers and in vitro-derived T-cell clones or lines. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues in unclear, given the significant difficulty in obtaining sufficient amounts of these cells for use on conventional glass-supported planar bilayers. Moreover, emerging data suggest that tissue resident T cells and T cells from lymphoid organs are significantly different in their phenotype and functional activity from those in peripheral blood, further solidifying the need to better understand the features of T cell synaptic interfaces in primary human T cells. To this end, we developed a new mini scale approach exploiting bilayers built into multichannel flow slides that allows us to perform imaging studies of T cell/bilayer interfaces with as few as 105 cells primary T cells isolated from human peripheral blood and lymphoid tissues without sacrificing the quality of the analysis. Our results indicate that while primary peripheral blood CD8+ T cells can share synaptic interface properties similar to in vitro derived T cell clones and lines, we found significant differences in the structure of the synaptic interface formed by primary lymphoid tissue compared to peripheral blood T cells, consistent with different efficiencies of these T cells.
A novel peptide pool with broad infectious antigen and MHC coverage for use as a positive stimulation control or as a means to elicit general T-cell responsiveness
Authors: Castro Aaron , Holenya Pavlo , Eckey Maren , Schulz Marco , Wenschuh Holger , Reimer Ulf , Tech Tatjana , Chan Kam , Janani Ramesh , Broaten Blake , Kern Florian
Published: January 2023

T-cell activation assays such as Elispot or Intracellular Cytokine Staining (ICS) require appropriate positive stimulation controls. Compared with conventional assay controls based on polyclonal stimulation (e.g. PHA/Ionomycin), peptide pools have the advantage of providing a more physiological, TCR-mediated signal. A frequently used positive control is the so-called ‘CEF’ pool, containing 23 selected class-I-MHC-presented T-cell-stimulating peptides from CMV, EBV, and Influenza A. As a result of frequent and widespread exposure, most if not all individuals have T-cells specific for at least one of these agents. However, the ‘CEF pool’ only covers the most frequent HLA alleles in European-Caucasoids but not other ethnic groups where its usefulness is limited. We, therefore, designed an extended pool referred to as ‘CEFX pool’ with significantly improved coverage of both infectious agents and HLA-alleles (176 peptides), which also contains numerous CD4 T-cell-stimulating peptides. This novel pool provides a much stronger and more universal positive control than the ‘CEF’ pool and was successfully tested in various assay formats including Elispot, ICS, and ELISA of stimulated T-cell supernatants. For specific questions, sub-pools for CD4 or CD8 T-cell stimulation or not containing CMV-derived peptides are available. Because of its broad antigen coverage we are currently exploring the novel pool’s potential for measuring/monitoring general T-cell responsiveness in clinical situations such as immunodeficiency, immunosuppression, or immunosenescence.
An efficient human whole blood workflow using CyTOF technology: a lyophilized 30-plex antibody panel coupled with automated data analysis
Authors: Li Stephen K.H. , Majonis Daniel , Bagwell C. Bruce , Hunsberger Benjamin C. , Baranov Vladimir , Ornatsky Olga
Published: January 2023

CyTOF® mass cytometry is a highly multiplexed, single-cell analysis platform that uses metal-tagged antibodies. A significant advantage of this technology is its ability to resolve more than 30 markers in a single panel without the need for compensation. Nonetheless, a highly multiplexed panel requires optimization of antibody titers, full panel verification, sample preparation, data acquisition, and analysis. Preparation of a staining cocktail from 30 individual tubes may be prone to errors. Not only is data analysis time-consuming for complex panels, it is difficult to interpret without expertise in immunology.

We have developed a lyophilized 30-plex immunphenotyping panel contained in a single tube, an efficient workflow, and an automated software solution for human whole blood analysis using mass cytometry. The panel focuses on T cell lineage while also capturing other relevant immune populations. Blood is added directly to the lyophilized antibody tube, followed by RBC lysis, wash, and fixation steps and finally data acquisition of stained samples on the Helios™ system. The dedicated software tool accepts FCS3.0 files and in under 10 minutes automatically generates reports on the number of live cells, percentage of specific cell populations, staining intensities, histograms, 2D dot plots, and tSNE graphs. The auto-calculated frequencies of populations are comparable to manual gating, with a validated correlation coefficient over 0.9. The panel and software tool enable researchers to streamline immunophenotyping of whole blood while accurately and reproducibly monitoring changes in immune cell subsets in patient samples.
Exploring the applicability of ctDNA technology as a diagnostic tool for improved Cancer care and management within the Pacific
Authors: Sika-Paotonu Dianne , Guilford Parry
Published: January 2023

For Pacific peoples living in New Zealand and in the Pacific region, the burden of cancer remains a public health concern of significance. The medical situation for cancer patients in New Zealand and also in the Pacific is of relevance given the health inequalities that exist for Pacific peoples and the global burden of cancer. This work sought to obtain clarity around the cancer diagnostic, management practices and treatments that exist for cancer patients in the Cook Islands. Circulating tumour DNA (ctDNA) is a novel cancer detection method and refers to small fragments of tumour DNA found in the blood circulation. These DNA fragments can be isolated and quantified to obtain data about the cancer’s size and progression. The possibility of utilizing a simple blood sample to allow detection and monitoring of cancer growth is highly desirable. It is anticipated this simple method of detecting and monitoring cancer will enhance the precision of cancer diagnostics and surveillance and also lead to improved cancer health outcomes for Pacific peoples in the Cook Islands. This work sought to investigate the potential utilization of ctDNA to support and improve the diagnosis, care and health outcomes of Pacific cancer patients in the Cook Islands. A high degree of interest was expressed by the medical, nursing, health and community research staff and lay persons in the development of ctDNA as a potential diagnostic tool within the Cook Islands. Of particular interest was the potential to monitor patients remotely using the technology rather than be inconvenienced by travelling far distances with their families to the Main Hospital. ctDNA remains a feasible tool with potential to support cancer patients in the Cook Islands.
High-throughput single-cell linking of antigen specificities with T cell receptor sequences using de novo generated DNA-linked MHC tetramers
Authors: Jiang Ning
Published: January 2023

Identification of correlated antigens recognized by T cell and its T cell receptor (TCR) sequences at single cell level is extremely useful to the understanding and treating immune-related diseases. Although CyTOF has pushed the number of antigenic peptides that can be interrogated at once to around 100, the destructive nature of CyTOF prohibits linking pMHCs recognized by a T cell with its TCR receptor sequences.

Here we describe a new technology, TetATCR-Seq for Tetramer Associated TCR sequencing, that is capable of de novo generating a large amount of peptides and mapping antigen recognition with single cell TCR sequences by DNA barcoded tetramers. It evolves three major steps: 1) de novo peptide generation, 2) DNA barcoded tetramer generation, and 3) single cell sequencing of tetramer DNA barcode and TCR sequences.

Using a published set of cancer neo-antigen sequences, we showed that these peptides and their wildtype counterparts can be quickly generated. Resulted tetramers can be used to clearly identify a population of neo-antigen only or wild type antigen only or double positive T cells. Subsequent single cell TCR sequencing showed that TCR paired a and b chain sequences can be obtained in more than 80% of single T cells. Simultaneous tetramer DNA barcode sequencing showed that many T cells are found to cross-reactive to both neo-antigen and its wild type counterpart, suggesting the necessity of strict screening for neo-antigen only TCR in adoptive cell transfer therapy.

In summary, TetaTCR-seq enables one to quickly survey antigen repertoire again TCR repertoire at single cell level for a large number of peptides. It is a useful technology to screen for T cell cross-reactivity and select neo-antigen specific TCR for cancer immunotherapy.
Multiplex immunoassay detection of autoimmune disease autoantibodies in serum and plasma
Authors: gilliam brooke e. , Pepin D , Xiao Qiang
Published: January 2023

Autoantibody formation is principal to the pathogenesis of a variety of autoimmune diseases. Dysregulated apoptosis and the subsequent defective clearance of cellular debris leads to the exposure of autoantigens and the generation of autoantibodies. The presence of autoantibodies may indicate disease activity, prognosis, and clinical associations related to a variety of autoimmune diseases, including systemic lupus erythematosus (SLE), Sjögren’s Syndrome, Scleroderma, Polymyositis/Dermatomyositis, and various overlap syndromes of these diseases. Here we report the development of a multiplex immunoassay to monitor 20 autoantibodies present in blood that are involved in a variety of autoimmune diseases: SSA/Ro60, SSA/Ro52, SSB/La, RNP, RNP/Sm, Sm, Ribosomal P, Proteinase 3, Myeloperoxidase, PCNA, β 2-Glycoprotein, CENP-A, CENP-B, Scl-70, Jo-1, C1q, PM/Scl-100, Ku, Mi-2, and PL-12. Using the 20-plex immunoassay, we measured expression of autoantibodies in serum and plasma from SLE patients with various disease manifestations and overlap syndromes, healthy controls, and CDC reference samples available for 11 autoantibodies. Additionally, control beads were included in the immunoassay. Levels of SSA/Ro60, SSA/Ro52, SSB/La, RNP, RNP/Sm, and Sm matched clinical laboratory features noted at the time of SLE sample collection. Additionally, anti-C1q antibody levels were associated with lupus nephritis. The CDC controls exhibited elevated MFI for the autoantibodies for which they were expected to react strongly. This study demonstrates the value of using multiplex technology to evaluate multiple autoantibodies, allowing for an extensive autoantibody profile to evaluate patients with various autoimmune diseases.
Evaluation of a B-Cell Biosensor Immunoassay for Rapid and Sensitive Detection of Ricin, a Harmful Plant Protein Toxin
Authors: Venkateswaran Kodumudi , Selvan Senthamil R. , Sarwar Jawad , Parameswaran Nishanth , Babu Prasanti , Koval Sophia , Venkateswaran Roma , Krishnan Krish VV. , Simpson Jeanette , Hodge David R.
Published: January 2023

Ricin is a carbohydrate binding protein produced in the castor bean seeds and is very toxic to humans. Ricin is one of the most frequently used toxin as a biothreat agent in many individual incidents. PathSensors has licensed the Cellular Analysis and Notification of Antigen Risks and Yields (CANARY) technology from Massachusetts Institute of Technology and developed Zephyr biosensor sandwich immunoassay for rapid detection of Ricin. In this assay, CANARY technology utilizes engineered B Cells that express ricin specific monoclonal antibody on the surface as biosensors. Luminescence, emitted from the B cells upon binding of the toxin, is measured as photons in a Luminometer to detect ricin in a suspicious sample. In this study, a detailed performance evaluation of this rapid ricin detection test was done in multiple phases using well characterized materials. The dynamic range of this assay was determined by testing wide ranging concentrations (0 to 100 ng/test) of ricin and limit of detection (LOD) was calculated by PROBIT regression analysis. Further validation of assay was performed by testing inclusivity, informational, near neighbor, lectin, and white powder panels, and also environmental aerosol filter sample testing. LOD for ricin was found to be 1 ng/test. Repeatability testing showed more than 99% sensitivity and 100% specificity. There was no false positive detection of environmental background panel and other potentially interfering materials except Ricinus communis agglutinin also known as RCA120. The validation indicates that the ricin biosensor test is user-friendly laboratory detection method which has great potential for use in national and international public health and environmental test laboratories.
The Benzathine Penicillin G (BPG) reformulation preferences study-edging closer towards a new Penicillin for Rheumatic Fever and Rheumatic Heart Disease
Authors: Sika-Paotonu Dianne , Tiatia Ramona , Sung Yun K. , Thornley Craig , Betty Bryan , Wineera-Parai Ranei , Eddie Barbara , Baker Michael , Puni Ronald , Dalton Estere , Taueetia-Suá Tua , Maloney Margaret , Sanchez-Felix Manuel , Spector Jonathan , Carapetis Jonathan
Published: January 2023

Acute Rheumatic Fever (ARF) is the autoimmune condition triggered by untreated Group A Streptococcal (GAS) infection of the upper respiratory tract (and possibly skin). Severe and recurrent untreated attacks of ARF can cause cardiac damage, known as Rheumatic Heart Disease (RHD). RHD remains a significant cause of morbidity and mortality globally and in Australia, New Zealand (NZ) and the Pacific Region, the disease burden of ARF and RHD amongst Indigenous and Pacific communities is one of the highest in the world, usually affecting children and young adults. The most effective recommended preventative measure for ARF requires painful monthly intramuscular injections of Benzathine Penicillin G (BPG) for 10 years or more. Known as secondary prophylaxis, these injections aim to prevent GAS infections that may lead to recurrence of ARF and either cause or worsen RHD. Adherence to secondary prophylaxis remains a challenge in many settings and improved understanding of barriers and a BPG reformulation that is more appropriate are urgently needed. This work sought to explore the BPG reformulation preferences and perspectives of predominantly Māori and Pacific children/teens in New Zealand currently receiving monthly BPG intramuscular injections, their families and healthcare providers using three software applications developed for this purpose. A total of 82 participants comprised of children/teens, family members and health professionals have thus far participated in this research project. This is the first time software applications have been successfully developed to collect qualitative and quantitative data on individual preferences for BPG formulations and dosing regimens in New Zealand.
Efficient isolation of particle-free human ILC2s from peripheral blood mononuclear cells
Authors: Poon Grace F.T. , Kyei Stephen K. , Antignano Frann , McQueen Karina L. , Peters Carrie E. , Takei Fumio , Valdez Yanet , Kokaji Andy I. , Woodside Steven M. , Eaves Allen C. , Thomas Terry E.
Published: January 2023

Group 2 innate lymphoid cells (ILC2s) have important roles in type-2 immune responses and are implicated in allergies, asthma, helminth infections and other metabolic diseases. To better understand their role in immunity, highly purified ILC2s are required. ILC2s are phenotypically defined as CD45+ lineage-negative CRTH2+ CD127+ CD161+, and comprise approximately 0.05% of peripheral blood mononuclear cells (PBMCs). Fluorescence activated cell sorting is the most common method to isolate ILC2s but it is both time-consuming and expensive because the cells are so rare. To address this issue, we have developed an immunomagnetic isolation method to obtain highly pure, particle-free ILC2s from human PBMCs in 2.5 hours. First, CRTH2 positive cells are labelled with a CRTH2-PE monoclonal antibody. These cells are then positively selected using an anti-PE antibody complex, EasySep™ Releasable RapidSpheres™ and an EasySep™ magnet. After separation, the magnetic particles are released from the positively selected cells and a second cocktail of antibody complexes and EasySep™ Dextran RapidSpheres™ is added to label non-ILC2s within the CRTH2 positive population. After a second separation, the released particles and unwanted cells are retained in the tube within the magnet, whereas the ILC2s are simply poured off and ready for use. With this method, the purity of ILC2s isolated from PBMCs was 84–95% (median 91%, n=18) with a recovery of 0.4–14.3×103 ILC2s per 108 starting PBMCs (median 2.46×103, n=18). The isolated ILC2s are functional, producing IL-13 upon in vitro stimulation with IL-33 and IL-2. Overall, EasySep™ Human ILC2 Isolation Kit allows researchers to easily and efficiently isolate highly pure and functional ILC2s.
Induction of neuroantigen-specific CD8+ T cell responses as a treatment for relapsing autoimmune demyelinating disease
Authors: Brate Ashley A. , Sinha Sushmita , Itani Farah R. , Pewe Lecia L. , Harty John T. , Karandikar Nitin
Published: January 2023

Multiple Sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system (CNS). We have shown that CNS-specific CD8+ T cells possess a disease suppressive function in MS and its animal model, experimental autoimmune encephalomyelitis (EAE). In humans, this suppressive function of CD8+ T cells is deficient during MS relapses and is restored during quiescence, suggesting that lack of CD8+ T cell mediated regulatory function may contribute to relapses in MS. We thus evaluated the role of CD8+ T cells in the context of the relapsing-remitting model (RR-EAE), using SJL mice. We demonstrate here that PLP178–191- and MBP84–104-specific CD8+ T cells ameliorated disease severity in an antigen-specific manner. Moreover, PLP178–191 CD8+ T cells reduced the number of relapses in PLP178–191-induced disease, even when transferred as a treatment during ongoing disease. We also probed the role of endogenously generated CNS-specific CD8+ T cell responses by using Listeria monocytogenes (LM) encoding CNS antigens to preferentially prime suppressive CD8+ T cells in vivo. Using this system, we show infection with LM expressing PLP175–194 endogenously induced disease suppressive CD8+ T cells that protected and treated PLP178–191 disease. Importantly, LM-PLP175–194 infection during disease remission of PLP139–151-induced EAE could effectively prevent relapses. These studies support a potential immunotherapeutic strategy using LM vaccination to endogenously prime disease regulatory CD8+ T cells.
Differential expression of CNS-specific proteins in progressive EAE point to potential biomarkers for progressive MS
Authors: Chase Carol Ann , Raphael Itay , Forsthuber Thomas
Published: January 2023

The lack of reliable markers for assessing the progression of multiple sclerosis (MS) from the relapsing-remitting to the secondary progressive form hinders the ability for therapeutic intervention and results in continued physiological deterioration. To begin to address the urgent need for biomarkers of progressive MS we investigated proteome changes over the disease course of progressive experimental autoimmune encephalomyelitis (EAE) in NOD mice as a preclinical model of the disease using a high-throughput quantitative proteomic technique pioneered in our lab. In this study, we utilized this established proteomic technique as well as bioinformatics tools to prioritize key proteins whose expression level in the CNS correlated specifically with the progressive phase of disease in the NOD EAE model. Hierarchical clustering of timepoints indicate that samples cluster based on progression of disease. Specifically, late time points cluster separately from naïve and peak time points. Importantly, statistical testing identified proteins with differential expression in the CNS of mice across the time course of NOD EAE, several of which are CNS specific. We are seeking to detect corollary changes in these CNS-specific proteins in the serum, pointing to a minimally invasive means of monitoring disease progress and measuring drug efficacy. Our studies will provide proof-of-concept for identifying homologous human biomarkers to guide treatment in individual patients. Furthermore, our results may provide insights into mechanisms that contribute to disease pathology and offer additional therapeutic targets for slowing the progression of MS.
The mechanism of preventing EAE via gene therapy
Authors: Keeler Geoffrey D. , Cote Isabelle , Gaddie Cristina D. , Hoffman Brad E.
Published: January 2023

Similar to Multiple Sclerosis, EAE is a complex disease in which immune tolerance is lost and antigen specific (Ag-sp) Tregs are known to play a role. We have developed a gene immunotherapy that has been shown to induce MOG specific Tregs, restore tolerance, and effectively prevent, and/or reverse, significant disease. However, the spatiotemporal profile of the induced Tregs is unclear. To identify the tissue specific populations of AAV8.MOG-induced specific Tregs, we used a transgenic Foxp3 reporter mouse with a MOG35–55/I-Ab tetramer. Two weeks prior to EAE induction, mice were tolerized via a single injection of vector. At the peak of disease, tissues were harvested and fluorescently labeled for phenotypic identification. FACS analysis revealed a higher proportion of Ag-sp Tregs in the cervical and inguinal lymph nodes (LN) and spleen, as compared to controls. In contrast, a higher proportion of Ag-sp effector cells were seen in control mice as compared to treated. Furthermore, a significant number of infiltrating effector T cells were isolated from the spinal cords of control mice, while very few T cells were recovered from treated mice. This aligns with cytokine data that was collected showing there to be higher production of IFN-γ in the spinal cord and spleen of control mice. These results suggest that AAV.MOG induced Ag-sp Treg populations accumulate in the peripheral organs, and may prevent effector cells from migrating to the spinal cord. This work provides new insights on tolerance induction mechanisms. These insights will be useful for the development of effective cell based therapies capable of treating autoimmune diseases.
Small molecule modulators of prion protein (PrpC) in Dendritic cells regulate inflammatory responses in a model of Multiple Sclerosis
Authors: Fallarino Francesca , Barreca Maria Letizia , Manni Giorgia , Scalisi Giulia , Biasini Emiliano , Gargaro Marco , Manfroni Giuseppe
Published: January 2023

Experimental autoimmune encephalomyelitis (EAE) is the most commonly used experimental model for human multiple sclerosis, an autoimmune disease driven by differentiated Th1 and Th17 cells, and inflammatory dendritic cells (DCs). Recent data show that EAE is worsened in mice lacking PrPC protein and disease exacerbation has been attributed to T cells, which would differentiate into more aggressive effectors, when deprived of PrPC.

Novel compounds, modulating PrPC activity, have been recently developed, although not tested in EAE models yet. Interestingly, by specific gene array studies, we found that PrPC was highly expressed in DCs and differentially expressed among selected DC subsets. Moreover, by using a series of computational, and biochemical assays we identified novel small molecules, able to modulate PrPC function in specific cell lines overexpressing PrPC. By employing such tools, we studied the impact of PrPC modulation in promoting regulatory DC subsets. To this aim, DCs were treated either with novel PrPC modulators or a reference PrPC binding molecule Fe (III)-TMPyP. Then, they were co-cultured with sorted naïve CD4+ T cells in vitro. Notably, we found a significant expansion of FOXP3+CD4+ T regulatory (Treg) cells in cultures containing DC subsets, which were pre-treated with the specific PrPC modulators. Moreover, in a EAE model, we showed that systemic administration of such PrPC regulators, resulted in significant reduction of disease severity, compared to untreated controls. Overall, our results suggest that PrPC modulation in selected DC subsets may represent a novel means to restrain inflammatory T cell effectors, resulting in reduced inflammation, demyelination, and axonal injury in a model of EAE.
Tumor necrosis factor receptor 2 promotes neuroprotection during chronic autoimmune neuroinflammation
Authors: Gomez-Rivera Francisco , Raphael Itay , Raphael Rebecca , Robinson Rachel R. , Nalawade Saisha A. , Forsthuber Thomas
Published: January 2023

Multiple sclerosis (MS) is an inflammatory autoimmune disorder affecting the central nervous system (CNS) which affects over 400,000 Americans and over 2.5 million people worldwide. Although most patients are initially diagnosed with relapsing-remitting MS, the majority of these patients later develop a chronic-progressive form of MS, for which there is no well-established mouse model. The most common genetic factor associated with MS susceptibility is the Human Leukocyte Antigen (HLA)-DR2b haplotype. Additionally, studies in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS, showed that lack of TNF signaling through its receptor TNFR2 leads to disease exacerbation and severe demyelination. Here, we developed a mouse model which expresses HLA-DR2b and lacks TNFR2, designated DR2bΔR2. Strikingly, DR2bΔR2 mice develop progressive EAE with pathology and clinical features observed in progressive MS patients. Adoptive transfer studies revealed that the clinical phenotype of EAE in DR2bΔR2 mice are largely dependent on TNFR2 expression in the CNS. Subsequently, we showed that DR2bΔR2 mice have a significant increase of lesions in the cerebellum, associated with reduced of oligodendrocyte progenitor cells (OPC) recruitment or function. Moreover, we showed that DR2bΔR2 mice are presented with chronic astrogliosis in demyelinating lesions. Our studies provide key insights into CNS repair and regulatory mechanisms controlled by TNFR2 during neuroinflammation and provide novel therapeutic strategies for treating progressive MS.
Selective inhibition of gelatinases mitigates T-cell receptor signaling in CD4+ T-cells resulting in reduced clinical severity in a murine model of Multiple Sclerosis.
Authors: Onwuha-Ekpete Lillian C. , Tokmina-Roszyk Dorota , Fields Gregg
Published: January 2023

Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that mediate the degradation of extracellular matrix components. The gelatinases, MMP-2 and MMP-9, have functions essential for normal physiological processes such as leukocyte trafficking, but their dysregulation is associated with various pathologies including Multiple Sclerosis (MS). During MS, CD4+ T-cells activated in the periphery penetrate the blood-brain barrier (BBB), recruit immune cells, initiate destruction of the myelin sheath, and cause axonal loss. Experimental Autoimmune Encephalomyelitis (EAE) is a well-established murine model of MS. In EAE, gelatinases are required for penetration of the BBB and generation of auto-antigens. Recent studies have demonstrated that they contribute to normal intracellular function of various cell types including antigen activated T-cells; however, their intracellular role in immune cell activation during EAE pathogenesis is not known. In our studies, we demonstrate that gelatinases are important for effective T-cell receptor (TCR) signaling. Upon activation, CD4+ T-cells lacking gelatinase activity demonstrated a reduced ability to express early activation markers, proliferate, produce cytokines, and undergo metabolic reprogramming. However, in the absence of TCR stimulation, CD4+ T-cells have no proliferative defects. In addition, RNA-seq analysis highlight key proteins impacted by gelatinase inhibition that are associated with the TCR signaling. Furthermore, EAE mice demonstrated reduced clinical severity following gelatinase inhibition. These results emphasize the importance of the gelatinases as therapeutic targets in CD4+ T-cell mediated autoimmune diseases.
Coordinated events following TLR-9 and CD122 signaling promote NF-kB/AKT/STAT5-dependent B-CLL clonal expansion
Authors: Mongini Patricia K. A. , Gupta Rashmi , Vergani Stefano , Allen Steven L. , Barrientos Jacqueline , Kolitz Jonathan , Rai Kanti , Chiorazzi Nicholas
Published: January 2023

B-cell chronic lymphocytic leukemia (B-CLL) clones express BCR specific for microbes and/or apoptotic cells and elevated TLR-9. In patients, B-CLL clonal expansion occurs within lymphoid tissues. We recently reported that CpG DNA (ODN-2006) and IL-15, found within stromal cells of CLL-infiltrated lymphoid tissues, manifest synergy in promoting in vitro B-CLL growth (J. Immunol 195:901–923, 2015). This study explores the mechanism responsible by using purified B-CLL from peripheral blood to assess the temporal requirements for IL-15; effect of ODN on expression of IL-15 receptors (IL-15Rα and CD122 (IL-2/15Rβ)); and IL-15-induced activation of AKT and STAT5. Experiments with CFSE-labeled B-CLL show that IL-15 is critical during a 20–36 h window after ODN priming. Neutralizing mAbs to IL-15 or CD122 fully abrogate IL-15-driven growth when added during this interval. RT-PCR/immunofluorescence studies show that ODN triggers rapid increases in mRNA/protein for IL-15Rα and CD122 that are blocked by IkBα inhibitor. Furthermore, ODN-primed B-CLL exposed to IL-15 manifest elevated pAKT(Ser473) and pSTAT5(Tyr694) levels. Both PI-3K/AKT and STAT5 pathways are functionally relevant, as indicated by growth abrogation by pharmacologic inhibitors, LY294002, pimozide or STAT5 Inh II. Importantly, activated STAT5 levels are high in cycling CFSE-labeled B-CLL; furthermore, extended divisions were blocked by delayed (day 4) administration of STAT5 inhibitors or neutralizing mAbs to IL-15/CD122. Together, findings show that ODN + IL-15-driven growth represents coordinated early functions of TLR-9 and CD122 as well as later CD122 signaling during clonal expansion. This synergy may help drive in vivo growth of B-CLL clones in patients.
TLR and CLR agonists induce anti-tumor B cell responses against peritoneal carcinomatosis
Authors: Dyevoich Allison , Haro Marcela , Haas Karen M.
Published: January 2023

Peritoneal carcinomatosis is a condition where primary tumors metastasize into the peritoneal cavity and induce malignant ascites. The median survival of patients is poor, averaging about twenty weeks post diagnosis. Due to the high mortality rate associated with this disease and the lack of curative treatments available, it is imperative that more effective treatments be developed for peritoneal carcinomatosis. The immune system plays an important role in tumor elimination, and one strategy currently being explored to treat malignancies involves leukocyte activation through pattern recognition receptors (PRRs), including Toll-Like Receptors (TLRs) and C-type Lectin Receptors (CLRs). Our data shows that mice treated with a TLR and CLR agonist are afforded significant protection when challenged with an aggressive ascites-forming mammary tumor cell line (TA3-Ha). However, when mice lack B cells, the protection is lost. Administration of the combined treatment results in a significant increase in proliferating, antibody-producing B cells within the visceral adipose tissue (VAT). Consistent with this, the treatment induces a significant increase in IgM and complement (C′) bound to tumor cells, both of which were determined to be critical players in the anti-tumor effects. In summary, our data demonstrates pathogen associated molecular patterns can induce rapid natural IgM production that elicits anti-tumor protection via C′ activation.
Myeloid derived suppressor cells (MDSC) and anti-MUC1 immunosurveillance in pre-malignancy and cancer
Authors: Ma Peiwen , Beatty Pamela , McKolanis John , Schoen Robert , Brand Randal , Finn Olivera J.
Published: January 2023

Recent work from our lab showed that in a colon cancer prevention trial of a tumor antigen MUC1 vaccine in patients with premalignant colon adenomas, those who did not make anti-MUC1 IgG, had higher pre-vaccination levels of circulating MDSC compared to vaccine responders. This was the first observation of increased MDSC in premalignancy. MDSC in premalignancy have not been compared to MDSC in cancer and our hypothesis is that there might be differences in the composition of various MDSC subpopulation and their immunosuppressive functions due to the shorter time of exposure to disease and differences in the disease microenvironment. We are testing this hypothesis on two cohorts of patient samples, premalignant and malignant colon and pancreas. In both cohorts we saw an increase in MDSC in premalignant and malignant disease over healthy controls. The colon premalignant group could be divided into MDSChighAnti-MUC1 IgGlow and MDSClowAnti-MUC1 IgGhigh confirming our previous observations that MDSC are impeding anti-MUC1 immune response in premalignancy as well therefore potentially facilitating progression to cancer. The pancreas cohort also showed increase in MDSC in patients with pancreatic cysts (IPMN), precursors to pancreatic cancer, as well as their suppression of anti-tumor immunity. Furthermore, we found increased levels of PGE2 and its metabolite in patients’ serum from both cohorts, which is closely related to MDSC proliferation and mechanism of suppression. While we do not see much difference in the MDSC phenotype and subpopulation composition, the immunosuppressive capacity of tumor MDSC appears to be greater. We are currently testing this observation in vitro in T cell suppression assays.
Synergy of an anti-HER2 ADC TAK-522 (XMT-1522) in combination with anti-PD1 monoclonal antibody (mAb) in a syngeneic breast cancer model expressing human HER2
Authors: Khattar Mithun , Traore Tary , Horton Kristin , Gallery Melissa , Brauer Pamela , Riceberg Jessica , Bodyak Natalya , Protopopova Marina , Zhang Qingxiu , Lowinger Timothy B. , Veiby Petter , Huszar Dennis , Wang Frank
Published: January 2023

Antibody-drug conjugates (ADCs) are a highly potent class of drugs that specifically target cancer cells expressing a tumor associated antigen. The ADC TAK-522 consists of a novel human IgG1 anti-HER2 monoclonal antibody and a novel, auristatin-based cytotoxic payload (Auristatin F-hydroxypropylamide, AF-HPA). An average DAR of twelve AF-HPA molecules is achieved via a biodegradable polymer conjugation platform. The free payload AF-HPA and TAK-522 induced immunogenic cell death (ICD) in vitro in multiple cell lines measured by cell surface expression of calreticulin using microscopy and flow cytometry. Furthermore, we developed a novel syngeneic breast cancer model expressing human HER2 at a relatively low antigen density. Treatment in this poorly immunogenic tumor model with TAK-522 but not Kadcyla showed significant inhibition of tumor growth in vivo. Importantly, a combination of anti-PD1 mAb and TAK-522 therapy substantially and synergistically enhanced the anti-tumor efficacy, resulting in complete responses in some mice. The frequency of complete responders was further increased when the two drugs were sequentially, rather than concurrently, administered such that TAK-522 administration was followed by anti-PD1 mAb therapy, suggesting an immunological mechanism with induction of immunogenic cell death by TAK-522, which may activate the adaptive immune system by releasing tumor specific antigens. TAK-522 is currently being tested in a phase-1b clinical trial in patients with advanced breast, lung and gastric cancer expressing HER2. Based on our data, TAK-522 represents a potential candidate for combination therapies with immune checkpoint modulators in patients with poorly immunogenic HER2 expressing tumors.
The immune response in fractionated radiotherapy schedules: Implications for combination immunotherapy
Authors: Battaglia Nicholas Griffin , Gerber Scott A. , Lord Edith M.
Published: January 2023

Radiotherapy (RT) is used to treat approximately half of patients with solid tumors. Though DNA damage induced by RT plays a role in limiting tumor growth, RT also elicits an adaptive immune response. This response has been well characterized in the context of high dose, ablative RT and is dependent on IFN-γ expressing CD8+ T cells. In contrast, little is known about the immune response following clinically relevant fractionated RT schedules. Additionally, recent work has demonstrated that increased utilization of glycolysis is essential for T cell effector function, particularly for increasing expression of IFN-γ, and competition for glucose in the tumor microenvironment may limit the anti-tumor immune response. Using the MC38 adenocarcinoma murine tumor model, we have examined the immune response following iso-effective ablative and fractionated RT schedules designed to have biologically equivalent doses. Our results indicate that IFN-γ levels and T cell activation are decreased whereas macrophage frequencies are increased after fractionated RT as compared to ablative RT, correlating with increased rates of tumor rebound. Interestingly, preliminary data shows that tumor cells and tumor associated macrophages have increased consumption of glucose as compared to T cells following RT, indicating that selectively targeting the metabolism of these populations may improve the anti-tumor T cell response by reducing competition for glucose. Further study of these populations’ metabolism following fractionated and ablative RT will provide insights as to how these pathways may be targeted to improve outcomes of existing treatment modalities. Supported by awards T32AI007285 from the NIAID and R01CA028332 from the NCI.
Immune effects of combinatorial repurposing drugs in refractory ovarian and colorectal cancer patients.
Authors: Marquez-Manriquez Juan P. , LUCERO-DIAZ PEDRO-ALEJANDRO , MONTIJO-FERNANDEZ LORENA , MATUTE-BRISENO JOSE-ANTONIO , MONTANO GEOBANNI TORRES , VERBURG MARK L. , Rosas-Delgado Martin-Orlando , CAMACHO-HERNANDEZ ALEJANDRO
Published: January 2023

Background

Some refractory cancer patients have a good Karnofsky despite refractory disease. We studied the potential immunomodulatory properties of approved drugs. We used fresh PBMC from refractory patients to study the immune effects of several drugs such as metformin, pidotimod, erdostein, esomeprazole, colchicine, bortezomib, gemcitabine, zoledronic acid and naproxen.

Methods

25 high-grade serous ovarian cancer (HGSOC) and 25 colorectal cancer (CRC) subjects were included after approved protocol. The inclusion criteria include Karnofsky 80–100%, complete CT scan, acute phase proteins, granzyme B ELISPOT and antigen-specific ELISA.

Results

We found after 24 months of treatment in HGSOC 80% of complete response (CR), 15% of stable disease (SD) and 5 % of pseudo-progressive disease (PPD). In CRC we had 50% of CR, 30% of SD and 20% of PPD. In the patients with CR there was an immunological correlation of CD8 persistent immune response by granzyme B ELISPOT in HGSOC and CRC (p=0.0001) and PR (p=0.001), respectively. The combination was well tolerated and after 10 months of stopping the treatment some patients have persistent CD8 specific immune response by ELISPOT and ELISA.

Discussion

The combination of daily oral metformin, colchicine, pidotimod, erdostein, and naproxen plus weekly low dose of intravenous (IV) esomeprazole (80 mg), bortezomib (0.5 mg/m2), gemcitabine (0.5 mg/m2) and zoledronic acid (100 mg) is clinically feasible, with limited clinical toxicity. We now understand the importance of preserving the CD8 immune response and the use of immune biomarkers such as antigen specific ELISA to improve the rational and generate new combinations to improve clinical outcomes in patients with refractory HGSOC and CRC.
Long Non-Coding RNAs Regulate Transcription of Lymphoma Oncogenes
Authors: Beri Nina , Pyfrom Sarah C. , Oltz Eugene M. , Payton Jacqueline
Published: January 2023

Non-Hodgkin Lymphoma (NHL) is the most common blood cancer in the U.S. Our analysis of gene regulation in >100 NHL and normal B cell samples revealed a distinct profile of long non-coding RNA (lncRNA) expression. Few lncRNAs have been characterized, and there are no guidelines for functional prediction or categorization. Therefore, we developed a novel computational approach: Correlative Recurrent Expression of Predicted Elements (CREPE), which integrates -omics data to create a correlative regression model to predict lncRNA function. CREPE analysis revealed several NHL-associated lncRNAs that may regulate the expression of signaling proteins downstream of B-cell receptor activation. We highlight a multi-exon lncRNA that is highly expressed in NHL and has a top-ranked CREPE transcriptional regulatory score: AC074289.1. Consistent with a role in regulating transcription, qRT-PCR of subcellular fractions demonstrate nuclear localization and chromatin association (3.5–5X : cytoplasm). Expression of AC074289.1 is cell-type specific with 5–10X higher levels in B lymphoid compared to T or epithelial cell lines. AC074289.1 expression positively correlates with a neighboring gene, PELI1, which encodes Pellino 1, an E3 ubiquitin ligase that modulates NF-κb signaling in immune cells. NF-κb signaling also plays a significant role in NHL pathogenesis. Notably, high PELI1 expression is associated with more aggressive NHL. In summary, our global -omics study of normal and NHL samples identified new gene regulatory connections between lncRNAs and potential NHL oncogenes, including AC074289.1 and PELI1. This novel approach will accelerate our understanding of lncRNA function and may uncover new therapeutic targets in NHL.