bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid modification of specificity and affinity of anti-influenza VHH antibody using two in vitro evolution methods
Authors: Kishimoto Hidehiro , Yoshida Maiko , Tsukahara Narutoshi , Matsubara Toshiyuki , Nakayama Hideorshi , Murakami Akikazu
Published: January 2023

Heavy-chain antibodies (HCAbs) were discovered in camelids in 1990s and the Ag-binding fragment of the HCAbs consists of a single domain known as a variable domain of the heavy chain of HCAbs (VHH). Because of the many favorable properties of VHHs, i.e. small size (12kDa-15kDa), high affinity, high-stability and well expression in microorganisms, they have become widely used in research and are beginning to show commercial potential. Most of the VHHs have been isolated from recombinant VHH phage display libraries from immunized camels or llamas and their successive panning has been the preferred method for the isolation of high-affinity specific binders, however, the procedure is time-consuming and costly. Therefore, we constructed semi-synthetic alpaca VHH phage display libraries having more than 20 billion repertoires. From the library, Ag specific VHH clones can be isolated within 10 days, and we have obtained hundreds of VHH clones against more than 30 different Ags. One of the anti-influenza HA (H1N1) VHH clone has neutralization activity. Unfortunately, the clone only binds to H1 sub-type of hemagglutinin. Therefore, we optimized two kind of in vitro evolutional methods to modify the specificity and affinity of the anti-H1 VHH clone. Random mutagenesis is commonly used for evolution of antibodies using the phage display method, but it is very difficult to obtain clones with greatly increased affinity. We have already constructed a rapid affinity improvement method using phage display system, CDR3 preservation method, thus we combined these two methods to create new rapid modification system of VHH antibodies. Here, we show an example in which specificity was changed from influenza H1 to H5, and further improved affinity.
Zika virus detection using a flow cytometry platform
Authors: Wood Steven C
Published: January 2023

Zika is an arthropod-borne virus related to other Flaviviridae (FV) members such as West Nile Virus, Yellow Fever Virus Japanese Encephalitis Virus and Dengue Virus All these FV infections can develop into indistinguishable clinical symptoms such as headaches, fever, body aches, and joint pains. However, in utero exposure to Zika results in microcephaly, fetal demise and Guillain–Barré syn-drome. These devastating complications necessitated the development of a specific and sensitive assay that distinguishes Zika infection from those caused by other FV. Development of a Zika specific immune-detection assay is challenging due to the presence of cross reactive antibodies. Therefore, we developed a high throughput Zika detection assay using a flow cytometry platform and Zika antigens as reference materials. Sera from Zika positive and negative patients were diluted 100, 1,000 and 10,000 fold and used for the initial standardization experiments. Minimal background fluorescence was detected at 10,000-fold dilution of sera, and therefore the assay was standardized at these optimal conditions. Flow cytometric analysis revealed the presence of anti-Zika antibodies in Zika patients and out of 10 patient samples tested, 9 were confirmed to be Zika positive. The ability of this flow cytometry based assay to detect anti-Zika antibodies was also confirmed using sera samples collected from Zika Ns1 and Env protein immunized mice. In summary, we report the development of a novel flow cytometry based Zika detection method that can be used for Zika counter measures. The rapid turnaround time of flow based assays as well as their ability to detect and distinguish antibody subtypes and antigens will improve diagnostic accuracy of Zika detection.
High Throughput pMHC-I Multimer Library Production Using Chaperone-Mediated Peptide Exchange
Authors: Overall Sarah , Toor Jugmohit S , Yarmarkovich Mark , Hao Stephanie , Nguyen Son , Sada-Japp Alberto , Betts Michael R , Maris John M , Smibert Peter , Sgourakis Nikolaos G
Published: January 2023

The development of pMHC-multimer technologies has been indispensable for the characterisation of T cell responses to individual viral and tumor peptide antigens. Given the large variety of T cell receptors expressed in polyclonal repertoires, peptide exchange technologies are becoming increasingly important in providing a high throughput means of generating pMHC-multimer libraries, containing a wide range of antigens to probe such repertoires. Here we present a simple method for the isolation of empty MHC-I molecules which can be readily used for high throughput multimer library preparation. We utilise the chaperone TAPBPR, which binds MHC-I molecules and stabilises them in a peptide-receptive conformation, to isolate empty MHC-I molecules through the combination of placeholder peptides and small molecule competitors. We demonstrate that empty H-2Dd:TAPBPR and HLA-A*02:01:TAPBPR complexes can be stored long term and subsequently loaded with peptides of choice in a high throughput fashion to afford pMHC-multimer libraries. The resulting pMHC-multimers show equivalent flow cytometric properties to multimers made from MHC-I molecules refolded in vitro with synthetic peptides and are significantly improved relative to multimers prepared using peptide exchange of conditional ligands. Using this simple system to generate a library of oligonucleotide-labeled pMHC-multimers, combined with our recently described multi-modal cellular indexing technology (ECCITE-seq), we can identify distinct T cell receptor sequences present in polyclonal repertoires, together with their antigen specificities.
A universal planar assay format for high sensitivity cytokine quantification in human serum and plasma
Authors: Lambert Jeremy , Mendes Muriel , Yan Yu-xin , Ball Andrew
Published: January 2023

Quantifying prospective biomarkers of immune signaling in biological samples requires high assay sensitivity. The Simoa Planar Array provides greater sensitivity than ELISA, but has a finite assay menu. This study aimed to develop a universal ‘homebrew’ planar immunoassay format for development of high sensitivity assays for any analyte, by using an anchor antibody/peptide tag system to develop a universal capture plate.

Anchor antibody specific for a peptide tag was microprinted in microwell plates. The peptide tag was conjugated to analyte-specific capture antibodies via maleimide chemistry, enabling high affinity binding of capture to anchor antibody. Bound sample antigen was sandwiched between the peptide-tagged capture and biotinylated detector antibodies; chemiluminescent detection was used to quantify signal.

This ‘homebrew’ approach successfully detected sub picomolar levels of cytokines and chemokines in human or mouse serum and plasma, including IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-22, ITAC, MIP1a and MIP3b. When compared to commercially available planar assays the universal planar assay format provided comparable LLOQ and LOD with sample correlation (R2) >0.97 for all analytes tested. The universal planar assay was used to successfully screen multiple antibody pairs for human and mouse biomarkers and to develop robust assays using mouse, rabbit or goat monoclonal and polyclonal antibodies.

This universal planar homebrew assay format combines the accuracy, reproducibility and high sensitivity of Simoa Planar Array technology with total analyte flexibility, and provides assay developers a simple and versatile method to rapidly develop robust high-sensitivity assays for any analyte of their choice.
Transcriptomic analysis of Paired Nasal and Bronchial brushes in Cystic Fibrosis Patients
Authors: Erb Carla J , Chen Kong
Published: January 2023

Cystic Fibrosis is a genetic disorder affecting approximately 30,000 people in the United States. CF mainly affects the lungs and digestive track, leaving individuals with CF more susceptible to lung infections. Transcriptomic analysis of bronchial brushing can be used to identify gene signatures of inflammatory lung diseases such as asthma and CF. However, due to the invasiveness of this technique, the application is limited in pediatric patients. We sought to determine whether nasal brushes could be used as an alternative for transcriptomic analysis of the airway by determination of the similarities and differences in nasal and bronchial gene expression, and whether there is a correlation between nasal transcriptomes and their bronchial counterparts. To address this, we compared five pairs of Nasal and Bronchial Brushes obtained from pediatric CF patients.

The initial principal component analysis (PCA) analysis showed distinct clustering of the bronchial and nasal data. A Pearson correlation test was run on each pair of nasal and bronchial data sets for all genes and revealed both weak and strong correlations with correlation coefficient value ranging from 0.244 to 0.872. To visualize the correlation of CF specific genes, we generatedheatmaps of a few subsets of genes chosen from published literature including top differentially expressed genes in CF vs non-CF. Our finding suggested that patients with overall higher correlation coefficient tend to have better correlations in top genes down-regulated in CF versus non-CF bronchial epithelium. However, further analysis and data set collection might be necessary due to the heterogeneous nature of these samples.
Human G-CSF knockin NOG mice support the differentiation of human neutrophils and recapitulate an emergency granulopoiesis after bacterial infection
Authors: Ito Ryoji , Katano Ikumi , Takahashi Takeshi , Goto Motohito , Takahashi Riichi , Ito Mamoru
Published: January 2023

Humanized mice, in which the human hematopoietic system is reconstituted in immunodeficient mice, are a useful animal model to study human hematology and immunology. Although human T or B lymphocytes significantly differentiated in human hematopoietic stem cells (HSC)-transferred humanized mice, myeloid lineage cells, especially granulocytes, do not fully develop in them. In this study, we generated a novel NOG mouse strain, human G-CSF knockin (KI) mouse, to reconstitute mature human neutrophils in their circulation. We constructed the KI vector that the human G-CSF gene is driven by CMV promoter and targeted murine G-CSF receptor locus to exclude the cross reaction to murine G-CSF receptor. Severe murine neutropenia was observed in hG-CSF KI mice due to the dysfunction of murine G-CSF receptor, and the human G-CSF cytokine was extremely produced in their serum. To analyze human hematopoiesis, we transferred human HSC into x-ray irradiated hG-CSF KI mice. During weeks 4–12 of transplantation, human neutrophils and monocytes were significantly developed in hG-CSF KI mice compared to conventional NOG mice. An emergency granulopoiesis was recapitulated in them, since human neutrophils were highly mobilized into PB after zymosan treatment or E. coli infection. In ex-vivo experiments, neutrophils produced ROS after LPS or PMA stimulation, suggesting these neutrophils are functionally matured. This strain is the first humanized mouse that mature human neutrophils developed in PB and is useful to study innate host defense against bacterial infections.
Simultaneous mRNA, protein, and immune-repertoire profiling of thousands of single cells
Authors: Chang Christina , Nakamoto Margaret , Lai Janice , Siddique Imteaz , Mortimer Stefanie
Published: January 2023

High-throughput single cell RNA-seq (scRNA-seq) has transformed our understanding of heterogenous immune populations. Advances in scRNA-seq, such as oligo-conjugated antibody technologies that enable protein expression profiling alongside mRNA, are expanding the range of molecules that can be profiled at the single cell level. Although sequencing the T-cell and B-cell immune repertoire can provide crucial insights into the complexities of the adaptive immune system, thus advancing discoveries in immunology and immune-oncology, the ability to extract this information from single cells requires new technology to profile regions of the mRNA missed by conventional 3′ scRNA-seq. Here we demonstrate a novel approach using the BD Rhapsody™ system to extract immune-repertoire information in addition to gene-expression information from the same cells. In a single workflow, we profiled a panel of 400 mRNA targets and 40 protein markers as well as the CDR3 region of T- and B-cell receptors in thousands of human PBMCs. After using the mRNA and protein information to robustly annotate different lymphocyte populations, we examined the T- and B-cell repertoires for differences in their clonal diversity. We were able to identify clonally expanded T cells and B cells and examine how their gene-expression profiles differ from that of non-expanded cells. This study demonstrates the power of combining mRNA, protein, and immune repertoire analysis at the single cell level to answer complex and important biological and clinical questions.

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

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Disruption of TOX transcription factors enhances CAR T cells function in solid tumors
Authors: Seo Hyungseok , Chen Joyce , Avalos Edahi Gonzalez , Castruita Daniela Samaniego , Moyado Issac Lopez , Wu Cheng-Jang , Das Arundhoti , Lu Li-Fan , Bhandoola Avinash , Rao Anjana
Published: January 2023

T cells expressing chimeric antigen receptors (CARs) have shown impressive therapeutic efficacy against leukemias and lymphomas, but have not been as effective against solid tumors because chronic stimulation with tumor antigens causes them to enter a hyporesponsive (“exhausted” or “dysfunctional”) state. Here we show that the high-mobility group (HMG)-box transcription factors TOX and TOX2 are highly expressed in CAR-expressing exhausted (PD-1highTim3high) CD8+ tumor-infiltrating lymphocytes (TILs). CAR TILs deficient in both TOX and TOX2 (TOX DKO) suppressed tumor growth and prolonged survival of tumor-bearing mice, compared to wildtype (WT), TOX-deficient or TOX2-deficient CAR TILs; they also showed increased effector function with increased TCF1 expression and decreased expression of the inhibitory receptors PD-1, LAG3 and CD160. Like NR4A transcription factors, TOX and TOX2 are critical for the transcriptional program of hyporesponsiveness downstream of NFAT. Disruption of TOX expression or activity could be a promising strategy for cancer immunotherapy.
Inflammasome Signaling Mediates Myeloid Immunosuppression within the Tumor Microenvironment
Authors: Lang Cara N , Kim Young
Published: January 2023

Myeloid Derived Suppressor Cells (MDSCs) are important mediators of immune evasion in cancer and have been associated with worse overall survival and progression free survival in patients with multiple types of solid tumors. While MDSCs were initially characterized to inhibit T cells and NK cells through the upregulation of iNOS and arginase-1 and production of Nitric Oxide, reactive oxygen species, TGF-β, and IL-10, there is now growing literature that MDSCs can promote cancer progression through other mechanisms. Here we propose that myeloid-intrinsic inflammasome activation plays a direct role in tumor progression. To investigate this, in vitro tumor proliferation studies were performed and show that CD11b+, Ly6C+ monocytic MDSCs isolated from tumor bearing mice promote inflammasome-dependent proliferation of multiple murine tumor cell lines. Mass cytometry and RNA sequencing data of human Head & Neck Squamous Cell Carcinoma patient samples indicate that the total myeloid immune infiltrate can differ greatly among patients. In cases where the T cell immune infiltrate is low, we propose that the infiltrating myeloid cells are utilizing inflammasome signaling to promote tumor progression. These results provide a greater understanding of the role of inflammasome signaling in MDSC-mediated immunosuppression within the tumor microenvironment which will allow for a better understanding of the function of MDSCs in cancer and identification of potential targetable components of MDSC signaling for cancer therapeutics.
The fate of leukemia-specific CD8+ T cell clones is dictated by antigen specificity and the mechanism of cognate antigen presentation
Authors: Chen Xiufen , Flood Blake , MacNabb Brendan , Blazar Bruce R. , Kline Justin
Published: January 2023

We previously demonstrated that high-affinity leukemia-specific CD8+ T cells are deleted in leukemia-bearing hosts in a manner that depends on cross-presentation of the antigen by splenic Batf3-lineage dendritic cells. Here, we generated a T cell receptor transgenic mouse strain (Tg101) that expresses TCR-α and -β chains from a CD8+ T cell clone specific for a native H-2Kb-restricted antigen on C1498 leukemia cells. In naïve mice, Tg101 T cells demonstrate no evidence of self-reactivity, arguing that the Tg101 antigen is leukemia-specific. In leukemia-bearing mice, high-affinity 2C CD8+ T cells specific for the H-2Kb-restricted model antigen, SIYRYYGL, encounter antigen rapidly (day 3–4) via its cross-presentation by splenic CD8a+ DCs, proliferate briefly, and are largely deleted by day 12. Conversely, antigen encounter by Tg101 CD8+ T cells in mice with leukemia is delayed (day 8–10), and primarily occurs in the liver - a prominent site of leukemia progression. Subsequently, Tg101 T cells expand, upregulate expression of co-inhibitory receptors, including PD-1, LAG-3, TIM-3 and TIGIT, and produce effector cytokines poorly, consistent with the acquisition of a dysfunctional state. Surprisingly, in vivo antigen encounter by Tg101 T cells occurs independently of CD11c+ cells, indicating that the Tg101 antigen is poorly cross-presented. Rather, the antigen is directly presented to Tg101 T cells by C1498 cells. Collectively, our data reveal that CD8+ T cell tolerance is effectively generated in leukemia-bearing animals. However, the nature of the leukemia antigen and the context in which it is presented are critical factors in dictating the underlying mechanism of tolerance that ensues. A search for the Tg101 antigen is ongoing.
Influence of immune-checkpoint inhibitor and HLA-G in patients with Cervical Cancer
Authors: CRISPIM-FREITAS JANAINA C. O. , Morais Rayanne S. P. , Oliveira Valéria S. , Sadissou Ibrahim , Palomino Gustavo M. , Cobucci Ricardo N. O. , Lira George A. , Carvalho Kleyton T. C. , Silva Emanuelly B. O. , Silva Norma L. , Donadi Eduardo A.
Published: January 2023

A better understanding of the role of Immune checkpoint inhibitors (ICI) and immunoregulatory genes HLA-G is essential for the development of immunotherapeutic strategies in cancer. This study aims to evaluate the influence of CTLA-4, PD-1 and polymorphisms of HLA-G and soluble HLA-G (sHLA-G) in patients with cervical cancer (CC), different degrees of Cervical Intraepithelial Neoplasia (CIN), and healthy control (HC). One hundred and seventy-four (174) non-pregnant Brazilian women, sexually active, older than 18 years, were evaluated. These patients were stratified according to the results of the anatomopathological examination in: CC group n=24, Low-grade squamous intraepithelial lesions (LSIL) n=66, High-grade squamous intraepithelial lesions (HSIL) n=84. HC group consisted of 47 women without cytological and/or colposcopic abnormalities detected. The peripheral blood of these patients was collected for analysis of CTLA-4, PD-1 and HLA-G polymorphisms using real-time PCR. The analysis indicated a significantly higher 14bpDel allele frequency in CC when compared to HC group (P=0.030, OR=1.71, 95%CI 1.01–2.74). Moreover, the 14bpDel/Del genotype frequency was also significantly higher in patients with CC group (P = 0.035, OR=2.23, 95%CI 1.05–4.97). Qualitative analysis revealed that the production of sHLA-G is detectable in 52.03% (n=77) of all patients groups and 34.29 %(n=12) in HC groups. This analysis revealed a trend of significance (P= 0.063; OR=2.08, 95%CI=0.96–4.48) in CC group. The polymorphisms of CTLA-4 and 14 bp HLA-G are associated with a significantly increased risk of CC. HLA-G 14bp Del/Del polymorphism, and the serum sHLA-G can act as a promising biomarker for might be a genetic risk factor for CC.
Patient-derived, vaccine-elicited MUC1 antibodies mediate immune effector functions against cancer cells
Authors: Miller Michelle L , Lohmueller Jason , Ricci Eric , Lu William , Finn Olivera J
Published: January 2023

Cancer cells undergo changes that the immune system recognizes, and this can be leveraged for therapy. Antibodies specific for such changes can exert anti-tumor effects via multiple mechanisms, including blocking oncogenic signaling, serving as Trojan horses carrying toxic compounds, coordinating innate immune engagement for cytotoxicity through cellular and non-cellular means, and promoting antigen uptake to jumpstart adaptive immunity for durable protection. We analyzed 12 high-affinity fully human IgG1 monoclonal antibodies that recognize a tumor-specific hypoglycosylated form of Mucin-1 (MUC1) that is overexpressed in >80% of all cancers. These antibodies are unique and different from humanized murine anti-MUC1 mAbs, having undergone selection and affinity maturation in individuals receiving the MUC1-100mer peptide vaccine (Lohmueller et al. Sci. Rep. 2016). Importantly, in all vaccinated individuals that generated antibodies to MUC1, there have been no adverse events in 8 years, increasing the likelihood that as therapeutic agents they would be safe. To test their immune effector capacity, we used these human anti-MUC1 antibodies in in vitro antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC) assays with different huMUC1-expressing cell lines, immune effector cells and serum. Several of the anti-MUC1 antibodies can mediate ADCC by NK cells and ADCP by macrophages, although there was very little or no CDC function. Notably, unlike in published studies on rituximab and alemtuzumab, the amount of antibody binding to cancer cells (MUC1) did not always correlate with increased effector function.
Bortezomib improves antitumor CD8+ T cell function by modulating miR-155 and its targets
Authors: Renrick Ariana N , Thounaojam Menaka , Chaudhuri Evan , Dash Chandravanu , Shanker Anil
Published: January 2023

The immunosuppressive tumor microenvironment dampens host antitumor immunity by multiple mechanisms including interfering with various cell signaling pathways that aid in the differentiation and function of immune cells. We have taken an immunotherapeutic approach in order to strengthen antitumor immunity in mice bearing solid tumors, specifically breast tumors. In doing so, we observed that treatment with bortezomib, an FDA-approved proteasome inhibitor, has the ability to increase CD8+T lymphocyte IFNγ secretion and expression of effector molecules, perforin, granzyme-B and the T-box transcription factor eomesodermin. In order to understand the molecular mechanism(s) of how bortezomib works to improve antitumor immunity we sought to explore its effects on miRNA expression as well as function. We found that treatment of wild-type or tumor-bearing BALB/c mice with bortezomib modulated the expression of various miRNAs in CD8+T cells. From miRNA array data, we identified miR-155 as one prominent modulator of antitumor CD8+T cell immune functions. miR-155 has been accredited to controlling CD8+T cell responses by regulating interferon signaling in viral infections and cancer. We are currently elucidating the mechanisms of bortezomib-mediated effects on miR-155 and its targets, such as suppressor of cytokine signaling 1 (SOCS1) and inositol polyphosphate-5-phosphatase (SHIP1) that are associated with T cell function. These data provide novel insights on using bortezomib not only as an agent to sensitize tumors to cell death, but also to provide lymphocyte-stimulatory effects that could overcome immunosuppressive actions of tumor on antitumor T cell function.
Getting in the groove: Editing of MHC-I antigen repertoires by molecular chaperones is governed by a network of protein dynamics
Authors: Sgourakis Nikolaos , McShan Andrew , Natarajan Kannan , Jiang Jiansheng , Park Jihye , Overall Sarah , Toor Jugmohit S , Kumirov Vlad , Flores-Solis David , Badstubner Mareike , Kovrigin Evgenii L , Bagshaw Clive R , Pallesen Jesper , Procko Erik , Margulies David H
Published: January 2023

Molecular chaperones TAPBPR (TAP-binding protein related) and tapasin associate with class-I major histocompatibility complex (MHC-I) molecules to promote optimization (editing) of peptide cargo. Using solution NMR, we investigate the molecular mechanism of peptide exchange performed by the 90 kDa chaperone protein complex. We identify TAPBPR-induced conformational changes on conserved MHC-I surfaces, consistent with our independently determined X-ray structure of the empty complex. Conformational dynamics present in the empty MHC-I are stabilized by TAPBPR in a peptide-deficient complex, and become progressively dampened with increasing peptide occupancy. Incoming peptides are recognized by the chaperoned groove according to the global stability of the final pMHC-I product, and anneal in a native-like conformation. Our results demonstrate an inverse relationship between MHC-I occupancy by peptide and the affinity of TAPBPR for such pMHC-I molecules, where the lifetime of transiently bound peptides controls the dynamic regulation of a conformational switch, located near the TAPBPR binding site, which triggers TAPBPR release. We further discuss the role of protein dynamics in shaping chaperone specificity towards different human and murine class-I MHC allotypes, and present the high-resolution cryoEM structure of a human A*02/TAPBPR complex with novel insights into the antigen editing mechanism. These results suggest a similar mechanism for the editing function of tapasin in the peptide-loading complex, and provide a molecular blueprint for the design of novel chaperones with tailored antigen editing functions.
The gut microbiota from lupus-prone B6. Sle1.Sle2.Sle3 (TC) induces inflammation via a mechanism other than bacterial translocation
Authors: Brown Josephine Michelle , Choi Seung-Chul , Morel Laurence
Published: January 2023

The lupus-prone microbiota from TC mice induces autoimmunity when transferred to germ-free B6 mice and broad-spectrum antibiotic treatment attenuates autoimmunity in TC mice, demonstrating a microbiota involvement in inflammation. Aged TC mice show colon inflammation relative to B6 controls Fecal blood was detected in approximately 30% of aged TC mice tested, whereas B6 mice were negative. Based on these findings, we sought to understand if compromised barrier integrity could account for the proinflammatory nature of the TC microbiota. Serum endotoxin and intestinal tight junction protein expression were similar between TC and B6 controls, and serum FITC levels were lower in TC mice following FITC-dextran gavage. Colony growth was found in about 30% of aged TC liver and mesenteric lymph node homogenates, but not in the tissues of pre-autoimmune TC mice. The TC bacterial colonies were identified as Staphylococcus xylosus, a non-pathogenic commensal. These findings suggest that TC mice do not have a compromised intestinal barrier, and that the bacterial translocation that occurs in a minority of TC mice only after the onset of autoimmunity is a consequence, rather than a cause of autoimmunity. Therefore, the inflammatory nature of the TC microbiota is likely related to immune activation starting in the gut itself through the production of specific metabolites.
Type II Cytokines in the Thymic Microenvironment Influence T cell Negative Selection in Type One Diabetes
Authors: Cattin-Roy Alexis N , Ukah Tobechukwu Kenneth , Zaghouani Habib
Published: January 2023

From an autoimmunity perspective type II cytokines, such as IL-4 and IL-13, are viewed as anti-inflammatory because of their ability to foster peripheral tolerance. This report provides evidence that credits these cytokines with yet another attribute that pertains to central T cell tolerance. Specifically, a subset of early thymic progenitors (ETPs) which expresses the IL-4Rα/IL-13Rα1 heteroreceptor (HR) adopts a pattern of lineage commitment with guidance from IL-4/IL-13 in the thymic microenvironment. Indeed, IL-4 and IL-13 direct HR+ ETPs towards the myeloid lineage by inhibiting their T cell potential and promoting maturation towards dendritic cells (DCs). Given that DCs can contribute to negative selection of self-reactive T cells, IL-4 and IL-13 in the microenvironment would impact the development of autoreactive T cells in two ways: diminished T cell generation from ETPs and elimination of self-reactive lymphocytes by DC-driven negative selection. This postulate is supported by findings indicating that in the C57BL/6 mouse strain, where IL-4 and IL-13 are readily available in the thymic microenvironment, HR+ ETPs give rise to myeloid cells that contribute to negative T cell selection leading to resistance against experimental allergic encephalomyelitis (EAE). In contrast, in NOD mice IL-4 and IL-13 availability is limited and HR+ETPs give rise to T cells; perhaps leading to diminished negative selection of T cells with diabetogenic potential and higher susceptibility to type 1 diabetes (T1D). These previously unrecognized observations extend the function of type II cytokines to central tolerance and offer a possible link of the environment to the development of autoimmunity as envisioned by hygiene hypothesis theories.
Involvement of CCR5 in bone resorption associated to apical periodontitis
Authors: De Rossi Andiara , Alves Sandra Yasuyo Fukada , Nelson-Filho Paulo , Silva Raquel Assed B , Silva Lea Assed Bezerra
Published: January 2023

The involvement of Cysteine-cysteine chemokine receptor 5 (CCR5) in the protection or stimulation of bone resorption associated with apical periodontitis that occur as consequence of endodontic infection has not yet been elucidated. The present study proposes to characterize the involvement of CCR5 in the genesis and progression of apical periodontitis, using animals deficient for this molecule. Experimental induction of periapical lesions was performed in CCR5 −/− (knockout) or wild type transgenic mice through the coronary opening of the lower 1st molars and exposure of the root canals to the oral environment for their contamination. After the death of the animals (at 0, 7, 21 and 42 days), the samples obtained in different experimental periods were evaluated by conventional microscopy and fluorescence (morphometry and characterization of the lesions), histoenzimology (TRAP for osteoclasts), immunohistochemistry, immunofluorescence and real-time PCR, to characterize the inflammatory cell in the presence or absence of CCR5 (neutrophils, macrophages and CD4 and CD8 lymphocytes), expression of cytokines (IL-1±, TNF-±, IFN-³, IL 4, IL-6 and IL-10), their ligands (RANTES and MIP- 1±) and factors involved in bone formation and resorption (RUNX2, NFATc1, ALP, RANK, RANKL, OPG and cathepsin K) and infection of tissues. Our results shows that CCR5 knockout mice developed more extense and severe inflammation and alveolar bone destruction at periapical region at 21 and 42 days after endodontic infection. It was concluded that CCR5 deficiency results in development of more severe periapical alveolar bone resorption associated to endodontic infection.
Reduced acetylation of histones H3 in peripheral blood mononuclear cells of patients with Primary Sjögren’s syndrome
Authors: Li Yan
Published: January 2023

Sjögren’s syndrome (SS) is an autoimmune disease characterized by an exocrine glands epithelium injury with dense lymphocytic infiltrates, mainly composed of activated T and B cells, involvement of the peripheral nervous, pulmonary, blood, kidney, skin, and joint. Present at the interface of genetic and environmental risk factors, histone acetylation is suspected to play a key role in SS. To clarify this point, we investigated the expression of histone deacetylases (HDACs1–10) and histone acetyltransferase (HAT-p300 CREEBP PCAF) by real time PCR in peripheral blood mononuclear cells (PBMCs) from SS patients. Elisa measured HDACs\HATs and histone H3/ H4 acetylation activity. Flow Cytometry and western blot verified histone acetylation level. The expression of HDACs1-10, p300, CREEBP and PCAF in PBMCs from SS patients was decreased in mRNA comparison with healthy controls (HCs). Total histone acetylation was reduced in PBMCs from SS patients, while no difference was observed in histone deacetylation. The extent of total histone H3, but not H4, acetylation in PBMCs from SS patients was decreased compared to that in HCs. An altered balance between HDAC and HAT activity was found in SS PBMCs. As a consequence, part of the pathogenic mechanisms in SS may be linked to epigenetic modifications, thus opening new therapeutic perspectives in SS.
Peripheral blood neutrophil and G-CSF levels are linked to aztreonam lysine use in humans with cystic fibrosis
Authors: Wright Nathan Darrell , Gordon John Robert
Published: January 2023

The neutrophil response contributes substantially to disease progression in cystic fibrosis (CF). Research was directed at understanding the circulating neutrophil in CF. Medical chart data and 30 blood samples were collected from volunteers with CF (ΔF508/ΔF508; mean age 29 years) while 21 blood samples were collected from age-matched controls. Isolated plasma was then analysed for G-CSF and CXCL1 and statistically compared with neutrophil numbers and medical chart data, while signalling pathways in these neutrophils were investigated using kinome array approaches.

Our results indicated that the CF group being treated with the inhaled β-lactam antibiotic aztreonam lysine had significantly increased G-CSF levels (p<.05) and isolated circulating neutrophil numbers (p<.01) when compared to the CF group without the antibiotic. Neutrophil numbers were also significantly increased in both the inhaled aztreonam lysine positive (p<.001) and negative (p<.05) CF groups when compared to the non-CF control group. Neutrophil numbers in CF displayed a significant (p<.05) positive correlation with G-CSF levels. We observed a significant (p<.05) negative correlation between disease progression in CF (observed as FVC%) and neutrophil numbers. Kinome analyses indicated several altered pathways that warrant further investigation. These results revealed a connection in CF between antibiotic treatment, inflammatory markers in the blood, altered neutrophil signaling, changes in circulating neutrophil numbers, and disease progression. From a clinical perspective these results identified a potential connection between inhaled aztreonam lysine, circulating neutrophil numbers and decreased lung function in our CF volunteers.
Potential role of myeloid-derived suppressor cells in pulmonary fibrosis
Authors: Liu Tianju , Rinke Andrew , Flaherty Kevin , Phan Sem Hin
Published: January 2023

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease without effective curative therapy. Recruitment of bone marrow-derived myeloid cells is implicated in lung fibrosis by promoting fibrosis via paracrine mechanisms. Recent evidence shows increased circulating myeloid-derived suppressor cells (MDSC) in IPF patients. More recently, the importance of MDSC is suggested by the observation of MDSC accumulation in IPF lungs. The objective of this study was to assess the accumulation/expansion of MDSC in bleomycin-induced mouse model of pulmonary fibrosis and human IPF, and investigate their potential role in lung myofibroblast differentiation. Flow cytometry analysis showed that MDSCs in lung were increased in a mouse model of lung fibrosis, as well as in peripheral blood samples from IPF patients. The increase of the monocytic subtype of MDSC (M-MDSC) was greater than that for the granulocytic subtype (G-MDSC) in the peripheral blood samples from patients with IPF. In vitro co-culture of normal mouse lung fibroblasts with sorted bone marrow (BM)-derived MDSCs using trans-well inserts revealed paracrine activation of lung fibroblasts by activated M-MDSC, but not GMDSC, as manifested by significantly elevated mRNA levels of α-smooth muscle actin and TGFβ mRNAs. Both quiescent M-MDSC and G-MDSC failed to stimulate fibroblast activation or myofibroblast differentiation. Interestingly, the level of TGFβ mRNA in M-MDSC was higher than G-MDSC. These findings indicated that in pulmonary fibrosis, BM-derived MDSCs were mobilized and recruited to the lung, and the recruited M-MDSC subpopulation subsequently played a paracrine role by activating resident lung fibroblasts and myofibroblast differentiation.
Simultaneous analysis of gene expression and T cell repertoire in peripheral blood mononuclear cells from patients with cystic fibrosis at the single cell level
Authors: Ferguson Annabel , Luo Jaidi , An Xiaojing , Pilewski Joseph M , Chen Wei , Chen Kong
Published: January 2023

Highly sensitive RNA sequencing (RNA-Seq) methods enables gene expression analysis of low-input samples and even single cells. Single-Cell RNA sequencing (scRNA-seq) is a powerful tool for studying the transcriptome and enables discovery of cellular differences usually masked by bulk sampling methods. The Chromium Single Cell Immune Profiling Solution developed by 10X genomics makes it possible to examine gene expression and T cell receptors simultaneously on a cell-by-cell basis. Patients with cystic fibrosis (CF) often suffer inflammatory lung diseases caused by chronic Pseudomonas aeruginosa (PA) colonization. This colonization could also skew the lymphocytes repertoire in peripheral blood mononuclear cells from CF patients. We investigated the use of scRNA-seq to assess the lymphocyte repertoire in CF patients with PA colonization and whether the single-cell transcriptomic changes correlate with preferential T cell receptor (TCR) usage. Our analysis did not find evidence for clonal expansion of T cells in peripheral blood of these CF patients, suggesting that PA colonization mainly elicits local adaptive immune responses in the lungs. However, this dataset did reveal a striking depletion of the population of T cells known as mucosal associated invariant T cells (MAIT cells) in CF patients. Within the MAIT cell population, we found an overrepresentation of certain genes that was observable in healthy but not CF samples. In this study we show the feasibility of simultaneous analysis of gene expression and lymphocytes repertoire in PBMCs. We recapitulate previous findings that implicate a protective role for MAIT cells against PA colonization, and also provide evidence for the involvement of particular genes in this effect.
The impact of IL-27 on human T cells is altered in multiple sclerosis patients
Authors: Clénet Marie-Laure , Laurent Cyril , Devergne Odile , Duquette Pierre , Prat Alexandre , Larochelle Catherine , Arbour Nathalie
Published: January 2023

Interleukin-27 (IL-27) exhibits pro- and anti-inflammatory properties. Although this cytokine dampens the severity of experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS), whether IL 27 contributes to the pathobiology of MS remains unclear. We detected the IL-27 receptor (IL-27R) on brain-infiltrating T cells in MS tissues suggesting that they are susceptible to this cytokine. Notably, beneficial effects of MS immunomodulatory therapies are associated with IL-27 induction. We compared blood samples from untreated MS patients to healthy donors (HC) for i) serum levels of IL-27 and expression of IL-27R by T cells and ii) effects of IL-27 on phenotype and functions of T cells. Reduced proportions of CD4 T cells but greater percentages of CD8 T cells expressed IL-27R in MS patients compared to HC. IL-27 triggered phosphorylation of STAT1 (pSTAT1) and STAT3 (pSTAT3) in all T cell subsets; similar levels of pSTAT1 were induced in T cells from MS and HC. In contrast, increased proportions of CD4 and CD8 T cells exhibited pSTAT3 in response to IL-27 compared to cells from HC. Moreover, addition of IL-27 to activated T cells significantly decreased the percentages of GM-CSF or IL-17 producing CD4 and CD8 T cells from MS patients but had a minimal impact on HC’s cells. Finally, sera from MS patients contained elevated amounts of soluble IL-27 and IL-27Rα (a natural IL-27 antagonist) compared to HC. Our results reveal that IL-27 has an enhanced impact on T cells from MS patients, which could contribute to dampen key immune responses (e.g. GM-CSF, IL-17) associated with this inflammatory/autoimmune disease. However, elevated in vivo levels of its natural antagonist could hinder IL-27-mediated anti-inflammatory properties.
Morphometric and cytologic characterization of tertiary lymphoid follicles in resected bowel of patients with Diverticulitis, Ulcerative Colitis, and Crohn’s Disease
Authors: Mauner Aaron Laurence , McNamee Eoin N
Published: January 2023

The presentation of ectopic Tertiary lymphoid structures (TLS) in peripheral tissues is a pathological hallmark for a number of Autoimmune and Immune mediated conditions, not least within the bowel wall of patients with Crohn’s disease. However, there remains limited understanding of the cellular constituents or the function of TLS during intestinal pathology. To address this, we characterized the location, size, frequency and cellular constituents of B-cell follicles within resected bowel tissues from patients with Diverticulitis (DIV), Crohn’s Disease (CD) or Ulcerative Colitis (UC) (n=9–10/group). Samples were formalin fixed, paraffin embedded, serially sectioned, and subsequently stained by multiplexed immunohistochemistry. Morphometric analysis was performed with Olympus CellSens software and PerkinElmer Vectra 3.0 Automated Quantitative Pathology Imaging System and statistical analyses performed using R Statistical Software. Results show that the number, size and anatomic location of large B-cell aggregates differed greatly between disease states, with both UC and DIV follicles localizing primarily in the mucosa; while samples from patients with CD presented with mature Ki67+ TLS in the deeper layers of serosa and muscle. Multicolor IHC shows that TLS in Crohn's disease have large intra-germinal center CD4+ T-cell populations while UC samples contained a marked increase in both monocytes and neutrophils compared with both CD and DIV. These data suggest that despite ectopic TLS being present in a number of pathologic conditions, their composition within the bowel wall is disease state dependent and suggests altered germinal center function.
Monocytes of Chronic Fatigue Syndrome Patients, their Family Members without CFS, and Unrelated Healthy Donors: Searching for Differences
Authors: Hudig Dorothy , Tokunaga Keli , Sung Alexander P , Guglielmo Michael J , Smith-Gagen Julie , Merica Rebecca , Redelman Doug
Published: January 2023

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is characterized by fatigue that does not improve with rest and worsens with exertion. ME/CFS may involve inflammation in the brain and/or be associated with viral infections. Increased numbers of pro-inflammatory monocytes in blood are a hallmark of infection. Biomarkers for, and information about, the CFS disease process are greatly needed. Here we queried circulating monocytes. In a pilot study, CFS patients and family members without CFS from three families were compared in order to reduce genetic and environmental variables. The patients conformed to the Fukuda 1994 standards. Unrelated healthy control subjects matched to the patients were included in the study. Blood monocytes were stained and analyzed by flow cytometry: counted with TruCountR beads, or stained after isolation and overnight culture of isolated peripheral blood monocytes. M1 pro-inflammatory monocytes are CD16A negative, while anti-inflammatory monocytes are CD16A positive; CD2 is a marker for dendritic monocytes, and changes in forward scatter could indicate in vitro activation. Notably, there were statistically significant differences between the lower percentages of M1 monocytes of the CFS patients compared to the controls; however, these differences were not significant between the patients and their family members without CFS. Also, the CD16A MFIs of the patients' monocytes were lower compared to controls but not to family members. One conclusion is that it is important to include family members without CFS as a control group in the search for biomarkers to diagnose CFS.
CD4+ T cell antigen discovery in sarcoidosis
Authors: Greaves Sarah A , Mitchell Angela , Falta Michael , Santos Radleigh , Pinilla Clemencia A , Fontenot Andrew P
Published: January 2023

Sarcoidosis is an inflammatory granulomatous disease that primarily affects the lung. It has a worldwide distribution and affects individuals of all ages, races and both sexes. Despite the prevalence of the disease and continued investigation, the cause of sarcoidosis remains unknown. Evidence suggests that CD4+ T cells in the bronchoalveolar lavage (BAL) of sarcoidosis patients are instrumental in disease progression. Löfgren’s syndrome (LS) is an acute form of sarcoidosis that is accompanied by a specific set of inflammatory symptoms. In patients with LS, disease susceptibility has been associated with expression of HLA-DR3 and an expansion of BAL CD4+ T cells expressing T cell receptor (TCR) a-chain variable region (TRAV) 12-1. We hypothesize that in HLA-DR3+ LS patients, TRAV12-1 CD4+ T cell clones accumulate and expand in the lung in response to unknown antigens. To address the antigen specificity of these expanded CD4+ T cells, we performed single cell PCR of BAL CD4+ T cells derived from DR3+ LS patients to identify related abTCRs expressing TRAV12-1 and generated T cell hybridomas expressing these disease-relevant abTCRs. Using unbiased decapeptide positional scanning libraries, we have identified several stimulatory mimotopes. We used a biometrical analysis to obtain candidate etiologic peptides derived from human, viral, pollen, and mycobacterial protein databases and have identified several exogenous peptides that stimulate TCRs derived from the BAL of patients with active LS. Once etiologic antigens are validated, we will synthesize HLA-DR3-peptide tetramers to allow for more sophisticated approaches in the diagnosis and prognosis of this disease.
Antigenic Function of 14-3-3ζ is a Novel Tool to Treat Rheumatoid Arthritis
Authors: Chakravarti Ritu , McGowan Jenna , Peter Cara , Zhang Youjie , Veerman Brent , Rahaman Rafid , Joe Bina
Published: January 2023

Autoantigens are an integral part of autoimmunity, but their role in health and disease are mostly unknown. We identified 14-3-3ζ as an autoantigen in patients with thoracic aortic aneurysm caused by large vessel vasculitis. Like others, neither the cause nor the consequence of newly-identified antigenic function of 14-3-3ζ protein is known. To address this, we investigated the antigenicity of 14-3-3ζ by studying ex vivo effects on human peripheral blood mononuclear cells (PBMC) polarization, and in vivo using a rat autoimmune disease model. Exogenous 14-3-3ζ promoted PBMC proliferation and T cell polarization towards increasing Th1 and Th17 populations. A specific increase in Th1 cells and IFN-γ production provided evidence for MHC class II presentation of 14-3-3ζ antigen. These 14-3-3ζ-induced responses of T cell polarization and cytokine secretion were inhibited by immunosuppressive agents. Our in-depth studies further revealed that 14-3-3ζ-induced phosphorylation of MAPK led to NF-kB activation in macrophages and PBMC. In vivo administration of recombinant 14-3-3ζ in rats triggered strong innate and adaptive immune responses with significant increases in IL-17, IFN-γ, and anti-14-3-3ζ antibody. Importantly, the immunization of Lewis rats with 14-3-3ζ protected them from rheumatoid arthritis in Pristane-Induced-Arthritis (PIA) model. These collectively demonstrate that the antigenic 14-3-3ζ by acting as a DAMP promotes IFN-γ and IL-17 levels, and confers an anti-arthritic effect. To the best of our knowledge, this is the first report describing the antigenic function of 14-3-3ζ and its physiologically relevant anti-arthritic role.