bluuebunny/crossref_metadata_2025 huggingface dataset:
Immune responses to the microbiome tune MHC class II antigen presentation by the intestinal epithelium to control gut pathology
Authors: Koyama Motoko , Mukhopadhyay Pamela , Schuster Iona S , Henden Andrea S , Hülsdünker Jan , Varelias Antiopi , Vetizou Marie , Kuns Rachel D , Robb Renee J , Zhang Ping , Blazar Bruce R. , Thomas Ranjeny , Begun Jakob , Waddell Nicola , Trinchieri Giorgio , Zeiser Robert , Clouston Andrew D , Degli-Esposti Mariapia A , Hill Geoffrey R.
Published: January 2023

The factors initiating pathology in the gastrointestinal (GI) tract are largely unknown. Graft-versus-host disease (GVHD) of the gastrointestinal (GI) tract is the principal determinant of lethality following allogeneic bone marrow transplantation (BMT). MHC class II-dependent GVHD is initiated by recipient alloantigens being presented to donor T cells that in turn undergo Th1 and Th17 differentiation to mediate pathology in the GI tract. Critically, the mechanisms that initiate disease, especially the relevant antigen presenting cells, remain unclear. Here, we identify the response of IL-12-secreting macrophages to the microbiome as the key driver of IFNγ secretion by mucosal type-1 innate lymphoid cells (ILC1). The latter controls MHC class II expression and antigen presentation by intestinal epithelial cells (IEC) specifically within the ileum. When these tightly regulated responses between microbiome and lamina propria lymphocytes are perturbed by inflammatory signals driven by pre-transplant conditioning, dramatic increases in antigen presentation by IEC occur in the ileum and lethal GVHD ensues. Conditional and Villin-specific deletion of MHC class II in IEC, or inhibition of IL-12 pre-transplant, completely prevent the initiation of lethal GVHD, identifying a readily translatable therapeutic strategy.
Kinase domain deleted engineered HER2 could elicit efficient tumor immune response
Authors: Jeon Insu , Koh Choong-Hyun , Kang Tae-Seung , Lee Jeong-Mi , Kang Yejin , Im Jiyoung , Shin Kwang-Soo , Song Boyeong , Kang Chang-Yuil
Published: January 2023

In tumor immunotherapeutic vaccines, it is important to select tumor-associated antigen because of immunogenicity and oncogenic properties of tumor antigens. Many vaccination platforms were developed to enhance immunogenicity of antigens. In addition, many strategies are using tumor antigens in artificially mutated or truncated forms to eliminate oncogenic function of antigens. On the other hand, as the T cell immune responses depend on the recognition of epitopes that are loaded on the MHC class I molecules, it is important to maintain the original size as much as possible. In this study, we engineered HER2 antigens with four different forms, K684, K965, K1001, and K1117 and tested immunogenicity in B cell and monocyte based vaccine models. In murine models, we observed antigen specific T cell response in the antigen size dependent manner and antigen specific antibody response in the antigen expression dependent manner. We also tested tumor therapeutic models with full-length HER2 expressing CT26. All of four antigens showed tumor suppression in full-length HER2 expressing CT26 tumor models and K1117 showed highest therapeutic effect. In addition, the K1117 showed significant therapeutic effect in HER2 intracellular domain expressing CT26 tumor models compared to the K684. Moreover, in human PBMC studies, the K1117 showed better CD4 and CD8 T cell responses than the others did. These data suggested that antigens containing wide epitope repertoire could show better therapeutic effect.
Higher PIK3C2B gene expression of H1N1+ specific B-cells is associated with lower H1N1 immunogenicity after trivalent influenza vaccination in perinatally HIV infected children
Authors: Cotugno Nicola , Zicari Sonia , Morrocchi Elena , De Armas Lesley R , Pallikkuth Suresh , Rinaldi Stefano , Rocca Salvatore , Manno Emma Concetta , Zangari Paola , Andrews Sarah F , Cagigi Alberto , Rossi Paolo , McDermott Adrian B , Pahwa Savita , Palma Paolo
Published: January 2023

Perinatally HIV-infected children (PHIV), despite effective antiretroviral treatment (ART), present chronic immune activation and suboptimal memory B-cell response to vaccinations compared to healthy controls (HC). Here we aimed at identifying frequency and transcriptional profiles of H1N1-specific B-cells (H1N1-Sp) in PHIV differentially responding to Trivalent Influenza Vaccination (TIV) and HC. H1N1-Sp were FACS-sorted from PBMCs collected 21 days after TIV, and analysed by multiplexed RT-PCR (Fluidigm). Differentially induced genes after H1N1 in vitro stimulation were further assessed on sorted memory B cells. H1N1 response was measured by hemagglutination inhibition assay (HIA) and B-cell ELISpot. 10 out of 19 PHIV were non-responders (NR) while all HC (n=8) correctly responded to TIV (≥4 fold change HIA and >80 ELISpot/106PBMCs). Our results showed no differences in H1N1-Sp frequencies and absolute counts between the groups. H1N1-Sp of PHIV showed higher expression of genes related to apoptosis susceptibility (PPP1R13B (logFC=12.77, p=0.01) and immune activation (CD28 (logFC=10.64, p=0.02) compared to HC. Among PHIV, lower PIK3C2B (logFC=−6.67, p=0.02) expression was found in vaccine responders (R) vs NR. In addition, only R showed a reduction in PIK3C2B expression after in vitro H1N1 stimulation in resting memory B cells (CD19+IgD-CD27+CD21+) (FC=−2.6, p<0.05). In conclusion, a gene expression perturbation, underlying immune activation, persists in H1N1-Sp from PHIV despite ART. Also, a higher expression of PIK3C2B may lead to a lower H1N1 immunogenicity after TIV. These preliminary data may inform future vaccination-adjuvants aiming at modulating PIK3C2B in order to improve vaccination outcomes in PHIV.
CD4 cell-derived IL-2 synergizes with influenza infection to exacerbate lung inflammation
Authors: Strutt Tara M , Gilchrist Ashley
Published: January 2023

Defining the most penetrating correlates of protective memory T cells is key for designing improved vaccines and T cell therapies. Here, we evaluate how interleukin (IL-2) production by CD4 memory cells, a widely held indicator of their protective potential, impacts recall responses against murine influenza A virus (IAV). Unexpectedly, we show that IL-2-deficient memory cells are more effective on a per cell basis at combating IAV than wild-type memory cells marked by strong IL-2 production. Improved outcomes orchestrated by IL-2-deficient cells include reduced weight loss and improved respiratory function that correlate with reduced levels of a broad array of inflammatory factors in the infected lung. Blocking CD70-CD27 signals that reduce T cell IL-2 production tempers the inflammation induced by wild-type memory CD4 cells and improves the outcome of IAV infection in vaccinated mice. Using a reductionist model, we show that IL-2 drives rapid and extremely potent lung inflammation involving NK cells and neutrophils, which can synergize with sublethal IAV infection to promote acute morbidity and mortality. These results suggest that IL-2 production is dispensable for optimal protective CD4 T cell recall responses and that the lung environment is particularly sensitive to IL-2-induced inflammation.
IFN-I blockade improves the immunogenicity of viral vector vaccines
Authors: Palacio Nicole M , Dangui Tanushree , Wang Yidan , Penaloza-MacMaster Pablo A
Published: January 2023

Type I interferons (IFN-I) are critical for antiviral immunity. Studies in the lymphocytic choriomeningitis virus (LCMV) model show that IFN-I signaling improves the control of acute infection, but IFN-I signaling can be immunosuppressive and impair the control of chronic infection. The role of IFN-I after vaccination, however, remains understudied. Vaccination is biologically distinct from natural infection, as immune responses with the former are elicited after in situ challenge with a low dose of an attenuated pathogen, resulting in a benign localized infection that induces immunological memory. To evaluate the role of IFN-I in this setting, we immunized C57BL/6 mice intramuscularly with LCMV-based vaccines expressing model antigens, including SIV Gag, co-administered with IFN-I receptor blocking antibodies. IFN-I blockade resulted in significant improvement of CD8 T cell and B cell responses. Furthermore, IFN-I blockade also improved the immunogenicity of other viral vectors, including rhabdovirus- and poxvirus-based vectors. Improvement of vaccine-elicited immunity was also observed in BALB/c mice using alternative immunization routes. Importantly, transient IFN-I blockade did not affect vaccine clearance, but it abrogated the influx of T regulatory cells (Tregs) to the site of immunization. Our data suggest a model in which IFN-I recruits Tregs to the site of immunization, resulting in in situ regulation of vaccine-induced immunity. Altogether, we show in multiple viral vector regimens, different MHC backgrounds, and different routes, that localized IFN-I modulation improves vaccine-induced immunity. Our findings may be important to develop more potent viral vector vaccines against infectious diseases.
Classical splenic dendritic cell subsets, cDC1 and cDC2, from C57BL/6 mice are more potent in stimulating the Th1 immune response than those from BALB/c mice during mousepox
Authors: Toka Felix N , Szulc-Dąbrowska Lidia , Koper Michal , Struzik Justyna , Gierynska Malgorzata
Published: January 2023

Classical (conventional) dendritic cells (cDCs) specialize in presenting antigen to naïve T cells, and are divided into cDC1 (CD8α+) and cDC2 (CD11b+) subpopulations based on their lineage, surface phenotype and characteristics. Despite differences, both cDC subsets can stimulate CD4+ T cells and promote polarization toward a T helper 1 (Th1) phenotype. Many studies have shown that C57BL/6 (H-2b) and BALB/c (H-2d) mice readily mount vigorous Th1 or Th2 responses, respectively, upon infection with different infectious agents, including ectromelia virus (ECTV), responsible for mousepox in mice. In this work, we compared the ability of cDC subsets from mouse strains with different susceptibility to mousepox (C57BL/6 – resistant and BALB/c – susceptible) to stimulate the Th1 cytokine immune response during ECTV infection. Results showed that splenic cDC1 and cDC2 from BALB/c mice highly express MHC class II, CD83 and/or CD86 than those from C57BL/6 mice at 5 days post infection with ECTV. Despite higher activation status, both subsets of cDCs from BALB/c mice produce low amounts of Th1-polarizing cytokines, including IL-12 and IFN-γ, than those from C57BL/6 mice. Moreover, splenic cDC1 and cDC2 cells, from ECTV-infected C57BL/6 mice, stimulated higher proliferation and production of IFN-γ and IL-2 by allogeneic CD4+ T cells of C3H (H-2k) mice in a mixed leukocyte reaction than BALB/c mice. Both subsets of BALB/c cDCs up-regulated genes engaged in cell maturation and activation compared to C57BL/6 cDCs. Overall, our data indicate that both, cDC1 and cDC2 promote CD4+ T cell differentiation into IFN-γ-producing Th1 subset, and so ensure development of a strong cell-mediated immune response and recovery of C57BL/6 mice from mousepox.
Expression of the lupus associated Pbx1-d isoform induces pro-inflammatory phenotypes in mesenchymal stem cells (MSCs) in a mouse model of lupus
Authors: Robusto Brian Andres , Zeumer Leilani , Morel Laurence
Published: January 2023

Systemic Lupus Erythematosus (SLE) is an autoimmune disease that causes chronic inflammation. It is most prevalent in women, especially in women from minority populations and is heterogeneous. In previous experiments, it has been discovered that mesenchymal stem cells (MSCs) from a lupus-prone mouse strain (Sle1a1) express a defective allele of Pbx1 (Pbx1-d), a gene that controls stemness in MSCs. Sle1a1 MSCs grow faster, differentiate quicker into osteoblasts than the B6 control, and have impaired immunosuppressive function. This data together with a significant decrease in the expression of genes associated with stemness and an increase in expression of genes associated with differentiation suggests that the Pbx1-d allele disrupts the immunoregulatory functions of MSCs. This could contribute to lupus pathogenesis. We aimed to see if Pbx1-d expression in Sle1a1 MSCs increases the expression of genes promoting inflammation and activates the innate immune system. 26 genes were selected that showed an expression fold change greater than 2 in RNA sequencing as compared to B6 control MSCs. Some of the genes belong to metabolic pathways, which we are currently validating by comparing mTOR activation, glucose metabolism and mitochondrial respiration between B6 and Sle1a1 MSCs. Preliminary results suggest a different production of inflammatory cytokines and chemokines by activated Sle1a1 MSCs. Further the supernatants of activated Sle1a1 MSCs favors Th17 polarization and impairs Treg polarization in vitro. MSC cell therapy has been considered to treat lupus. Our studies provide unique insights on how a lupus susceptibility gene impairs MSC direct functions and their interactions with T cells.
The effects of germline STAT3-activating mutations from autoimmunity and lymphoid malignancy on mouse and human T cells
Authors: Masle-Farquhar Etienne , Payne Kathryn , Singh Mandeep , Rao Geetha , Al-Eryani Ghamdan , Jara Christopher , Jackson Katherine , Brink Robert Adrian , Tangye Stuart , Reed Joanne , Goodnow Christopher C
Published: January 2023

Signal transducer and activator of transcription 3 (STAT3) regulates gene expression downstream of cell surface cytokine and hormone receptors. Heterozygous germline loss of function STAT3 mutations lead to the primary immunodeficiency hyper-IgE syndrome while somatic activating STAT3 mutations recur in human solid organ and immune malignancies. Germline heterozygous activating STAT3 mutations result in early-onset autoimmunity with aspects of immunodeficiency. Affected individuals share characteristics with autoimmune lymphoproliferative syndrome and immunodysregulation polyendocrinopathy enteropathy X-linked syndrome, including reduced T regulatory cell numbers. They present with variable autoimmune symptoms including type 1 diabetes, rheumatoid arthritis, gut enteropathies and autoimmune cytopenias. Whilst many effects of STAT3 loss of function on immune cells have been described, the mechanisms behind autoimmunity and immunodeficiency in patients with activating STAT3 mutations remain unclear.

Here, we present a detailed characterisation of T cell development and maturation in young and old mice on two different backgrounds with Crispr-engineered germline activating mutations in two domains of STAT3. We use mixed chimeras, flow cytometry, T cell receptor deep sequencing and high throughput single-cell transcriptomics to reveal cell-extrinsic and -autonomous roles of overactive STAT3 in T cells in autoimmunity and immune malignancy. To our knowledge, this is the first report of mice with Stat3 germline activating mutations identical to those in autoimmunity or malignancy. We validate our key findings in humans with gain of function germline STAT3 mutations and childhood-onset autoimmunity.
Sleep-loss activates NLRP3 inflammasome-activity in astrocytes by increasing electroencephalogram slow-waves and activates microglia to increase NREM sleep amounts
Authors: Zielinski Mark R , Prabhala Rao H , Niznikiewicz Madaleine M , Desrosiers Gretchen
Published: January 2023

We previously found that NLRP3 inflammasome activity is a critical mechanism regulating sleep and slow-waves. Microglia and astrocytes play a major role in sleep regulation and altering slow-waves. We investigated the contribution of NLRP3 inflammasome activation in microglia and astrocytes on sleep and slow-waves after sleep loss. For this study, C57BL6/J mice were sleep deprived or allowed to sleep ad libitum. NLRP3 inflammasome-activity related molecules (inflammatory cytokines - interleukin (IL)-1 beta (IL-1β) and IL-18) from the cortex were determined in microglia and astrocytes using flow-cytometry. Additionally, NLRP3 floxed mice were crossed with CX3CR1- or GFAP-CRE mice to target microglia or astrocytes, and sleep and slow-waves were assessed by polysomnography. In CD11b+ cells (i.e., microglia), a significant enhancement (p<0.05) in NLRP3+, NLRP3+IL-1β+, and NLRP3+IL-18+ cells were found after sleep deprivation compared to controls. In GFAP+ cells (i.e., astrocytes), we found a significant enhancement (p<0.05) in NLRP3+ cells and NLRP3+IL-1β+ cells compared to controls but a near significant increase in NLRP3+IL-18+ cells (p=0.09). Mice with inhibited NLRP3-activity in microglia exhibited a significant attenuation in increased NREM sleep amounts after sleep deprivation, although slow-wave enhancements remained unaffected. Conversely, mice with inhibited NLRP3-activity in astrocytes had a significant lack of increased slow-waves after sleep deprivation, although they had significant enhancements in NREM sleep amounts. Our data indicate that NLRP3 inflammasome-activity in microglia contributes to increases in NREM sleep amounts after sleep loss while astrocytes contribute to slow-wave activity.
Anti-inflammatory effects of β-cry on 5FU-induced cytokine production in hOMF
Authors: Oseko Fumishige , Yamamoto Toshiro , Sato Yoshiki , Yamamoto Kenta , Adachi Keiji , Akane Mitsunori , Nishigaki Masaru , Kanamura Narisato
Published: January 2023

Objective

Beta-cryptoxanthin (β-cry), a carotenoid abundant in satsuma mandarin, has anticancer and antioxidant effects. We previously reported the anti-inflammatory effects of β-cry on the periodontal ligament. However, the effects of β-cry on oral mucositis, an adverse event due to cancer chemotherapy, remain unclear.

Fluorinated pyrimidines are frequently employed for oral cancers, with a high incidence of stomatitis. In the present study, we examined the effects of 5-fluorouracil (5-FU) and β-cry on oral mucosal fibroblasts.

Materials & Methods

Human oral mucosal fibroblasts (hOMF) were cultured with 5FU (1 mg/mL) and/or β-cry (100 nM). Then, a cell proliferation test was performed by WST assay and the expressions of inflammatory cytokines and apoptosis-related genes were examined by real-time RT-PCR.

Results

The proliferation of hOMF was inhibited by the addition of 5-FU, and the inhibition was suppressed with β-cry. Inflammatory cytokines, such as IL-1β, IL-6, and IL-8, and the ratio of Bax/Bcl-2 in hOMF were increased with 5-FU, and the increase was suppressed with β-cry.

Conclusion

Beta-cry may have anti-cytotoxic and anti-inflammatory effects on oral mucositis caused by 5-FU, suggesting that it may provide a novel method for preventing oral mucositis due to cancer chemotherapy.
Developing & Validating an Approach for Diagnosing and Prognosticating Cancer from Biochemical Motifs in T-cell Receptors
Authors: Ostmeyer Jared L , Cowell Lindsay G , Christley Scott
Published: January 2023

Immune repertoire deep sequencing allows profiling T-cell populations and enables novel approaches to diagnose and prognosticate diseases by identifying T-cell receptor sequence patterns associated with clinical phenotypes and outcomes. Our study objective is to develop a method to diagnose and prognosticate cancer using T-cell receptors sequenced from tissue biopsies. To determine how to profile the specificity of a T-cell receptor, we analyze 3D X-ray crystallographic structures of T-cell receptors bound to antigen. We observe a contiguous strip typically 4 amino acid residues in length from the complimentary determining region 3 (CDR3) lying in direct contact with the antigen. Based on this observation, we extract 4 residue long snippets from every receptor’s CDR3 and represent each snippet using biochemical features encoded by its amino acid sequence. The biochemical features are combined with information about the abundance of the snippet or the receptor and scored using a logistic regression model. Each logistic regression model is fitted and validated under the requirement that at least one positively labelled snippet appears per tumor and no positively labelled snippets appear in healthy tissue. Using a patient-holdout cross-validation, we fit logistic regression models to distinguish colorectal tumors from healthy tissue matched controls with 93% accuracy, breast tumors from healthy tissue matched controls with 94% accuracy, ovarian tumors from non-cancer patient ovarian tissue with 95% accuracy (80% accuracy on a blinded follow-up cohort), and regression of preneoplastic cervical lesions over 1 year in advance with 96% accuracy. In conclusion, immune repertoires can be used to diagnose and prognosticate disease.
Formation of human lymphoid organoids and their immunological function
Authors: Watanabe Takeshi , Kobayashi Yuka , Kawamoto Hiroshi
Published: January 2023

We previously reported that the artificially synthesized and immunologically active lymphoid tissues (aLTs) could be stably constructed in mice by implantation of lymphoid stromal cell-embedded biocompatible scaffold (collagen sponge) as well as by applying the chemokine/lymphokine-trapped gel-embedded scaffold. The aLTs possessed the structures and functions resemble to the secondary/tertiary lymphoid organ. The antigen-specific immune response was effectively induced in the aLTs. In the present study, we have aimed to generate the aLTs composed of human lymphoid cells and human stromal cells expressing LTbR, VCAM-1, ICAM-1 and several lymphoid chemokines. The spheroids were formed as the scaffolds from the stromal cells. Then, the stromal cell spheroids were transplanted into renal subcapsular space of the immunodeficient mice together with human PBMCs absorbed in the collagen sponges. After 3 weeks, the secondary lymph node-like three-dimensional organoids (LTOs) containing clusters of the human T and B cells and the scattered human DC cells were stably formed. Next, the human LTOs were constructed by using the PBMCs prepared from healthy donors who had received once the vaccination of varicella zoster virus (VZV). The immunodeficient mice carrying human LTOs were immunized with inactivated VZV. The GC-like B cell proliferation and the production of the anti-VZV specific antibody as well as human IFNg was remarkedly induced in the LTOs. These data indicate that the immunologically functional human LTOs could be generated by applying human PBMCs with the appropriate human lymphoid stromal cells. Currently, induction of immune responses against various protein antigens as well as human cancers is under investigation.
Single-center report of antibody deficiencies in Azerbaijan
Authors: Fazal Nadeem , Nasrullayeva Gulnara , Mammadova Vafa , Aliyeva Gunay , Mollayeva Nushaba
Published: January 2023

The most common type of Primary Immune Deficiencies (PID) are Antibody Deficiencies (AD) characterized by the recurrent bacterial infections. Usually the immune disbalance is observed as a decrease in B-cell function, hypogammaglobulinemia and deficiency of IgG and IgA or all three IgG, IgA and IgM antibodies. Early onset acute or chronic infections in gastrointestinal, respiratory and urinary tract are the most common symptoms of AD. The aim of the study was to analyze the data of 126 pediatric and adult patients diagnosed as AD in our PID center between 2010–2019. Immune status of the patients was examined by phenotyping of immune cells in peripheral blood, measurement of serum IgM, IgG, IgA and IgE levels, evaluation of phagocytic activity by NBT, and detection of CIC by photometric method. Additional abdominal CT and ultrasound examinations were also performed. From 126 pediatric and adult patients registered in PID Center, Baku, Azerbaijan, 9 patients were diagnosed with sIgA deficiency, 26 were common variable immune deficiency, 9-Bruton disease and 2 patients with Hyper IgM syndrome. Patient cohort included 5 adults and 41 between 12 months to 11 years age. At the time of the study, 37 patients were receiving intravenous immune globulin (IVIG) as a replacement therapy (31 patients were on regular IVIG). IVIG therapy was associated with decreased incidence of infections and overall improvement in well-being of the patients. It is important to consider hypogammaglobulinemia in patients with a history of recurrent and severe infections. We recommend that patients’ immune status should be examined including evaluation of serum immunoglobulin levels and B and T-cell subsets.
The role of Thymic Stromal Lymphopoietin (TSLP) in protection from Type II diabetes
Authors: Choa Ruth , Wada Shogo , Meng Hu , Pai Ruth-Anne Langan , Okumura Mariko , Tohyama Junichiro , Nace Arben , Hopkins Christian , Haldar Malay , Seale Patrick , Cotsarelis George , Seykora John , Fitzgerald Garret , Arany Zoltan , Kambayashi Taku
Published: January 2023

Within the past 30 years, obesity and associated disorders such as Type II diabetes are rapidly becoming world-wide epidemics. Type II diabetes, caused by the loss of insulin sensitivity, accounts for $327 billion annually in the U.S. Recent studies have shown that the immune system plays a critical role in regulating the metabolic function of adipose tissue. Importantly, Type II diabetes is a disease of chronic low-grade inflammation, and adipose T regulatory cells (Tregs) can help maintain insulin sensitivity by suppressing the inflammation caused by monocytes, CD8 T, and Natural Killer (NK) cells.

We have previously reported that topical application of the Vitamin D3 analog MC903 induces a two-fold increase in the proportion and absolute number of Tregs systemically. MC903 induces production of the cytokine Thymic Stromal Lymphopoietin (TSLP) from skin keratinocytes, and TSLP stimulates proliferation of Tregs through activation of dendritic cells. In a model of diet-induced Type II diabetes, we found that mice on high fat diet treated with MC903 compared to vehicle demonstrate improved metabolic parameters, including increased glucose tolerance and decreased insulin resistance, decreased fasting glucose and insulin, and lower liver triglycerides. Concordantly, treatment of mice with a TSLP-expressing adeno-associated virus also induced similar results, suggesting that TSLP is sufficient to improve insulin sensitivity. Importantly, there was a robust increase in Tregs and decrease in CD8 T cells and NK cells in the adipose of MC903 and TSLP-treated mice. Together, our results suggest that activation of TSLP signaling may be a therapeutic immunotarget for improving insulin sensitivity and preventing Type II diabetes.
MAIT cell function in response to airway epithelial cells is dysregulated in the context of cigarette smoke and COPD
Authors: Harriff Melanie , Larson Emily , Leiser Margalit , Heisler Chelsea
Published: January 2023

MAIT cells are a subset of innate-like lung-resident CD8+ T cells that are thought to play a role in early immune responses to airway infections. These cells exhibit immediate effector function following recognition of bacterial small molecule metabolites presented on the MHC Class I-like molecule, MR1. The impact of cigarette smoke and the development of COPD on MR1 and MAIT cell function is unknown. To address this, we obtained primary airway epithelial cells from the lungs of human subjects with COPD (n=6) or healthy controls (n=6). These cells were treated or not with cigarette smoke extract (CSE), infected with S. pneumoniae, and then used as antigen presenting cells in an IFN-γ ELISPOT assay with a MAIT cell clone. We found that MAIT cells produced more IFN-γ when incubated with uninfected cells from COPD lungs compared to healthy lungs, suggesting that the development of COPD could result in an increase in antigen-independent MAIT cell responses. In contrast, only treatment with CSE impacted antigen-dependent MAIT cell responses to S. pneumoniae-infected airway epithelial cells. Collectively, the differences in MAIT cells responses could not be explained by differences in bacterial infection or MR1 expression in the airway epithelial cells between groups. Dysfunctional MAIT cell responses in the airways could contribute to COPD-associated inflammation by inappropriately activating pro-inflammatory pathways. Furthermore, reduced early recognition of bacterial infection by MAIT cells in the context of cigarette smoke could contribute to persistent colonization of the lower airways and exacerbations.
Modulated MAPK signaling during mast cell differentiation promotes an impaired inflammatory phenotype
Authors: Hicks Natalie Jade , Crozier Robert W.E. , Den Hartogh Danja J. , MacNeil Lori A. , MacNeil Adam J.
Published: January 2023

Mast cells are innate immune cells derived from hematopoietic stem cells. Mechanisms controlling IL-3-directed mast cell differentiation are incompletely defined. MAPK contributes to IL-3R-mediated signaling, yet specific contributions to maturation of functional mast cells are undetermined. We aimed to evaluate the role of the MAPK signaling nodes—JNK, ERK, and p38—in mast cell differentiation, and explore contributions to epigenetic modifier expression. Bone marrow-derived mast cell (BMMC) cultures were established from C57BL/6 mice and differentiated for 7 weeks in the presence or absence of MAPK inhibitors (JNK, JNK-IN-8; ERK, SCH772984; p38, GW856553X). Following inhibitor withdrawal (5 days), phenotypic and functional characteristics of IgE-sensitized BMMCs were assessed using flow cytometry, ELISA, and β-hexosaminidase release assays. Cells differentiated under JNK inhibition showed a significant impairment in the secretion of IL-6 (p<0.05) and IL-13 (p<0.05) and a non-significant impairment in TNF and CCL1 24 hrs post allergen challenge. Degranulation was also significantly decreased (by 25%, p<0.05) in JNK-inhibitor-cultured BMMCs. Expression of c-kit and level of sensitization by IgE was unaffected by impaired JNK signaling. Treatment with p38 and ERK inhibitors did not affect receptor expression, cytokine secretion, or degranulation. Interrogation of MAPK-dependent modifiers of histone acetylation by qPCR and western blot showed that JNK-mediated contributions to mast cell functional responses were independent of HDACs and HATs examined. These results position JNK as a critical contributor to normal mast cell differentiation and potential target in modulating mast cell phenotype.
Altered natural killer cell responses linked to allergen sensitization in patients with atopic dermatitis
Authors: Ohayon David E , DeVore Stanley B. , Biagini Myers Jocelyn M. , Kroner John , Stevens Mariana , Waggoner Stephen N , Hershey Gurjit K Khurana
Published: January 2023

Atopic dermatitis (AD) is a common chronic inflammatory disease associated with skin barrier dysfunction. AD often precedes the development of atopic co-morbidities, including food allergy, asthma and allergic rhinitis. The contributions of natural killer (NK) cells to pathogenesis of AD and progression to allergic disease are unclear. Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples collected from n=82 children participating in the Mechanisms of Progression of Atopic Dermatitis to Asthma (MPAACH) study, a cohort of toddlers with AD. The children underwent skin prick testing to 11 aeroallergens and 6 foods, and wheezing was defined as one or more episodes of wheezing in the past 12 months. We used flow cytometry to evaluate the phenotype of NK cells in patient PBMC from MPAACH subjects. Our analyses revealed that CD56brightNK cells derived from sensitized children expressed significantly less NKG2D (73.8 ± 3.0 % versus 82.3 ± 2.7 %, respectively, p=0.020) and more TIM-3 (440 ± 462 versus 353 ± 391 median fluorescence intensity, respectively, p=0.085) compared to non-sensitized children. Increased expression of TIM-3 on CD56neg NK cells was also associated with wheezing (47.5 ± 0.51 % versus 19.2 ± 0.40 %, p=0.020). Collectively, these observations suggest that changes within the NK-cell repertoire may promote progressive development of AD and atopic co-morbidities in early life.
IFN-γ synergistically activates IL23A gene in LPS-stimulated macrophages
Authors: You Chaelin , Kang Kyuho
Published: January 2023

The cytokine IL-23 is expressed by activated myeloid cells and contributes to the development of Th17-mediated immunity. Dysregulation of IL-23 is associated with chronic inflammatory diseases, but the molecular mechanisms underlying the regulation of IL23A gene in human macrophages, are largely unknown. Unlike murine macrophages, TLR4 signaling alone did not strongly induce expression of IL23A gene but was synergistically activated by IFN-γ priming. To identify human macrophage-specific enhancers in the IL23 gene locus, we used an integrated epigenomic approach including ATAC-seq and ChIP-seq. We found a putative enhancer which is located about 2 kb upstream of the IL23A transcription start site and binds macrophage-lineage-determining factors, such as PU.1 and C/EBP. In this putative IL-23 enhancer, histone acetylation and chromatin accessibility are synergistically increased by LPS stimulation in IFN-γ-primed macrophages. IFN-γ induced coordinate occupancy of STAT1 and IRF1 at IL23A promoter and enhancer. The expression of eRNA and the recruitment of chromatin looping-associated factors cohesin and CDK8-mediator kinase at IL23A enhancer and promoter were increased by IFN-γ priming. The functional relevance of IL-23 enhancer will be tested by eRNA-targeted knockdown and enhancer deletion experiments. These results identify the critical enhancer region in IFN-γ-mediated synergistic activation of TLR4-inducible IL-23 production in human macrophages.
Epigenetic control of the protocadherin-gamma locus provides a potential source of cell surface codes in B cell differentiation
Authors: McDermott Nicole L , Saadi Amel , Cocco Mario , Care Matthew , de Tute Ruth , Owen Roger , Doody Gina , Tooze Reuben
Published: January 2023

Cellular adhesion plays an important role in determining homing and retention of cells to specific locations. Antibody-secreting plasma cells (PCs) depend on access to niche microenvironments for survival, but the factors that control the adhesion behaviour of different PCs are incompletely understood. We noted that cadherin superfamily protocadherin (PCDH) genes showed a dynamic pattern of regulation during differentiation of human PCs.

Clustered PCDHs are split into 3 gene loci (α, β and γ). Choice of variable first exons, which encode the extracellular domain is determined by selective promoter usage. This is controlled by CTCF and enhancer looping. In neurons, PCDH expression generates single cell identity codes important for synapse formation.

Tracking differentiation from B cell to PC stage, ATAC-seq demonstrated selective accessibility over the PCDHG but not PCDHA or PCDHB loci. ChIP-seq for CTCF, H3K4me3 and H3K27ac revealed active promoter marks and CTCF interactions within the PCDHG locus. Flow cytometry confirmed heterogenous expression of PCDHG family members during PC differentiation in line with mRNA patterns. Consistent with post-translational regulation, PCDHG surface expression was additionally increased by ADAM protease inhibition.

We conclude that the B cell lineage shows selective epigenetic accessibility at the PCDHG locus and expression of PCDHG family members upon differentiation to the PC state. This is coupled with patterns of CTCF occupancy implicated in regulating surface identity codes. We conclude that epigenetic control of the PCDHG locus provides a potential source of cell surface codes amongst differentiating PCs.
The role of microRNA-142 in B cell activation and effector functions
Authors: Graham Natalie Marie , Wang Wei-Le , Reyes Estefany Y. , Boldin Mark P.
Published: January 2023

Control of gene expression by microRNA (miRNA) has recently emerged as a critical mechanism that regulates B cell activation and function. However, the role of specific miRNAs in this process is unclear. Using germline knockout (KO) mice, we have previously shown that miR-142 ablation impairs humoral immunity despite significant expansion of the B cell compartment, suggesting that miR-142 is critical for B cell effector function. We found that mice lacking miR-142 cannot generate specific antibody responses upon antigen challenge and display impaired germinal center (GC) formation and plasma cell differentiation. To more precisely dissect the role of miR-142 in B cell effector responses, we developed an activated B cell-specific miR-142 KO mouse by breeding conditional miR-142 KO (miR-142fl/fl) mice with mice expressing Cre recombinase driven by activation-induced cytosine deaminase (AIDCre). AID is required for class switch recombination and somatic hypermutation, and is induced in B cells upon antigen encounter. To label activated B cells in miR-142fl/flAIDCre mice, we bred them to Ai6 mice that carry a loxP-flanked STOP cassette, allowing for the expression of fluorescent ZsGreen1 reporter protein in the presence of Cre recombinase. FACS analysis of Peyer’s patches from naive miR-142fl/flAIDCreZsgreen+/− mice revealed a significant decrease in GC B cells, while the number of ZsGreen1+ activated B cells increased, suggesting an aberrant B cell activation and GC response. We will discuss at the meeting the role of miR-142 in promoting humoral responses to antigen by presenting the findings from our ongoing studies of B cell differentiation and effector function in immunized miR-142fl/flAIDCreZsgreen+/− mice.
IL-2 signals promote acute and selective apoptosis of B cells in Peyer’s Patches
Authors: McKinstry Karl Kai , Singh Ayushi , Dhume Kunal
Published: January 2023

Interleukin-2 (IL-2) is a pleiotropic cytokine demonstrated to be therapeutically effective in treating cancer, but its clinical use can drive severe side effects including gastrointestinal toxicity. Similar symptoms are observed in inflammatory diseases such as Crohn’s Disease. Mounting evidence indicates roles for IL-2 in Crohn’s Disease, but the underlying mechanisms are unknown. Most murine studies aimed at determining IL-2’s in vivo actions on immune cells in the steady state have focused on secondary lymphoid organs. Here, we systemically administered IL-2:anti-IL-2 antibody (clone S4B6) complexes to mice for three consecutive days and compared impacts on major leukocyte populations in secondary lymphoid organs versus in the gut and gut-associated lymphoid tissues. While we confirmed established patterns of IL-2-induced CD8 T cell and NK cell proliferation and expansion in the spleen and peripheral lymph nodes, these subsets were not impacted in the gut. Unexpectedly, we observed a sharp IL-2-dependent decline in Peyer’s Patch cellularity. This correlated with a specific reduction in B cells, while B cells in secondary lymphoid organs were not changed. IL-2 signals promoted selective and acute apoptosis in Peyer’s Patch B cells and phenotypic analysis revealed that IgD+ B cells were most impacted. Our results highlight the importance of fully elucidating systemic impacts of IL-2 complex administration, a widely used approach to modulate T cell and NK cell activity in diverse experimental settings, to properly interpret outcomes. This work may also aid in defining novel ways in which IL-2 contributes to the etiology, severity, and differential presentation of gastrointestinal conditions such as Crohn’s Disease.
Standardized Approach for Monitoring Plasma Cell Disorders
Authors: Johansson Ulrika , Li Na , Golts Natalie , Saksena Suraj
Published: January 2023

Multi-color flow cytometry using consensus surface markers (CD138, CD38, CD19, CD45, CD27, CD56, CD81, and CD117), intracellular kappa and lambda, as well as other auxiliary markers is routinely used for the identification and characterization of plasma cells. Here, we describe the development of a modular 10-color plasma cell panel for immunophenotyping of the plasma cell compartment. The panel was developed as an 8-color dried backbone, manufactured in a single test-per-tube, ready to use format. The dried backbone can be supplemented with liquid (drop in) reagents, which provides the flexibility of using intracellular kappa and lambda to examine clonality or using CD38-multi-epitope and/or anti-p63 (VS38c) reagent(s). The single test-per-tube format is ideally suited for MRD applications, where sample size is limited and acquisition of millions of events is needed for data analysis. In addition, the dried reagent tubes have a long shelf life (12 months at 20–25°C vs. 24 hours for liquid cocktails with multiple BV dyes in BSB buffer at 2–8°C). Furthermore, utilization of multiple high-performing polymer dyes in the panel affords high resolution of the relevant cell populations.

The dried plasma cell tube(s) were tested on bone marrow samples from healthy and diseased subjects in conjunction with intracellular staining for kappa and lambda. Our results demonstrate that the 10-color plasma cell panel can be used for identification of the plasma cell compartment (based CD38 and CD138 expression), assessment of aberrant marker expression (CD19, CD27, CD81, CD117, CD45, and CD56) for the identification of normal and abnormal plasma cells, and examining clonality in multiple patient samples (Kappa and Lambda).
Photobiomodulation (PBM) regulates nitric oxide (NO) production by peripheral blood mononuclear cells (PBMC) isolated from Multiple Sclerosis (MS) patients
Authors: Tolentino Miguel , Cho Chi C , Lyons Jeri-Anne
Published: January 2023

Multiple sclerosis (MS) is an autoimmune disease characterized by myelin destruction, neurodegeneration and mitochondrial dysfunction. High production of nitric oxide (NO) by iNOS contributes to the generation of reactive nitrogen species, leading to increased nitrosative stress and mitochondrial dysfunction linked to MS progression. Current therapies focus on the suppression of the immune system in early stages, but they are not effective in chronic stages where nitrosative stress takes control over pathogenic mechanisms. Thus, new therapies that reduce nitrosative stress and offer neuroprotection at any stage are needed. Photobiomodulation (PBM) has demonstrated efficacy to improve neurodegenerative disorders. Our lab showed that PBM at 670nm reduced clinical severity in the C57BL/6 mouse experimental autoimmune encephalomyelitis model of MS by down-regulation of nitrosative stress, apoptosis, and pro-inflammatory cytokines, and up-regulation of anti-inflammatory cytokines. Likewise, PBM modulated cytokine expression by peripheral blood mononuclear cells (PBMC) and CD4+ T cells isolated from MS patients. Here we show that PBM reduces NO production by PBMC from MS patients. PBMC were isolated by gradient centrifugation, activated with Phytohaemagglutinin (PHA) and received PBM treatment. As surrogate for NO, nitrite production in supernatants was measured by fluorometry. The most effective PBM doses at decreasing nitrite were 830nm (10J/cm2) and pulsed 640nm (3J/cm2). The expression of Interferon-γ correlated with nitrite expression at these same doses. PBM effect was dependent on the disease modifying drug that subjects were taking. The results show the potential of PBM on NO regulation in MS patients.
Characterization of Immune Phenotypes in Peripheral Blood of Adult Renal Transplant Recipients using Mass Cytometry (CyTOF)
Authors: Kowli Sangeeta , Martinez Olivia M , Lebrec Herve , Minocherhomji Sheroy , Maecker Holden T.
Published: January 2023

Background

Chronic immunosuppressive therapy in transplant patients can be associated with opportunistic infections and risks of cancer development. There is an important need to better measure the immune status of patients in a transplant setting to improve patient monitoring and guide development of appropriate immunosuppressive agents and regimen.

Results

In this study, CyTOF was performed to comprehensively characterize the immune differences between 10 renal transplant recipients and 11 age-matched healthy donors, longitudinally over 6 months. Overall, the immune cells of transplant patients showed signs of increased CD8+ T cell activation/differentiation. Higher levels of CD57 on CD8+ T cells were observed. CD8+ T cells and their memory subsets also showed increased production of MIP-1β and IFNγ. Further, CD107a and Granzyme B expression were also increased in CD8+ T cells and CD56hi NK cells, while Tregs had decreased IL-10 production. These changes are consistent with a more stimulated immune system, despite the administration of immune suppressants to the patients. However, there were also some signs of immune suppression in the transplant patients consistent with the treatment regimen. γδT cells showed decreased TNFα and IFNγ expression, while MIP-1β expression was reduced in NKT, CD56hi NK and γδT cells. CD8+ T memory cell subsets also showed decreased expression of IL-2, IL-17 and GM-CSF.

Conclusions

This mosaic pattern of functional changes highlights the importance of comprehensive immune monitoring of these patients. Such information will aid in optimizing individual treatment strategies and development of improved immunosuppressants that specifically target overly activated populations.
Immunoproteomic identification of in vivo response toward residual antigenic proteins in xenogeneic heart valve biomaterials
Authors: Shortreed Nicholas A , Panicker Anjali J , Dalgliesh Ailsa J , Gates Katherine V , Griffiths Leigh G
Published: January 2023

Valve replacement is an important therapeutic approach for management of valvular heart disease, accounting for over 200,000 surgeries annually worldwide. Deficiencies of current heart valve prostheses led the NHLBI cardiac surgery working group to highlight the need for improved heart valve leaflet biomaterials. Recently, our group has engineered a novel method to eliminate antigens from bovine pericardium (BP) extracellular matrix (ECM) scaffolds, while retaining biomaterial structure-function properties and regenerative capacity. However, despite reducing BP-ECM scaffold antigen burden by >92%, graft-specific humoral IgG response persisted at ~30% of the level associated with native BP in a rabbit model. In this study, we sought to determine the identities of residual antigens present in antigen removed BP-ECM scaffolds. New Zealand White Rabbits were subpannicularly implanted with either native, glutaraldehyde fixed, or antigen-removed BP-ECM scaffolds (n = 6 per group). Rabbit serum, collected pre- and at 56 d post-implantation, was used to generate IgG affinity chromatography columns. Native BP protein extracts were loaded onto columns, and captured antigens were identified by LC-MS/MS. A total of 259 antigens were identified in native BP rabbits. The glutaraldehyde fixed and antigen-removed rabbits responded to ~4% and ~12% of these same antigens, respectively. A small subset of antigens were uniquely identified in each of the treatment groups, not identified in native BP. These results inform ongoing efforts to eliminate antigens from xenogeneic tissues in development of ECM scaffolds, and may provide insight into the relative immunogenicity of individual xenoantigens in the context of tissue engineering.