bluuebunny/crossref_metadata_2025 huggingface dataset:
Low-dose neoadjuvant chemotherapy dominates Ki67− quiescent tumor dormancy for an effective immunotherapy of breast cancer
Authors: Aqbi Hussein , Coleman Cara , Idowu Michael , Manjili Masoud
Published: January 2023

Breast cancer mortality is mainly due to distant recurrence of the disease. The disease recurrence is usually because of the establishment of tumor dormancy following successful treatment of early stage breast cancer. In current clinical practice, dormant tumor cells are not actively treated, because they do not respond to chemotherapy or radiation therapies. Immunotherapeutic targeting of tumor dormancy is also challenging because of the induction of tumor immunoediting, leading to tumor escape and relapse. Recently, we have characterized Ki67− quiescent and Ki67low indolent types of tumor dormancy. We demonstrated that quiescent, but not indolent, dormant tumor cells fail to undergo immunoediting and remain highly susceptible to immunotherapy. Here, we sought to develop neoadjuvant therapies for dominating a quiescent type of tumor dormancy as the best target for immunotherapy. We showed that a low dose 5-FU+Adriamycin+Cyclophosphamide (FAC) dominated Ki67− quiescent type of tumor dormancy in the neu-overexpression mouse mammary carcinoma (MMC) tumor cells. These dormant cells were effectively controlled by immunotherapy in FVBN202 transgenic mouse model of spontaneous breast cancer. Since the antibiotic azithromycin has been shown to effectively eliminate senescent cells, we also sought to determine its efficacy against dormant tumor cells. We demonstrated that azithromycin specifically induced apoptosis in FAC-induced dormant tumor cells. All together, the results suggest that use of less toxic, low dose chemotherapy combined with azithromycin in a neoadjuvant setting could lead to the elimination of dormant tumor cells by immunotherapy.
PD-1 expressions on monocytes from patients with hepatocellular carcinoma differs depending on the progression of the cancer
Authors: OHAMA Hideko , Asai Akira , Tsuchimoto Yusuke , Fukunishi Shinya , Higuchi Kazuhide
Published: January 2023

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death in the world. Multifocal HCC often arise synchronously or metachronously, leading high mortality rates in HCC patients. Therefore, it is necessary to diagnose the progression for HCC and to appropriately treat it accordingly. In this study, we tried to analyze for immunological characters of monocytes isolated from several stages of HCC patients and examined whether these characters can be used as a new tumor marker for HCC.

From 2017 August to 2018 December, 35 patients of advanced-stage HCC and 33 patients of early-stage HCC were enrolled. Monocytes from peripheral blood of these patients were stained for anti-PD-1 antibody, anti-PD-L1 antibody and anti-PD-L2 antibody. These cells were analyzed for flow cytometry. Five healthy donors were used for the control groups.

In the results, monocytes from early-stage HCC patients were not express PD-L1 (2.9 ± 1.1%), and express few PD-L2 (43.4 ± 13.5%) and PD-1 (45.2 ± 15.1%). However, monocytes from advanced-stage HCC patients were expressed PD-L1 (75.3 ± 20.7%) and PD-L2 (74.7 ± 15.8%), but not PD-1(1.4 ± 1.1%). As the controls, monocytes from healthy donors were not expressed PD-L1 (21.7 ± 5.5%), PD-L2 (31.7 ± 23.8%) and PD-1 (11.6 ± 6.4%). When CD8− cells from advanced-stage HCC patients were co-cultured with monocytes from syngeneic patients, the turmoricidal activities of these cells were suppressed(15.7±7.5%). In contrast, CD8− cells from early-stage HCC, were co-cultured with syngeneic monocytes, kept turmoricidal activities.

In conclusions, PD-L1 expression in monocytes was increased, as HCC progressed and may be using a new marker of the classification for the progression in hepatocellular carcinoma.
Repression of osteopontin by IRF8 regulates a novel immunosuppressive checkpoint
Authors: Klement John David , Paschall Amy V , Redd Priscilla S. , Ibrahim Mohammed L. , Lu Chunwan , Yang Dafeng , Celis Esteban , Abrams Scott I , Ozato Keiko , Liu Kebin
Published: January 2023

While current immune checkpoint inhibitors (ICIs) have shown efficacy in a wide variety of human cancers, many patients and cancer types fail to respond to ICI immunotherapy, suggesting the presence of other immunoinhibitory checkpoints. We report here that a deficiency in the transcription factor interferon regulatory factor 8 (IRF8) led to a loss of function of cytotoxic T lymphocytes (CTLs) in both an allograft tumor and a peptide vaccine model. However, a T cell-specific knockout and a mixed chimera model demonstrated that IRF8 suppressed CTL activation through a CTL-extrinsic mechanism. This was found to be through IRF8-dependent suppression of the matricellular protein osteopontin (OPN). During colorectal cancer (CRC) development, IRF8 is downregulated in both tumor-associated CD11b+Ly6CloLy6Ghi myeloid cells and colon epithelial cells, where it represses OPN expression by binding to its promoter. Accordingly, in both mouse CRC models and human CRC patients, transcriptional downregulation of IRF8 was accompanied by an upregulation of OPN at the transcriptional and protein level. Enhanced levels of OPN were found to negatively correlate with patient survival. In vitro stimulation of mouse and human CTLs in the presence of OPN led to decreased activation and interferon gamma secretion. Similarly, deletion of spp1, which encodes for OPN, from murine CT26 colon adenocarcinoma cells caused increased susceptibility to CTL-mediated lysis and enhanced CTL activation. As such, tumor and myeloid-derived OPN may function as a novel immune checkpoint to restrain host CTL activation.
Examining the role of mouse Ly-6A as immune checkpoint inhibitor
Authors: Capozzoli Benjamin Daniel , Mallon Francine , Bamezai Anil K
Published: January 2023

Discovering new immune checkpoint inhibitors is critical for extending the current strategy of cancer immunotherapy to more cancer patients where blocking a single immune checkpoint inhibitor is insuffcient. We hypothesized that the Ly-6A protein is an immune checkpoint inhibitor. Ly-6A, a member of the Ly-6 supergene family, is a cell surface receptor on CD4+T cells with cell-cell adhesion and cell signaling properties. Naïve CD4+T cells show low expression of Ly-6A protein, while the expression of this protein is highly upregulated in activated and clonally expanding CD4+T cells. While overexpression of Ly-6A on CD4+T cells results in their inhibited antigen-specific clonal expansion of CD4+T cells, consistent with that is CD4+T cells from Ly-6A−/− mice show moderate hyper-responsiveness. To examine the immune inhibitory and potentially immune checkpoint inhibitory role of Ly-6A protein we have transplanted B16 melanoma tumors into Ly-6A−/− mice. We have observed that Ly-6A−/− mice often have smaller tumor volumes or absence of a tumor within the same time frame that C57Bl/6 mice develop consistently large tumors. FACS analysis has also revealed that Ly-6A−/− mice also have more activated TILs compared with the C57Bl/6 mice as well as more active T cell populations in the non-draining and draining lymph nodes. These early results suggest that Ly-6A may behave as an immune checkpoint inhibitor. Additional experiments with B16 cell line, another tumor line (MC38 adenocarcinoma), and antibody blockade of Ly-6A are underway.
Multidimensional single T cell analysis reveals altered T cell biology upon treatment of murine tumor with checkpoint blockade immunotherapy
Authors: Rao Samhita , Kornberg Adam , Lin Sijie , Gharib Karim , Koganti Lahari , Garcia Javier Alejandro Garcia-Vilas , Masuda Kazuya , Han Arnold
Published: January 2023

Checkpoint blockade immunotherapy has shown great success in certain types of cancers, but are ineffective in most cancers. We do not understand why these therapies fail.

Here, we seek to better understand functional changes in T cells in responsive tumors through single cell TCR sequencing and high dimensional phenotypic analysis within single tumor-infiltrating T cells. Through surgical biopsy, we analyze the same tumor longitudinally before and after treatment, enabling us to track the fate of individual T cell clones.

Our data show that the CD4/CD8 ratio decreases upon treatment. T cells expressing inhibitory markers such as PD1, CTLA4, diminish post treatment. We see distinct clusters of effector CD4 and CD8 T cells in treated tumors, including effector CD4 populations with Tfh, Th1 and Th2 phenotypes. CD4 Tregs are greatly diminished upon successful treatment, and diminish through clonal contraction. Furthermore, TCR acts as a unique marker for T cell ancestry, which links T cell function to T cell specificity. Based on TCR clonality, we observe that expanded T cell clones before treatment do not share the same TCRs with expanded T cell clones after treatment. This implies new CD8 T cell clones are recruited into the tumor upon treatment.

From our data, we will be able to identify T cell clones that are phenotypically distinct and expanded in responsive tumors which is critical in understanding what makes for a successful T cell response after immunotherapy.
Monitoring PD-1 Signaling in Tumor Infiltrating Lymphocytes
Authors: Bu Xia , Juneja Vikram R. , Mahoney Katheleen M. , Renolds Carol G. , Mcguire Kathleen A. , Maleri Seth , Hua Ping , Zhu Baogong , Klein Sarah R , Greenfield Edward , Armand Philippe , Ritz Jerome , Sharpe Arlene , Freeman Gordon J
Published: January 2023

Programmed death (PD)-1 pathway blockade is a successful strategy for cancer immunotherapy. However, only a limited number of patients respond. There is great need to identify predictors of therapeutic response to PD-1 immunotherapy. One challenge is that PD-1 expression can mark activated T cells, which are susceptible to PD-1-mediated inhibition, but does not indicate whether a PD-1-mediated immunoinhibitory signal is being delivered. Currently there are no methods to detect active PD-1 signaling and, in turn, successful PD-1 inhibition. Here, we describe a novel antibody that detects PD-1 signaling. This antibody detects phosphorylation of the immunotyrosine switch motif (ITSM) in the intracellular tail of both human and mouse PD-1 (phospho-PD-1). Using this anti-phospho-PD-1 mAb, we show that PD-1+ tumor infiltrating lymphocytes (TILs) in MC38 murine colorectal tumors have high levels of phosphorylated PD-1, particularly in PD-1+TIM-3+TILs. Upon PD-1 blockade, PD-1 phosphorylation is markedly decreased in TIM-3+CD8+ TILs prior to tumor clearance. We also observed decreased phospho PD-1 levels in T cells from peripheral blood of human lymphoma patients following treatment with PD-1 and LAG-3 mAb. These data demonstrate that phosphorylation of the ITSM of PD-1 marks dysfunctional T cells that may be rescued with PD-1 blockade. Analysis of phospho-PD-1 may serve as a potential biomarker for effective PD-1 immunotherapy.
Interferon alpha promotes extrinsic apoptosis of keratinocytes following exposure to ultraviolet B radiation
Authors: Estadt Shannon N , Moore Tyson , Kahlenberg J. Michelle
Published: January 2023

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by sensitivity to ultraviolet radiation (UVR). UVR can trigger cutaneous and systemic disease flares, yet mechanisms driving this are not well characterized. Type I interferons (IFNs) are expressed at higher levels at baseline in non-lesional skin of SLE patients and promote increased death of SLE keratinocytes (KCs) after UVR. The manner in which they regulate cell death after UVR is currently unknown. This study explores cell death pathways activated in KCs by type I IFNs and UVR. We treated immortalized human KCs (N/TERTs) overnight with IFNα prior to UVB exposure. Four hours post UVB, Annexin V (AV) and propidium iodide (PI) and cleaved-caspase-3 (CC3) staining were used to measure cell death via flow cytometry. IFNα significantly increased the percentage of CC3+ and AV+PI− cells compared to treatment with UVB alone. Further, IFNα treatment promoted apoptosis at a lower dose of UVB (20 mJ/cm2). Pre-treatment with RIPK1, RIPK3, or caspase-1 inhibitors had no effect on death suggesting this enhanced death does not involve necroptosis or pyroptosis. Use of a pan-caspase inhibitor or a caspase-8 inhibitor significantly reduced AV+PI− cells following IFNα and UVB treatment while treatment with a caspase-9 inhibitor did not, suggesting enhanced activation of the extrinsic apoptosis pathway. These data suggest that photosensitive responses exhibited by SLE patients are likely due to type I IFN priming of keratinocytes that sensitizes cells to undergo increased extrinsic apoptosis after minimal exposures to UVB. Continued investigation into mechanisms by which this occurs will be crucial for developing novel preventative strategies for photosensitivity.
Dysregulation of type I IFN and IL-4R signaling regulates the development of BCR VH and VL repertoire encoding anti-Sm autoantibodies in SLE
Authors: Mountz John D. , Gao Min , Liu Shanrun , Chong Zechen , Wu Qi , Yang PingAr , Chatham W. Winn , Hsu Hui-Chen
Published: January 2023

The most specific predictor for systemic lupus erythematosus (SLE) and lupus nephritis (LN) is the presence of anti-Smith (Sm) autoAb. Mechanisms leading to selection bias for anti-Sm development is unclear. We found that anti-Sm levels in SLE correlated with increased endogenous IFN-β and interferon-stimulated genes (ISGs) in combination with low (IL-4R) network genes. Using flow cytometry analysis of 47 SLE patients, we found that anti-Sm+ patients exhibited an increased ratio of IFN-β+ activated naive (aNAV) B cells and a decrease in IL-4R+ resting naive (rNAV) B cells. Using a combined 10X single-cell transcriptomics and BCR usage analysis, anti-Sm+LN+ISGhiIL4Rlopatients (Sm+) were compared with anti-Sm−LN− ISGloIL4Rhi patients (Sm−). BCR heavy chain analysis showed that while B cells from Sm+ patients exhibit greater usage of IGHV1-2, IGHV4-34, and IGHV4-39 genes, Sm− SLE patients exhibit greater usage of IGHV3-30, IGHV3-33, and IGHV4-31 genes. Preferential Ig light chain usage for ISGhi B cells was IGLV1-51, IGLV3-21, IGLV6-57, IGKV1-5, IGKV3-15 and IGKV 6-21. Dominant heavy-light chain pair usages for the Sm+ patients were IGHV4/IGKV1 and IGHV4/IGKV3. IGHV4-34, which is intrinsically autoreactive and the target of the 9G4 anti-idiotype Ab, was found to be expressed by the ISGhiIL4Rlo B cells derived from the same Sm+ patients. The results indicated that increased levels of ISGs and decreased levels of IL-4R pathway genes are the two dominant signatures and the underlying mechanisms leading to selection and activation of anti-Sm+ B cells. Verification that the B-cell IFN-β and IL-4R phenotypes are associated with race (AA vs. EA), repertoire (anti-Sm+) and disease (LN+) would lay the basis for targeted rational therapies.
AIM2 deficient mice develop severe Systemic Lupus Erythematosus pathogenesis due to elevated type I interferon signaling
Authors: Meng Guangxun , LU Ailing , WU Shuxian
Published: January 2023

Systemic lupus erythematosus (SLE) often involves kidney damage, and the inflammasome-caspase-1 cascade had been demonstrated promoting renal pathogenesis. However, our present work found that deficiency of Aim2 led to enhanced nephritis in pristane-induced experimental lupus model. We further found that increased type I interferon (IFN-I) signal in the Aim2−/− mice was in tandem with exaggerated SLE. Moreover, adaptive immune cells were also involved in the interstitial nephritis in the glomeruli of Aim2−/− mice. Of note, even without pristine challenge, Plasmacytoid dendritic cells in the kidney of Aim2−/− mice were significantly more abundant compared with control animals. A transcriptome analysis revealed that Aim2 deficiency led to enhanced expression of IFN-I-induced genes in the kidney at different developmental stages. Taken together, our study demonstrates a critical role for AIM2 in suppressing IFN-I production and development of SLE.
Type 1 interferons promote allo-antibody responses to RBC transfusion in a lupus mouse model
Authors: Gibb David R , Madany Emaan , Kadi Najwa El , Pandya Sumaarg , Ng Kessandra , Jefferies Caroline Anne
Published: January 2023

Red blood cell (RBC) transfusion exposes recipients to hundreds of unmatched minor RBC antigens. This exposure can lead to production of alloantibodies that can cause potentially fatal hemolytic events. Prior studies have reported that inflammatory states, including autoimmune disease, in the transfusion recipient can promote alloimmunization. Recent studies reported that pro-inflammatory type 1 interferons (IFNα/β) regulated RBC alloimmunization in multiple mouse transfusion models. Interestingly, patients with systemic lupus erythematosus (SLE) have an increased frequency of alloimmunization and express an IFNα/β gene signature. Thus, we tested the hypothesis that IFNα/β activation in lupus promotes RBC alloimmunization by utilizing the pristane-induced lupus mouse model. Pristane significantly induced serum IFNα and expression of multiple interferon-stimulated genes (ISG) in peritoneal fluid cells, including inflammatory macrophages. Following transfusion with allogeneic RBCs expressing the KEL antigen, pristane-treated WT mice produced significantly elevated levels of anti-KEL IgM and anti-KEL IgG, compared to untreated mice. Pristane-treated mice lacking the IFNα/β receptor (IFNAR1−/−) or IFNα/β-inducing transcription factors (IRF3/7−/−) mice failed to produce ISGs and produced significantly lower levels of anti-KEL IgG compared to WT mice. In conclusion, pristane induction of a lupus-like phenotype promoted alloimmunization to the KEL RBC antigen in an IFNα/β-dependent manner. These results warrant further investigation of the role of IFNα/β in alloimmunization to other RBC antigens and the contribution of IFNα/β responses to the increased frequency of alloimmunization in patients with SLE.
Complement C1q synergizes with PTX3 in promoting NLRP3 inflammasome over-activation and pyroptosis in rheumatoid arthritis
Authors: Wu Xunyao
Published: January 2023

Excessive inflammatory cytokines play crucial roles in the pathogenesis of rheumatoid arthritis (RA), however, the underlying mechanism remains unclear. In this study, we demonstrated that pentaxin 3 (PTX3), an essential component of innate immunity, was elevated in RA and preferentially bound to CD14+ monocytes. C1q promoted the binding and resulted in increased cell proliferation, activation and caspase-1-related late apoptotic cells (7-AAD+annexin V+), as well as enhanced release of inflammatory cytokines including TNF-α, IL-1β and IL-6. Serum from RA patients, compared with healthy controls, induced gasdermin D (GSDMD)-dependent pyroptosis in monocytes, and this ability was associated with disease activity. Moreover, PTX3 synergized with C1q to promote pyroptosis in RA-serum pre-incubated monocytes by coordinately enhancing NLRP3 inflammasome over-activation and inducing GSDMD cleavage, cell swelling with large bubbles, caspase-1-dependent cell death and inflammatory cytokine release including IL-6. On the other hand, IL-6 promoted PTX3 plus C1q-induced pyroptosis in both normal and RA serum pre-incubated monocytes. These findings collectively implicated an important role of IL-6 in driving PTX3 plus C1q-mediated pyroptosis in RA and shed lights on a potential new treatment strategy targeting pyroptosis-mediated persistent inflammatory cytokine release.
Organic compound farnesol as possible inhibitor of inflammasome complex, in murine macrophages and mouse model of multiple sclerosis
Authors: Sell Lacey B , Ramelow Christina C. , Strawn Kevin D. , Hevrin Theresa , Kirby Trevor O. , Gibson K. Michael , Roullet Jean-Baptiste , Ochoa-Repáraz Javier
Published: January 2023

Many advancements in the understanding of multiple sclerosis (MS) have been made through the use of laboratory models. One commonly used model is experimental autoimmune encephalomyelitis (EAE), a mouse model characterized by central nervous system (CNS) inflammation and demyelination, allowing for symptoms resembling some of the most prominent features of the human disease. Although the exact etiology of MS is still being investigated, experiments with EAE have shown that the NLRP3 inflammasome complex of the innate immune system is critical and necessary for disease development. The inflammasome complex can be assembled in all innate immune cells, including microglia and astrocytes in the CNS. Dysregulation of inflammasome activity can result in uncontrolled inflammation, which underlies many chronic diseases, and metabolic and autoimmune disorders such as MS. Our lab has shown that farnesol a 15-carbon organic sesquiterpene and primary alcohol, reduced EAE disease severity and onset and also decreased T-cell infiltration into the CNS. However, the mechanisms of its action have yet to be fully defined. Therefore, in-vitro work on murine macrophages is being conducted to investigate how farnesol may be potentially affecting the pathway of the inflammasome complex and providing this protection. Furthermore, since farnesol is a quorum-sensing molecule that impacts biofilm formation, other studies of ours are aimed at evaluating how farnesol affects the gut-brain axis and specifically the gut microbiome of EAE mice.
Effects of using nanogel-cross-linked porous gel with cell adhesion factor for cell transplantation
Authors: Sato Yoshiki , Yamamoto Kenta , Takizawa Shigeta , Adachi Keiji , Oseko Fumishige , Amemiya Takeshi , Kishida Tsunao , Yamamoto Toshiro , Mazda Osam , Kanamura Narisato
Published: January 2023

Objective

Transplantation of engineered three-dimensional (3D) tissue may provide therapeutic benefits to patients with various diseases. To achieve this goal, appropriate 3D scaffolds and cells are required. We report a cell transplantation experiment in which a cell adhesion factor is incorporated into a porous crosslinked nanogel to attach cells.

Materials and Methods

RGDC and fibronectin were incorporated to attach cells to the porous cross-linked nanogel. RGD-conjugated, Fibronectin-coated, and non-coated NanoCliP-FD matrices were soaked in the cell suspension, and air bubble was pushed out of the matrices with tweezers to allow infiltration of the cell suspension into the matrices. The adhesion between the porous crosslinked nanogel and the cells was evaluated by staining and WST assay. Thease samples were used for cell transplantation.

Results

When different cell adhesion factors were incorporated into the porous cross-linked nanogel, it became clear that the adhesion modes were different. When cells were transplanted using a complex in which cells and porous crosslinked nanogel were adhered, tissue recovery was observed.

Discussion

By incorporating a cell adhesion factor into the porous crosslinked nanogel, it became possible to use it as a cell transplant material. Cells attached to the porous cross-linked nanogel are considered to be very useful for tissue recovery.
Functional differences in behavior of monocyte/macrophage from HIV+ patients versus healthy subjects
Authors: Lozano Javier , Chow Dominic C , Ndhlovu Lishomwa C , Siriwardhana Chathura , Liu Jocelyn , Irei Jason , Nagamine Lorna , Shikuma Cecilia , Boisvert William A
Published: January 2023

Increased risk of atherosclerotic cardiovascular diseases is common in HIV+ adults on stable antiretroviral treatment (ART). A key step in the development of the atherosclerotic plaque is the transmigration of monocytes to the plaque area and their differentiation into macrophages and eventually foam cells. The objective of this study was to determine if the propensity of monocytes to transmigrate as well as their ability to take up and efflux cholesterol is affected by HIV infection. Monocytes (transmigration) and differentiated macrophages (lipid metabolism) from HIV+ ART treated patients were compared against those from matched controls as a single point cross-sectional study. Monocyte transmigration was assessed by transwell assay using MCP1 as a chemoattractant, whereas lipid uptake was measured by flow cytometry analysis of internalized acetylated LDL and cholesterol efflux by tracking tritiated cholesterol excreted from the cell after exposure to HDL. Interestingly, isolation of monocytes via negative selection with magnetic beads revealed that the yield of monocyte was significantly lower from HIV+ patients compared to controls. While no significant differences were found in cholesterol uptake or efflux parameter, the ability of monocyte to transmigrate was slightly increased in HIV patients compared to controls suggesting that the chemotactic function of monocytes may be partly responsible for the differences in the atherosclerosis risk in patients with HIV.
Human eosinophils and neutrophils biosynthesize novel 15-lipoxygenase metabolites from 1-linoleoyl-glycerol and N-linoleoyl-ethanolamine
Authors: Archambault Anne-Sophie , Tinto Francesco , Martin Cyril , Dumais Elizabeth , Rakotoarivelo Volatiana , Laviolette Michel , Silvestri Cristoforo , Kostrzewa Magdalena , Ligresti Alessia , Boulet Louis-Philippe , Di Marzo Vincenzo , Flamand Nicolas
Published: January 2023

BACKGROUND

The endocannabinoids 2-AG and AEA are lipids regulating many physiological processes, notably inflammation. The endocannabinoidome includes other monoacylglycerols (MAG) and N-acyl-ethanolamines (NAE) such as 1-linoleoyl-glycerol (1-LG) and N-linoleoyl-ethanolamine (LEA). Endocannabinoid hydrolysis inhibitors are now being tested as potential anti-inflammatory agents. By increasing MAG and/or NAE levels, these inhibitors will likely increase the levels of their metabolites. Herein we investigated whether 1-LG and LEA were substrates for the 15-lipoxygenase pathway, which is strongly involved in asthma and its severity. We thus assessed how human eosinophils and neutrophils biosynthesized the 15-lipoxygenase metabolites of 1-LG and LEA. Linoleic acid (LA), a well-documented substrate of 15-lipoxygenases, was used as positive control.

RESULTS

We synthesized the putative 15-lipoxygenase metabolites of 1-LG and LEA using Novozym435 and soybean lipoxygenase. Eosinophils, which express the 15-lipoxygenase-1, metabolized LA, 1-LG, and LEA into their 13-hydroxy derivatives. This was almost complete after 5 minutes. Substrate preference of eosinophils was LA>LEA>1-LG. Human neutrophils, which express the 15-lipoxygenase-2, also metabolized LA, 1-LG, and LEA into their 13-hydroxy derivatives. This was maximal after 30 seconds. Substrate preference was LA≫1-LG>LEA. Importantly, the new 15-lipoxygenase metabolites we disclose were found in tissues from humans and mice.

CONCLUSIONS

We successfully showed that human eosinophils and neutrophils transforms 1-LG and LEA into novel 15-lipoxygenase metabolites. How these new metabolites modulate the inflammatory cascade is now being explored.
The Use of Moodle Apps and Khan Academy Short Videos to Engage Students to Learn Microbiology/Immunology
Authors: Fazal Nadeem , Fadhil Shams , Edquiban Marianne , Olk Sandra
Published: January 2023

The purpose of this research project was to look at the use of technology in Immunology education. There have been reports that non-conventional ways do not effectively deliver the content matter to the students. We used Moodle and online resources to evaluate ways to enhance student engagement and participation. In the past we have investigated and published innovative methods to accomplish this goal. In the current study we used pre-lectures as a tool to enhance student learning and performance. The intent was to enforce and supplement classroom teaching and not introduce an alternative. During the spring semester a didactic course of Immunology is taught to first year pharmacy students at Chicago State University. Prior to this investigation we surveyed and determined that students are challenged with this course of Immunology. Other than being introduced to Immunology for the first time, they struggle with getting used to workload in a professional school and time management. Since our preliminary survey showed that students do not always have enough time to watch one-hour long lecture videos, we decided to use short and topic-focused videos <10 minutes. Students could listen driving to school, work, or during the 10-minute breaks between classes. For the purpose of this research project we selected reliable online resources that offered information via short lecture videos. Usage data showed that Khan Academy videos were among the top choices among students. We selected Immunology short videos and provided links to via Moodle, which allowed us the pertinent usage data over the entire semester. Our data showed that those students who listened to pre-lectures Immunology videos performed significantly better in exams than those who did not.
Concept maps as a dynamic tool for teaching scientific thinking
Authors: Thompson Scott Beckett , Pugh-Bernard Aimee
Published: January 2023

Scientific thinking often involves understanding interrelated and multidimensional topics. We have employed the concept map as an active learning method for teaching a diversity of concepts in an undergraduate scientific thinking course. Here we describe our implementation of concept maps and how it can be used as tool for instruction, assessment and team building. A concept map is a diagram used to organize ideas and depict their relationships, typically constructed of information presented in boxes with labeled lines drawn between them to indicate connections. In our educational implementation, students write key concepts from assigned reading onto sticky notes and work in small groups using these notes and dry-erase markers to create a concept map on a white board. The modular nature of this approach enables students to dynamically edit and rearrange concepts both during the initial construction and throughout a class period to incorporate new ideas. Students have created concept maps on topics including the scientific method, the diagnostic process for physicians and scientific research articles. When students have disagreements on ideas, the collaborative and flexible nature of these concept maps enable a constructive resolution and the addition of alternative thoughts and ideas. Additionally, throughout the semester the sophistication of the concept maps evolves thus serving as qualitative assessment of student growth. Concept mapping enables students to collaboratively work towards understanding scientific ideas and to think critically about the relationships between them. Visualization of how students organize knowledge facilitates the assessment of student growth and the diversity of understanding within a class.
Appropriate Contextualisation of Immunology based STEM and Health teaching for undergraduate Medical Sciences Education
Authors: Sika-Paotonu Dianne , Chandra Pratik , De Silva Hashini , Hermann Zsófia , Lowry Fionnuala , Papalii Sophie , Rajakumaran Priyanka , Simpson Emma , Walton Michaela , Mitchell Maxine
Published: January 2023

Clinically relevant examples are needed to support knowledge application of Immunology teaching content within undergraduate Medical Sciences Education frameworks. The appropriate contextualisation of these Immunology teaching examples is essential, particularly when presenting health related issues that are of significance and relevance to Māori and Pacific population groups living in New Zealand (NZ) and the Pacific Region. Acute Rheumatic Fever (ARF) is an autoimmune condition that results from untreated Group A Streptococcal (GAS) infection of the throat (and skin). Repeated or severe episodes of ARF that remain untreated can lead to Rheumatic Heart Disease (RHD) or permanent heart damage. Māori, Pacific and Indigenous Aboriginal population groups are affected disproportionately by ARF and RHD. This work involved Immunologically related ARF and RHD teaching examples were presented to 4th year undergraduate Medical Science Education students that were contextualized, and relevant to current health priorities for Māori and Pacific population groups. Sound understanding was demonstrated by students participating in these classes. Strong interest in Immunologically related ARF/RHD research and teaching examples with positive feedback expressed by students within written and verbal teaching feedback platforms. A subset of students were motivated to undertake research placements to contribute to the NZ based ARF and RHD related Penicillin reformulation work. Overall, the contextualisation of the Immunologically related ARF/RHD research and teaching examples supported knowledge application and helped raise awareness of health issues for Māori and Pacific population groups living in NZ and the Pacific Region.
Graduate training in T cell immunotherapy and CAR-T cell therapy
Authors: Sterner Rosalie , Hedin Karen E. , Hayden Richard E. , Nowakowski Grzegorz S. , Wyles Saranya P. , Greenberg-Worisek Alexandra J. , Terzic Andre , Kenderian Saad S.
Published: January 2023

The objective was to evaluate the impact of an interdisciplinary graduate level course on students’ knowledge and interests in immunotherapy and CAR-T cell therapy. It was a one week course conducted in November 2018. Students were surveyed before and after the course. The survey included questions regarding background, self-perceived knowledge/confidence in skills, and interests/predicted behaviors. Students were assigned to work in interdisciplinary teams to develop a research proposal. A total of 25 students taking the course for graduate level credit were surveyed. Of these, all 25 (100%) completed the surveys. Students came from variable backgrounds and were at different stages of graduate training. After completion of the course, there was a statistically significant increase in self-perceived knowledge of immunotherapy (mean±standard deviation score of 3.6±0.9 post-course vs 2.6±1.0 pre-course, on a 5 point Likert scale, P<0.001), knowledge of the benchtop research to clinic translational process (3.7±0.8 post-course vs 3.0±0.9 pre-course, P<0.001), confidence in critical reading skills (4.3±0.6 post-course vs 4.0±0.5 pre-course, P=0.008), confidence in immunotherapy focused grant writing skills (3.6±0.9 post-course vs 2.8±1.1 pre-course, P<0.001), interest in working in interdisciplinary teams (4.8±0.4 post-course vs 4.6±0.6 pre-course, P=0.02), and interest in attending immunotherapy educational events (3.5±1.3 post-course vs 3.2±1.3 pre-course, P≤0.05). In conclusion, this course helps trainees to build knowledge and interest in the fields of immunotherapy and CAR-T cell therapy and can potentially serve as a platform for future educational courses in interdisciplinary, translational research.
Bridging statistics and life sciences education for immunologists: Exploring a significantly impactful collaboration
Authors: Singh Jastaranpreet , White Bethany , Tong Lilin
Published: January 2023

Statistical tests and graphs are an important part of any immunological research publication or presentation. However, not all immunologists have the statistical expertise to choose the best methods to evaluate and represent their experiments, and the formal statistics training required by undergraduate immunology curricula is often quite limited. How can we prepare future immunologists for statistical practice in research? As a collaborative effort between the Department of Statistical Sciences and the Human Biology Program at the University of Toronto, a second-year undergraduate course was developed to integrate statistics instruction with research design to improve the quantitative training of life sciences students. In a Scholarship of Teaching and Learning (SoTL) study conducted within the course, student attitudes and self-efficacies for statistics, as well as their abilities to recognize and handle problems related to statistical practice in life sciences research, were assessed through surveys administered at the beginning and end of the course (n=126). Here, we present our team-teaching model and strategies for promoting meaningful connections between statistics and life sciences research through course design, activities and assessments. We also highlight results from our SoTL study, which revealed that 77% of students felt more confident about choosing the correct statistical procedure, while 74% indicated that the course helped increase their confidence with respect to results interpretation. We hope that insights from this teaching partnership and our SoTL findings will help inform future course offerings to better prepare our students to effectively engage with statistics in immunological research.
Citrullination modulates MHC class II antigen processing and presentation by revealing cryptic epitopes
Authors: Curran Ashley M , Jang Yura , Thomas Mekha A , Girgis Alexander A , Crawford Jonathan D , O’Meally Robert N , Cole Robert N , Na Chan Hyun , Darrah Erika
Published: January 2023

Cryptic peptides, hidden from the immune system under physiologic conditions, are revealed by changes to MHC class II processing and can facilitate loss of tolerance to self-antigens. Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by immune responses to citrullinated self-antigens, in which arginine residues are converted to citrullines; however, the initiating events in RA remain poorly understood. We investigated the hypothesis that citrullination exposes cryptic epitopes by modifying protein structure and proteolysis in a manner sufficient to activate a previously ignorant repertoire of self-reactive T cells. We first utilized in vitro proteolytic mapping to define the effect of citrullination on four well-defined RA autoantigens-. We then used a natural antigen processing assay to elucidate the molecular mechanisms of citrullination-modulated processing and evaluated autoantigen-specific T cell responses against the unique peptide repertoire. We show that citrullination alters MHC class II processing and presentation of RA autoantigens, resulting in the destruction of dominant epitopes and the generation and presentation of cryptic epitopes, composed primarily of native peptide sequences. The citrullination-dependent repertoire stimulates T cells from RA patients with anti-citrullinated protein antibodies more robustly than the native repertoire and control T cells. The generation of this unique repertoire is achieved through altered protease affinity and protein destabilization, rather than direct presentation of citrulline-containing epitopes, suggesting a novel paradigm for the role of protein citrullination in the breach of immune tolerance in RA.

Supported by a grant from the Rheumatology Research Foundation and by the Luke Evnin and Deann Wright Fellowship
Aquaporin 4 Is A Mediator Of Essential Dendritic Cell Function
Authors: Nicosia Michael , Beavers Ashley , Yamamoto Yosuke , Thompson Trevor , Zindrick Thomas , Valujskikh Anna
Published: January 2023

Aquaporins are transmembrane water channels implicated in a range of physiologic functions. We previously reported that Aquaporin 4 (AQP4) is expressed by T cells and treatment with a small molecule AQP4 inhibitor (AER-270) significantly delays T cell mediated mouse heart allograft rejection. Our findings did not exclude a role for antigen presenting cells (APCs), and the purpose of this study is to investigate the requirement for AQP4 in dendritic cell (DC) functions.

Stimulation with AQP4−/− spleen APCs with the addition of antigenic peptide, resulted in reduced frequencies of IFNγ producing MAR T cells compared to WT APCs. We confirmed that splenic DCs express AQP4. Bone marrow derived dendritic cells (BMDCs) from WT and AQP4−/− mice were stimulated with LPS or protein antigen, the absence or inhibition of AQP4 resulted in no defects in up-regulation of maturation markers following stimulation. WT and AQP4−/− BMDCs were pulsed with Lucifer Yellow, FITC-dextran and OVA-AF647 to assess antigen uptake with no observable defect after AQP4 inhibition or in AQP4−/− BMDCs. However, AQP4−/− BMDCs pulsed with OVA protein had reduced expression of SIINFEKL peptide-MHC-I complexes. In contrast, when pulsed with OVA-derived SIINFEKL peptide, AER-270 treated WT BMDCs or AQP4−/− BMDCs showed no change in peptide-MHC complex expression suggesting a role for AQP4 in antigen processing. Consistent with this, the absence or inhibition of AQP4 resulted in decreased DQ-OVA signal, indicating that AQP4 is required for the antigen loading into the lysosome.

Our data indicate that AQP4 plays a critical role in the processing of antigens by dendritic cells that impacts their ability to prime T cells, and can be therapeutically targeted in a transplant setting.

Supported by grants from NIH (R56AI152368-01)
Dynamic features of tapasin as revealed by structures of two tapasin/Fab complexes
Authors: Taylor Daniel Kyle , Jiang Jiansheng , Boyd Lisa F , Cresswell Peter , Mage Michael G , Margulies David H , Natarajan Kannan
Published: January 2023

Intracellular loading of major histocompatibility complex class I (MHC-I) molecules occurs within the peptide loading complex and is accomplished through the concerted action of several proteins, including the chaperone/catalyst tapasin. In the absence of tapasin, peptide loading is compromised, resulting in decreased cell surface expression of some, though not all, MHC-I allelomorphs. To gain a mechanistic understanding of tapasin’s function we have produced recombinant Fab fragments of two anti-tapasin antibodies, PaSta1 and PaSta2, and investigated their binding to human recombinant soluble tapasin by surface plasmon resonance methods and X-ray crystallography. Both antibody Fabs bind with nanomolar affinities characterized by slow off rates. PaSta1 and PaSta2 recognize distinct epitopes as tapasin captured on a surface immobilized with one PaSta Fab binds the other PaSta Fab with nanomolar affinity. However, tapasin captured on a PaSta2 Fab surface is not able to bind peptide/HLA-B*44:05/beta-2 microglobulin complexes while tapasin captured on a PaSta1 Fab surface can, indicating that PaSta2 sterically competes for the MHC-I binding site. This finding was echoed in the structural characterization of PaSta Fab-tapasin complexes by X-ray crystallography. PaSta1 binds to an extended N-terminal region of tapasin, a site accessible even in the tapasin/ERp57 complex. By contrast, PaSta2 binds at the junction of the N-terminal and C-terminal domains of tapasin, where tapasin may interact with HLA-I molecules. Comparison of conformations of tapasin in complex with PaSta1, PaSta2, HLA-B*44:05, and ERp57 reveals the dynamic nature of tapasin, a versatile chaperone and catalyst.

Supported by the intramural research program of the NIAID, NIH
Autophagy in antigen presenting cells enhances the adjuvant effect by promoting antigen processing.
Authors: Hashimoto Kahoko , Ootomo Takuya , Sonobe Tsubasa , Kurosaki Naoko , Hasegawa Akihiro , Nakayama Toshinori
Published: January 2023

Previously, we proposed that LPS or IFNγ treated DC and macrophage induced cell proliferation, cytokine production, antigen presentation and autophagy function were detected. Among DC subset, CD8 positive DC enhances autophagy function with cross presentation but the similar effect was not shown in macrophages. To understand the difference, we studied that how DC are enhanced antigens processing through ubiquitin proteasome pathway, as well as autophagy pathway. Three different types of DC in vitro cultured mice DC with GM-CSF, DC with GM-CSF and IL-4, or GM-CSF and IL-15 were prepared, these DCs or macrophages were stimulated by using LPS or IFNg before antigen uptake. GM-CSF induced DC or GM-CSF and IL-15 induced DC showed enhanced antigen processing with active autophagy function, however GM-CSF and IL-4 induced DC showed active antigen presenting within 60 min, still autophagy function was not detected. Contrary to active antigen processing, GM-CSF and IL-4 - DC did not show inducible effect after adjuvant treatment, but GM-CSF or GM-CSF and IL-15-DC increased autophagy function after adjuvant treatment that was assessed by using fluorescent labeled phagosome or lysosome function analysis. Additionally, we investigated gene expression pattern related to autophagy such as ATG5 or ATG18 by q-PCR, and western blotting. Cultured DC with GM-CSF and IL-15 indicated prominent active autophagy related gene expression pattern. To induce more effective immune response on vaccination or immune therapy, effect of adjuvant treatment for autophagy function in DC and macrophage are required.
MATERNAL AUTOANTIBODIES AS A POSSIBLE ASD-RISK BIOMARKER
Authors: Celis Nora Alexandra Ramirez , Van de Water Judy A
Published: January 2023

Maternal autoantibody-related ASD (MAR ASD) is a subtype of autism in which pathogenic maternal autoantibodies (IgG) can cross the placenta, access the developing brain, and cause neurodevelopmental alterations and behaviors associated with autism in the exposed offspring. We previously reported maternal IgG response to eight proteins (CRMP1, CRMP2, GDA LDHA, LDHB, NSE, STIP1, and YBOX) and that reactivity two or more specific proteins (MAR ASD patterns) can be used to predict ASD risk in a discovery study.

Objective

In the current study, we aimed to validate the previously described MAR ASD patterns (CRMP1+GDA, CRMP1+CRMP2, NSE+STIP1, CRMP2+STIP1, LDHA+YBOX, LDHB+YBOX, GDA+YBOX, STIP1+YBOX, and CRMP1+STIP1) and their accuracy to predict ASD risk in a prospective cohort.

Methods

We used plasma from mothers of autistic children (ASD= 540) and general population controls (not verified as typically developing) (GP=420) collected by the Early Markers of Autism (EMA) study. We evaluated maternal IgG reactivity against each of the eight fetal brain proteins by ELISA and used Fisher Exact Test (p < 0.05) to compare the association for each pattern with child outcome (ASD vs GP).

Results

We found reactivity to at least one protein in more than 50% of the samples in the case and control groups, suggesting that reactivity to a single antigen is not diagnosis-specific. However, when we tested the nine previously identified MAR ASD patterns, we found highly-specific reactivity in 10% ASD group vs 1% of the GP controls and increased the odds for an ASD outcome nearly 8-fold (p< 0.001, OR 7.81, 95% CI from 3.32 to 22.43) demonstrating that the MAR ASD patterns are highly specific for the case group and could be used to assess ASD risk prior to symptoms.

Supported by EPA2P01ES011269-11, 83543201, R01ES015359, NICHD funded IDDRC P50 (P50HD103526), CONACYT-UC MEXUS Doctoral Fellowships 2015.