bluuebunny/crossref_metadata_2025 huggingface dataset:
Investigations of Salmonella Motility by the Defensin HD6
Authors: Akahoshi Doug T , Natwick Dean , Collins Sean , Bevins Charles L
Published: January 2023

Antimicrobial peptides are key effector molecules throughout nature responsible in part for the defense against microbial pathogens and influencing the composition of colonizing microbes. Defensins are the major antimicrobial peptides produced by Paneth cells in the mammalian small intestine and are essential for maintaining homeostasis between host and gut microbes. While other defensins have potent microbicidal activity, human Paneth cell alpha defensin 6 (HD6) lacks such activity, yet provides protection from invasion by enteric bacterial pathogens. Using a transgenic mouse model expressing HD6 in Paneth cells, we reported the importance of HD6 in preventing the translocation of pathogenic Salmonella Typhimurium (STM) across the small intestinal epithelium (doi:10.1126/science.1218831); however, the mechanism underlying the inhibition of invasion is not fully understood. We hypothesize that HD6 binds to extracellular bacterial protein structures, such as flagella, and inhibits swimming motility through a process of direct binding and self-assembly. Using a semi-solid agar based motility assay, we demonstrate Salmonella radially migrate from the site of inoculation at a slower rate when in the presence of HD6. In ongoing studies using live epifluorescence microscopy to observe GFP-expressing STM in the presence of HD6, we observe a dramatic decrease in STM motility in a concentration dependent manner with some STM appearing to be completely immobilized, but without changes in bacterial viability. With these findings in mind, we plan to expand our investigation to include both structural variants of HD6 and mutants of STM to more clearly define the key elements of peptide and bacteria that mediate this activity.
Dynamics of migration patterns of polymorphonuclear myeloid-derived suppressor cells during tumor progression
Authors: Fu Shuyu , Patel Sima , Mastio Jerome , Dominguez George A , Torres Kevin Alicea , Nefedova Yulia , Zhou Jie , Gabrilovich Dmitry I
Published: January 2023

Although recruitment of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) to the tumor site is well documented, little is known about their migration patterns within bone marrow in tumor progression. We found that the activation of neutrophils migration in cancer is a two-phase process. The first phase was characterized by the accumulation of a previously uncharacterized population of neutrophils that lack immunosuppressive activity but display a potent ability to spontaneously migrate, whereas the later phase is associated with accumulation of neutrophils with typical features of PMN-MDSC with low migratory activity in the bone marrow of mice with various subcutaneous (ectopic) tumors and in mice with advanced orthotopic lung cancer. Mechanistically, PM-LCs displayed increased metabolic flux through oxidative phosphorylation and glycolysis and have more ATP than that of control neutrophils. However, PMN-MDSC were indistinguishable from neutrophils from tumor-free mice in their metabolic activity. In line with studies of mice, CD15+ neutrophils exhibited greater spontaneous migration than that of PMN-MDSCs from the same patient with cancer. These results elucidate the dynamic changes that neutrophils undergo in cancer.
Focal tumor ablation by irreversible electroporation augments immunotherapy to promote tumor growth control and formation of tumor antigen-specific tissue resident memory CD8+ T cells
Authors: Burbach Brandon J , O’Flanagan Stephen D , Rollins Meagan R , Slaughter Joseph R , Young Katharine M , Song Meredith , Shao Qi , Azarin Samira M , Bischof John C , Shimizu Yoji
Published: January 2023

Memory CD8+ T cells populate non-lymphoid tissues (NLT) and can protect against malignancies, but little is known about the establishment of tumor-specific tissue resident memory CD8+ T cells (TRM) by therapeutically initiated immune responses. We used a transplantable mouse model of prostate carcinoma and MHCI tetramers to track and quantify tumor-specific CD8+ T cells. Tumor challenge led to systemic expansion of endogenous, naïve tumor antigen-specific CD8+ T cells and established a small population of TRM (CD44hi, CD62Llo, CD69+, CD103+) in several NLTs. Previously, we demonstrated that debulking primary tumors using irreversible electroporation (IRE) induced expansion of tumor-specific T cells. We hypothesized that IRE would cooperate with checkpoint blockade to increase antigen-specific TRM and promote tumor clearance in vivo. Combination treatment with IRE followed by α-CTLA4 promoted a robust 50-fold expansion of proliferating (Ki67+) tumor-specific T cells in blood, a 10-fold increase of TRM in NLT, and was associated with complete remission of residual tumor. Conversely, monotherapy allowed tumor outgrowth and failed to boost T cell numbers, resulting in small populations of PD-1hi tumor-specific CD8+ T cells. Similarly, anti-PD-1 alone or following IRE was also ineffective, suggesting that combined IRE/α-CTLA4 therapy targets newly primed T cells. IRE also enhanced antigen presentation by XCR1+ dendritic cells, promoting T cell priming in regional lymph nodes. These results indicate that combining α-CTLA4 checkpoint immunotherapy with focal tumor ablation, such as IRE, capable of releasing high quantities and qualities of endogenous tumor antigens may serve as a potent in situ tumor vaccination strategy.
ITK regulates antigen-specific CD8+ T cell-derived IL-10 production and modulates influenza-induced pulmonary immunopathology
Authors: Huang Weishan , Solouki Sabrina , August Avery
Published: January 2023

Influenza (flu) infection is a leading cause of respiratory disease and death worldwide, causing 3-5 million cases of severe illness and more than 250,000 deaths during an average flu season. Severe life-threatening influenza infections in humans are accompanied by an aggressive pro-inflammatory response and an insufficient anti-inflammatory immunity. The production of the immunosuppressive cytokine IL-10 by virus-specific T cells is critical in limiting the immunopathology during flu infection, however, our understanding of how this immunomodulatory event is regulated at the molecular level is very limited. Using mouse models that report the production of IL-10 through green fluorescent protein, OTI CD8+ T cells and influenza A viruses that express the OTI antigen ovalbumin (OVA), we find that the T cell receptor (TCR) responsive tyrosine kinase, Interleukin-2 Inducible T cell kinase (ITK), is a critical regulator of the ability of CD8+ T cells to develop into IL-10-producing cells in an antigen specific manner. The absence of ITK resulted in increased morbidity and mortality in murine model post influenza A infection, accompanied by a severe impairment of IL-10 production by CD8+ T cells in the airway. ITK kinase activity is required for its role in promoting IL-10-producing CD8+ T cell differentiation, by regulating the levels of expression of transcription factors including IRF4, Blimp1, AHR and cMAF. Our data suggest that ITK regulates CD8+ T cell production of IL-10 during influenza A infection, and modulates the development of pulmonary immunopathology. Targeting ITK signaling maybe a promising strategy in modulating airway inflammation during viral infections.
Herpesvirus entry mediator promotes Th1 responses in the cornea during ocular HSV-1 infection
Authors: Park Seo Jin , Riccio Rachel , Kopp Sarah , Longnecker Richard
Published: January 2023

Herpes simplex virus-1 (HSV-1) is a major cause of infectious blindness in the developed world. Ocular herpetic disease is characterized by persistent inflammation in the corneal tissue even after the virus has successfully been cleared, which causes irreversible damage to the cornea. It is unknown what molecular mechanisms drive chronic inflammation following ocular HSV-1 infection. Herpesvirus entry mediator (HVEM), a TNFR superfamily member, mediates both stimulatory and inhibitory signals in T cells by interacting with its functional ligands, LIGHT and B and T lymphocyte attenuator (BTLA). Mice lacking HVEM exhibit a significant decrease in Th1 cells, PMNs, and monocytes in the cornea 14 days post-intracorneal HSV-1 infection as well as a decrease in Th1 and Th17 cells in the draining lymph nodes. We hypothesize that HVEM contributes to exacerbation of ocular disease by stimulatory co-signaling through interaction with its ligands. We observe that LIGHT deficient (LIGHT−/−) and BTLA deficient (BTLA−/−) mice alone do not show significant difference in infiltrating immune cells or corneal pathology compared to wild type (WT) mice. Interestingly, mice lacking both BTLA and LIGHT (BTLA−/−LIGHT−/−) exhibit significant reduction in Th1 cells and improvement in corneal pathology. We show that stimulatory HVEM signaling mediated by both LIGHT and BTLA promotes chronic inflammation during ocular HSV-1 infection.
Measurement of MHC-peptide interaction through cell-surface expression and stability assay improves screening of potential CD4+ T-cell epitopes
Authors: Miyadera Hiroko , Nagai Hideaki , Yoshiyama Takashi , Tokunaga Katsushi , Hoshino Yoshihiko
Published: January 2023

Introduction

Accurate prediction of T-cell epitopes have remained a bottleneck for developing peptide vaccines for infections and cancer immunotherapy, especially for major histocompatibility complex class II (MHC II). We previously found that cell-surface expression level of the covalently linked peptide-MHC II complex is a good indicator of MHC-peptide interaction (Miyadera et al. 2015 J Clin Invest). We named this assay as “delta MHC (dMHC) assay” and validated its utility in the prediction of CD4+ T-cell epitopes.

Method

dMHC assay was performed using engineered fibroblast cells that stably express DRA- or DQA1. The cell line was transduced with pMXs-IG (Kitamura et al. 2003 Exp Hematol) that carries DRB1/3/4/5- or DQB1-peptide fusion construct. Cell surface HLA expression was measured by flow cytometry and normalized to the internal control GFP (Miyadera et al. 2015 J Clin Invest).

Results and Discussion

We conducted systematic binding analysis for HLA class II (27 alleles: HLA-DRB1,3,4,5, and HLA-DQ) and nine peptides derived from Mycobacterium tuberculosis antigens CFP-10 and ESAT-6 that contain potential CD4+ T-cell epitopes (Nagai et al. 2014 J Immunol Res). The cell surface HLA expression level in the presence of the test peptide were normalized to the expression level of the negative control peptide (g9). The normalized expression level (dMHC-g9) showed overall good correlation with the predicted binding affinity (IC50). Moreover, dMHC-g9 outperforms IC50 in predicting potential T-cell epitopes. These data indicate that dMHC assay may be suitable as a platform for large scale analysis of MHC-peptide interaction and data sharing, which should facilitate improved prediction of CD4+ T-cell epitopes.
Histotripsy initiates local and systemic immunological response and reduces tumor burden in breast cancer
Authors: Hendricks Alissa Danielle , Howell Justin , Schmieley Rebecca , Kozlov Shawn , Simon Alex , Coutermarsh-Ott Sheryl L , Vlaisavljevich Eli , Allen Irving C
Published: January 2023

Histotripsy is an emerging, non-invasive, and non-thermal tumor ablation method that has been shown to ablate tissues with millimeter precision guided by real-time ultrasound imaging feedback. Histotripsy ablates tissues by focusing high pressure ultrasound pulses to create cavitation, liquefying tissue. By utilizing this mechanism, preliminary data has suggested that Histotripsy could be used to both ablate a primary tumor and to stimulate the immune system to treat metastatic disease. However, there has not been work done to date on investigating the interactions of the immune system with Histotripsy. For the current work, we hypothesized Histotripsy treatment of a 4T1 tumor in a murine model would stimulate local and systemic immune system responses with an improvement on disease burden. Here, we show that Histotripsy was able to reduce the size of the treated tumor, number of metastases, and change the cytokine profiles. Treated tumors had an average of 15% reduction in volume in 24hrs following treatment and showed increases in necrosis and innate immune cell infiltration. Analysis of early response revealed increased pro-inflammatory cytokines, correlating with increased anti-tumor microenvironments. At a long term follow up, Histotripsy decreased disease burden through primary tumor size and metastasis number. These results demonstrate Histotripsy can stimulate an immunological effect with the increased anti-tumor microenvironment in the treated area one day after treatment, and overall disease morbidity in the long term. Histotripsy has promise as an immunomodulating tumor ablation method for breast cancer, and, once established, has the potential to be utilized in different tumor types as well.
Weighted Gene Co-expression Network Analysis of CXCL13, CXCR5, and associated genes in multiple myeloma
Authors: Adebayo Olayinka Omoyitola , Young Corey D , Carey Kaylin , Dill Courtney D. , Nunez Sha’Kayla K , Griffen Tiara L , Jain Sanjay , Lillard James W
Published: January 2023

Multiple myeloma (MM) is characterized by the neoplastic proliferation of plasma cells. In 2018, there were approximately 30,770 new cases diagnosed and about 12,770 dead. Characterizing its molecular phenotypes is important for effective treatment of MM and predicting relapse. CXCL13-CXCR5 interactions are involved in malignancy cell homing, adhesion, signal transduction, and calcium flux, all of which promote MM progression. Here, we analyzed RNA-sequence data from matched normal, primary and relapsed MM cases. Patient control mRNA samples matched primary tumor and relapse samples were acquired from the database of Genotypes and Phenotypes to evaluate the mRNA expression patterns of CXCL13, CXCR5 and associated genes in MM. Bioinformatics tools were to integrate published genomic data from MM patients (n=480) and identify genes associated with CXCL13-CXCR5 signaling. DESeq analysis was used to determine differentially expressed genes between normal tissues, primary tumor, and relapse groups. Weighted gene co-expression network analysis identified clusters of genes significantly associated with the molecular phenotypes of MM. Notably, gene expression is driven by NFAT and JUN, known to be activated by the CXCL13-CXCR5 axis, and plasma cell signaling pathways significantly correlated with select MM molecular phenotypes and patient survival. Ingenuity pathway analysis was performed to analyze upstream regulators, gene interaction and canonical pathways. Taken together, our data show CXCR5-CXCL13 signaling networks are significantly expressed and associated with MM pathogenesis and plasma clonality. This study provides a better understanding of the heterogeneous nature of MM and the role of CXCL13-CXCR5 axis in MM.
A pan-allele Class I MHC prediction tool in mice enables tumor immunology studies
Authors: DeVette Christa Innae , Andreatta Massimo , Welm Alana , Nielsen Morten , Hildebrand William H
Published: January 2023

An understanding of tumor-presented Class I MHC peptides is a prerequisite to effective cancer immunotherapy, and more tools are needed to elucidate immune interactions in breast cancer. One tool, the mouse mammary tumor virus (MMTV)-Polyoma Middle T Antigen (PyMT) mouse, models the development of spontaneous metastatic breast cancer in vivo. Surprisingly, little data exists for the MHCI of MMTV-PyMT tumors, which express the H2-q haplotype. To develop the MMTV-PyMT tumor model for tumor immunology, we present an open-access Class I MHC peptide prediction tool for multiple murine haplotypes (including H2-q): NetH2pan (http://www.cbs.dtu.dk/services//NetH2pan/). H2-d/k/b peptides were obtained from the IEDB, while H2-q peptides were eluted from MHC: HeLa and 721.221 cell lines transfected with PyMT and soluble H2-Kq/Dq. Cells secreting these MHC I were expanded, peptide-bearing MHC I affinity purified, and presented peptides analyzed by mass spectrometry. The identified peptide sequence data were used to develop a predictive algorithm to enable in silico candidate peptide searches. Validation of the algorithm using peptides eluted from tumors demonstrated a positive predictive value (34%) improved from other MHC I prediction tools. For a candidate antigen PyMT, the 5 PyMT-peptides eluted from tumors were in the top 25 predicted. Three of 5 peptides were immunogenic as determined by IFNγ ELISpot and peptide:MHCI tetramers in vaccinated mice, and correlated with tumor prevention. These data show that NetH2pan can predict immunogenic epitopes with high-fidelity and that these peptides can be utilized for vaccine development. These studies enable the MMTV-PyMT model for mechanistic studies involving tumor-T cell crosstalk.
Δ42PD-1 inhibitor acts as a PD-1-independent immunotherapy against human hepatocellular carcinoma
Authors: TAN ZHIWU , Chiu Mei Sum , Yan Chi Wing , Yang Xinxiang , Wong YIK CHUN , Man Kwan , Cheng Alfred Sze-Lok , Chen Zhiwei
Published: January 2023

Pathogen infection-induced chronic inflammation is a key contributor to both carcinogenesis and progression of hepatocellular carcinoma (HCC). Persistent toll-like receptor 4 (TLR4) signaling activation can lead to inflammatory responses and chronic inflammation in the tumor microenvironment. Previously, Δ42PD1, a PD1 isoform, has been shown to directly interact with its cognate receptor TLR4 and contribute to innate immune activation and intestinal pathogenesis during acute HIV-1 infection. Here, we aim to determine whether such interaction may also occur in HCC to promote TLR4 signaling activation and in turn induce inflammatory responses, contributing to HCC progression. We studied the relationship between TLR4, Δ42PD1, pro-inflammatory cytokines and tumor development in humanized mouse models and HCC patients. In consistence with others’ findings, TLR4 signal was shown to be associated with chronic inflammation and HCC tumor progression. Furthermore, we demonstrated that Δ42PD1 activated TLR4-mediated inflammatory responses in HCC cells via cJUN MAPK transcriptional activation. Notably, Δ42PD1-TLR4 pathway activation likely formed a feedback loop to induce Δ42PD1-expression CD3+ T cells, a new phenotype of T cell exhaustion, further contributing to tumor development. Importantly, administration of a Δ42PD1-specific monoclonal antibody CH101 interrupted Δ42PD1-TLR4 pathway and displayed therapeutic efficacy in both subcutaneous and orthotopic HCC models. Together, our results demonstrated the importance of Δ42PD1-TLR4-mediated inflammatory responses in promoting HCC and provided conclusive evidence that targeting Δ42PD1-TLR4 pathway reduced inflammatory responses to arrest HCC development.
Cell-intrinsic programmed death ligand-1 (PD-L1) inhibits cytotoxic chemo, promotes DNA damage repair, and enhances ATM/ATR signaling following exposure to DNA damaging agents in bladder, melanoma, and ovarian cancer cells
Authors: Kornepati Anand V , Zhang Deyi , Hambright Heather G. , Kari Suresh C. , Deng Yilun , Clark Curtis A. , Gupta Harshita B. , Chakravarty Rekha , Hu Yanfen , Li Rong , Curiel Tyler J.
Published: January 2023

We have shown that cancer cell-intrinsic PD-L1 signals increase mTORC1 and unbundle actin. To test if tumor cell PD-L1 affects the DNA damage response (DDR) we made PD-L1KO mouse ID8agg (ovarian) and B16 (melanoma), and human bladder RT4 cancer lines. The DNA damaging agent gemcitabine (gem) reduced PD-L1KO ID8agg and RT4 viability in vitro vs. control (con) (ID8agg: 29X vs. 1.3X; RT4: 2X vs 1.1X). 4h gem increased phospho (p)-γH2AX, a marker of DNA double strand breaks: 2X in PD-L1KO vs. con RT4. P-γH2AX stayed abnormally high after gem removal, further showing defective DDR in PD-L1KO. 24h gem also induced more p-γH2AX in PD-L1KO vs. con ID8agg, and 48h gem induced more apoptosis in PD-L1KO vs. con RT4. Cisplatin, a DNA damaging agent distinct from gem, reduced PD-L1KO ID8agg viability 9X vs. 2X in con. No viability difference was seen in cisplatin treated PD-L1KO vs. con RT4. Thus, PD-L1 chemo resistance effects are cell type and/or agent specific. To define DDR mechanism(s), we found that 24h gem decreased p-Chk2 (ATM DDR pathway), due to reduced total Chk2, but not p-Chk1 or total Chk1 (ATR DDR pathway) in PD-L1KO vs. con RT4. 20 Gy X-rays (DNA damage distinct vs. gem), reduced p-Chk1 in PD-L1KO but not con B16 and induced less p-RPA32 in PD-L1KO vs. con B16, suggesting defects specifically in homologous recombination mediated DNA repair. Thus, cell-intrinsic PD-L1 DDR control is insult and/or cell type specific. RT4 DDR effects were independent of mTOR or actin. DDR affects immunotherapy so PD-L1 cell-intrinsic DDR control could be exploited in treatment or response biomarkers. DDR inhibitors, chemo or radiation combos could improve anti-PD-L1 efficacy in distinct cancers, subjects of ongoing work, as is testing tumor cell intrinsic PD-1 effects.
PD1 blockade modulates Ca2+ fluxes in T cells of head and neck cancer patients
Authors: Gawali Vaibhavkumar Shantaram , Chimote Ameet A , Chirra Martina , Janssen Edith , Wise-Draper Trisha , Conforti Laura
Published: January 2023

Immunotherapy offers promising results in head and neck cancer (HNC) albeit it faces the problem of high resistance. PD1 (programmed death receptor-1) and its ligand (PD-L1/PD-L2) interaction reduces T cell receptor (TCR)-stimulated Ca2+ fluxes. Ion channels regulate Ca2+ entry in T cells downstream to the TCR (store-operated Ca2+ entry, SOCE). We hypothesized that anti-PD1 antibody (Pembrolizumab, Pembro) increases SOCE. We studied the effect of Pembro and PD-L1 on SOCE in CD8+ peripheral blood T cells (PBTs) of HNC patients and healthy donors (HD) using Thapsigargin (TG). Additionally, we tested the effect of Pembro on KCa3.1, Kv1.3 and Ca2+ release activated Ca2+ (CRAC) channels in PBTs of HNC patients using patch-clamp electrophysiology. TCR-mediated Ca2+ fluxes were assessed in PD1-expressing NFAT-reporter Jurkat cells. In these cells, Pembro increased the number of cells that responded to TCR stimulation with a sustained increase in Ca2+ (30%, p<0.001). Pembro increased SOCE by 55% (p<0.02) in PBTs of a subset of HNC patients (5/9). Baseline SOCE (before Pembro) was lower in the HNC PBTs that positively responded to Pembro compared to untreated HD PBTs (p<0.006). PD-L1 decreases the Ca2+ response, which was reversed by Pembro in HD PBTs (p<0.05). Pembro did not alter the expression of ion channels, but significantly increased KCa3.1 and Kv1.3 conductance in a subset of patients (5–6/8) while it increased divalent free (DVF) currents through CRAC channels only in 2/5 patients. These data suggest that Pembro enhances SOCE in T cells by modulating ion channels activity. This mechanism may contribute to the improved functionality of T cells in cancer patients undergoing immunotherapy. (DoD CA160714; 2-R01-CA95286)
Single Cell RNA-sequencing Identifies Distinct Fibroblast Subsets in a Human 3D Skin Model of Systemic Sclerosis
Authors: Kosarek Noelle Noreen , Wheeler Tamar , Toledo Diana Maria , Huang Mengqi , Pioli Patricia A , Whitfield Michael
Published: January 2023

Systemic sclerosis (SSc) is a multisystem autoimmune disease characterized by skin and organ fibrosis, autoantibody formation, and vascular injury. Due to the systemic nature of this disease, 30–40% of patients die within the first 10 years after diagnosis. There are currently no FDA-approved disease-modifying therapies for SSc and available treatments fail to target the origins of skin and organ fibrosis 1. There is a need to identify targetable cell populations and molecular mediators responsible for disease. Here we report single cell RNA-sequencing (scRNA-seq) profiles of 11,140 human fibroblasts, macrophages, and keratinocytes from eight 3D SSc human skin equivalents 2. We isolated 956 macrophages, 606 keratinocytes, and 9578 fibroblasts from this model system. The keratinocyte cluster was identified by high expression of keratin genes such as KRT6A and KRT16, while the macrophage cluster was identified by upregulation of HLA-DR, CD163, and CD206. The fibroblast population was composed of six different subpopulations distinguished by differentially expressed features. Three of the fibroblast subpopulations were driven by genes encoding collagen proteins such as COL1A1 and COL3A1, while the other three subpopulations were driven by genes encoding mitochondrial and ribosomal components, metal and ion binding proteins, and lipoproteins. These distinct fibroblast subpopulations may represent targetable cell types for therapeutic intervention.
Effects of a-lysophosphatidylinositol (LPI) on kinase activation and cytoskeletal re-arrangements leading to migration in mast cells
Authors: Aguilar Lizbeth Magnolia Martinez , Ibarra-Sánchez Alfredo , Macías-Silva Marina , González-Espinosa Claudia
Published: January 2023

Mast Cells (MCs) initiate allergic responses but also participate in innate inflammatory reactions triggered by infection, tissue damage and cancer. MCs are recruited to affected sites where they release preformed and neo-formed mediators of inflammation. Bioactive lipids (such as sphingosine-1-phosphate, S1P) have demonstrated to induce MC chemotaxis. Recently, α-lysophosphatidilinositol (LPI) has awakened scientific interest due to the discovery of its endogenous receptor, the orphan receptor GPCR55. This receptor has been shown to couple to Gα12/13 pathway, controlling actin polimeryzation and cell migration. In this study, we aimed to characterize the role of LPI in the activation and migration of MCs. Using murine bone marrow-derived MCs, we observed that low concentrations of this lipid (10 nM and 100 nM) induces maximal ERK 1/2 phosphorylation at 15 and 10 minutes, respectively. Also, we found that LIMK (an important G12/13 and Rho-dependent kinase) is phosphorylated after 5 and 10 min after 10 nM or 100 nM LPI, respectively. Both ERK 1/2 and LIMK phosphorylation patterns were reproduced with the selective agonist of GPCR55, O-1602, at equi-potent concentrations to those of the LPI. Using Boyden chamber assays, we observed that LPI provocked higher chemotaxis of MCs when compared with S1P. Finally, using confocal microscopy, we observed that LPI triggered depolymerization of cortical actin, as well as the formation of filopodia between 15 and 30 minutes post stimulation. We concluded that GPR55 expressed in MCs induces important changes on MAPK and LIMK activation and its ligand, LPI, is a potent chemotactic factor of MCs.
Complement protein C1q modulates endothelial cell functions in atherosclerosis
Authors: Monsibais Alisha N , Espericueta Victoria , Megliola Leilani , Fraser Deborah A
Published: January 2023

Atherosclerosis is a chronic inflammatory disease characterized by the development of plaque in arteries. Interactions between monocytes, endothelial cells and lipoproteins play a major role in the disease pathogenesis. C1q is traditionally known for activation of the complement cascade, leading to inflammation and clearance of foreign targets. However, we previously showed that C1q bound to modified lipoproteins (e.g. oxLDL) plays a protective role by polarizing monocytes and macrophages towards an anti-inflammatory state during engulfment and increases survival and efferocytic function of macrophage foam cells. Additionally, C1q modulates macrophage chemokine production in in vitro and in in vitro models of atherosclerosis. Here we investigate C1q modulation of both endothelial cell chemokine production, and functions affecting monocyte migration. Human aortic endothelial cells (HAEC) were incubated with oxLDL +/− C1q. RNA and supernatants were collected for chemokine gene regulation and protein analysis, respectively. Monocyte adhesion and transendothelial migration were also measured. The data show that C1q modulates chemokine gene and protein expression, increases monocyte adhesion and transendothelial migration and enhances HAEC monolayer permeability. Thus, C1q interactions with HAEC in hyperlipidemic conditions may result in increased availability of phagocytes for clearance in the early atherosclerotic lesion. However in later stages infiltrating cells may contribute to disease progression through an increased proinflammatory environment and complement activation. Understanding timing and molecular mechanisms in atherosclerosis will be vital for the development of novel treatments.
Inhibition of trans-endothelial migration of HIV-1 infected myeloid cells using virus expression dependent anti-ALCAM CRISPR-Cas9
Authors: Kaminski Rafal , Dabrowski Konrad , Luongo Francesca Paola , Myhlyk Maksym , Ebo Sunday , Rom Slava , Young Won-Bin , Burdo Tricia
Published: January 2023

Migration of HIV-1 infected monocytes across the endothelial barrier plays an essential role in establishing and maintenance of viral reservoir in the brain and continues despite antiretroviral therapy (ART). Using the CRISPR-Cas9 gene-editing platform, we created an inducible knockout for activated leukocytes cell adhesion molecule gene (ALCAM/CD166), an HIV-1 host dependency factor shown to play a critical role in CNS entry of infected myeloid cells. First, using lentiviral delivery, we developed several ALCAM knockout clones in monocytic U937, and their latently infected with HIV-1 equivalent: U1 cells. As expected, ALCAM−/− myeloid cells showed markedly reduced adhesion to and transmigration through monolayers of endothelial cells. Next, using AAV6 delivery, we replicated these results in primary human monocytes from three different healthy donors. Finally, to limit CRISPR-Cas9 editing to HIV-1 infected cells, we placed Cas9 expression under the control of minimal HIV-1 Tat responsive LTR promoter (−80/+66). HIV-1BAL-GFP infection of AAV6/adeno-LTR-CRISPR-anti-ALCAM treated CD4+ T cells, monocytes, and monocyte-derived macrophages (MDMs) resulted in the induction of Cas9 expression and CRISPR mediated cleavage of exon 1 of ALCAM gene in Tat expression dependent manner and inhibition of their transendothelial migration. We demonstrated the feasibility of using CRISPR gene editing to disrupt a single CAM gene, i.e, ALCAM to disable adhesion and transmigration ability of HIV-1 infected myeloid cell line cells, primary monocytes, and macrophages. Moreover, we provided data proving the achievability of creating an HIV expression dependent CRISPR platform to restrict Cas9 cleavage activity only to HIV infected cells.
IL-4 regulates resident memory CD8+ T cell formation and persistence
Authors: Mora-Buch Rut , Akbaba Hasan , Chasse Alexandra Y , Sama Andrea M , Mani Vinidhra , Bromley Shannon K
Published: January 2023

Tissue resident memory CD8+ T cells (TRM) persist long-term in lymphoid and non-lymphoid tissues to provide rapid, local defense against pathogens and tumors. Tissue cytokines drive the formation and persistence of CD8+ TRM. However, whether the local microenvironment can inhibit TRM formation is largely unknown. Individuals suffering from conditions associated with T helper 2 (Th2)-type immune responses, such as atopic dermatitis, asthma, and some cancers, present impaired anti-viral or anti-tumor responses. We hypothesize that Th2 cytokines inhibit the formation and/or persistence of CD8+ TRM. We demonstrate that Th2 cell supernatant downregulated TGF-β-induced CD103 expression on in vitro- and in vivo- activated CD8+ T cells in an IL-4- and Stat6-dependent manner. Moreover, IL-4 induced Eomes expression by TGF-β-stimulated CD8+ T cells. In vivo, more CD8+CD103+ TRM populated the skin of Il4ra−/− and Stat6−/− mice compared to wild-type mice at homeostasis. Additionally, CD8+ T cell activation in the presence of IL-4 impaired subsequent CD8+ TRM formation; IL-4-exposed CD8+ T cells expressed decreased CD103 in a mouse model of cutaneous inflammation, and these cells failed to persist within the skin at memory time-points. These data suggest that IL-4 counters TGF-β-mediated signals to inhibit CD8+ TRM formation/persistence and may allow identification of novel treatments to boost anti-tumor immunity and anti-viral responses in patients with type 2 inflammation.
Hot Spot-type Questions offered via ExamSoft to learn Immunology
Authors: Fazal Nadeem , Olk Sandra , Edquiban Marianne , Fadhil Shams
Published: January 2023

There are many ways to promote meaningful student learning of Immunology. Hot Spot question format is a new type of active learning tool. It indicates that having planned repetition over an extended period of time leads to better retention by including images with text during instruction. This leads to long-term retention and improved student outcomes. In Hot Spot, users indicate the answer by clicking a specific area of an image. First year pharmacy students participated in a 3-hour long Immunology workshop. The students reviewed a video and handout explaining Hot Spot before coming to the workshop. Hot Spot questions containing basic Immunology were prepared for the workshop. A class of sixty two students were given ExamSoft online platform to take a pre-workshop quiz. The students were split into small learning groups and were given half an hour to discuss the pre-workshop content of basic Immunology. After half an hour, the students were randomly asked to pick up a partner to take ExamSoft quiz assessment again in pairs. The difference between pre- and post-workshop was that the students were paired in post-workshop quiz and were allowed to discuss concepts of Immunology with their partners. This pairing exercise and use of Hot Spot type assessment enhanced learning of basic concepts of Immunology and was evident from post-workshop quiz grades. The students not only did better on assessment grades but the post-workshop student feed back surveys also greatly commended this type of learning method. Therefore, we recommend learning basic Immunology through workshops, pairing of students and use of Hot Spot assessments. We also advocate teaching all basic pharmaceutical sciences via interactive educational tools for enhancing student learning.
Optimization of a T cells education protocol to treat hematological cancers
Authors: MATHIEU Cedric , MINGUY Annabelle , TROTTIER Jessica , SANCHEZ-DARDON Jaime , DAVE Vibhuti , ROY Denis-Claude
Published: January 2023

Allogeneic hematopoietic stem cell transplantation is the sole curative treatment for patients with high-risk hematological cancers. Most of its activity relies on the ability of engrafted T cells to recognize minor histocompatibility antigens (MiHAs) expressed on malignant cells resulting in their elimination. However, donor lymphocytes can also recognize MiHAs expressed on non-hematopoietic cells leading to graft-versus-host disease. Hence, the expansion of T lymphocytes displaying potent cytotoxic activity toward MiHA(s) preferentially expressed on leukemic cells would be greatly beneficial for engrafted patients. Using a proteo-genomic approach, novel MiHAs that are predominantly expressed on hematopoietic cells have been identified. We previously showed that these novel MiHAs can be exploited for ex vivo expansion of the antigen specific T cells. However, the frequencies of these MiHA specific T cells are extremely low. Thus it is challenging to generate an optimal ex vivo expansion of antigen specific T cells to a single MiHA. In this context, we aim to improve this complex manufacturing process by using simultaneously multiple MiHAs to enhance the probability of obtaining response to at least one MiHA. This approach allowed us to expand T cells populations with distinct MiHAs specificities. Further, upon restimulation, MiHA specific T cells express high levels of inflammatory cytokines Interferon-γ and Tumor Necrosis Factor-α thus suggesting their functionality. In conclusion, multipeptide T cell stimulation allows for simultaneous expansion of functional T cells specific for several MiHA. This could be translated in improved therapeutical approach reducing the probability of immune escape of malignant cells.
QuickSwitch™: a validated platform for both screening MHC binding SARS-CoV-2 peptides and making SARS-CoV-2 specific MHC class I and II tetramers.
Authors: Poluektov Yuri O , Delcommenne Marc , Daftarian Pirouz M , George Marybeth
Published: January 2023

The success of SARS-CoV-2 vaccines is measured by their ability to mount long-lasting memory responses. To this end, it is important to identify viral protein fragments, or peptides, that generate the best immune response and lead to the development of memory T cells. For this purpose, researchers need a system that would allow them to rapidly test the ability of an antigenic peptide to be presented by the immune system and to identify T cells that can be stimulated by such a peptide in the context of the right MHC molecule. The QuickSwitch™ platform contains two modules for performing both functions. First a large number of SARS-CoV-2 peptides, including mutant peptides were incubated with QuickSwitch™ monomers or tetramers and exchanged against their exiting peptides in the first module. Peptide binding affinities to various human MHC Class I and Class II alleles, which are correlated with their exchange rates, were determined by the capture binding assay, the second QuickSwitch™ module. MHC tetramers built with SARS-CoV-2 high affinity MHC binding peptides were then used for T cell staining. Cell staining indicated that tetramers generated with the QuickSwitch™ platform were able to stain and identify T cells specific for the tested peptides. Further, peptides that bound to multiple MHC alleles, including potentially dominant peptides, were also detected. In conclusion, since QuickSwitch™ allows the screening of MHC binding SARS-CoV-2 peptides and, at the same time, the generation of corresponding tetramers, this platform is a useful tool for developing SARS-CoV-2 vaccines, monitoring T cell populations from vaccinated subjects and assessing the durability of mounted responses.
B cells licensed by DCs are primary antigen presenting cells for DNA plasmid upon passive uptake
Authors: Rastogi Ichwaku , McNeel Douglas G
Published: January 2023

Dendritic cells are considered the primary cells involved in professional antigen presentation. However, our group and others have shown that B cells are similarly capable of presenting antigens leading to activation and proliferation of antigen-specific T cells. In this study, we aimed to understand the mechanism of presentation of antigen, encoded by plasmid DNA when delivered to APCs via passive uptake. We used plasmid DNA encoding ovalbumin, splenic B cells from Bl6 mice, DCs from Flt3L treated Bl6 mice, and CD8 T cells from OT1 mice that are restricted for the SIINFEKL-specific T-cell receptor. We have previously reported that only B cells are capable of transcribing the encoded antigen, whereas DCs and macrophages degrade the plasmid DNA upon passive uptake. In our current study we show that B cells also translate the encoded antigen, but can present the antigen to CD8 T cells only in the presence of DCs. We utilized a trans-well assay to show that physical contact between B cells and DCs is required for successful antigen presentation and that live DCs are essential, as replacing them with DC lysate or DC supernatant or both was not adequate. We also show that the presence of DCs promotes B cell survival, mostly through CD40-CD40L signaling by using CD40 KO mice and that B cells are primary antigen presenting cells in this co-culture by using MHC I KO mice. We also show that exposure of B cells to CD40 or CD40L, which is one of the identified interactions between B cells and DCs, was not sufficient for B cells to present antigen in the absence of DCs. In summary, our data suggests that B cells and DCs communicate through a currently unknown cell-cell interaction that licenses B cells for antigen presentation after passive uptake of plasmid DNA.
Repertoire size and predictability of influenza A virus matrix protein (M158–66)-specific CD8+ TCR
Authors: Li Jiangyuan , Cifello Jeffrey , Lu Jian , Weng Nan-Ping
Published: January 2023

Influenza A virus (IAV) is a major human pathogen that causes seasonal infections worldwide. IAV-specific CD8+ T-cell response provides an essential part of immune protection against IAV infections. In HLA-A2+ humans, the dominant epitope on IAV is matrix protein M1 residues 58–66 (GIL GFVFTL, referred to as GIL). Here, we investigated the TCR repertoire size, predictability, and signaling strength of GIL-specific CD8+ T cells healthy adults. We analyzed over 40,000 GIL-tetramer positive CD8+ T cells by scRNAseq with TCR sequences and identified over 10,000 unique abTCRs sequences. Next, we developed a machine learning algorithm (an ensemble random forest models) to further predict the probability of GIL-binding TCR (ranging from 0.1 to 1 with 1 being highest binding confidence) using reported GIL-TCRs. To confirm the predicting accuracy, we expressed 72 TCR with different RF predicting score and showed that TCR with a RF score greater than 0.8 showed 100% GIL-binding (30 different TCRs). Using RF model and filtering, we identified over 4,000 unique GIL-specific TCRs with less than 800 were previously reported. Overall, our study revealed TCR repertoire size of GIL-specific CD8+ TCRs and indicated the features of GIL-binding TCRs are identified by our ML model to accurately predict this TCR specificity, which paces way to investigate frequency and quality of antigen specific TCR repertoire size and function in the general T cell population.
Reverse Immunology: An approach to identify Mtb antigens recognized by T cells
Authors: Jaiswal Swati , Williams Travis , Gideon Hannah P , Hughes Travis K , Tu Ang Andy , Nyquist Sarah K , Shalek Alex N , Fortune Sara M , Flynn JoAnne L , Behar Samuel
Published: January 2023

T cells plays important role in controlling the infection. Previous research findings have suggested that many immunodominant antigens are not similarly immunogenic. We attempt to discover novel antigen that can be developed as a potential vaccine candidate. Low dose Mycobacterium tuberculosis (Mtb) infection in Cynomolgus macaque results in human like granuloma formation. It is comprised of T cells, B cells, NK cells, and several other cells. Upon FDG-PET scan it has been shown that some of these granulomas are permissive or progressive in nature and some are restrictive or controlling.

Our aim is to discover Mtb antigens that can be recognized by the T cells present in the granuloma. To investigate our hypothesis, we have infected Cynomolgus macaque with low dose of Mtb. T cells were isolated from the low and high burden granuloma. They were subjected to scRNA sequencing. Using bioinformatic tools and algorithms we have identified high value TCRs from controlling granulomas. The TCRs were reconstructed and transfected in TCR deficient Jurkat cells with NFAT promoter and luciferase reporter gene. The Mtb ORFeome library was cloned and expressed in E. coli. For antigen preparation, IPTG induced recombinant E. coli were killed by Peracetic acid or heat-killed and presented to the high value TCRs expressed in the reporter Jurkat cells by autologous BLCLs. The recognition was measured using luciferase activity.

The selected unique TCRs show remarkable luciferase activity in response to PMA and Ionomycin stimulation. We believe that the antigen recognized by these TCRs will be equally immunogenic and can be developed as the new target for the vaccine against tuberculosis.

Supported by BAA-NIAID-NIHAI201700104
H-2Kb and H-2Db class I molecules on cerebral endothelium differentially modulate CD8 T cells dynamics and pathological outcomes in experimental cerebral malaria
Authors: Fain Cori E , Zheng Jiaying , Jin Fang , Ayasoufi Katayoun , Chen Maggie , Dropik Abigail , Khadka Roman , Tritz Zachariah Peter , Hansen Michael , Johnson Aaron J
Published: January 2023

Immune mechanisms of neuropathology in human cerebral malaria are not fully understood. Using the Plasmodium berghei ANKA model of experimental cerebral malaria (ECM), we sought to define the contribution of cerebral endothelial cell (CEC) MHC class I antigen presentation, to the pathogenesis and lethality of ECM. CD8 T cell interactions with class I molecules on CECs has been put forward as a putative mechanism for ECM. However, the importance of this interaction has not been confirmed molecularly in vivo. We therefore hypothesized that conditional ablation of antigen presentation by H-2Kb and H-2Db class I molecules specifically on brain endothelium would result in controlled modulation of activation and entry of parasite-specific CD8 T cells, attenuating pathology. To test this, we crossed endothelium-specific CDH5-Cre mice with our novel H-2Kb and H-2Db LoxP mice to create endothelium-specific cKO of either or both class I molecules. 2-photon imaging revealed that each discreet class I molecule influenced differential T cell dynamics at the neurovasculature during lethal stage of disease. Furthermore, CD8 T cell activation and infiltration of the brain was distinctly altered, per class I interaction, and vascular permeability at the blood-brain barrier was attenuated, resulting in improved survival of H-2Kb and H-2Db endothelial cKO mice. Together these data demonstrate that parasite-specific CD8 T cells undergo Ag-dependent engagement with H-2Kb or H-2Db class I molecules on CECs in order to coordinate activation, entry, and induction of neuropathology. These studies indicate that modulation of CEC class I restricted antigen presentation may hold therapeutic benefit in cases of severe malaria.

Supported by Ruth L. Kirschstein National Research Service Award Individual Predoctoral Fellowship F31 1F31NS116924-01. NIH R01 5R01NS103212-05.
mTORC2 contributes to murine lupus associated immunopathology
Authors: Zeng Hu , Zhou Xian , Li Yanfeng , Davidson Anne
Published: January 2023

The development of systemic lupus erythematosus (SLE) is associated with overactivation of the type I interferon (IFN) pathway, lymphopenia, and increased follicular helper T (Tfh) cell differentiation. However, the cellular and molecular mechanisms of action of type I IFN in SLE remains incompletely understood. Here we show that type I IFN activates the mechanistic target of rapamycin complex 2 (mTORC2) in T cells to promote T cell lymphopenia. mTORC2 also promotes Tfh differentiation and disrupts Treg homeostasis. Inactivation of mTORC2 greatly ameliorated the immunopathology in a lupus-prone mouse model, associated with reduced Tfh differentiation, normalization of Treg homeostasis and reduced T cell glucose metabolism. These data indicate that mTORC2 acts downstream of type I IFN and costimulatory receptor ICOS, to promote T cell lymphopenia and Tfh differentiation in murine lupus development, suggesting that inhibition of mTORC2 could limit lupus disease progression.

Supported by Lupus Research Alliance Mayo Foundation for Medical Education and Research R01AR077518