bluuebunny/crossref_metadata_2025 huggingface dataset:
Combination B-lymphocyte Depletion Treatment with Anti-CD20 and BAFF Blockade is No Superior to BAFF Blockade Alone in The NOD Model of Type 1 Diabetes
Authors: Wang Qiming , Serreze David V
Published: January 2023

While type 1 diabetes (T1D) is an autoimmune disease in which autoimmune destruction of pancreatic β cells is ultimately mediated by T-cells, in the NOD mouse model, and also likely humans B-lymphocytes also play a key pathogenic role. B-lymphocyte depletion either through antibody targeting CD20 or blocking their access to BAFF elicits strong T1D protection in NOD mice. However, whether the combined strategy of targeting both CD20 and BAFF could further improve therapeutic efficacy for T1D remained unknown. Currently humans at high future T1D risk can only be identified at late prodromal stages of disease indicated by markers such as insulin autoantibodies (IAA). We show BAFF blockade treatment commenced in already IAA positive NOD mice prevented progression to overt T1D, whereas anti-CD20 was ineffective as a mono- or co-therapy at this disease stage. While BAFF blockade depletes the majority of islet infiltrating B-lymphoctes, a memory-like subset remained, whose potential diabetogenic capacity needs further exploring. Although BAFF blockade treated mice are protected from T1D development, the severity of insulitis was not reduced. One complicating factor underlying the failure of anti-CD20 is that NOD mice developed neutralizing antibodies against the injected anti-CD20, and the degree of B-lymphocyte rebounding correlates with the titer of neutralizing antibodies present in the sera. The dual directed immunotherapy does not either improve the protection offered by BAFF blockade only, or reduce the severity of insulitis, despite the greater depletion on various B-lymphocyte subsets and further reduction on those that survived of MHC and CD40 molecules respectively involved in antigen presentation, and class switching.
Identification and characterization of an antagonistic anti-mouse CD40 antibody
Authors: Ralph Kerry L. M. , Panzenbeck Mark , Xiao Haiguang , Frego Lee , Bigwarfe Tammy , Waltz Erica , Grimaldi Christine , Last-Barney Kathy , Souza Don , Brodeur Scott , Nabozny Gerald , Fine Jay S. , Distler Jorg H. W. , Ramanujam Meera
Published: January 2023

Targeting CD40-CD40L interactions has been an interesting drug concept for the treatment of autoimmune diseases given this pathway’s role in the development of both humoral and cell mediated immune responses. While preclinical blockade of CD40L is well documented in various autoimmune animal models of disease, limited data exists for CD40 blockade due to lack of a truly antagonistic anti-mouse CD40 tool antibody (Ab). Here we describe the in vitro and in vivo characterization of a fully antagonistic anti-mouse CD40 monoclonal Ab (BI CD40-1); a chimeric rat Fv anti-mouse CD40 mAb engineered with a mouse IgG2a Fc containing mutations to abrogate Fcγ receptor binding. BI CD40-1 blocks molecular CD40-CD40L interactions (IC50 = 0.25 nM) and exhibits potent binding to CD40 expressed on mouse B cells (EC50 = 0.42 nM ± 0.08). In vitro profiling of BI CD40-1 using a mouse splenocyte proliferation assay confirmed potent antagonistic activity (IC50 = 0.27 nM ± 0.09) as well as absence of any agonistic properties (stimulation index < 2 @ 67 nM). Administration of BI CD40-1 to mice prior to ovalbumin (OVA) immunization resulted in dose-dependent blockade of OVA-specific IgG responses (100, 100, 74, 0% inhibition at 10, 3, 1, and 0.3 MPK; day 13) correlating with similar dose dependent receptor occupancy and inhibition of B cell activation as measured by ex vivo CD54 upregulation. Prophylactic dosing in cGVHD model of scleroderma showed dose dependent protection in disease development as seen by decreased dermal thickness, myofibroblast counts and collagen deposition. BI CD40-1 represents a novel fully antagonistic anti-mouse CD40 mAb, an important tool for mechanistic understanding of the therapeutic value of targeting of CD40 in inflammatory diseases.
Molecular interactions of small molecule inhibitors targeting cytoplasmic TIR domains.
Authors: Snyder Greg A , Irwin Gregory , Mistry Pragnesh Dilipkumar , VanBesien Richard , Flecther Steven , MacKerell Alexander , Brown Lindsey , Xiao Tsan Sam , Wintrode Patrick , Vogel Stefanie
Published: January 2023

Regulation of Toll-like receptor (TLR) signaling using small molecule agonists and antagonists have been widely sought after for controlling inflmation and disease. Most studies have focused on therapeutic targeting of TLR extracellular ligand binding domains. Recently, Computer-aided drug design (CADD) screens specifically targeting intracellular Toll-Interleukin-1 receptor resistance domains have identified and functionally characterized several small molecule and peptide inhibitors which protect against lethality in animal models of infection, inflammation and disease. We have sought to structurally characterize the molecular interactions of CADD derived small molecule inhibitors with respective TLR-TIR domains by determining the X-ray co-crystal structures of TLR2 –TIR domain in the presence of C29 and its substructure O-vanillin. Positive electron density is observed near the BB loop and residue Ile 685 of native TLR2-TIR in the presence of the small molecule inhibitor (C29) exhibit compared with the native apo form of this structure. However, the presence of (S-(DIMETHYLARSENIC) CYSTEINE) in this crystallization condition complicate analysis. To rule out the effects of DMSO and (S-(DIMETHYLARSENIC) CYSTEINE) we sought to examine small molecule inhibitor C29L substructure (o-Vanillin) in a second crystal form of TLR2 and which contains a more simplified crystallization condition grown in the presence of 1mM of C29L. This co-crystal exhibits positive electron density located in an around the BB loop compared with apo forms of this structure. Additionally, in this recent structure form we observe additional DD loop density previously not defined in the originally reported crystal form.
IFN-β suppresses GM-CSF production by CD4+ T cells by inhibiting STAT5 signaling
Authors: Rasouli Javad , Mari Elisabeth Rose , Ciric Bogoljub , Rostami Abdolmohamad
Published: January 2023

Multiple sclerosis (MS) is an autoimmune disease characterized by accumulation of immune cells in the central nervous system (CNS). Granulocyte-macrophage colony-stimulating factor (GM-CSF) is necessary for development of CNS autoimmunity in animal models of MS, suggesting that GM-CSF also plays an important role in the pathogenesis of MS. We previously reported that untreated MS patients have higher frequencies of GM-CSF+ CD4+ T cells in their peripheral blood, and that IFN-β therapy normalized their numbers. Since IFN-β suppresses GM-CSF expression by CD4+ T cells in MS, we sought to determine the mechanisms of its action. It has been shown that IFN-β activates multiple JAK/STAT signaling pathways in T cells, and that GM-CSF expression in T cells is largely dependent on STAT5 signaling. Here, we show that in vitro IFN-β strongly suppressed GM-CSF production by T cells from healthy donors, untreated and IFN-β-treated MS patients, as well as patients diagnosed with other neurological diseases. IFN-β did not increase T cell apoptosis in our cell culture; hence, reduced production of GM-CSF by T cells was not due to pro-apoptotic effects of IFN-β. IFN-β significantly decreased the phosphorylation of STAT5 in total CD4+ T cells, including GM-CSF-producing CD4+ T cells. In summary, our findings suggest that IFN-β suppresses GM-CSF production by CD4+ T cells by inhibiting STAT5 signaling.
FGL2 promotes tumor progression via inducing TIGIT expression on T cells in tumor microenvironment of glioma
Authors: Yan Jun , Gabrusiewicz Konrad , Xia Xueqing , Heimberger Amy B. , Li Shulin
Published: January 2023

Glioblastoma multiforme (GBM) is the most common primary brain tumor with a median survival of only 14.6 months. Our previous work shows that FGL2 promotes glioma tumor growth and the expression levels of FGL2 positively correlated with glioma grade in patients. T cell Ig and ITIM domain (TIGIT) is an inhibitory receptor expressed by activated T cells, Tregs, and NK cells. Here we found that there is a statistically significant positive correlation between FGL2 and TIGIT mRNA expression level in both low grade glioma database (TCGA, provisional) and glioblastoma multiforme database (TCGA, provisional). Future, FGL2 and TIGIT are coordinately expressed by human tumor-infiltrating lymphocytes of glioma as detected by flow cytometry. In addition, TIGIT expression on T cells which suppresses their function, can be induced by initial T cell activation or FGL2 in vitro. Induced TIGIT expression upon initial T cell activation is suppressed in FGL2KO T cells which has higher tumor-killing ability than WT T cells. Therefore, our results show that FGL2 induces TIGIT expression in brain-infiltrated lymphocytes which suppresses anti-tumor immune response in glioma. By this mechanism, FGL2 promotes tumor progression of glioma.
Role of Prostate apoptosis response-4 tumor suppressor in the survival and growth of Chronic Lymphocytic Leukemia
Authors: McKenna Mary Kathryn , Nooti Sunil K , Alhakeem Sara Samir , Gachuki Beth W , Frissora Frank , Greene Joseph T , Byrd John C , Muthusamy Natarajan , Rangnekar Vivek R , Bondada Subbarao
Published: January 2023

Chronic Lymphocytic Leukemia (CLL), the most common adult leukemia in the western world, is characterized by accumulation of clonally expanded CD5+CD19+ B lymphocytes in blood and secondary lymphoid organs with impaired apoptotic mechanisms. Prostate apoptosis response-4 (Par-4) is a pro-apoptotic tumor suppressor protein, which is silenced by promoter methylation in more than 30% of all cancers. It is also secreted and induces apoptosis selectively in many types of cancer cells but not in normal cells. Here we characterized the role of Par-4 in CLL cells using a murine model. The Eμ-Tcl1 mouse serves as an excellent model to study CLL as they develop a CLL like disease by 9–13 months of age, due to B cell specific over-expression of the oncogene, T cell Leukemia 1 (Tcl1). Adoptive transfer of primary CD5+CD19+ CLL cells from the Eμ-Tcl1 CLL mice into recipient syngeneic mice leads to the development of a CLL like disease within 3–5 weeks of transfer. Surprisingly Eμ-Tcl1 CLL cells constitutively express more Par-4 than normal B-1 or B-2 cells in mice. These CLL cells also secrete Par-4 which is functional in being cytotoxic to prostate cancer cells. We showed that Eμ-Tcl1 CLL cells have constitutively active B-cell receptor signaling and that inhibition of BCR signaling causes a decrease in Par-4 expression and increases apoptosis. Interestingly, we have found that shRNA mediated knockdown of Par-4 in human CLL cell lines, results in their reduced growth. These results suggest that intrinsic Par-4 may play a pro-survival rather than pro-apoptotic role in CLL. We are currently investigating the mechanisms underlying this novel role of Par-4 and regulation of its expression in CLL cells using CRISPR system to knockout Lyn and Btk in CLL cells.
PD-1 expression is not indicative of T cell exhaustion in breast cancer tumors.
Authors: Egelston Colt , Avalos Christian , Lee Peter P.
Published: January 2023

PD-1 expression on tumor infiltrating lymphocytes (TILs) has been implicated to be associated with T cell exhaustion. Indeed, we have found that PD-1 is expressed on a majority of both CD4 and CD8 T cells in the tumors of breast cancer patients. To determine if patient T cells have an exhausted functional phenotype, we compared patient peripheral blood mononuclear cells (PBMCs), tumor draining lymph nodes (TDLNs), TILs, and healthy donor PBMCs. We examined the cytokine production capacity in response to non-specific stimulation, followed by intracellular flow cytometry for production of IFNγ and TNFα. In the majority of patients, we found TIL memory T cells at least as potent as circulating memory T cells in producing both IFNγ and TNFα. Furthermore, PD-1+ T cells did not show any significant reduction in cytokine production compared to PD-1− T cells. Additionally, immune phenotyping by flow cytometry shows a lack of other checkpoint molecules, such as LAG-3, TIM-3, and CTLA-4 on TILs. This data suggests that PD-1+ T cells in breast cancer tumors may not suffer from exhaustion and that PD-1 expression alone does not indicate T cell exhaustion.
The adhesion molecule PSGL-1 is a checkpoint regulator that promotes T cell dysfunction in melanoma tumors
Authors: Tinoco Roberto , Carrette Florent , Barraza Monique L , Bradley Linda M
Published: January 2023

The tumor microenvironment can promote tumor growth by suppressing effector T cell responses. Using a melanoma model that recapitulates aspects of human disease, we investigated the role of the adhesion molecule, P-selectin glycoprotein ligand-1 (PSGL-1) in tumor infiltrating lymphocytes (TILs). We found that PSGL-1 was highly expressed on TILs that also expressed high PD-1 and TIM-3 inhibitory receptors. PSGL-1-deficiency did not impact T cell recruitment in tumors but instead resulted in a greater TIL accumulation. Furthermore, PSGL-1-deficient T cells had reduced PD-1 expression and increased production of effector cytokines that resulted in delayed tumor growth. We did not find differences in Treg recruitment but instead had an increased effector to Treg ratio in tumors. Our findings show that PSGL-1 does not have a nonredundant role in the trafficking of TILs into melanoma tumors, but can act as an immune checkpoint inhibitor to limit anti-tumor T cell responses in the tumor microenvironment thereby promoting tumor growth.
Inflammasome-independent role of AIM2 in mediating protection against colorectal tumorogenesis
Authors: Karki Rajendra , Man Si Ming , Zhu Qifan , Kanneganti Thirumala-Devi
Published: January 2023

Colorectal cancer is a leading cause of cancer-related deaths. Mutations in the innate immune receptor AIM2 are frequently identified in patients with colorectal cancer. AIM2 inflammasome is activated by double stranded DNA and inflammasomes are central regulators of intestinal homeostasis. The expression of the AIM2 is reduced in several types of tumors compared with healthy tissues, highlighting a previously undefined function for this sensor in the development of colorectal cancer. Here, we found that Aim2-deficient mice were hypersusceptible to colonic tumor development. Production of inflammasome-associated cytokines and other inflammatory mediators were largely intact in Aim2-deficient mice, however, intestinal stem cells were prone to uncontrolled proliferation. Aberrant Wnt signaling expanded a population of tumor-initiating stem cells in the absence of AIM2. Therapeutic modulation of AIM2 expression has the potential to prevent colorectal cancer.
The role of Signal Transducer and Activator of Transcription protein 5 (STAT5) in non-small cell lung cancer (NSCLC)
Authors: Friedrich Juliane Michaela Margarete M , Heim Lisanne , Springel Rebekka , Mittler Susanne , Gafencu Dumitrita , Hartmann Arndt , Sirbu Horia , Finotto Susetta
Published: January 2023

Signal Transducer and Activator of Transcription protein 5 (STAT5a/b) belongs to a family of T cell transcription factors. The different isoforms of STAT5 are activated by several cytokines and tyrosine kinases, which led to the assumption, that both STAT5 isoforms regulate cell growth. Interleukin (IL)-2, a major cytokine regulating immunocompetent cell survival, proliferation and differentiation can activate STAT5. Interestingly, by contrast to other T cell subsets, T regulatory cells (Tregs) express the IL-2 receptor constitutively. It is considerable that Tregs are immunosuppressive and thus are able to suppress the function of certain effector T cells, eg CD4, CD8 and natural killer T cells. Tregs are highly dependent on IL-2 and thus are able to consume IL-2 more efficiently than effector cells, which results in a depletion of IL-2 from effector cells and leads to a disorder in the immune response. We thus asked about the role of STAT5 in NSCLC.

We measured increased STAT5 protein levels by Western Blot in lung tumor tissue compared to control lung tissue derived after lung surgery from patients who suffered from lung cancer, especially adenocarcinoma (ADC). This finding was confirmed by STAT5 immunohistochemistry (IHC) staining, where a stronger signal for STAT5 was detected in lung tumor tissue compared to lung control region, in a cohort of ADC patients (n=10). Moreover, preliminary FACS analysis of freshly isolated cells from the resected lung of one patient with NSCLC showed strongly upregulated pSTAT5 levels in tumor infiltrating CD4+ T lymphocytes compared to CD4+ lymphocytes derived from respective lung control region.

Thus unrevealing the role of STAT5 in lung tumor should help to improve lung cancer immunotherapy.
Inflammasome/IL-1 pathways in tumor microenvironments
Authors: Guo Beichu , Zhang Jinyu , Fu Shunjun
Published: January 2023

Emerging evidence indicates that chronic Inflammation plays important roles at all stages of tumor development including initiation, growth, invasion and metastasis. IL-1β and other IL-1 family members are major mediators in an inflammatory response. Clinical studies reveal increased concentration of IL-1β protein in tumor tissues, and such increase is associated with poor prognosis for cancer patients. While chronic inflammation is associated with tumor development, the mechanism of such connection remains not completely understood. Our preliminary data have demonstrated that inflammasome and IL-1β play a critical role in promoting tumor growth and metastasis. An inflammasome is a multimolecular complex, composed of a NOD-like protein (NLR), the adaptor protein ASC, and caspase-1, which is essential for the cleavage of pro-IL-1β into its active form. Using orthotropic breast cancer and transgenic mouse models, we found that breast cancer was associated with inflammasome activation. Our results show that mice deficient for inflammasome components had much slow tumor growth and lung metastasis. Compared to wildtype mice, inflammasome deficient mice have low levels of IL-1 in primary tumor tissues and metastastic lungs. Furthermore, our data reveal that pharmacologic disruption of inflammasome/IL-1β pathway diminished tumor growth and metastasis in vivo. These results suggest that the activation of inflammasomes and production of IL-1β in vivo may provide an inflammatory tumor microenvironment to promote tumor growth and metastasis.
MUC1 promotes an inflammatory microenvironment aggravating colitis-associated tumorigenesis in mice through up-regulation of pro-inflammatory cytokines.
Authors: Cascio Sandra , Faylo Jacque , Sciurba Joshua , Xue Jia , Finn Olivera J
Published: January 2023

MUC1 is a transmembrane glycoprotein aberrantly expressed in human adenocarcinoma as well as chronic inflammatory conditions. We previously demonstrated that the tumor form of MUC1, in association with p65, upregulates expression of pro-inflammatory cytokines, including IL-6 and TNF-alpha, by modulating their transcription in epithelial cancer cells. Using azoxymethane/dextran sulfate sodium (AOM/DSS) murine model of inflammation driven colon carcinogenesis, we explored mechanism underlying MUC1/p65-induced transcription of IL-6 and TNF-alpha and its significance for the microenvironment of colitis-associated cancer. MUC1.Tg mice showed higher tumor incidence, decreased survival, greater body weight loss and shorter colon length. Consistent with our previous in vitro data, expression of NF-kB-dependent cytokines was higher in MUC1.Tg mice compared to WT. Moreover, we discovered that MUC1/p65 complex modulates IL-6 and TNF gene expression by regulating the crosstalk of post-translational modifications on their promoters. To understand the significance of MUC1/p65-modulated cytokines in progressive colitis that gives rise to colon cancer, we analyzed infiltration of inflammatory cells into the inflamed colon tissues. ELISA assay and gene expression analyses demonstrated that the treatment with AOM/DSS in the presence of human MUC1 on the colonic epithelia resulted in a significant upregulation of M1 type macrophage-associated genes, including IL-6, TNF-alpha and iNOS, whereas expression of M2 type macrophage markers, such as Arginase 1, Ym1 and IL-10, were drastically reduced compared to WT mice. Thus our findings reveal a pro-inflammatory role for MUC1 in colitis-related carcinogenesis.
Brain Slice Culture Demonstrates Expedited Cell Traficking in the Presence of Fractalkine in a Medulloblastoma Tumor Microenvironment Model
Authors: Tomaszewski William H. , Dorand Rodney Dixon , Petrosiute Agne , Huang Alex
Published: January 2023

20% of childhood brain tumors are medulloblastomas (MB), and the five-year survival rate can be as low as 60%. The interaction between MB and native immune cells are not well understood, and identifying methods of crosstalk could produce better outcomes for patients. Fractalkine (FKN) is a ligand for the receptor CX3CR1 that plays an inhibitory role in the brain’s immune system by keeping microglia quiescent. Normally, FKN is surface bound and expressed by neurons and astrocytes, but under inflammatory conditions FKN is secreted to serve as a chemoattractant. Preliminary work in our lab shows that FKN is produced by MB and may act as a decoy to maintain microglial quiescence leading to immune escape. Therefore we investigated FKN signaling and microglial kinetics ex vivo utilizing organotypic brain slice culture coupled with real time 2-photon laser scanning microscopy. To differentiate microglia from MB, CX3CR1+/GFP or CX3CR1GFP/GFP knock-in mice were sacrificed, and the brain was removed and sectioned. Slices were collected and cultured on inserts in an incubation chamber during imaging. To determine baseline activity, CX3CR1+/GFP and CX3CR1GFP/GFP brain slices were imaged over 48 hours. MB tumor cells, labelled with SNARF-1, were added to the cultures and imaged over the same time course. Images were processed to differentiate the motility of microglia and tumors. We found that microglia from CX3CR1+/GFP moved slower than microglia from CX3CR1GFP/GFP at baseline and in the presence of tumor. Furthermore, we found that MB moved faster in CX3CR1GFP/GFP slices, suggesting that fractalkine plays a role in the migration of MB.
MIF is necessary for late-stage melanoma patient MDSC immune suppression and differentiation
Authors: Yaddanapudi Kavitha , Rendon Beatriz , Rayyan Numan , Richie Jamaal , Kim Eun Jung , Wise Ashley , Mitchell Robert
Published: January 2023

Patients diagnosed with unresectable stage IV melanoma have an average survival of 6–10 months; a survival time that hasn’t changed significantly in the last 30 years. Despite some encouraging improvements in response rates and overall survival in patients receiving Ipilimumab (anti-CTLA-4) and/or Nivolumab (anti-PD-1) immunotherapies, the majority of patients will ultimately progress. Myeloid-derived suppressor cells (MDSCs) are potently immunosuppressive innate immune cells that accumulate in advanced cancer patients and actively inhibit anti-tumor T lymphocyte responses; therefore, can contribute to immunotherapeutic resistance. Increased circulating MDSCs in stage III/IV melanoma patients predict patient mortality and directly correlate with reduced melanoma antigen-specific T cells. Our results demonstrate that circulating monocytic MDSCs are found in significantly higher numbers in chemotherapy naïve late stage melanoma patients and the immunosuppressive activity of these cells is highly reliant upon the innate immunomodulatory cytokine, macrophage migration inhibitory factor (MIF). Moreover, inhibition of MIF results in a functional reversion from immune suppressive MDSC to an immunostimulatory dendritic cell (DC)-like phenotype that is at least partly due to reductions in MDSC prostaglandin E2 (PGE2). These findings indicate that monocyte-derived MIF is centrally involved in melanoma MDSC induction and immune suppressive function and that therapeutic targeting of MIF may provide a novel means of inducing anti-tumor immune responses in late stage melanoma patients.
ubiquitinated proteins enriched by Vx3 conjugated Alpha-alumina nanoparticles induce efficient anti-tumor response
Authors: jinjin zhao , Shen Yanfei , Wang Lixin
Published: January 2023

Weak and ineffective therapy of malignant tumor threatened thousands of people. Immune therapy is one of the most promising methods for tumor treatment due to little side effects. Our previous study has shown that ubiquitinated proteins from tumor cells enriched by Vx3 (A7) protein have an effective anti-tumor efficacy through establishing mice tumor models. However, it was difficult to enrichment ubiquitinated proteins in this methods. Here, we reported that such kind of problem could be solved through a microparticle precipitate method by using a-Al2O3 nanoparticles. Briefly, a-Al2O3 was first functionalized with (3-Aminopropyl) triethoxysilane, and subsequently with glutaraldehyde, as proved by the Fourier transform-infrared (FT-IR) analysis. Finally, the Vx3 (A7) protein was conjugated with the aldehyde-functionalized a-Al2O3 via Schiff ‘ s base reaction. Using Vx3 (A7) protein conjugated with a-Al2O3 could effectively enrich much more ubiquitinated proteins compared with our previous study as indicated by western blot. Interestingly, the ubiquitinated proteins that enriched by this new strategy show a high tumor specific immune response and an effective anti-tumor efficacy through in vivo experiments. It could be concluded that this ubiquitinated-protein conjugated to a-Al2O3 nanoparticles are considered to be a good choice in cancer immunotherapy.
Rapamycin directly inhibits T cell lymphoma proliferation at high doses and facilitates immune-independent toxin-mediated killing at a low dose in vivo.
Authors: Liu Yang , Pandeswara Srilakshmi , Dao Vinh A , Padrón Álvaro , Hurez Vincent , Curiel Tyler J
Published: January 2023

Hyperactive mammalian target of rapamycin (mTOR) signaling that drives tumor growth makes mTOR inhibition an attractive cancer therapeutic strategy. mTOR inhibitors are approved to treat certain carcinomas, but effects on T cell hematologic malignancies, which also express hyper-activated mTOR, are less reported. We studied effects of the mTOR inhibitor rapamycin (Rapa) in mouse EL4 T cell lymphoma. Typical pharmacologic Rapa doses (1–8 mg/kg) significantly reduced tumor burden and improved survival in subcutaneous EL4 challenge independent of interferon (IFN)-γ or host T cells which we showed was by direct suppression of tumor cell proliferation. Though we found EL4 refractory to many anti-cancer immunotherapies, its growth was significantly slowed by regulatory T cell depletion with denileukin diftitox (DD) in vivo in a T cell-dependent fashion. However, typical Rapa doses inhibited T cell activation and tumor infiltration in vivo and failed to boost DD treatment effects as expected. Low dose (LD, 75 μg/kg) Rapa reportedly improves anti-tumor immunity and we found it increased potentially beneficial CD8+ T cells with a central memory phenotype (CD44hiCD62L+) in EL4 challenge, but without clinical effect. LD Rapa significantly enhanced DD treatment efficacy, but surprisingly, DD + LD Rapa treatment effects were independent of T cells, IFN-γ or perforin. Instead, Rapa up-regulated EL4 IL-2 receptor expression (DD binding site) in vitro and in vivo, apparently facilitating direct DD tumor cell killing. We thus show an unexpected mechanism for mTOR inhibitor in cancer immunotherapy and show distinct, biphasic mechanisms of action at typical pharmacologic doses versus LD Rapa that can be exploited clinically.
The clinical-stage TLR5 agonist, Entolimod, mediates antimetastatic activity by stimulating a CXCR3-dependent NK-DC-CD8+ T cell axis
Authors: Brackett Craig M , Kojouharov Bojidar M. , Gollnick Sandra O. , Burdelya Lyudmila G. , Abrams Scott I , Gudkov Andrei V.
Published: January 2023

Activation of an anticancer innate immune response is an attractive immunotherapeutic opportunity that is challenged by insufficient safety of systemically administered innate immune modulators. Unusual tissue specificity of expression of TLR5 determines a uniquely safe profile of cytokines induced by the sole TLR5 agonist flagellin. Entolimod, a pharmacologically optimized flagellin derivative, was initially developed to treat and prevent acute radiation syndrome with demonstrated efficacy in rodents and non-human primates and safety in human healthy volunteers. In addition, Entolimod demonstrated antitumor effects in mouse models of uveal melanoma, lymphoma, breast, and colorectal carcinoma. Entolimod’s mechanism of action involves activation of NF-κB-, AP-1-, and STAT-3-driven immunomodulatory pathways in hepatocytes that initiates a cascade of cell-cell signaling events that mobilize innate and adaptive immunity to the liver. This includes CXCR3-dependent blood-borne NK cell homing followed by DC activation and antitumor CD8+ T cell memory formation. These results define systemically administered TLR5 agonists as an organ-specific immunoadjuvant enabling efficient antitumor vaccination that does not depend on identification of tumor-specific antigens. Therefore, Entolimod has strong promise as a safe, effective and broadly applicable immunotherapeutic agent against liver metastases, which are currently a major cause of cancer-associated mortality. Recent completion of a phase I trial of Entolimod in patients with advanced metastatic solid tumors has opened the opportunity to test whether efficacy demonstrated in animal cancer models could be translated into immunotherapy of human tumors.
Vaccines Bearing Engineered HIV-1 gp41 Transmembrane and Cytoplasmic-tail Domains Elicit Rare Reactivities Targeting the C-terminal Subdomain of the MPER
Authors: Scott Jamie Kathleen
Published: January 2023

Numerous vaccine strategies have failed to induce neutralizing (Nt) antibodies (Abs) against the HIV-1 gp41 MPER, the target of several broadly NtAbs. Factors contributing to this include an incomplete understanding of native MPER structure, and restricted/transient exposure of key Nt sites. Here, we describe novel MPER DNA and MPER-peptide liposome vaccines (PLVs) that best elicit Abs against N- and C-terminal (N- & C-) subdomains of the MPER; these subdomains are targeted by the 2F5 and 4E10/10E8 NtAbs, respectively. DNA-prime, PLV-boost immunization of rabbits and guinea pigs (GPs) with vaccines that optimize exposure of the N-terminal subdomain, elicited low-level 2F5-like reactivities that were moderately elevated with boosting. Co-immunization with the vaccines increased durability of these responses, with preliminary results showing moderate Nt activity from GPs (but not rabbits; however, GP pre-immune sera exhibited high Nt background). DNA and PLV vaccines were optimized to expose both the N- and C-subdomains. DNA vaccines were engineered to (i) encode an N-terminal coiled-coil to support a trimeric structure, (ii) express the native gp41 TMD, and (iii) truncate the cytoplasmic tail. Lipids and peptides for PLVs were designed to optimally expose the C-subdomain. Prime-boost and co-immunization of rabbits with the DNA vaccines and mixed N- and C-subdomain PLVs elicited Abs targeting both Nt sites of the MPER, but no Nt activity. Similar immunizations are underway in GPs. Together, our results illustrate the importance of (i) the TMD in eliciting Abs against the C-subdomain, (ii) co-immunization, and (iii) perhaps of GPs as a model, for eliciting NtAb responses.
Lipid nanoparticles incorporated with Ebola glycoprotein for induction of humoral immunity against Ebola infection.
Authors: Bazzill Joseph D , Cooper Christopher L , Fan Yuchen , Bavari Sina , Moon James J
Published: January 2023

The recent outbreaks of Ebola virus in Africa and isolated incidents in the U.S. have increased global attention and concern for this pathogen. The highly contagious nature and severe mortality rate of Ebola infection highlight the urgent need for an effective vaccine. To cull its spread, many vaccines are currently being tested in pre-clinical and clinical settings. Here, we report the development of a vaccine nanoparticle system that can safely elicit antibody responses and protect animals against Ebola viral challenge. We incorporated Ebola glycoprotein (GP) into lipid-based nanoparticles, called Interbilayer-crosslinked multilamellar vesicles (ICMVs), that were produced by fusing and crosslinking functionalized phospholipids. The resulting nanoparticles exhibited GP loading efficiency of 45–60 % with the particle diameter of approximately 350 nm. We observed strong binding of the murine mAbs 6D8 and 13C6, recognizing GP epitopes, on GP-ICMVs, indicating surface-bound GP in its natural configuration on ICMVs. C57BL/6 mice were immunized on days 0 and 21with 10 ug GP plus 2.5 ug monophosphoryl lipid A, a Toll-like receptor 4 agonist, in either ICMV or soluble formulations. Compared with the soluble GP+MPLA control, GP-ICMVs elicited 10-fold and 3-fold higher GP-specific IgG sera titers at two weeks post prime and boost, respectively. Vaccinated mice were challenged with 1000 p.f.u. of mouse adapted Ebola virus on day 28 after vaccination. All mice immunized with ICMVs survived up to 21 days post challenge, compared with 60 % survival for mice immunized with the GP+MPLA formulation. These preliminary experiments show strong elicitation of protective immunity however more research is needed to determine real world efficacy.
Evaluating the immunostimulatory characteristics of LicKM, a novel carrier protein.
Authors: Parent Michelle A , Chabot Ashley , Chichester Jessica
Published: January 2023

The key objective of vaccination is the induction of an effective pathogen-specific immune response that leads to protection against infection and/or disease caused by that pathogen. New vaccine technologies have resulted in second-generation recombinant vaccines containing highly purified antigens with improved tolerability and safety profiles. Unfortunately, the immune responses they induce are suboptimal without the help of adjuvants. Here, utilizing a non-toxic bacterial carrier molecule from Clostridium thermocellum a modified Lichenase (LicKM) and based upon preliminary results we hypothesize, that this serves a dual purpose; as both a mechanism to carrier dominant immunogenic epitopes for presentation to antigen presenting cells during vaccination and as an adjuvant enhancing immunity. This thermostable enzyme contains a catalytic domain loop structure separating it into two regions; an N-terminal and C-terminal region. Target protein sequences are expressed as either N or C terminal fusions; and this molecule, LicKM, may contain a single or double fusion. Currently, we are investigating the mechanism by which this molecule enhances the host humoral and cell-mediated response to vaccination. At this time, we have determined that LicKM physically binds to cells associated with the immune response. Additionally, murine vaccination in the presence of LicKM enhances antibody isotype titer production and possibly enhances the cellular responses. Our goal is to understand the immunostimulatory capability of this protein for its possible use to enhance future vaccination protocols.
Increased protection and decreased lung immunopathology induced by the ID93/GLA-SE vaccine candidate following infection with Mycobacterium tuberculosis HN878.
Authors: Baldwin Susan L , Reese Valerie A. , Huang Po-wei D. , Beebe Elyse A. , Podell Brendan K. , Reed Steven G. , Coler Rhea N.
Published: January 2023

Mycobacterium tuberculosis (M. tuberculosis) HN878 causes death and extensive lung pathology in infected C57BL/6 mice. A clinically relevant isolate of M. tuberculosis increases the possibilities of assessing the efficacy of experimental TB vaccines against human TB strains in a relatively inexpensive and immunologically intact small animal model. This model will also allow for the measurement of survival in mice and hence the determination of long-lived effects of M. tuberculosis vaccines. In this study, we show that the ID93/GLA-SE tuberculosis vaccine candidate elicits protection against M. tuberculosis HN878 by reducing the bacterial burden in the lung and spleen, and prevents the extensive lung pathology that is induced by M. tuberculosis HN878 in C57BL/6 mice. Future studies utilizing this model will allow the use of specific knock-out mice on the C57BL/6 mouse background to critically examine key immunological factors that are responsible for long-lived, vaccine-induced immunity and prevention of pulmonary immunopathology induced by a virulent clinical isolate of M. tuberculosis.
Modulation of Protective Efficacy of Fracisella tularensis Immunogens by Differential Cultivation in Mueler Hinton Broth Versus Brain Heart Infusion Medium
Authors: Kumar Sudeep , Sunagar Raju , Pham Giang , Franz Brian J , Gosselin Edmund J
Published: January 2023

Francisella tularensis (Ft) can survive in a variety of habitats with a remarkable ability to adapt to changing environmental conditions. For example, Ft expresses a distinct set of antigens (Ags) in the macrophage, which are not expressed in Ft grown in vitro with Mueler Hinton Broth (MHB) medium. In contrast, Ft grown in Brain Heart Infusion (BHI) medium exhibits an antigenic profile more closely resembling that of in vivo growth conditions. In addition, studies also suggest an alteration in immunological properties of Ft when grown in BHI versus MHB medium. Thus, we investigated the protective efficacy of BHI and MHB-grown Ft, when inactivated and used as an immunogen to protect against Ft LVS challenge. Inactivated Ft (iFt) generated in MHB (iFt-MHB) was found to have superior protective efficacy as compared to that of the iFt generated in BHI (iFt-BHI). The enhanced protection generated by iFt- MHB-immunized mice was also associated with lower bacterial burden and sepsis-like conditions in the lungs and spleens of iFt-MHB versus iFt-BHI-immunized mice. Further evaluation of early immunological events revealed, in the case of iFt-MHB immunogen, superior Ag binding to Ag-presenting dendritic cells (DCs), increased MHC class II expression by DCs, and enhanced Ag presentation by DCs, as apposed to the use of iFt-BHI. Also, iFt-MHB induced increased levels of Ft-specific IgG as well as IFNg and IL17 production versus that of iFt-BHI. These studies thus demonstrate the critical importance of considering bacterial growth medium in Ft vaccine development.
Increase in Incidence of Clostridium Difficile infection in aged models and the need for novel vaccines for the prevention of disease
Authors: Kutzler Michele A. , Baliban Scott M , Bernui Mariana
Published: January 2023

Clostridium difficile associated disease (CDAD) constitutes a large majority of nosocomial diarrhea cases and is mediated by the effects of two secreted toxins, toxin A (TcdA) and toxin B (TcdB). Key toxin-neutralizing epitopes have been discovered within the carboxy-terminal receptor-binding domain (RBD) of the toxins, which has generated interest in developing the RBD as a vaccine target. The elderly mount an impaired humoral response to C. difficile toxins leading to more severe and recurrent disease; however, it has yet to be determined what underlying mechanisms lead to lower anti-toxin titers, thus higher incidence of severe and recurrent disease. While numerous platforms have been studied, very few data describe the potential of DNA vaccination against CDAD. We created highly optimized plasmids encoding the RBD from TcdA and TcdB and immunized C57BL/6 mice and rhesus macaques intramuscularly followed by in vivo electroporation. In these animal models, vaccination induced significant levels of anti-RBD antibodies within the serum and feces that could neutralize toxins in an in vitro cytotoxicity assay. Moreover, mice that were actively immunized with the DNA vaccine or given passive transfer of immunized non-human primate sera were protected from a lethal intraperitoneal challenge of purified C. difficile toxins. Finally, immunized mice were significantly protected following orogastric challenge with strains of C. difficile spores that were homologous (VPI 10463; n=10/10) and heterologous (hypervirulent UK1; n=4/8) strains to our vaccine antigens. These data demonstrate the robust immunogenicity and efficacy of a TcdA/B RBD-based DNA vaccine in preclinical models of acute toxin-associated disease.
Guinea pig infection with the intracellular pathogen Rhodococcus equi
Authors: Bordin Angela Ilha , Gressler Leticia Trevisan , Sule Preeti , Alexander Ellen Ruth , Conner Meagan , Brake Courtney , Edwards John , Cirillo Jeffrey , McMurray David , Cohen Noah
Published: January 2023

Rhodococcus equi is an opportunistic intracellular pathogen that causes pyogranulomatous pneumonia in foals and immunocompromised people, and is clinically and microbiologically similar to tuberculosis (TB). Currently, no model exists of R. equi pneumonia other than intra-bronchial infection of foals with R. equi. Because infection of foals is labor-intensive and costly, a guinea pig (GP) model of R. equi pneumonia would be of great benefit for evaluating novel approaches for control and prevention of this disease (and possibly TB). Our objective in this study was to develop a GP model of R. equi pneumonia. Groups of 6 GPs were infected with either 101, 102, 103, or 104 CFU of virulent R. equi strain using a Madison aerosol chamber, and groups of 6 GPs were infected with either 106, 107, or 108 CFU of virulent R. equi intratracheally. GPs were observed daily for clinical signs of pneumonia, and were euthanized on days 1, 3, 7, 14, or 35 post-infection (PI). Necropsy was performed and lungs were weighed, homogenized with PBS, and plated onto selective agar to determine bacterial load. No clinical signs of the disease or gross pathologic changes were observed regardless of the inoculum or mode of infection; histopathological findings of lungs are pending. Viable bacteria recovered from the lungs increased on day 3 PI, but decreased on day 7. No bacteria were recovered from animals euthanized either 2 or 5 weeks PI. Despite being highly susceptible to the agent of TB, GPs appear to be resistant to pulmonary infection with virulent R. equi at high doses. These remarkable findings suggest that this model might reveal novel mechanisms of resistance to natural infection with R. equi, which could inform the development of prophylactic or therapeutic approaches.
A Critical Combination of Bacterial Dose and Virus-Induced Alveolar Macrophage Depletion Leads to Pneumococcal Infections During Influenza
Authors: Smith Amber M
Published: January 2023

Coinfection with pneumococcus during influenza A virus infection is characterized by rapid, uncontrolled bacterial growth, a rebound in viral titers, and a robust inflammatory response. Several factors contribute to influenza-pneumococcal pathogenicity, including aberrant immune responses, tissue destruction, and pathogen strain and dose. By analyzing pathogen kinetics with a mathematical model, we predicted that bacterial establishment and growth is driven by a defect in clearance by alveolar macrophages (AMs), the first line of defense against pneumococcal invasion. This prediction was tested and confirmed in an experimental study, which suggested that these cells are depleted during influenza. Remarkably, both our model prediction and the follow-up experiments agreed that the AM population is reduced by ~85–90% 7d post-influenza infection (pii). Analyzing our coinfection model in more depth allowed us to quantify an initial dose threshold for successful bacterial infection that depends on the level of AM depletion and predict how the threshold changes throughout an influenza virus infection. To validate this prediction, we infected groups of mice with pneumococcus at 1, 3, 5, 7, 9, or 11d pii and at a dose either higher or lower than the predicted threshold. Within 4h after inoculation, bacterial loads decline for doses below the threshold, increase for doses above the threshold, and are dichotomous for doses close to the threshold. These results give insight into the conditions necessary for a secondary bacterial infection to establish during influenza infection and the probability of a successful secondary bacterial infection occurring with seasonal and pandemic influenza virus strains.
Differential Tbet, Gata3 and RORγt expression in mature and neonatal regulatory T cells during influenza virus infection
Authors: Heil Luke , Oliphant Samantha , Hollifield Melissa , Garvy Beth A
Published: January 2023

Neonates respond poorly to influenza infection and have trouble clearing the virus. In contrast to adult mice, pups display reduced cytokine production and poor migration of cells into the alveolar space during infection, resulting in an interstitial pneumonia and delayed clearance of virus. Pups also have a large population of regulatory T cells (Tregs) in their lungs compared to adults. Tregs suppress immune reactions and their abundance in pups could explain the poor response to infection. However, we found that pups that lacked Tregs failed to clear influenza infection, suggesting that pups require Tregs to clear the virus. We hypothesize that there is a functional difference between adult and pup Tregs that may explain this finding.

Tregs express the transcription factor Foxp3, and it is essential for their function, but they may also express T helper subset transcription factors such as Tbet, Gata3 or RORγt. The function of this co-expression is incompletely understood, but they may play a role in establishing functional subsets of Tregs. We investigated whether Tregs from adult mice expressed different transcription factors than Tregs from pups during infection.

We performed flow cytometry on cells from the lungs of neonatal and adult mice infected with influenza virus to examine the prevalence of Tregs that co-expressed Foxp3 with Tbet, Gata3 or RORγt. We found that Tregs in pups tended to express Gata3 more frequently than Tregs from adults. In contrast, adult Tregs were more likely to express Tbet or RORγt than pups. We are investigating whether this differential expression of transcription factors has functional consequences in modulating the response to influenza virus.
Lipoxygenase-derived lipid mediators effectively regulate influenza-induced immunopathology.
Authors: Pernet Erwan , Downey Jeffrey , Coulombe François , Allard Benoit , Martin Jim , Divangahi Maziar
Published: January 2023

Despite the worldwide application of vaccination and other antiviral interventions, influenza A virus (IAV) infection still remains a serious threat to humans. The success of IAV infection is linked to its ability to cause lower airways infections, which activates alveolar macrophages and often leads to a cytokine storm, pneumonia and respiratory failure. At this stage, uncontrolled host inflammatory response is the major cause of death. Therefore, understanding the mechanisms of immune regulation during IAV infection is critical in preventing IAV-induced immunopathology and mortality. Although cytokines have been extensively studied in immunity to IAV infection, little is known about the role of eicosanoids. We have recently demonstrated that cyclooxygenase-derived PGE2 plays a deleterious role in protection against IAV infection by inhibiting antiviral type I interferon (IFN). Herein, we aimed to investigate the potential contribution of lipoxygenase (LOX)-derived lipid mediators in IAV-immunity using the 5-LOX (Alox5−/−) deficient mice. Our data indicate that despite reduced pulmonary IAV titers, Alox5−/− mice are more susceptible to infection and exogenous administration of stable lipoxin A4 restore protection to IAV in Alox5−/− mice. This increased susceptibility was coupled with enhanced immunopathology and decreased pulmonary function. Remarkably, the production of type I IFN and IL-10 were significantly reduced in the lungs of IAV-infected Alox5−/− mice as well as IAV-infected Alox5−/− macrophages. Collectively these findings identified a protective role of the LOX-derived lipids against IAV-induced immunopathology and may pave the way for novel anti-influenza treatments using stable bioactive lipids.