bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of anti-CTLA-4 and anti-PD-1 on memory T-cell differentiation and resistance to tumor relapse
Authors: Mok Stephen , Duffy Colm R. , Allison James P.
Published: January 2023

Clinical studies have shown that αPD-1 therapy mediates a greater response rate compared to αCTLA-4 according to RECIST criteria. However, it may not be as durable as αCTLA-4. 25% of patients who initially responded to αPD-1 have tumor relapse within 24 months. In contrast, the 10-year survival rate of patients treated with αCTLA-4 is at least ~25% suggesting a durable response. The goal of this study is to understand the relationship between memory T-cell formation in tumor models mediated by checkpoint antibodies and tumor relapse.

Our results have shown that αCTLA-4 could induce a superior memory antitumor response during tumor rechallenge on mice compared to that treated with αPD-1 after 80 days. We also showed that this memory antitumor response was antigen-specific. These antigen-specific memory T-cells in mice that were treated with αCTLA-4 had better proliferative potential with cytokine producing function and more CD62L+CD44+ memory T-cells were converted back to CD62L-CD44+ effectors during rechallenge. We found that αCTLA-4 and αPD-1 facilitated the development of CD4 and CD8 memory T-cells respectively. We also demonstrated that the memory antitumor activity mediated by the combination of αCTLA-4 and αPD-1 overlapped with αCTLA-4 alone suggesting the effect of the combined treatment is dominated by αCTLA-4.
Bifidobacterium can mitigate intestinal immunopathology in the context of immune checkpoint blockade
Authors: Wang Feng , Sun Shan , Yin Qian , Chen Liang , Davis Mark M.
Published: January 2023

Antibodies that attenuate immune tolerance have been used to effectively treat cancer, but they can also trigger severe autoimmunity. To investigate this, we combined anti-CTLA-4 treatment with a standard colitis model to give mice a more severe form of the disease. Pre-treatment with an antibiotic, Vancomycin provoked an even more severe, largely fatal form, suggesting that a gram-positive component of the microbiota had a mitigating effect. We then found that a commonly used probiotic, Bifidobacterium could largely rescue the mice from immunopathology without an apparent effect on anti-tumor immunity, and this effect may be dependent on regulatory T cells.
Autophagy-deficient breast cancer shows early escape from dormancy and recurrence following chemotherapy
Authors: Aqbi Hussein F. , Smith Timothy , Butler Savannah , Idowu Michael , Payne Kyle K. , Manjili Masoud H.
Published: January 2023

Breast cancer patients who respond to cancer therapies often end up with a distant recurrence of the disease. It is not clear why people with the same type of breast cancer respond to treatments differently, some escape from dormancy and relapse earlier than others. In addition, some tumor clones respond to immunotherapy while others do not. Here, we investigated the role of autophagy in expediting or delaying recurrence of neu overexpressing mammary carcinoma (MMC) following adriamycin (ADR) chemotherapy, and in affecting response to immunotherapy. We took two strategies, including a transient blockade of autophagy with chloroquine (CQ) which transiently blocks fusion of autophagosome and lysosome, and a stable knockdown of autophagy, which inhibits the formation of autophagosome in MMC. We found that while CQ prolonged tumor dormancy, a stable knockdown of autophagy resulted in early escape from dormancy and recurrence. Interestingly, MMC with a stable knockdown of autophagy contained an increased frequency of ADR-induced polyploidy cells which rendered MMC resistant to immunotherapy. On the other hand, a transient blockade of autophagy did not affect the sensitivity of MMC to immunotherapy. Our observations suggest that while chemotherapy-induced autophagy may facilitate tumor relapse, cell intrinsic autophagy delays tumor relapse by inhibiting the formation of polyploidy tumor cells. Our findings are consistent with other reports showing that autophagy-deficient cells contain polyploid nuclei because of increased levels of reactive oxygen species. Our results also suggest that responsiveness of breast cancer patients to neoadjuvant therapies might be determined by tumor intrinsic autophagy.
Chemotherapeutic drugs induce expression of CS1 (CD319) in multiple myeloma cells resulting in enhanced NK cell mediated killing.
Authors: Malaer Joseph D. , Mathew Porunelloor A.
Published: January 2023

Elotuzumab (Empliciti), a humanized monoclonal antibody against CS1, has been demonstrated an effective drug for multiple myeloma (MM). During clinical trials, elotuzumab in combination with chemotherapeutic drugs was more effective than antibody treatment alone. However, the underlying mechanism for this is not yet known. We hypothesize that chemotherapeutic drugs induce the surface expression of CS1 on MM cells making them more susceptible to natural killer (NK) mediated killing and therefore increasing the effectiveness of elotuzumab. NCI-H929 (multiple myeloma cell line) cells were incubated with lenalidomide and anti-CS1 mAb, or doxorubicin and dexamethasone in order to determine if surface and mRNA expression of CS1 changes. Surface expression of CS1 was determined via flow cytometry, while mRNA expression was determined by RT-qPCR. Our results indicate that treatment with anti-CS1 mAb in combination with lenalidomide or treatment with doxorubicin and dexamethasone increased the cell surface expression of CS1; however, these drugs reduced the expression of CS1 mRNA. Additionally, chemotherapeutic drug incubation increased NK cell effector function against NCI-H929 cells. These results, in conjunction with previous clinical trials, may indicate a novel mechanism in which chemotherapeutic drugs increase surface CS1 expression and therefore increase the effectiveness of anti-CS1 mAb therapy.
Approaches to reverse interleukin-10 mediated immunosuppression in chronic lymphocytic leukemia
Authors: Rivas Jacqueline R. , Alhakeem Sara S. , Eckenrode Joseph , Zhang Yinan , Collard James P. , Rangnekar Vivek M. , Muthusamy Natarajan , Thorson Jon S. , Leggas Markos , Bondada Subbarao
Published: January 2023

B cell Chronic Lymphocytic Leukemia (CLL), the most common leukemia in the western world, is characterized by an accumulation of abnormal B cells in the blood and lymphoid organs. Broad immunosuppression is a serious complication for these patients, with infections and secondary cancers recognized as the leading causes of morbidity and mortality. Although the immune suppression is in part due to clonotypic CLL B cells crowding immune organs, immune responses may also be suppressed by the production of soluble mediators. We and others found that CLL cells produce interleukin-10 (IL-10), a cytokine that suppresses T cell effector function. We hypothesized that this IL-10 may reduce anti-tumor T cell responses. In our studies we use human cells as well as the Eμ-Tcl1 mouse model for U-CLL, in which the oncogene Tcl1 is expressed under the immunoglobulin VH promoter and μ-enhancer. Previously, we found that IL-10R−/− T cells control CLL growth better than WT T cells in the Eμ-Tcl1 adoptive transfer model. We also saw that CLL IL-10 production is dependent on BCR signaling and activation of the transcription factor Sp-1. IL-10 production can be blocked by mithramycin (MTM), an Sp-1 inhibitor. However, MTM is not used routinely as an anti-cancer agent due to its short half-life and associated toxicities, so we prepared and tested novel analogues of MTM to reduce these effects. One of these analogues, MTM23, shows promise as it has similar potency to MTM, but does not interfere with T cell gamma-interferon production or viability. This approach is novel as current therapies for CLL do not target IL-10, or address the immunosuppression seen in CLL patients. IL-10 blockade could therefore be a strategy for harnessing anti-tumor T cells to treat CLL in humans.
A clonotype-independent pre-TCR complex and its downstream signals can support transformation of thymic T cells.
Authors: Hathcock Karen S. , An Joseph BH. , Hodes Richard J.
Published: January 2023

The kinase ataxia telangiectasia mutated (ATM) communicates between molecules that sense DNA damage in the form of double stranded breaks (DSB) and effector mechanisms that maintain genomic integrity. Persistent DSBs can lead to transformation and ATM-deficiency is associated with increased lymphoid malignancies in both humans and mice. ATM-deficient mice exclusively develop thymic T cell lymphomas with high penetrance. We explored the requirement for specific components of the TCR and its signaling pathway for the induction of these thymic lymphomas. We generated multiple strains of ATM-deficient mice in which mature T cell are absent by deletion of CD3ɛ, an invariant component of the pre-TCR complex; by deletion of RAG or TCRβ, in which recombining TCR variable components are deleted; or by deletion of LAT, an important component of pre-TCR and TCR signaling. These strains all showed a similar and severe developmental arrest at thymic DN3. Despite this arrest, mice deficient in RAG or TCRβ, exclusively developed thymic T cell lymphomas. In contrast, mice deficient in CD3ɛ or LAT, developed no or few T cell tumors, respectively, but developed B cell lymphomas. These findings suggest the importance of a clonotype-independent TCR complex and its downstream signals in transformation of T cells. Experiments are in progress to ask if survival of ATMKO T cell lymphomas after transformation is similarly dependent on CD3ɛ and LAT signaling.
Multiple defects in NK cells of surgically resectable pancreatic cancer patients can be reversed by ex-vivo stimulation
Authors: Jeon Seunghyun , Lim Seon Ah , Kim Jungwon , Shin Min Hwa , Kwon Joonha , Lee Kyung-Mi
Published: January 2023

Tumor infiltrating lymphocytes (TILs) are found in advanced pancreatic ductal adenocarcinoma (PDAC) patients and shown to correlate with overall survival (OS) rate. Although majority of TILs consisted of CD8+/CD4+T cells, the presence of NK cells and their role in the pathogenesis of PDAC remains elusive. We performed comprehensive analyses of TIL and PBMC to explore the role of NK cells in the newly developed PDAC patients. Multiple defects in cell migration and effector function were observed in NK cells of patients’ peripheral blood. Extremely low frequencies of NK cells (<0.5%) were found in TILs of PDAC patients which was attributable not to low expression of tumor chemokines, but rather lack of the CXCR2 chemokine receptor. Forced expression of CXCR2 on patients’ NK cells rendered them capable of trafficking PDAC tumors. Furthermore, patients’ NK cells exhibited impaired cell-mediated killing against autologous PDAC tumor cells and failed to proliferate within hypoxic tumor microenvironment. Importantly, these defects can be overcome by ex-vivo activation of NK cells from PDAC patients. When the proliferative capacity of NK cells in vitro was used to stratify patients on the basis of cell expansion, a clear difference in DFS and OS was observed. Patients whose NK cells proliferated <250 fold experienced significantly lower DFS and OS than those expanded ≥250 fold. Multiple defects are observed in NK cells of PDAC patients. Ex-vivo activation of NK cells restores tumor reactivity and provides a therapeutic modality while their fold expansion can be a potentially significant prognostic indicator of OS and DFS in PDAC patients.
Notch-mediated crosstalk of antitumor CD8+T and NK cells
Authors: Thomas Portia , Uzhachenko Roman , Shanker Anil
Published: January 2023

A large majority of immune-oncology research efforts focus on T-cell immunotherapy. However, limited clinical efficacy has been achieved with adoptive T-cell effectors alone. In addition, research has shown that the sole use of adoptive T-cells leads to the development of tumor escape variants and “occult cancer”. Conversely, tumor-infiltrating natural killer (NK) cells have been shown to cause elimination of these escape variants. These observations indicate that the interaction between the innate and adaptive immune effectors is indispensable for effective tumor immunity. Notch is a major signaling system important for communication between neighboring cells. Our previous studies show that it plays a crucial role in the differentiation and antitumor function of CD8+T cells. However, its role in NK cell activation and antitumor function is not clear. Thus, we analyzed Notch receptor-ligand system on NK cells and in the context of their crosstalk with CD8+ T cells. Our data indicate that inhibiting Notch signaling in NK cells decreases NK cell activation. In addition, Notch intracellular domain (NICD) expression is increased upon NK activation, suggesting that the Notch signaling pathway may be involved in NK activation. Our data also show that activated CD8+T cells activate naïve NK cells through direct physical contact. Transmembrane notch receptor expression is decreased during this interaction suggesting that the Notch receptors may be involved in CD8+T cell – NK cell crosstalk. Experiments are underway to further understand the relevance of Notch signaling pathway in innate-adaptive immune crosstalk. The findings will help develop novel strategies for adoptive cell immunotherapies, specifically against solid tumors.
Memory CD4+ T cells guide memory B cell adaptability to drifting influenza vaccination
Authors: Gage Emily , Van Hoeven Neal , Coler Rhea
Published: January 2023

Influenza A annually infects 5–10% of the world’s human population resulting in an estimated one million deaths. Unlike other infectious agents, where either infection or vaccination against the disease confers long-term immunity, influenza causes annual epidemics and re-infects previously exposed individuals as a result of antigenic drift in the surface glycoprotein hemagglutinin (HA). Due to antigenic drift, the human immune system is simultaneously exposed to both novel and conserved parts of the influenza virus through vaccination and/or infection multiple times throughout life. Preexisting immunity by infection or vaccination influences subsequent neutralizing antibody responses, the correlate of protection for influenza. To understand how preexisting immunity augments future responses to drifted influenza immunization, we established mouse models, sequentially infecting mice with a H1N1 strain and then immunizing with a second drifted H1N1 strain. Mice previously infected with A/CA have increased neutralizing antibody response (nAb) to A/PR upon immunization with A/PR HA. This increase in nAbs was dependent on CD4+ T cell and memory B cell, correlating with CD4+ T cell reactivity conserved across both influenza HA’s. We also found increased germinal center PR8-specific B and T follicular helper cells post vaccination in the draining lymph node. These results suggest conserved MHC Class II restricted epitopes within HA are critical for B cells to adapt to drifting influenza and could be leveraged to boost out subsequent neutralizing antibody responses. Understanding the mechanism by which preexisting immune responses shape future responses is essential to optimize and leverage vaccination strategies.
Pattern recognition receptor agonists enhance protection elicited by a single, low dose protein vaccine in an immunization route-specific manner
Authors: Lampe Anna T. , Hain Jacey L. , Farris Eric J. , Pannier Angela K. , Brown Deborah M.
Published: January 2023

Despite advances to increase vaccine efficacy, influenza A virus (IAV) remains a significant health risk. Current vaccines against IAV rely on the generation of strain specific antibodies that fail to provide protection against heterosubtypic IAV. Vaccination may be improved through the use of vaccine adjuvants to amplify antigen presenting cell (APC) activation and enhance T and B cell priming. Combined adjuvants that trigger multiple pattern recognition receptors (PRR) are a promising strategy to better mimic live infection. In this study, the efficacy of combining CpG and MPLA, which activate TLR9 and TLR4 respectively, as vaccine adjuvants was examined. It was observed that single, low dose hemagglutinin (HA) protein vaccines containing combination CpG and MPLA delivered intramuscularly (i.m.) or intranasally (i.n.) elicit superior protection against lethal challenge compared to vaccines containing single agonists, as indicated by lower mortality and significantly lower morbidity. Early systemic cytokine responses are being investigated to elucidate the mechanism behind this improved vaccine efficacy. However, it was observed that i.m. administration of combination agonist vaccines induced higher IgG2c antibody responses compared to single agonist vaccines, while minimal levels of antibodies were observed in mice vaccinated i.n. In addition, T cell analysis following lethal infection indicated that i.n. vaccination induces distinct activation profiles compared to i.m. Taken together a comparison of the antibody and T cell profiles of mice vaccinated i.m. and i.n with combined TLR9 and TLR4 agonists suggests that route of vaccination can dictate immune response and may modify effective correlates of protection.
Antibody levels to Influenza A are greater in adults with Trisomy 21 but are less functional compared to disomic adults.
Authors: King Scott , Kusher Samantha , Jacobs Brennan , James Stephanie F.
Published: January 2023

Adults with Trisomy 21 (Down syndrome; DS) are particularly susceptible to respiratory infections which is a primary cause of death in adults with DS. Due to medical advances there has been a rapid increase in the lifespan of people with DS, creating a critical gap in our understanding of immunity in these adults. Although no studies to date have identified the pathogens responsible for the majority of respiratory infections observed in adults with DS, data from previous influenza epidemics suggest that adults with DS have significantly higher rates of morbidity and mortality as a result of influenza infection. Their high rates of pneumonia and subsequent death may result from an impaired ability of B cells to generate antibodies of sufficient quantity and function to control infections. Data evaluating the effectiveness of immunizations in adult individuals with DS is inconclusive, although it has been established that those with DS have decreased B cell counts. Herein, we hypothesized that adults with DS will have an attenuated antibody response following immunization compared to typical adults. Antibody concentrations to influenza A were evaluated using a standard ELISA, and antibody titer was measured using a hemagglutination inhibition assay. Our results show adults with DS have significantly increased IgG antibody concentration to Influenza A compared to age matched controls (p=0.005). However, when evaluated by a hemagglutination inhibition assay (to A/HongKong) adults with DS had lower levels of inhibition compared to typical adults (p=0.02). These data indicate that although adults with DS can develop an antibody response to influenza, this response may not be as protective against disease as in typical, disomic individuals.
Finding the balance between protective and pathologic vaccine induced immunity using LCMV specific T lymphocytes
Authors: Luna Evelyn
Published: January 2023

Vaccination remains one of the most effective strategies for preventing human diseases caused by infectious pathogens, yet developing an efficacious vaccine for persistent viral infections such as HIV remains a difficult aim of much research. Vaccines expressing pathogen T cell epitopes has been a widely employed strategy to try and improve the efficacy of vaccines against chronic viral infections. However, selectively engaging T cells has the potential to increase rather than prevent pathology during subsequent infection, consequently shifting a mild disease to a lethal one. Thus, determining the immune parameters that lead to protection versus those that mediate pathology is critical for developing a safe and effective vaccine against persistent viral infections. Using lymphocytic choriomeningitis virus (LCMV) we investigated how the precursor frequency of CD8 T cells affects the balance of protection vs. pathology during chronic viral infection. We found that the balance is based largely on the number of T cells responding to the pathogen. Intermediate numbers of responding CD8 T cells resulted in maximum pathology. We have also observed that increasing vaccine-induced CD4 T cell responses, unlike CD8 T cells, never leads to protection, but instead high host mortality. We have now began to study the interaction of CD8 and CD4 T cells during infection to determine what effect CD4 T cells have in protective versus pathologic vaccine-induced immunity and what exactly mediates such responses.
An multivalent vaccine elicits protective Th17 response via activation of C-type lectin receptor- and Card9-mediated signal against pulmonary Coccidioides posadasii infection
Authors: Hung Chiung-Yu , Zhang Hao , Campuzano Althea , Ostroff Gary , Yu Jieh Juen
Published: January 2023

Coccidioidomycosis is a potentially life-threatening fungal infection that is responsible for ~150,000 infections annually in the United States. Development of a vaccine against Coccidioides infection is urgent. We have created a multivalent coccidioidal antigen (rCpa1) that contains promiscuous human T cell epitopes. The rCpa1 was formulated with 5 types of experimental adjuvants including yeast cell-wall particles and CpG adjuvant. The rCpa1 formulated with glucan chitin particles (GCPs) derived from a non-pathogenic yeast demonstrated the best protective efficacy for susceptible C57BL/6 and HLA-DR4 transgenic mice against this disease compared to the other 4 tested vaccines. This is the first report of a subunit vaccine that can provide a comparable degree of protection to a live, attenuated vaccine against coccidioidomycosis. The GCP-rCpa1 vaccine elicited a robust and durable Th17 memory response. Depletion of Th17 cells significantly reduced the protective efficacy of this vaccine, indicating that Th17 cells play an indispensable role. Dectin-1 and Dectin-2 molecules can bind to the GCP-rCpa1 vaccine particles. Macrophages derived from mice lacking expression of Dectin-1, Dectin-2 and downstream immune adapter CARD9 secreted reduced amounts of IL-1β and IL-6 upon stimulation with GCP-rCpa1 that are essential for Th17 development. Furthermore, vaccinated Dectin-1−/−, Dectin-2−/− and CARD9 −/− mice recruited significantly reduced amounts of Th17 cells to the lungs compared to C57BL/6 mice and succumbed to coccidioidomycosis. Taken together, protection against Coccidioides provided by GCP-rCpa1 vaccine requires Dectin-1- and Dectin-2-CARD9-mediated signaling to activate Th17 immune response.
Memory T cell responses in survivors of the 2013–2016 West African Ebola outbreak
Authors: Sullivan Brian Martin , Sakabe Saori , Hartnett Jessica , Goba Augustine , Momoh Mambu , Sandi John Demby , Schieffelin John S. , de la Torre Juan Carlos , Grant Donald S. , Garry Robert F. , Oldstone Michael B.
Published: January 2023

Primary infection with Ebola virus protects against subsequent challenge, thus an optimal vaccine should generate immune responses similar to those found in disease survivors. To this end, we characterized CD8 and CD4 T cell responses in Ebola survivors to uncover the immunogenicity of seven of eight Ebola virus proteins. T cell responses of 33 Sierra Leonean Ebola survivors were examined. Nearly all individuals studied harbored nucleoprotein (NP)-specific CD8 T cell responses. Moreover, NP-specific T cells responded more strongly and were more numerous in 65% of NP responders compared to responses to other EBOV proteins in the same individual. Conversely, we found glycoprotein (GP) CD8+ T cell responses in only 37% of EBOV responders. GP responses were also weaker and less numerous than other responses in the same individual. Specificity of the CD4+ T cell responses were more evenly distributed. Altogether, our data indicate that NP is among the most immunogenic EBOV proteins while only VP30 was less immunogenic than GP in our cohort of Sierra Leonean survivors. An optimal vaccine designed to elicit both humoral and cell mediated immune responses in a broad human population should contain both NP and the GP.
Dynamics of adaptive natural killer cells in longitudinal analysis of CMV vaccine recipients
Authors: Gyurova Ivayla E. , Schlums Heinrich , Bernstein David , Bryceson Yenan , Waggoner Stephen N.
Published: January 2023

Classical vaccine efforts focusing on the induction of B- and T-cell memory have proven ineffective in the protection against pathogens such as cytomegalovirus (CMV). Thus, CMV remains a significant public health threat. There is clear evidence that patients lacking natural killer (NK) cells suffer recurrent, severe infections with multiple herpesviruses, including CMV, highlighting the importance of NK cells in controlling this pathogen. In addition, several studies describe the expansion and persistence of phenotypically and functionally distinct murine (Ly49H+) and human (NKG2C+, FcRγneg, EAT-2neg, SYKneg) NK-cell subsets with putatively enhanced antiviral effector function upon CMV infection. Therefore, one potentially pioneering approach to creating an efficacious CMV vaccine would include the induction of such NK cell adaptations. To test this, we used longitudinal PBMC samples collected during a vaccine trial with HCMV gB in MF59 adjuvant administered to CMV negative adolescent females. We observed several patterns of transient and sustained elevations in the frequency of FcRγneg, EAT-2neg, and SYKneg subsets at various time points despite absence of detectable CMV infection in any trial participants. Surprisingly, these patterns were apparent in participants regardless of administration of vaccine or placebo. In contrast to previous cross-sectional studies, we present evidence that these CMV-reactive adaptive NK cells exhibit continuous oscillations in the blood of CMV negative individuals, suggesting that they may react to unknown environmental or inflammatory cues.
Rig-I-Like Receptors (RLRs) regulate humoral immunity to West Nile Virus (WNV) infection.
Authors: Schenten Dominik , O’Ketch Marvin
Published: January 2023

Rig-I-like Receptors (RLRs) comprise a family of pattern recognition receptors that recognizes microbial RNA in the cytosol. RLR activation induces an anti-viral state in infected cells and leads to the release of proinflammatory cytokines and interferons. RLRs are therefore important mediators of innate immunity to many viral infections. However, the role of RLRs in the regulation of adaptive immunity is still poorly understood. Infection of MAVS-deficient mice, the essential signaling adaptor for RLRs, with West Nile Virus (WNV) results in a defective adaptive immune response. While this finding suggests a role for RLRs in the regulation of adaptive immunity to WNV, it is difficult to interpret due to a high WNV viremia in the absence of a MAVS-dependent innate immune response. In order to overcome this caveat, we have infected MAVS-deficient mice with a mutant form of WNV that is unable to assemble infectious virions and is therefore limited to a single round of infection. Here, we show that MAVS-deficient mice display increased numbers of antigen-specific CD4 T cells as well as an enlarged germinal center (GC) B cell compartment. Importantly, these mice fail to produce an effective neutralizing antibody response to WNV despite normal levels of WNV-specific antibodies. Together, these findings suggest that RLR-dependent signals regulate humoral immunity to WNV.
Endothelial dysfunction in the pathogenesis of acute Lassa Fever
Authors: Horton Lucy Elise , Sullivan Brian , Garry Robert F. , Grant Donald S. , Oldstone Michael B.
Published: January 2023

Lassa fever (LF) is an acute disease characterized by enhanced vascular permeability resulting from infection with Lassa virus (LV), a non-lytic, Old World arenavirus. Animal models of arenavirus-induced vascular damage suggest that cytotoxic CD8+ T cells serve a critical role in the destruction of vascular endothelium by destroying LV infected endothelial cells as well as impairing platelet aggregation. Using plasma samples from acute LF patients, we sought to identify predictive biomarkers associated with endothelial integrity and platelet aggregation in fatal versus non-fatal LF cases by testing platelet function and measuring hemostasis markers with ELISA. We discovered a significant increase in plasminogen activation inhibitor-1 (PAI-1), endothelial protein C receptor (EPCR) and thrombomodulin in LF Ag+ cases compared to LF IgM+ and febrile non-LF controls. In addition, platelets from uninfected controls displayed decreased aggregation when mixed with plasma from 28 of 33 acute LF patients compared to plasma from uninfected controls. Our results demonstrate that severe LF (determined by Ag positivity) is associated with platelet dysfunction and abnormal regulation of coagulation, and suggest that similar to the LCMV Clone 13 model, immune-mediated endothelial activation leading to loss of barrier function, impaired hemostasis, and altered thrombogenicity may be an important mechanism in the pathogenesis of acute LF. Studies are underway to relate these biomarkers to the state of T cell activation in this cohort of patients.
West-Nile virus replicon particles infect 293T cells expressing DC-SIGNR
Authors: Kunkel Hannah L. , Budimlic Emin , Keilty John , Schmitz Madilyn R. , Madigan Amanda , Crowson Nicole , Leonard Nathan L. , Schwarzinger Victoria R. , Meyer Alyssa J. , Soderlund Mary , Martinez Osvaldo
Published: January 2023

West-Nile virus (WNV) is an arbovirus usually transmitted to humans via a mosquito vector. Less than one percent of cases result in a serious neurological illness such as meningitis or encephalitis Studies have shown that WNV infection efficiency is enhanced by expression of DC-SIGNR on target cells, which normally do not express DC-SIGNR. To investigate WNV tropism, we established 293T kidney epithelial cell lines that stably express vector or DC-SIGNR. We demonstrated uniformly high levels of DC-SIGNR expression in selected cells. Virus replicon particles (VRPs), replication-incompetent viral particles containing necessary structural proteins for infection and a viral plasmid including a GFP reporter are used to safely and conveniently study viral entry. Entry assays using WNV (NY99) VRPs as well as a variant of WNV (NY99) which contains the beta-lactamase enzyme show significant entry into DC-SIGNR expressing cell lines, but not in controls that do not express DC-SIGNR. Future studies will focus on discovering which regions of the DC-SIGNR surface glycoprotein are required for WNV entry.
Cytomegalovirus blockade of TRAIL-mediated type 1 ILC defenses regulates viral persistence
Authors: Picarda Gaelle Genevieve Alice , McDonald Bryan , Dodard Garvin , Verma Shilpi , Ghosh Raima , Morabiti Rachid , Thiault Nicolas , Brossay Laurent , Griffith Thomas S. , Benedict Chris A.
Published: January 2023

Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) regulates cellular apoptosis and inflammation, and growing evidence indicates a key role in antiviral defense. Cytomegalovirus (CMV, a b-herpesvirus) establishes a lifelong persistent/latent infection that depends upon viral subversion of host innate and adaptive defenses. We have shown that both human and mouse CMV inhibit signaling by the TRAIL death receptors (DR) through the use of specific viral proteins. MCMV m166 blocks TRAIL-DR expression and promotes early viral replication by evading TRAIL-mediated natural killer (NK) cell defenses. After one week of systemic replication, MCMV spreads to the salivary glands (SG), the major mucosal organ responsible for horizontal transmission of the virus. We show that a MCMVΔm166 mutant replicates at lower absolute levels in the SG, but, surprisingly, persists for much longer times. Recent evidence suggests a role of type 1 Innate Lymphoid cells (ILC1) in control/clearance of MCMV. We investigated the role of the 2 main ILC1 populations (conventional and tissue resident (tr) NK cells) in controlling MCMV SG persistence. Depleting ILC1 prior to infection, or infection of TRAIL−/− mice, restored MCMVΔm166 SG replication to normal levels. In addition, SG trNK cells display a unique phenotype and express high levels of TRAIL during MCMV infection when compared to their conventional counterparts. Together our data reveal a key role for m166 inhibition of TRAIL-DR expression in promoting viral persistence by blocking ILC1 effector functions mediated by TRAIL, highlighting this TNF-family cytokine as an important factor regulating innate defenses in mucosal tissues.
Structural characterization of broadly neutralizing alphavirus antibodies
Authors: Basore Katherine , Earnest James T. , Diamond Michael S. , Fremont Daved H.
Published: January 2023

Mayaro is a mosquito-borne single-stranded positive RNA alphavirus endemic to tropical regions of South America. It is closely related to other arthritogenic alphaviruses, such as Chikungunya, Semliki Forest, Ross River, and Sindbis viruses. Although there are currently no available vaccines or therapies for alphaviruses, previous studies demonstrate that cross-protection between different alphaviruses could be mediated by antibodies that map to conserved epitopes. For example, a class of monoclonal antibodies against Chikungunya were recently shown to neutralize other alphaviruses by blocking viral entry and egress, all with epitopes on the surface E2 glycoprotein. A panel of antibodies generated against Mayaro infection and boosted with recombinate E2 protein were also found to be cross-neutralizing against other alphaviruses. Currently, epitope mapping and structural studies are being performed on these antibodies in order to determine the vital residues for binding, as well as to determine the mechanism of cross-neutralization.
A nuclear receptor involved in cholesterol metabolism regulates herpesvirus latency and reactivation
Authors: Reese Tiffany A. , Tao Lili , Lowe Alexandria , Wang Guoxun , Zarek Christina M.
Published: January 2023

Herpesviruses are a component of our virome, and infect virtually everyone. They establish chronic latent infection that is punctuated with periods of viral reactivation. Reactivation is thought to drive herpesvirus pathologies, including lymphomas and tumors. However, the signals that promote herpesvirus reactivation are poorly understood. We previously showed that parasite co-infection could induce herpesvirus reactivation through the induction of the cytokines IL-4 and IL-13, and activation of Stat6. Stat6 directly binds to a viral promoter and induces virus reactivation, suggesting that the virus has evolved to sense immune signals from the host to modulate reactivation. We sought to identify other factors that induce herpesvirus reactivation through the modulation of inflammation. We found that peroxisome proliferator-activated receptor (PPAR)-a, a ligand-activated nuclear receptor, is upregulated during g-herpesvirus infection. PPAR-a is activated by endogenous fatty acids to regulate cholesterol metabolism. PPARs also regulate inflammation, although these functions are less well understood. We found that agonist activation of PPAR-a induces herpesvirus reactivation. PPAR-a activation dampens type I interferon signaling, by inhibiting genes that modulate the DNA sensing pathway. These data suggest that in addition to evolving cytokine-sensitive promoters, herpesviruses induce cellular machinery that sense fatty acids as a means of regulating viral reactivation.
MDA5, a Janus-faced dsRNA sensor in Coxsackievirus-accelerated autoimmune diabetes
Authors: Tse Hubert Ming , Burg Ashley R. , Chen Yi-Guang
Published: January 2023

While microbial infections can trigger autoimmune diabetes, ultimately, innate immune activation and the synthesis of free radicals, proinflammatory cytokines, and Type I interferons contribute to pancreatic β-cell destruction in Type 1 diabetes (T1D). We previously demonstrated that Coxsackievirus B3 (CB3) infection of Non-Obese Diabetic (NOD) mice can accelerate T1D, partly due to the induction of oxidative stress and antiviral signaling pathways including melanoma differentiation-associated protein 5 (MDA5). Single-nucleotide polymorphisms within Ifih1, the gene encoding for MDA5, is associated with multiple autoimmune diseases including T1D, but the molecular mechanism contributing to innate immune dysregulation is not known. Since T1D is a chronic proinflammatory autoimmune disease involving MDA5, we hypothesized that aberrant MDA5 expression and signaling can contribute to diabetogenic viral-accelerated T1D. NOD mice mutated in Ifih1 that resulted in loss of MDA5 expression (NOD.Ifih1_m1) or an in-frame truncation in the helicase domain of MDA5 (NOD.Ifih1_m4) exhibited a delay in spontaneous T1D. Interestingly, CB3-infected NOD and NOD.Ifih1_m1 mice displayed accelerated virus-induced T1D, but NOD.Ifih1_m4 mice were significantly (p < 0.001) delayed. CB3-infected NOD.Ifih1_m4 mice were efficient in pancreatic viral clearance and exhibited decreases in circulating Type I interferons in contrast to NOD and NOD.Ifih1_m1 mice. The protective Ifih1_m4 mutation may establish an acute level of Type I interferons sufficient to inhibit CB3 replication without mediating bystander activation of autoreactive T cells in autoimmune diabetes.
Salmonella infection induces autoantibody production in MyD88-deficient host: Identification of candidate bacterial proteins with homology to murine antigens
Authors: al-Ramadi Basel K. , Issac Jincy M. , Mohamed Yassir A. , Bashir Ghada , Al-Sbiei Ashraf , Conca Walter , Khan Taj A. , Iqbal Asif , Cabral-Marques Otavio , Fernandez-Cabezudo Maria J.
Published: January 2023

Persistent bacterial infections have been causally linked to the development of autoimmune diseases, but the precise mechanisms underlying this remain largely unknown. Of note, we demonstrated that systemic infection by attenuated Salmonella enterica serovar Typhimurium induces a breakdown in B cell tolerance in MyD88-deficient mice, as evidenced by the production of autoantibodies to nuclear and cytoplasmic antigens and immune complex deposition in the kidneys. The splenic niche of infected MyD88−/− mice revealed increased counts of activated myeloid cell subpopulations as well as CD4+ and CD8+ T cells. Moreover, this was associated with increased Th21 cell differentiation and the appearance of CD4+ T cells co-expressing IFN-g/IL-4 and IFN-g/IL-10. Importantly, infection with other bacteria (e.g. Acinetobacter baumanii, Streptococcus agalactiae or Escherichia coli) was unable to trigger the autoimmune phenomenon. We reasoned that the autoimmune response could be triggered by a process of molecular mimicry between mouse and Salmonella proteins. Bioinformatic analysis was used to identify potential Salmonella antigens with a high degree of homology to mouse proteins. This analysis revealed several candidate bacterial proteins with high homology to murine immune system proteins. The identified bacterial proteins were all uniquely expressed by Salmonella but not by the other three bacterial species that were incapable of inducing the autoimmune response in MyD88−/− mice. Therefore, our data suggest a mechanism for pathogen-specific induction of autoantibody production in the absence of MyD88 signaling pathway and provide important clues concerning the genotype-environmental factor correlations.
Transcription factor T-bet drives the pathogenesis of TH17-mediated neuroinflammation
Authors: Loo Tiffany Taylor , Shinoda Akane , Lazarevic Vanja
Published: January 2023

Emergingdata demonstrate that exposure to pro-inflammatory cytokines IL-12 and IL-23 convertsnon-pathogenic IL-17A-producing CD4+ TH17 cells intopathogenic effectors, though the molecular mechanism remains undefined. Here, we show that T-bet, a transcription factor normally associated with IFN-γ-producing TH1 cell differentiation, is an IL-23-induced critical regulator of pathogenic TH17 cells. Using the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS), we report that IL-23-treated T-bet-deficient TH17 cells were not pathogenic and failed to induce neuroinflammation. We found that T-bet expression in TH17 cells was required to disrupt the blood-brain barrier (BBB) upon migration to the CNS. Following in vivo reactivation, T-bet-sufficient TH17 cells co-expressed IFN-γ and GM-CSF cytokines, upregulated expression of myelo monocytic-specific chemokines (Ccl1, Ccl3, Ccl4 and Ccl5), and attracted neutrophils, inflammatory monocytes and dendritic cells to the CNS. Conversely, T-bet-deficient TH17 cells failed to cross the BBB and invade CNS tissue, showing decreased cytokine and chemokine expression and reduced myelomonocyte recruitment. Interestingly, pharmacological BBB disruption via adjuvant administration bypassed the requirement for T-bet expression in TH17 cells, resulting in disease induction by T-bet-deficient TH17 cells. Collectively, these data demonstrate that autoreactive TH17 cells permeabilize the blood-brain barrier in a T-bet-dependent manner, resulting in a cascade of damaging inflammatory infiltrates.
How big is a big effect size? A nonparametric measure of effect size when modeling cytokines
Authors: Qeadan Fares , Dwivedi Sunita , Erdei Esther , Beswick Ellen
Published: January 2023

Cytokines play a major role in driving immune responses in cancer and other diseases. There is a critical need to understand how they interact and regulate one another. In this study, we examine new approaches to understand meaningful networks between cytokines and their relevance to human disease.

Effect size (ES) is a way of assessing the difference between two groups with respect to a continuous measure such as cytokines. Its use, relative to p-values, gives easier understanding to whether a difference is actually clinically important or not. Results of a study can be statistically significant but still be too small to be of any practical value, or statistically insignificant but still be too large to not be of any practical value; especially when multiple comparisons adjustments are applied. As opposed to p-values, ES is independent of sample size, and thus has recently attracted the attention of researchers.

When modeling cytokines, we recommend the use of the A measure of stochastic superiority A = [#Y1 > Y2) + 0.5#(Y1=Y2)]/n1n2, due to Delaney and Vargha (2002), which is appropriate for the nonparametric Wilcoxon Test. The ES of interest from this measure is A/1 – A which gives the odds that an individual in one group will score higher than an individual in the other group. Our simulation indicated that odds= 1.253, 1.763, and 2.500 are equivalent to Cohen’s d = 0.2 (small), 0.5 (medium), and 0.8 (large).

We applied this measure on a panel of 26 cytokines identified in 29 subjects (15 with Metastasis and 14 without Metastasis) with gastric cancer, and revealed 11 cytokines with large ES, five of which with non-significant Sidak adjusted p-values: (from largest to smallest) G-CSF, IL-6, IL-8, MCP-1, IL-10, TNF-a, GM-CSF, MIP-1a, IL-1b, MIP-1b, and VEGF.
BAFF antagonists designed by computer-aided drug design inhibited the interaction of BAFF with its receptors
Authors: Sun Jian , Zhang Xiaojuan , Zhen Zhao , Hao Xiafei
Published: January 2023

BAFF is a critical factor for B cell maturation and survival, and its overexpression is involved in pathogenesis of autoimmune diseases. As blockade of BAFF activity significantly improves the symptoms of autoimmune diseases such as SLE and RA, BAFF is a promising therapeutic target for treating these diseases. Currently, most BAFF antagonists are antibodies against BAFF or soluble BAFF receptor-IgG1 Fc fusion proteins. Small molecules such as peptides as antagonists to block the biological activity of cytokines have been investigated intensively. Based on the crystal structures of the protein–protein complexes, protein mimics can be designed as antagonists with computer-aided drug design (CADD) to block the cytokine-receptor interactions. Since the crystal structures of BAFF and its receptors are known, we designed BAFF antagonist peptides with CADD. The peptides could inhibit the binding of BAFF with its receptors BCMA, TACI and BR3. In addition, BAFF-binding peptides originated from CADD were compared to the peptide selected from the phage display library. Both kinds of peptides could inhibit the binding of BAFF to its receptors, a peptide from the phage display library showed two-fold inhibition effect on BAFF binding to its receptors. 3-copy vs 1-copy of peptides were also studied and the results indicated that increasing the copy numbers of peptides in peptide-Fc fusion proteins did not significantly enhance the bioactivities of the peptides. Therefore, BAFF antagonist peptides designed by CADD could bind to BAFF in a dose-dependent manner and inhibit BAFF biological activity significantly, which might be potential agents for autoimmune disease therapies.
Production and characterization of monoclonal antibodies specific for chicken interleukin-4
Authors: CHAUDHARI ATUL , Kim Woohyun , Lillehoj Hyun H.
Published: January 2023

Interleukin-4 is an important Th2 type cytokine in mammals which regulates growth and differentiation of B Lymphocytes. On the mammalian counterparts, the function of IL-4 orthologue in chickens (chIL-4) is less understood. Although the source of chIL-4 is not well established, it has been shown to have an integral role in mediating Th2 type immune responses in chickens. Despite its significant role, no monoclonal antibodies (mAbs) against chIL-4 have been developed till date. Therefore, in the present study we used recombinant yeast-expressed chIL-4 as an immunogen to develop mAbs against chIL-4 to establish the basis for understanding the function of chIL-4 in regulating Th2 mediated immune responses in chickens. We developed two mAbs against chIL-4 and characterized their specificity by western blot, enzyme linked immunosorbent assay (ELISA) and intracellular staining (ICS). These antibodies displayed specific binding with the recombinant chl-IL-4 as well as endogenous chIL-4 by western blot, ELISA and ICS. Further, these antibodies neutralized the inhibitory effect of chIL-4 on lipopolysaccharide (LPS)-induced nitric oxide (NO) production and expression of inducible nitric oxide synthase (iNOS) in avian macrophage-like HD11 cells, thus confirming that these antibodies can block the chIL-4 bioactivity. In conclusion, the mAbs developed in the present study can be useful for understanding the role of chIL-4 in the basic concept of Th1 versus Th2 T cell subsets in chickens.