bluuebunny/crossref_metadata_2025 huggingface dataset:
Combined nanoemulsion and RIG-I agonist adjuvants-activation of diverse pathways for a broad spectrum influenza vaccine.
Authors: Wong Pamela T , Goff Peter H , O’Konek Jessica J , Landers Jeffrey J , Janczak Katarzyna W , Leroueil Pascale R , Palese Peter , Baker James R
Published: January 2023

Current influenza vaccines are limited by low immunogenicity, short duration of protection, and narrow cross-strain efficacy. Broad-based cellular immune responses and mucosal immunity are key to an effective vaccine. However, most single adjuvants exhibit only some of these qualities. Here we combine two adjuvants, an oil-in-water nanoemulsion (NE), and a synthetic RNA from Sendai virus (IVT DI), each individually shown to elicit protective immunity to influenza via distinct mechanisms. Intranasal (IN) immunization with NE and influenza hemagglutinin induces mucosal and systemic antibodies, activates NF-κB via TLR signaling, and induces a balanced TH1/TH2/TH17 cellular response. NE is also an antigen carrier, facilitating cellular uptake and processing. IVT DI activates the RIG-I receptor inducing type-I interferon and TH1 immunity. We sought to determine if combining the complementary properties of NE and IVT DI improves the strength of the immune response and affords new activities not seen with the single adjuvants. Combined adjuvant was evaluated with inactivated H1N1 virus (PR8) as antigen. While IN coadmin of NE and IVT DI did not improve humoral responses in mice, cellular recall responses were much enhanced relative to either adjuvant alone. Separate sequential admin of NE and IVT DI (2 wks apart) resulted in very high humoral and mucosal antibody responses and increased cytokine production. Interestingly, the cytokine pattern for sequential vs. coadmin of NE and IVT DI was distinct. Notably, both methods elicited a TH17 response not seen with IVT DI alone. Combining complementary adjuvants may be a promising approach for generating a broader, more optimal immune response for influenza when used with diverse HA antigens.
Ex vivo evaluation of HIV envelope specific human naive B cells
Authors: DeBuysscher Blair L , Steach Holly R , Stamatatos Leonidas , Taylor Justin J.
Published: January 2023

Recent isolation of HIV-1 broadly neutralizing antibodies (bNAbs) has led to vaccine strategies focusing on inducing bNAb responses. Interestingly, not all infected individuals develop bNAbs. Current vaccine candidates regrettable, only induce antibodies that are protective against narrow subsets of strains. This could be because vaccine design is based largely on assumptions that the desired B cells are present in the host, will respond, and are protective. However, recent results suggest that a limited number of B cells respond to vaccination and may require multiple rounds of somatic hypermutation to gain neutralizing breadth. We aim to study pre-vaccination B cell repertoires in hopes of illuminating HIV-1 specific B cell responses to vaccination. Using an HIV envelope (Env)-tetramer enrichment method, we have begun to assess human Env-specific B cells in the blood of HIV-unexposed individuals. This approach allows for careful analysis of the population of naive B cells able to bind to various Env variants, allowing us to compare the germ-line antibody sequences utilized by these cells to the predicted germline sequences of bNAbs. In the course of this work, we have found naive B cells able to cross-react with structurally distinct Envs derived from different clades. Ongoing work is focused on gauging whether naive Env-cross-reactive B cells express antibodies able to broadly neutralize HIV, or can gain this ability after somatic hypermutation.
Predicting vaccine efficacy against tuberculosis using immunologic and radiologic measures in the non-human primate model
Authors: DiFazio Robert M , Maiello Pauline , Tomko Jaime , Frye Lonnie , Bigbee Carolyn , Chedrick Chelsea , Updike Cassaundra , Lin Philana , Agger Else Marie , Andersen Peter , Flynn JoAnne L
Published: January 2023

The only licensed vaccine (BCG) for tuberculosis (TB) has variable efficacy and does not protect against pulmonary TB. New vaccines are in development and in human clinical trials. However, vaccine trials are long and expensive without useful surrogate markers of protection. A problem thus emerges: what does a protective response to TB look like? This project’s goal is to identify markers of vaccine efficacy that can further be developed as new clinical endpoints. We posit that exploiting differences in vaccine outcome (protected vs. unprotected) will identify novel markers that correlate with outcome, and that serial PET/CT will predict outcome early in infection, as well as identify sites of protection. Cynomolgus macaques vaccinated with BCG and a multistage vaccine (H56 in CAF01) have more sterile thoracic lymph nodes (LN) 6 months post-Mtb challenge, compared to unvaccinated controls, indicating that LN play an important role in H56-conferred protection. BCG/H56 prevents dissemination but not establishment of infection; vaccinated animals also have fewer CFU per granuloma than controls. Total FDG avidity by PET scan in lungs (a surrogate for inflammation and disease) and individual granuloma FDG avidity of vaccinated animals are significantly lower than controls at both early and late time points. Total FDG avidity can also be used to predict vaccine efficacy, as the animals protected by BCG/H56 had less FDG in lungs than unprotected vaccinated and latent controls at early time points post challenge, and will be further developed as a surrogate marker of protection at early time points in pre-clinical trials.
Phenotypic characterization of islet antigen-specific CD4 T cell subsets in mice of varying susceptibilities to Type 1 Diabetes
Authors: Martinov Tijana , Pauken Kristen E , Spanier Justin , Heffernan James R , Osum Kevin C , Fife Brian T
Published: January 2023

Type 1 diabetes (T1D) results from lymphocyte-mediated destruction of insulin-producing pancreatic beta cells. Islet-reactive CD4 T cells are critical mediators of disease. Here, we investigated the phenotype and regulation of insulin-specific and bulk CD4 T cells in diabetes-prone (NOD) and diabetes-resistant (NOR or B6.g7) mice. We utilized dual color tetramer staining with enriment techniques to enumerate and phenotype insulin-specific CD4 T cells in NOD, NOR and B6.g7 mice. To test whether there are inherent differences in the CD4 T cell compartment between the strains, we stimulated sorted CD4 T cells in vitro with αCD3/CD28 and performed transcriptional analysis of >40 targets. Additionally, we analyzed Th1/Th2/Th9/Th17/Th22/Treg cytokine production. Both insulin-specific CD4 T cells and bulk polyclonal CD4 T cells displayed a strong Th1 skewing in NOD and not in NOR or B6.g7 mice, indicating that different disease susceptibilities could be in part due to inherent differences in CD4 T cell activation. Experiments are underway to determine the transcriptional profile of insulin-specific CD4 T cells in these strains using an unbiased approach, and identify the peripheral tolerance mechanisms underlying diabetes resistance.
The role of the orphan nuclear receptor NR4A1 in erythro-myelopoiesis.
Authors: Mumau Melanie , Golec Sophia , Vanderbeck Ashley , Lynch Elizabeth , Punt Jennifer A , Emerson Stephen
Published: January 2023

Studies of hematopoietic stem cells (HSCs) have advanced our understanding of the intracellular signals and microenvironmental interactions that influence HSC fate. Our lab is interested in the role of orphan nuclear receptor NR4A1, an immediate response gene sensitive to external stimuli, in HSC development. NR4A1 regulates the development of specific, mature hematopoietic cell lineages from both the innate and adaptive immune system including patrolling monocytes, a mature myeloid cell subset. More recently, we have shown that NR4A1 expression also identifies a subpopulation of myeloid-biased long-term HSCs in the bone marrow. Given that NR4A1 directs both immature and differentiated cell types, we investigated its role in myeloid cell maturation. Within the bone marrow and spleen progenitor cell compartments, we find that NR4A1 is exclusively expressed by myeloid progenitors and not by more restricted megakaryocyte, erythroid, or common monocyte progenitors. Nr4a1−/−mice exhibit skewed myeloid progenitor cell populations, exhibiting a 2-fold increase in CD105+CD150− erythroid progenitors and cKit+Ter119+CD71+ pro-erythroblasts in the spleen but not in the bone marrow. In vitro cultures of Nr4a1−/−splenic myeloid progenitors also show that NR4A1 restricts the frequency of erythroid cells, yet bone marrow transplant studies show similar erythroid progenitor reconstitution from both WT and Nr4a1−/− donors. Taken together, our data reveal a new role for NR4A1 as both a direct and indirect modulator of erythropoiesis.
RNA binding protein HuR posttranscriptionally regulates antigen-specific T cell proliferation and allergen induced airway inflammation
Authors: Ellis Jason S , Techasintana Patsharaporn , Glascock Jacqueline J , Ridenhour Suzanne , Atasoy Ulus
Published: January 2023

RNA binding protein HuR (ELAVL1) governs transcript stability of IL-4, IL-13 and GATA-3 mRNAs, thereby controlling Th2 differentiation. We hypothesized that HuR plays a role in antigen induced T cell activation and initiation of allergic airway inflammation. We conditionally ablated HuR in T cells prior to activation (distal lck-Cre ROSA HuRfl/fl mouse). Half of mature CD4+ T cells were HuR deficient in these mice. To determine the effects of HuR KO in T cells, mice were immunized with OVA, boosted 10 days later, and antigen specific proliferative responses were measured in vitro. Despite being fully capable of proliferation upon anti-CD3/CD28 stimulation, YFP+ (HuR KO) T cells were unable to proliferate in response to antigen while YFP− control cells had no proliferative defects. We then used the OVA challenge model of airway inflammation to ascertain effects of HuR KO in vivo. HuR KO mice had significantly less total cellular inflammation, as well as significant reductions in BAL IL-13. To determine the mechanism of suppression, we conducted in vitro activation experiments. HuR KO CD4+ T cells were sorted into YFP+ and YFP− pools and activated. YFP+ T cells had significant decreases in IL-4, IL-5, IL-13 but not IFNγ protein. Gata-3, IL-4 and IL-2Ra (CD25) transcription were significantly reduced. KO T cells have reductions in pSTAT5 signaling which is required for robust Th2 differentiation. While only 50% of CD4+ T cells lack HuR, there was total suppression of Th2 differentiation in airway inflammation. These data elucidate a critical role for HuR in regulating Th2 differentiation and allergic airway inflammation.
Aeroallergen-induced IL-33 predisposes to respiratory virus-induced asthma by dampening anti-viral immunity
Authors: Phipps Simon
Published: January 2023

Frequent viral lower respiratory infections in early-life are an independent risk factor for asthma onset. This risk, and the development of persistent asthma, is significantly greater in children who later become sensitized. Here, we sought to elucidate the pathogenic processes that underlie the synergistic interplay between allergen exposures and viral infections. Mice were inoculated with a Pneumovirus (Pneumonia virus of mice; PVM) and exposed to low-dose cockroach extract (CRE) in early- and later-life, and airway inflammation, remodeling and hyperreactivity assessed. Mice were treated with anti-IL-33 or apyrase to neutralize or block the release of IL-33. PVM infection or CRE exposure alone failed to induce disease whereas PVM/CRE co-exposure acted synergistically to induce the features of asthma. CRE exposure during virus infection in early-life induced a biphasic IL-33 response and impaired IFN-a/l production, which in turn increased epithelial viral burden, airway smooth muscle growth, and type-2 inflammation. These features were ameliorated when CRE-induced IL-33 release was blocked or neutralized, while substitution of CRE with exogenous IL-33 recapitulated the phenotype observed in PVM/CRE co-exposed mice. Mechanistically, IL-33 down-regulated viperin and IFN regulatory factor 7 gene expression, and rapidly degraded IRAK1 expression in pDC, leading to TLR7 hypo-responsiveness and impaired IFN-a production. We identify a hitherto unrecognized function of IL-33 as a potent suppressor of innate antiviral immunity, and demonstrate that IL-33 contributes significantly to the synergistic interplay between respiratory virus and allergen exposures in the onset and progression of asthma.
Oncostatin M is produced by neutrophils, and promotes epithelial barrier dysfunction in mucosal airways disease
Authors: Pothoven Kathryn L , Norton James E , Suh Lydia A , Carter Roderick G , Welch Kevin C , Shintani-Smith Stephanie , Conley David B , Grammer Leslie , Peters Anju T , Liu Mark C , Kato Atsushi , Kern Robert C , Tan Bruce K , Schleimer Robert P
Published: January 2023

We have previously shown that Oncostatin M (OSM) was elevated in nasal polyp (NP) tissue from chronic rhinosinusitis (CRS) patients, and in BAL fluid following segmental allergen challenge (SAC) in allergic asthmatics (AA). Levels of OSM detected in vivo were sufficient to impair epithelial barrier function in ex vivo cultured airway epithelium. To determine which cell type(s) was producing OSM, NP sections were stained for OSM and hematopoietic cell specific markers. OSM showed co-localization with neutrophil elastase(n=10), but not with markers for eosinophils, macrophages, T cells or B cells (n=3–5). OSM mRNA correlated with mRNA for neutrophil markers CD16 (r=.61, p<.01) and cathepsin G (r=.56, p<.01) in mRNA from CRS patients and controls. OSM also correlated with the percent neutrophils in the BAL of AA following SAC (r=.50, p<.05), but did not correlate with total cell counts or eosinophil counts. Flow cytometric analysis of cells isolated from NP (n=10) showed that 14±6% of CD45+ cells were OSM+, and 85±6% of OSM+ cells were CD16+Siglec 8-, indicating neutrophil lineage. GM-CSF and FSTL-1 have been shown to induce OSM, and both were found to be elevated in NP. Additionally, GM-CSF, alone or in combination with FSTL-1, was sufficient to induce both OSM mRNA in ex vivo cultured blood neutrophils and OSM protein in the culture supernatants. These data suggest that neutrophils are the major producers of OSM in NP, and may potentially mediate epithelial barrier dysfunction in mucosal airways disease.
A heterozygous RAB27A mutation causes delayed cytolytic granule polarization and hemophagocytic lymphohistiocytosis in 2 unrelated teenagers
Authors: Cron Randy Q , Zhang Mingce , Bracaglia Claudia , Prencipe Giusi , Bemrich-Stolz Christina J , Beukelman Timothy , Dimmitt Reed A , Chatham Winn , Zhang Kejian , Li Hao , Walter Mark R , De Benedetti Fabrizio , Grom Alexei A
Published: January 2023

Frequently fatal, primary hemophagocytic lymphohistiocytosis (HLH) occurs in infancy resulting from homozygous mutations in natural killer (NK) and CD8 T cell cytolytic pathway genes. Secondary HLH presents after infancy and may be associated with heterozygous mutations in HLH genes. We report 2 unrelated teenagers with HLH and an identical heterozygous RAB27A mutation (259 G>C). The contribution of this Rab27A missense (A87P) mutation on NK cell cytotoxicity was studied by cloning it into a lentiviral expression vector prior to introduction into the human NK-92 cell line. NK cell degranulation (CD107a expression), target cell conjugation, and K562 target cell lysis was compared between mutant and wild-type (WT) transduced NK-92 cells. Polarization of granzyme B to the immunologic synapse and interaction of mutant Rab27A (A87P) with Munc13-4 were explored by confocal microscopy and proximity ligation assay (PLA), respectively. Over-expression of the RAB27A mutation had no effect on cell conjugate formation between the NK and target cells but decreased NK cell cytolytic activity and degranulation. Moreover, the mutant Rab27A protein was predicted to disrupt binding to WT Munc13-4 by crystal structure modelling, and decreased interaction of Rab27A (A87P) with WT Munc13-4 was shown by PLA in situ and by co-immunoprecipiation in vitro. Finally, Rab27A (A87P) over-expression in NK-92 cells delayed granzyme B polarization toward the immunologic synapse, as noted by confocal microscopy, with a resulting increase in interferon-γ production, a cytokine responsible for HLH. This heterozygous RAB27A mutation blurs the genetic distinction between primary and secondary HLH by contributing to HLH via a partial dominant-negative effect.
Hepatic Stellate Cells Directly Inhibit B Cells via Programmed Death-Ligand 1
Authors: Li Yan , Lu Lina , Qian Shiguang , Fung John J , Lin Feng
Published: January 2023

We previously demonstrated that mouse hepatic stellate cells (HSCs) suppress T cells via programmed death-ligand 1 (PD-L1), but whether HSCs exert any effects on B cells, the other component of the adaptive immune system, remains unknown. In this study, we found that mouse HSCs directly inhibited B cells and that PD-L1 was also integrally involved. We found that (a) HSCs inhibited the upregulation of activation markers on activated B cells; (b) HSCs inhibited the proliferation of activated B cells and their cytokine/immunoglobulin production in vitro; and (c) pharmaceutically or genetically blocking the interaction of PD-L1 with programmed death-1 (PD-1) impaired the ability of HSCs to inhibit B cells. To test the newly discovered B-cell-inhibitory activity of HSCs in vivo, we developed a protocol of intrasplenic artery injection to directly deliver HSCs into the spleen. We found that local delivery of wild-type HSCs into the spleens of mice that had been immunized with 4-hydroxy-3-nitrophenylacetyl-Ficoll, a T-cell-independent antigen, significantly suppressed antigen-specific IgM and IgG production in vivo, whereas splenic artery delivery of PD-L1-deficient HSCs failed to do so. In conclusion, in addition to inhibiting T cells, mouse HSCs concurrently inhibit B cells via PD-L1. This direct B-cell-inhibitory activity of HSCs should contribute to the mechanism by which HSCs maintain the liver’s immune homeostasis.
Step-wise formation of germinal center B cells is completed by a requisite phase of Th cell disengagement and CD40 signaling abstinence
Authors: Zhang Ting-ting , Gonzalez David , Haberman Ann M
Published: January 2023

There are conflicting reports on the role of CD40 signaling in the initiation and maintenance of germinal centers (GC). CD40 signaling invokes the nuclear translocation of RelB, a transcription factor that promotes IRF4 expression. We examined the early stages of GC B cell differentiation without any phenotypic preconceptions by the adoptive transfer of tagged antigen-specific B cells. As expected, B cells were first observed to express intermediate levels of BCL6 within the interfollicular (IF) zone shortly after immunization. However, these GC B cell precursors had only a partial GC phenotype and co-expressed RelB and IRF4, transcription factors known to repress BCL6 transcription. This initial co-expression of mutually antagonistic transcription factors is short-lived: follicular BCL6hi GC B cells with diminished levels of RelB and IRF4 emerge shortly thereafter, coupled with the disappearance of the immediate precursors as a function of cell division. Interestingly, potentiating T cell help at the GC initiation stage blocks this transition and diverts the fate of GC immediate precursors towards plasma cells, suggesting that ongoing T cell engagement discourages GC B cell formation. Consistent with this idea, we found that reducing the amount of antigen at the time of immunization accelerates GC B cell formation. Similarly, discontinuing T-cell derived factors in an in-vitro culture system promotes the generation of BCL6hi RelB− IRF4− cells with GC B cell properties. Taken together these results suggest that CD40 signaling in B cells is necessary to generate the immediate precursors of GC B cells, but transition to the BCL6hi follicular state is completed by a period of abstinence from Th cell CD40L.
TLR9 limits mouse and human B cell responses
Authors: Sindhava Vishal , Oropallo Michael , Moody Krishna , Naradikian Martin , Zhou Lin , Higdon Lauren , Green Nathaniel , Nündel Kerstin , Stohl William , Schmidt Amanda , Kambayashi Taku , Marshak-Rothstein Ann , Cancro Michael
Published: January 2023

TLR9 can augment B cell activation, but also prevents autoimmune pathogenesis through an unknown mechanism. Herein we describe a cell-intrinsic mechanism that limits the extent of B cell activation by TLR9 ligands delivered by the B cell antigen receptor. This self-limiting response requires that TLR agonists be linked with B cell receptor ligands, as it is not observed with independent B cell receptor and TLR9 ligation. The underlying mechanism involves p38 dependent G1 cell cycle arrest that precedes intrinsic mitochondrial apoptosis, and is shared by all pre-immune murine B cell subsets and CD27− human B cells. B cells can be rescued from this death mechanism by survival or inflammatory cytokines, and preferentially switch to IgG2c producing cells both in vitro and in vivo. These findings reveal a previously unappreciated system of crosstalk that limits responses to antigens containing TLR9 ligands, and suggest that circumventing this response-limiting process can promote sustained autoantibody production.
Type I interferon and TNF signaling plays key synergistic role in programmed necrosis
Authors: Brault Michelle , Stetson Daniel B , Oberst Andrew
Published: January 2023

Pathogen detection and downstream signaling events are important for protection against disease and clearance of infection. Upon sensing pathogen-associated molecular patterns (PAMPs), cells often produce interferons (IFNs), which drive multiple transcriptional changes, allowing a cell to enter an antiviral state. Intriguingly, the IFN-α/β receptor (IFNAR) has recently been shown to be important in triggering the cell death program necroptosis (or programmed necrosis), although the mechanism is unclear. Unlike apoptosis (which relies on caspase activation), necroptosis requires the activation of receptor interacting kinase 3 (RIP3) and leads to the swelling and bursting of the cell. Death receptor or Toll-like receptor signaling can activate RIP3 in certain cellular contexts, leading to cellular destruction by necroptosis. Furthermore, RIP3-deficient mice are highly susceptible to multiple viral infections, highlighting RIP3’s antiviral role. How IFN signaling interfaces with programmed necrosis remains to be discovered. My studies have illuminated the requirement of TNFα production downstream of IFN signaling to trigger necroptosis. We believe promoting necroptosis through the production of TNFα may be an underappreciated role of the IFN-driven antiviral response.
TLR3 signaling is impaired in small airway epithelial cells following cigarette smoke exposure.
Authors: Duffney Parker , Nogales Aitor , Thatcher Thomas , Martinez-Sobrido Luis , Phipps Richard P , Sime Patricia J
Published: January 2023

Mainstream and second hand cigarette smoke exposures increase the risk of respiratory viral infections, especially in children and those with underlying lung disease. Epithelial cells are the primary targets for viral infection and recognize foreign pathogens through multiple Toll-like receptors (TLR). TLR3 recognizes double stranded RNA, a common intermediate in virus replication, and activates antiviral signaling pathways. We hypothesized that cigarette smoke exposure impairs TLR3 signaling in airway epithelial cells leading to an increase in susceptibility to viral infection. Primary human small airway epithelial cells were cultured at the air-liquid interface and exposed to whole cigarette smoke. Following cigarette smoke exposure, cells were treated with the TLR3 ligand polyinosinic-polycytidylic acid (poly I:C), a mimic of RNA viral infection. Levels of interferon gamma induced protein 10 (IP-10) and antiviral interferon bioactivity were measured in culture supernatants. TLR3 cleavage was investigated using the cathepsin inhibitor z-FA-FMK and protein lysates were analyzed by western blot. Our data shows that cigarette smoke exposure causes a decrease in the production of IP-10, as well as a decrease in the interferon bioactivity of culture supernatants. Western blot data indicates that cigarette smoke inhibits cleavage of TLR3. Inhibitor studies confirmed that cleavage of TLR3 was important for the production of IP-10 and interferons. These data show that cigarette smoke disrupts the production of anti-viral signaling molecules by disrupting cleavage and activation of TLR3, as well as provides a mechanistic explanation for increased susceptibility to viral infections in smokers.
Augmentation of salivary IgA production in healthy subjects by fucoidan-agaricus mix ingestion
Authors: Miyazaki Yoshiyuki , Nakamizo Masaji , Kawahara Kazuhiro , Takeuchi Hideaki , Tachikawa Daisuke , Yamada Koji
Published: January 2023

Fucoidan is a series of sulfated polysaccharides derived from brown algae, and have reported to have various biological activities. Previously, we demonstrated that fucoidan derived from Cladosiphon okamuranus and Undaria pinnatifida effectively augmented anti-tumor immunity in combination with Agaricus blazei mycelia extract. In this study, we evaluated the capacity of the fucoidan-agaricus mix (FAM) to enhance oral mucosal immune function. Fifteen healthy volunteers (mean age, 41.2 years old; range, 22–56 years old; 8 males, 7 females) ingested 4 capsules each containing 250 mg FAM powder every day for 12 weeks. As a result, the mean of secretory rates of salivary secretory immunoglobulin A (sIgA) tended to be increased by the FAM administration for 4 and 12 weeks as compared with the initial value. The enhanced salivary sIgA secretion was more distinctly observed in subject group whose initial values of salivary sIgA secretory rate were lower than the total average. Furthermore, the intake of FAM led to significant augmentation in the salivary sIgA secretion (by 1.3-fold) in the group of subjects under 40 years of age. On the other hand, concanavalin A-induced blastogenic response of peripheral blood mononuclear cells and serum IgA concentration was not elevated during this trial. Therefore, it was suggested that FAM stimulated functional maturation rather than expansion of B lymphocyte. The intake of FAM did not significantly affect NK cell activities. In addition, the safety of FAM consumption was confirmed because no abnormal findings were observed in general clinical tests. From the results, it was suggested that intake of FAM was useful in augmentation of oral mucosal immune defense via enhancing salivary sIgA production.
The development of WX486, a novel inhibitor of BTK, to treat rheumatoid arthritis in China
Authors: Xu Yong , Tu Ronghua , Chen Hailiang , Yue Yang , Huang Lu , Wang Xuehai , Wu Chengde , Shen Chunli , Shen Liang , Wu Dongdong , Wu Jiahu , Wang Lexi , Li Jian , Chen Shuhui
Published: January 2023

BTK inhibition has been shown to be a potential approach to treat rheumatoid arthritis. We sought to develop an oral small molecule inhibitor in China market for rheumatoid arthritis. Our medchem group discovered a novel PCC WX486 which demonstrated superior in vivo anti-arthritic activity in two animal models. We studied WX486’s metabolic pathway in different species, and evaluated its genotoxicity, MTD, 7 day acute toxicity and 28 day GLP toxicity in rats and dogs. Our studies have shown that WX486 is effective in preclinical animal models, and with high safety window suitable to be developed for rheumatoid arthritis. IND for this indication will be filed with CFDA in early 2016.
Modulation of tumor vasculature promotes tertiary lymphoid organogenesis.
Authors: Weinstein Aliyah M , Chen Lu , Storkus Walter J
Published: January 2023

The tumor vasculature has been shown to have pro-tumorigenic effects, leading to the use of anti-angiogenic drugs as therapy. However, the importance of tumor vasculature in recruiting immune cells to the tumor microenvironment (TME), leading to the formation of tertiary lymphoid structures (TLS) within the TME, has also been shown. TLS are aggregates of immune cells that develop at site of chronic inflammation and function as local sites of immune priming. The presence of TLS in the TME is a positive prognostic marker in many human tumors, suggesting that modulation of the tumor vasculature to promote increased TLS formation may serve as a novel therapeutic modality for solid tumors.

We have previously shown that intratumorally-injected dendritic cells (DC) engineered to overexpress the Type 1 transactivator Tbet (DC.Tbet) slow tumor progression in a mouse model dependent on T and NK cell recruitment to the TME. Our recent work indicates that DC.Tbet treatment of established murine sarcomas and colon carcinomas leads to an upregulation by d5 following treatment of peripheral node addressin (PNAd) on CD31+ vascular endothelial cells, marking high endothelial venules (HEV) similar to those found in lymph nodes. Dense infiltrates of T cells and DC surround these HEV; however, T cells and DC are observed proximal to tumor vasculature before PNAd upregulation is observed, indicating that while HEV mark mature TLS, PNAd does not initiate TLS formation. Instead, we observe that DC.Tbet but not unmodified DC express CCL21, CXCL13, lymphotoxin (LT) α, and LIGHT. In vivo these can recruit and activate DC, T cells, NK cells, and B cells, which can modulate the vasculature via LT signaling axes to upregulate PNAd and propagate immune cell recruitment to TLS.
Impact of microbiome manipulation on age-related inflammation and immune system dysfunction
Authors: Kodroff Lauren Hillary , Norton Elizabeth B
Published: January 2023

The aging process can be described as a disease characterized by increasing inflammation and declining immune function. Gut microbiota may impact this process, as it is altered with both disease and advanced age. We examined if gut microbiota from young animals could reduce inflammation, cellular senescence, and immune system dysfunction in older animals. To do so, we performed fecal microbiota transplants (FMT) from young (14 weeks) to groups of young, middle-aged (50 weeks), and old (100 weeks) Balb/c mice. Groups (n=5) were pre-treated with ampicillin for 3 weeks followed by oral gavage with freshly homogenized fecal material (FMT groups) or PBS and euthanized 3 weeks later. Fecal samples were collected over time for microbial analyses. Small intestines, cecum, and colons were collected for gross and microscopic analyses. To examine changes to systemic immunity, splenic and whole blood B- and T-cells were stained for surface and intracellular marker expression (i.e. activation levels, senescence) by flow cytometry.

Overall, PBS control groups showed classic age-related effects. This included signs of molecular dysfunction in oldest mice with altered levels of the NfkB inhibitor IkBz and increased senescence subsets (P16INK4a+, β-gal+). A single FMT treatment had the most dramatic effects vs. PBS controls in older mice compared to other age groups. While treatment significantly reduced IkBz and microbial-sensing TLR2 expression, FMT did not have visible impact on cellular senescence. These results reinforce a critical role for the microbiome in age-related immune decline and suggest a regimen of FMT from healthy young subjects may be a potential strategy to lower chronic inflammation and improve immune function in the elderly.
A multivariate approach to data analysis of vaccine clinical trials.
Authors: Coccia M , Nozay F , De Mot L , Deyati Avisek , Jogert E , van der Most R , van den Berg R
Published: January 2023

Despite significant progress in prevention, diagnosis and treatment, Malaria and Tuberculosis (TB) remain major health challenges. In 2014 WHO estimated that ≈438000 people died from Malaria and ≈1.5 million from TB, mainly in resource-poor countries. Vaccines represent a cost-effective and efficient method of preventing infectious diseases. The development of vaccines for TB and Malaria would significantly contribute to reducing disease burden, particularly with the emergence of drug-resistant pathogens. GSK’s Malaria vaccine, Mosquirix™ (RTS,S/AS01), received a positive opinion from European regulators for the prevention of Malaria in young children in sub-Saharan Africa. GSK’s candidate vaccine for TB (M72/AS01) induces robust TB-specific CD4 T-cell responses in humans, and it is undergoing phase IIb clinical trials. System biology approaches can support vaccines at different stages of development by identifying molecular signatures that drive responses to vaccination. Here, we describe the application of systems vaccinology to the analysis of gene expression data from clinical trials for Malaria and TB candidate vaccines. Our analysis aimed to identify early predictors of vaccine efficacy in Malaria clinical trials and to detect signatures associated with reactogenicity in the early development of a TB vaccine. To better capture the multidimensional nature of the data, we used multivariate analysis approaches such as Partial Least Squares regression. Additionally, biological interpretation of our results allowed us to pinpoint biological processes linked with response to vaccination, advancing our understanding of vaccine mode of action.
The impact of cytomegalovirus on naive lymphocytes in a novel non-human primate model.
Authors: Willis Erin L. , Eberle Richard , Wolf Roman F. , White Gary L. , McFarlane Dianne
Published: January 2023

Immunosenescence causes changes in the immune system, such as losses of naïve T-cells, which can lead to immune impairment in the elderly. Cytomegalovirus (CMV) is a common chronic herpesvirus that has been implicated in immunosenescence, although its direct effects have remained unclear. Therefore, we wanted to test the impact of CMV on immunosenescence in a recently validated baboon model. To assess the effects of CMV on the immune system over time, we determined lymphocyte populations and CMV load in age-matched baboon groups representing variable CMV exposure and shedding pressures: specific pathogen free (SPF; CMV−); small-group conventionally housed (SMConv; CMV+); and large-group conventionally housed (LGConv; CMV+) baboons (7animals/group). All baboon groups showed a decrease in naïve cells over time (P≤0.5). However at Year 1, between-group comparisons revealed 50% fewer CD8+ naïve cells in LGConv animals compared to SPF and SMConv. In Year 2, the number of naïve cells in SMConv was intermediate to that of SPF and LGConv; and by Year 3, CD8+ naïve cells were decreased in both Conv groups compared to SPF (P≤0.5). Preliminary results also revealed that a greater proportion of LGConv baboons shed CMV compared to SMConv. These data indicate that all baboons, independent of pathogen status and environmental conditions, show signs of immunosenescence at this age (9.6–11.6 years); however, LGConv baboons appear to be aging more quickly. Ongoing work includes the addition of more animals, further characterization of viral shedding and load, and infection of SPF baboons with CMV to help verify these differences. Results of this project will lead to a better understanding of the role of CMV in immunosenescence.
‘Role of IL-28A in Herpes Stromal Keretitis’.
Authors: Jaggi Ujjaldeep
Published: January 2023

Ocular infection with herpes simplex virus (HSV) can result in a chronic immune-inflammatory lesion that is a significant cause of human blindness. A key to controlling SK lesion severity is to identify cellular and molecular events responsible for tissue damage. Interferons are key cytokines in the establishment of a multifaceted antiviral response. Although all IFNs are important mediators of antiviral protection, their roles in antiviral defence vary. Interferon lambda which is a type III cytokine is capable of inducing an antiviral response both in vitro as well as in vivo. This family consists of three structurally related interferon lambda subtypes called IFN-λ (IL-29), IFN-λ2 (IL-28A) and IFN-λ3 (IL-28B). Here, we show that IFN-λ2 (IL-28A) has a prominent role in HSK. The binding of IFN-λ to their cognate receptors induces a common signaling cascade that results in the activation of STAT1 and STAT2, which promotes MX1, OAS1 and IRF7 which in turn mediate the antiviral activity of the type-III IFNs. Here we show that IL-28A appears to specifically target neutrophil function and cause a major reduction in the total number of CD4 and γδT cells and also in TH17 cells in DLN. Also, we found that the serum levels of IL-1β were reduced at the early stage of treatment. Administration of IL-28A effectively reduces the HSK lesions and can prove to be a very reliable therapeutic agent.
Human astrocytes and microglia-like cells constitutively express the novel cytosolic viral DNA sensor cGAS
Authors: Martins Angelica N , Jeffries Austin M , Marriott Ian
Published: January 2023

Evidence is accumulating that the innate immune response to HIV-1 within the central nervous system (CNS) plays a critical role in neuroAIDS disease pathology. Microglia and astrocytes are uniquely positioned to respond to CNS pathogens, and these cells produce key inflammatory, neurotoxic, and antiviral mediators following exposure to retroviruses such as HIV-1. This virus infects glia but the mechanisms by which these cells perceive intracellular replicative HIV-1 have not been defined. However, it has become apparent that innate immune sensing of nucleic acids underpins many physiological and pathological responses to viral pathogens. Cyclic GMP-AMP synthase (cGAS) has been identified as a cytosolic DNA sensor and this molecule has recently been demonstrated to have a key role in sensing replicative HIV-1 DNA intermediates and eliciting responses in human monocytic cell lines. Furthermore, cGAS and its downstream adaptor molecule STING have been found to be expressed by murine microglia and astrocytes. Here, we have characterized cGAS mRNA and protein expression by human glial cells. Microglial and astrocytic cell lines and primary human astrocytes were found to constitutively express robust mRNA and protein levels of cGAS and such expression could not be elevated further by transfection with viral nucleic acid motifs. Importantly, cGAS mRNA and protein expression was maintained despite the successful establishment of pseudotyped HIV-1 infection. As such, the identification of robust levels of cGAS in both microglia and astrocytes that are maintained even in the face of HIV-1 challenge may indicate an important mechanism by which human glial cells can respond to this pathogen.
Gp96 initiates DNA methylome remodeling in dendritic cells and facilitates interaction with T cells.
Authors: Kinner Lauren , Sedlacek Abigail L , Watkins Simon C , Binder Robert J
Published: January 2023

Tumor-derived immunogenic heat shock protein gp96 represents a promising vaccine strategy for the treatment of cancer. It is well established that immunization with gp96 elicits Th1 and CTL favoring conditions leading to rejection of the tumor from which gp96 is derived. It is unclear, however, how higher doses of gp96 leads to priming of Treg and suppression of the anti-tumor effect. Previous studies have confirmed that gp96 is endocytosed by CD91+ dendritic cells (DCs), and suggest that uptake by plasmacytoid DCs (pDCs) contributes to suppressive responses. Here, we focus on the molecular events which control gene transcription and drive this dose-dependent dichotomy in responding DCs. Our data demonstrates that stimulation of CD91+ conventional DCs (cDCs) and pDCs with gp96 results in accumulation of nuclear DNA methyltransferase (DNMT) punctae. These events correlated with methylome remodeling following gp96 immunization in vivo. Gene ontology analysis revealed that the bulk of differential methylation occurred within gene sets involved in cell-cell adhesion, signaling, and angiogenesis. Specifically, methylation and expression of target gene Nrp1 is modified in gp96-conditioned pDCs but not cDCs, suggesting that these genetic events are cell type specific. Finally, we show that gp96 stimulation facilitates increased pDC-Treg interaction in a Nrp1-dependent manner. These findings demonstrate a previously unknown role of pDCs in driving the tolerogenic response to gp96 via methylome remodeling, and can be applied to current clinical trials to improve their efficacy.
The lack of NK and mCD8+ T cells in IL-15KO mice confers resistance to septic shock
Authors: Guo Yin , Luan Liming , Bohannon Julia , Patil Naeem , Fensterheim Benjamin , Hernandez Antonio , Wang Jingbin , Sherwood Edward
Published: January 2023

Septic shock remains one of the leading causes of death in the USA. However, the immunological mechanisms of septic shock are still not well understood. Interleukin (IL)-15 is an essential mediator for natural killer (NK) and memory (m) CD8+T cell survival, as indicated by the deficits of these cells in IL-15 knockout (KO) mice. As NK and CD8+T cells are implicated in the pathogenesis of septic shock, we hypothesized that IL-15KO mice will be resistant to septic shock due to their lack of NK and mCD8+T cells. IL-15KO mice showed a significant survival advantage over wild type (WT) control mice during septic shock caused by cecal ligation and puncture (CLP) or LPS challenge. IL-15KO mice displayed less sepsis-induced hypothermia and attenuated plasma IL-6 production in our sepsis models. Treatment with low-dose IL-15 superagonist (SA) regenerated NK and mCD8+T cells in IL-15KO mice and reestablished mortality in these mice during CLP- and LPS-induced septic shock. However, if NK and CD8+T cell regeneration was prevented by anti-asialoGM1 and anti-CD8α prior to IL-15 SA treatment, IL-15 SA failed to reestablish sepsis-induced mortality in IL-15 KO mice. When given to WT mice 4 days prior to CLP or LPS challenge, M96, an IL-15 neutralizing IgG, caused 80.8% depletion of splenic NK cells and conferred protection against septic shock. However, when given shortly prior to CLP or LPS challenge, M96 did not deplete splenic NK cells and failed to confer protection against septic shock. In conclusion, IL-15 contributes to the lethality of septic mice by maintaining NK and mCD8+ T cell populations, but does not play a direct role in septic shock. Thus, NK and mCD8+ T cells may represent effective therapeutic targets for septic shock.
Identifying key regulators of CD4 T cell function in the Mycobacterium tuberculosis-infected lung using systems analysis
Authors: Delahaye Jared L , Cohen Sara , Plumlee Courtney , Plaisier Christopher , Baliga Nitin , Urdahl Kevin
Published: January 2023

Mycobacterium tuberculosis (Mtb) infection is the leading infectious killer worldwide and an effective vaccine remains elusive. Although CD4 T cells are known to be important for protection, the specific requirements for a protective CD4 response are unclear. Our lab has shown that differences in antigen exposure drive significant phenotypic variation within Mtb-specific CD4 T cells. CD4 T cells specific for the Mtb protein Ag85B, which is only expressed early during infection, remain capable of robust cytokine production. Conversely, T cells specific for ESAT-6, which is expressed for the duration of infection, exhibit blunted cytokine production. To identify regulators that govern this T cell exhaustion, we employed a systems biology approach. We performed RNA-seq on unstimulated and stimulated Ag85B and ESAT-6-specific CD4 T cells isolated from the lungs of Mtb-infected mice and used cMonkey, a biclustering algorithm, to create gene clusters based on expression patterns and identify transcription factors predicted to regulate the clusters. This analysis revealed eight transcription factors that were themselves differentially expressed between Ag85B and ESAT-6-specific CD4 T cells and also predicted to regulate clusters of differentially expressed genes. We are currently focusing our analysis on two of these transcription factors, VDR and Lyl-1. Mtb infection of WT:VDR−/− mixed bone marrow chimeras have validated the systems-based predictions by showing that VDR regulates distinct processes in Ag85B-specific compared to ESAT-6-specific CD4 T cells. Understanding the disparate regulatory pathways operant in functional vs. exhausted T cells may inform new avenues for host-directed therapy of tuberculosis.
Age-associated defects impair the optimal development of a protective immune response to Clostridium difficile in the context of infection in a novel aged murine model
Authors: Bernui Mariana Estrella , Kutzler Michele A.
Published: January 2023

Clostridium difficile infections (CDI) is a major source of mortality in the US, especially among the elderly. Protection against CDI is mediated by anti-toxin host antibodies (Ab). Although it is known that elderly mount an impaired anti-toxin response, it is yet to be determined whether their lower Ab levels are due to defective germinal center (GC) priming during acute CDI that leads to defective B cell proliferation and maturation. To test our hypothesis, we developed an antibiotic mucosal spore challenge CDI model and infected young (4mo.) and aged (>18mo.) mice with UK1 hypervirulent C. diff spores. By day 4 post-infection 100% of the aged had succumbed to infection compared to only 10% of young mice. Following a sublethal spore challenge a significant decrease of total PD1+CXCR5+ Tfh cells was detected in aged compared to young at Day 12 and 21 PI (p< 0.05 and p< 0.03, respectively). We also observed a marked decline of total and antigen-specific CD19+PNA+CXCR5+ B cells in our aged cohort (p< 0.05). We re-challenged with a lethal dose 1 month later. Only 50% of the aged vs 100% of the young survived. At Day 7 post re-challenge (D37) there was a decrease in total GC B cells (M= 7.2% SD+/−1.8 in aged vs M=15.8% SD+/−3.3 in young) and no change in Tfh cells (M= 7.5% SD+/−0.7 in old vs M=8.2% SD+/−2.1 in young). Further, the frequency of anti-toxin-specific B cells was significantly decreased in the aged (p= 0.02). The frequency of toxin-specific IgG, IgM and IgA ASC, and serum and mucosal toxin-specific Ab were markedly reduced in the aged. Our data suggests aged mice are more susceptible to CDI due to a defect in early GC formation, leading to an impaired antigen-specific Ab response. Overcoming this could prevent age-associated recurrence and severe CDI.
Cardiac dysfunction during Francisella novicida bacterial infection; an instructive sepsis model to study heart function
Authors: Rajaram Murugesan , Makara Michael , Hoang Ky , Ganesan Latha Prabha , Crouser Elliott , Gunn John , Turner Joanne , Schlesinger Larry S , Mohler Peter
Published: January 2023

Francisella novicida (Ft.n) is a gram negative intracellular pathogen that causes severe sepsis syndrome. Sepsis patients with cardiac dysfunction have significantly higher mortality. Although several pathways have been associated with myocardial damage in sepsis, the precise cause(s) remains unclear and treatment options are limited. We have shown that Ft.n infection causes rapid dissemination, inflammation and sepsis in the mouse model, however, the basis for cardiac dysfunction during Ft.n infection is unknown. In our Ft.n sepsis model we have observed ECG abnormalities such as R’ wave formation, prolonged QRS and PR intervals, left ventricular wall thickening and increased cardiac output, similar to sepsis patients. Notably, we detected microlesions in the myocardium, and also seen in nosocomial Streptococcus infection and in sepsis patients. We have also determined that Ft.n-mediated microlesions are due to cardiomyocyte apoptosis due to increased infiltration of leukocytes and expression of inflammatory mediators (TNFα, IL-1β, IL-8 and SOD2). Finally we have found increased expression of miR155 and rapid degradation of HSF-1 in the heart suggesting that these processes regulate myocaridal inflammation and apoptosis. In conclusion, we have developed and used an Ft.n infection model to study the pathogenesis of heart dysfunction during sepsis.