bluuebunny/crossref_metadata_2025 huggingface dataset:
IRF-8 levels constrain aberrant myelopoiesis and improve immune control in the metastatic tumor microenvironment (TUM4P.901)
Authors: Netherby Colleen , Waight Jeremy , Miller Austin , Bogner Paul , Liu Kebin , Abrams Scott
Published: January 2023

Abnormal myelopoiesis is common in patients with diverse malignancies, including breast cancer. The myeloid compartment is essential for the induction of host immunity, thus alterations in myelopoiesis may help explain the failure to control neoplastic disease. Therefore, understanding the molecular basis of myeloid dysfunction may improve the efficacy of cancer immunotherapies. Given that interferon regulatory factor-8 (IRF-8) is crucial for myeloid differentiation, we hypothesized that changes in this master regulator underlie a novel mechanism for the altered myelopoiesis and myeloproliferative phenotypes observed in cancer, such as the accumulation of myeloid-derived suppressor cells (MDSC). We recently showed that tumors downregulate IRF-8 expression in myeloid progenitors in a cytokine-driven STAT3 or STAT5-dependent manner, resulting in MDSC generation. This suggested that IRF-8 expression functions as a ‘rheostat’ which directs the fate of the tumor-induced myeloid response. To test this, we examined the impact of modulating IRF-8 levels in two mouse mammary tumor models of lung metastasis using a newly designed genetic gain-of-function approach. In both models, we observed a significant reduction in lung metastases and the frequency of MDSC in IRF-8 transgenic mice compared to wild-type controls. Altogether, our results reveal an unrecognized role for IRF-8 in the host response against metastasis, which may lead to new therapeutic targets in cancer immunotherapy.
Genomic ablation of HMGB1 in dendritic cells mitigates their capacity to both promote carcinogenesis and effectively confer anti-tumor immunity as vaccines (TUM4P.919)
Authors: Vernon Philip , Liang Xiaoyan , Wang Wenqian , Schapiro Nicole , Lowe Devin , Billiar Timothy , Zeh Herbert , Lotze Michael
Published: January 2023

HMGB1 is the prototypical damage associated molecular pattern (DAMP) molecule. It plays a critical role in many aspects of DC biology. To study the role of DC HMGB1 in tumor immunity, DC-specific HMGB1-/- mice (DCH) were generated. In both subcutaneous and liver metastasis models of pancreatic and colorectal cancer respectively, DCH mice exhibited inhibited tumor growth. DC HMGB1 is also critical for carcinogenesis; bone marrow from DCH mice was transplanted into mice expressing pancreas-specific oncogenic Kras (KC). KC mice given DCH bone marrow exhibited lower frequencies of malignant pancreatic and less pro-tumor inflammatory infiltrate. Genomic loss of HMGB1 also negatively impacted other aspects of DC functionality. Mice inoculated subcutaneously with pancreatic and melanoma cancer cell lines and then vaccinated with bone-marrow derived KO DCs pulsed with tumor antigen were unable to achieve therapeutic protection in both models. Although both WT and KO DCs were able to stimulate allogeneic lymphocyte proliferation, KO DCs skewed responses away from Th1/Th2 phenotypes, and favored Treg/Th17 polarization. KO DCs also expressed lower levels of both RAGE and TLR4, suggesting these signaling pathways may be impaired in DCH mice, thus accounting for our observations.
Negative impact of myeloid-derived suppressor cells on CD8 effector T cell trafficking within the tumor microenvironment (TUM4P.924)
Authors: Ku Amy , Muhitch Jason , Abrams Scott , Evans Sharon
Published: January 2023

The success of immunotherapy and, unexpectedly, chemotherapy and radiation hinges on cytotoxic T cells gaining access to tumor targets. These observations have prompted interest in developing strategies to improve T cell trafficking in tumors. Here, we report that the ability of tumor vessels to respond to IL-6-dependent preconditioning regimens that boost CD8 effector T cell homing is temporally and inversely related to the expansion of myeloid-derived suppressor cells (MDSC) within the tumor microenvironment. Using real-time intravital imaging and immunofluorescence histology, IL-6 therapies were shown to convert vessels from T cell-low to -high recruitment sites in murine tumors with minimal MDSC infiltration (i.e., CT26 colorectal, B16 melanoma, EMT6 mammary tumors). This conversion requires induction of the ICAM-1 trafficking molecule on tumor vessels. Conversely, mammary (4T1, AT-3 and PyMT-MMTV) and pancreatic (Pan02) tumors with high MDSC burdens were refractory to IL-6 therapies, but became responsive after acute MDSC depletion. To further investigate contributions of MDSC to poor trafficking, IL-6-responsive tumors were admixed at a ratio of 2:1 with MDSC from tumor-bearing mice. Sustained elevation of MDSC in admixed tumors resulted in failure to support increased T cell trafficking in response to IL-6-dependent therapy. Taken together, these findings identify a novel role for MDSC in immune evasion by limiting T cell trafficking at tumor vascular loci.
Neutrophil recruitment into premalignant lesions differentially influences tumor growth versus progression. (TUM4P.928)
Authors: Blaisdell Adam , Daikoku Takiko , Tay Chin Siean , Dey Sudhansu , Erlebacher Adrian
Published: January 2023

Neutrophils possess a diverse arsenal of enzymatic and chemical weapons that can inflict damage upon pathogens and host cells alike. Most tumors harbor neutrophils even from the earliest stages of progression (premalignancy), wherein these weapons could, in theory, either kill a tumor cell or enhance its malignant potential via mutagenesis. However, the interaction between neutrophils and premalignant tumor cells has not been well characterized. To this end, we utilized a mouse model of endometrial carcinoma with defined stages of premalignancy and malignancy that faithfully represent human endometrial cancer progression. We observed robust neutrophil infiltration within premalignant lesions, accompanied by elevated expression of G-CSF, Il1a, Il1b and CXCR2 ligands. Blockade of G-CSF signaling substantially reduced tumor-associated neutrophil numbers and had profound and contrasting effects on tumor growth versus progression. Neutrophil-depleted lesions were larger and contained an increased number of proliferating tumor cells, but concomitantly showed less dysplasia and a reduced amount of DNA damage within tumor cells. Additionally, neutrophil recruitment into premalignant lesions relied heavily upon IL-1 signaling to induce the expression of G-CSF and CXCR2 ligands. These data establish a double-edged sword paradigm for neutrophils in the premalignant tumor microenvironment as agents of both tumor cell death and malignant potentiation.
Produce immunogenic monocyte derived-dendritic cells by Toxoplasma gondii proteins (TUM4P.904)
Authors: Boghozian Roobina , Mirzaei Reza , Hadjati Jamshid
Published: January 2023

Background: DCs based immunotherapy is considered as a promising approach for activating immune responses against tumor antigens. It has been believed that TLR ligands derived from microorganisms are more potent in activating DCs. In this study, the effects of Toxoplasma gondii (T.gondii) protein and DNA components on human DCs maturation were evaluated. Method: T.gondii Tachyzoites were obtained by intraperitoneal passage in mice. Protein and DNA components were extracted by relevant kits. Subsequently, protein lysate was fractionated by increasing amounts of ammonium sulfate and after that by ion-exchange chromatography. Protein fractions and DNA components were added to human monocyte derived DCs. After 20h, phenotype markers (CD80, CD83, CD86 and HLA-DR) on DCs were determined by flow cytometry. Also, IL-12p70 and IL-10 concentration in DCs supernatants were evaluated. Ultimately, the effects of different fractions on phagocytosis activity of DCs were compared. Results: We found that T.gondii protein components are more potent in activating DCs than DNA components. Moreover, there is a considerable difference in phenotype markers and phagocytic activity between DCs matured by different protein fractions. Also, the ability of some protein fractions is higher in activating DCs to produce IL-12p70 and lower IL-10. Conclusion: The protein fractions with high ability can be used to produce potent DCs for instructing immune responses against tumor antigens.
Effect of blocking different TGF-β isoforms on tumor immunity (TUM5P.937)
Authors: Terabe Masaki , Robertson Faith , Mizra Amer , Berzofsky Jay
Published: January 2023

TGF-β is a pleiotropic cytokine with three isoforms that have significantly different physiological functions. This cytokine has potent immunosuppressive activity and has been shown to play critical roles in tumor development. TGF-β1 is also necessary for the development of T cell subsets as well as MDSC. We have previously reported that blockade of TGF-β by a monoclonal antibody 1D11, which neutralizes all three isoforms of TGF-β, improves immunosurveillance and vaccine efficacy in mouse tumor models. However, the role of individual isoforms of TGF-β is unknown. In this study, we examined the role of different TGF-β isoforms in tumor immunity by using anti-TGF-β with unique specificities in two tumor models, a lung metastasis of the BALB/c CT26 colon carcinoma and a subcutaneous TC1 lung epithelial cell tumor in B6. In the CT26 model, significant reduction of tumor burden was seen through the dual blockade of TGF-β1 and 2, or inhibition of all three TGF-β isoforms. In the TC1 model, the mice were treated with a peptide vaccine when the tumor size reached at least 5mm in diameter with/without anti-TGF-β. Although the vaccine alone induced significant regression of tumors, both types of anti-TGF-β facilitated the vaccine efficacy. These results suggest that blockade of only TGF-β1 and 2 is sufficient to facilitate natural immunosurveillance or enhance vaccine-induced tumor immunity. Currently we are investigating the effect of TGF-β isoform blockade on T cell subsets.
Antibodies targeting the Rhizopus oryzae invasin, CotH3, enhance neutrophil killing (VAC6P.952)
Authors: Lin Lin , Gebremariam Teclegiorgis , Ibrahim Ashraf
Published: January 2023

Mucormycosis is a lethal fungal infection that afflicts the immunocompromised host such as those suffering from hyperglycemia, ketoacidosis, other forms of acidosis, neutropenia, and those receiving corticosteroids. Rhizopus spp. are the most common pathogens isolated from patients with mucormycosis. Previously, we identified R. oryzae CotH3 as a surface protein which mediates fungal invasion of host cells during mucormycosis. We found rabbit polyclonal anti-CotH3 antibodies to significantly block host cell invasion by R. oryzae and protect mice from R. oryzae infection. However, it is unclear if CotH3 antibodies enhance phagocyte killing of the fungus. Here, we used flow cytometry and mouse neutrophil killing assays to determine the binding and opsonophagocytic killing capacities of varying concentrations of anti-CotH3 antibodies against R. oryzae, respectively. Anti-CotH3 antibodies showed high-affinity binding (>99%) to R. oryzae even when low concentration of the antibodies were used (e.g. 3mcg dose). CotH3 antibodies also widely bound to other Mucorales known to cause mucormycosis including Mucor circinelloides, Lichtheimia, Rhizomucor, and Cunninghamella. Neutrophil kill assay has shown Anti-CotH3 antibodies to dramatically enhance mouse neutrophil killing by > 2-fold increase vs. control with preimmune antibodies (P<0.04). Our studies warrant the further development of monoclonal antibodies targeting CotH3 protein as a novel therapy against mucormycosis.
Approaches for sampling human antibodies for repertoire analysis and reagent development (VAC6P.953)
Authors: Chen Zhifeng , Kannan Harilakshmi , Derstine Lauren , Qiu Ping , Prokop Michael , Touch Sinoeun , Su Bin , Dubey Sheri , Wang I-Ming , ter Meulen Jan , DiStefano Daniel , Vora Kalpit
Published: January 2023

Purpose: Majority of vaccines rely on Human humoral response for protection. Sampling antibody response is useful for understanding protection correlates of vaccine and isolating reagent antibodies for lifecycle management. We used 3 approaches delineated below to isolate Rubella neutralizing antibodies from plasmablast B-cells separated from human subjects recently vaccinated with MMRII vaccine. Direct B cell cloning: Plasmablast B cells isolated from donors at day 7 after vaccination were used directly as templates in the RT-PCR amplification of antibody genes. Antigen-specific Fabs were isolated following molecular cloning and ELISA screening. Deep Sequencing: RNA extracted from plasmablast B cells was used as template in RT-PCR amplification. The PCR products of antibody genes were subjected to deep sequencing, retrieved antibody VH, VL and VK sequences were rank ordered based on the frequency and, top antibody sequences in each category were expressed as Fabs and tested. Immune Library Construction/Phage Display: Antibody heavy and light chain variable regions from plasmablast B cells were cloned into bacterial phage display vectors to make immune libraries, specific Fabs were identified following bio-panning. Result: All together 25 specific antibodies against Rubella were isolated through the above approaches, the best Fab ELISA binder had an EC50 of 3.5ng/mL (0.07nM). Two of the Fabs demonstrated neutralizing activity with EC50’s of 1µg/mL (20nM) and 4µg/mL (80nM).
Long-lived bone marrow plasma cells are inhibited by protein A in Staphylococcus aureus infection. (VAC6P.955)
Authors: Keener Amanda , Thurlow Lance , Richardson Anthony , Vilen Barbara
Published: January 2023

Long-lived plasma cells are now recognized as major contributors to protection against bacterial infection through their constitutive secretion of antibody. Staphylococcus aureus is a growing concern due to expansion of antibiotic resistance and causes recurring infections without inducing long-lived immunity. Little is known about the nature and longevity of the adaptive response to S. aureus. Protein A on the surface of S. aureus promotes virulence and inflammation which can alter B cell function and hinder long-lived plasma cell development. Although similar B cell responses were induced by WT and protein A-deficient infection and secondary challenge, long-term antigen-specific antibody levels waned after challenge with WT S. aureus, but were maintained after challenge with the protein A-deficient mutant. This suggests that long-lived B cell immunity is disrupted by protein A. We developed an ELISpot assay to detect S. aureus-specific antibody secreting cells and found that they accumulated in the bone marrow niche after protein A-deficient, but not WT challenge. The absence of bone marrow plasma cells after WT challenge was not a result of defects in germinal center or memory B cell formation. Our work indicates that protein A interferes with migration and/or survival of long-lived bone marrow plasma cells after S. aureus infection. It also provides insight into the requirements for successful development of long-lived immunity to bacterial pathogens.
NLRP3-dependent IL-1β production by FcγR-mediated Syk kinase signaling confers protective immunity in Francisella tularensis vaccination (VAC7P.966)
Authors: Periasamy Sivakumar , Duffy Ellen , Harton Jonathan
Published: January 2023

Pneumonic tularemia is a fatal disease caused by Francisella tularensis (Ft), a category ‘A’ biothreat agent with no FDA approved vaccine. Previous studies have shown that inactivated-Ft (iFt) complexed with anti-Ft LPS mAb (iFt-mAb) elicits protective immunity against lethal Ft challenge. Other vaccine studies have also shown that IL-1β and NLRP3 play a role in adaptive immunity. The AIM2 inflammasome is critical for innate immunity to Ft, but NLRP3 appears to be unnecessary. We investigated the role of inflammsomes in immune responses to iFt-mAb and found that NLRP3 is essential in this vaccine strategy. An iFt-mAb vaccination completely failed to protect IL-1RKO and NLRP3KO mice against lethal Ft challenge compared to Wt mice. Importantly, Ft-specific antibodies were significantly lower in both KO mice than Wt mice after vaccination and challenge. NLRP3-deficient mBMDM or THP-1 cells produced significantly less IL-1β compared to WT cells after iFt-mAb treatment. To probe the mechanism by which iFt-mAb activates NLRP3 and IL-1β, the role of FcγR and TLR2 signals were explored. Internalization of iFt-mAb and activation of NLRP3 were FcγR-dependent, while NLRP3-dependent IL-1β production required TLR2 and FcγR-signaling through Syk kinase. Our results highlight the role of IL-1β and NLRP3 in successful iFt-mAb vaccination that elicits better antibody and cellular responses. Specifically, FcγR signaling is required for NLRP3 inflammasome activation in this vaccine strategy.
Predicting vaccine efficacy against tuberculosis using immunologic and radiologic markers in the non-human primate model (VAC7P.974)
Authors: DiFazio Robert , Gideon Hannah , Coleman Teresa , Maiello Pauline , Frye Lonnie , Tomko Jaime , O'Malley Melanie , Johnston Paul , Fillmore Daniel , Rodgers Mark , Bigbee Carolyn , Chedrick Chelsea , Lin Philana , Flynn JoAnne
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 immunologic markers that correlate with outcome, and serial PET/CT will predict outcome early in infection. Vaccine-induced protection could work at any point in early infection: movement of bacilli from airway to parenchyma; formation of primary lesions; migration of bacteria to LN; and development of secondary lesions. In both our rhesus and cynomolgus macaque models of infection, prime-boost vaccination with BCG followed by viral-expressed or protein antigens protects against TB disease better than BCG alone or no vaccine. However, granuloma formation is not prevented and the vaccines do not reduce initial granuloma numbers. However, total PET signal, as a surrogate for inflammation and disease, in lungs of vaccinated animals is significantly lower at early time points. Future work will develop the predictive markers to elucidate early immune mechanisms that confer protection in the lungs.
Pentameric gH complex is the primary target of neutralizing antibodies generated in cytomegalovirus vaccine immunized rabbit (VAC7P.982)
Authors: Tang Aimin , Freed Daniel , Tang Qi , Li Fengsheng , Meng Weixu , Wang Dai , An Zhiqiang , Fu Tong-Ming
Published: January 2023

Human cytomegalovirus (HCMV) belongs to the herpesvirus family and can cause severe diseases in immune-compromised individuals and birth defects in babies from in utero infection. Understanding the protective immunity is the pre-requisite in developing an effective vaccine against HCMV. Humoral immunity can provide protection against HCMV infection and that such a protection comes from antibodies primarily targeting antigens of pentameric gH complex. To support a vaccine concept based on the pentameric gH complex, we generated and characterized 45 monoclonal antibodies (mAb) from a rabbit immunized with an experimental vaccine virus in which the expression of pentameric gH complex restored. From 45 mAbs, we identified 25 mAbs with neutralizing activities. We demonstrated that binding affinity to virions is not correlated with neutralizing activity and potent neutralizing mAbs (Elite neutralizer) preferentially bind to the pentameric gH complex. Using phylogenetic analysis, we further demonstrated that these antibodies are grouped into 26 distinct B cell lineages and the neutralizing and binding mAbs were largely segregated into different groups. In addition, neutralizing antibodies have longer complementarity-determining region (CDR) 3 than those non-neutralizing mAbs. In conclusion, the pentameric gH complex is the primary target of neutralizing antibodies by vaccination with a HCMV vaccine.
A polyantigenic genotype 1a/1b consensus hepatitis C virus DNA vaccine induces broadly reactive HCV-specific cellular immune responses in both mice and non-human primates (VAC7P.985)
Authors: Latimer Brian , Toporovski Roberta , Yan Jian , Pankhong Panyupa , Khan Amir , Sardesai Niranjan , Jacobson Jeffrey , Weiner David , Kutzler Michele
Published: January 2023

Chronic HCV is a surreptitious disease that can lead to cirrhosis and cancer decades following infection. Each year, 3-4 million individuals are infected. However, there are currently no vaccines available to prevent chronic HCV infection. From patients who acutely resolve (A.R.) infection, a strong and broadly reactive cytotoxic T lymphocyte (CTL) response is associated with viral clearance. With in vivo electroporation (E.P.), DNA vaccines induce strong CTL responses, making them well suited for an HCV vaccine. Our vaccine strategy maximizes recipients' opportunity to induce a broad T cell response with genotype 1a/1b consensus sequences of multiple HCV antigens. Immunogenicity of the HCV vaccine was assessed in mice and non-human primates (NHP). Animals were immunized three times, two weeks apart for mice and six weeks apart for NHP as an intramuscular injection followed by E.P. In mice, interferon-γ (IFNγ) responses spanned all HCV antigens and with total values exceeding 6,000 spot-forming units per million mononuclear cells (SFU) within both the spleen and liver by IFNγ ELIspot. Similarly in NHP, immune responses up to 4,680 SFU per million PBMCs were detectable by IFNγ ELISpot. Flow cytometry revealed HCV-specific CD4+ and CD8+ T cell responses by cytokine staining and significant frequencies of cytolytic CTL compared to baseline. Here, we have demonstrated the magnitude and breadth of HCV-specific immune responses elicited by an HCV DNA vaccine in mice and NHP.
A systems biology approach revealed distinct host responses between ALVAC-SIV and NYVAC-SIV vaccines during an evaluation of immunogenicity in rhesus macaques (VAC7P.992)
Authors: Yang Yajie , Gordon Shari , Doster Melvin , Liyanage Namal , Chang Jean , Williams Chris , Agy Michael , Mclain Randy , Agricola Brian , Law Lynn , Franchini Genoveffa , Palermo Robert , Katze Michael
Published: January 2023

Though only modestly effective, RV144 is the first HIV vaccine clinical trial to show efficacy against heterosexual transmission. It tested ALVAC-HIV (a canarypox vector expressing HIV Gag, Pro and gp120) in the prime-boost combination with AIDSVAX B/E (a HIV bivalent gp120). To understand the immune correlates and inform future trials, we repeated this regimen in a rhesus model and contrasted two different pox vectors. Our study consisted of 24 animals in four groups: two initially receiving ALVAC-SIV or the vaccinia vector NYVAC-SIV (both expressing SIV Gag, Pol and Env) as the prime, with later addition of SIV gp120 and alum boosts; and two control groups receiving empty ALVAC or NYVAC and alum only. Microarray analysis of whole blood samples showed that as early as 6 hours after the initial immunization, the gene expression features of the ALVAC-SIV group were distinct from the animals receiving NYVAC-SIV, with innate immunity pathways activated in an ALVAC-SIV-specific manner. By 24 hours post vaccination, ALVAC-SIV induced more differentially expressed (DE) genes than the other three groups relative to the pre-immunization baseline, and NYVAC-SIV and NYVAC control induced similar numbers of DE genes. We will relate these very early host responses to the differences in immunogenicity observed over the course of immunization. The impact of these early events on the later outcomes will be determined once the protective efficacies of the two vaccines are determined.
Identification of Chlamydia trachomatis proteins recognized by T cells from high-risk women resistant to reinfection and/or protected from ascending infection. (VAC7P.961)
Authors: Darville Toni , Zheng Xiaojing , O'Connell Catherine , Picard Michele , Wiesenfeld Harold , Hillier Sharon , Flechtner Jessica
Published: January 2023

Murine and human data indicate IFNγ-producing Chlamydia-responsive CD4 T cells are key to protection from genital tract disease, and chlamydial proteins have been identified that induce protection in mice. A preventative vaccine against C. trachomatis requires the identification of T cell antigens that induce protection in humans. We analyzed peripheral blood CD4 and CD8 T cell responses from high-risk women defined as “resistant” or “susceptible” based on negative evidence for reinfection or detection of repeated reinfection during a 12-month follow-up period. Autologous APCs preincubated with E. coli clones expressing individual chlamydial ORFs were coincubated with CD4 or CD8 T cells and IFN-γ measured by ELISA. CD4 T cells from resistant women recognized 16 proteins significantly more frequently when compared to susceptible women, and only 2 of 16 were among a subset of immunodominant CD4 proteins suggesting restricted MHC recognition. No proteins recognized by CD8 T cells met immunoprotective criteria. Five CD4 and 5 CD8 antigens were more frequently recognized by women who had cervical infection but no evidence of endometrial infection by PCR. Thus, although protection from reinfection appears dependent on CD4 T cells, both CD4 and CD8 T cells may contribute to disease prevention. The use of samples from persons with evidence of natural immune protection for antigen discovery should advance development of a vaccine against C. trachomatis.
A West Nile virus NS4B P38G mutant induces a differential innate cytokine response in human cell lines. (VAC8P.1005)
Authors: Xie Guorui , Luo Huanle , Mann Brian , Wicker Jason , McBride Jere , Tesh Robert , Barrett Alan , Wang Tian
Published: January 2023

West Nile virus (WNV)-induced neurological disease has become a public health concern. No vaccines have been approved for human use. We have previously shown that an attenuated WNV, the nonstructural(NS)4B-P38G mutant, induced stronger innate cytokine and T cell responses than wild-type WNV. Mice immunized with this mutant were all protected from subsequent lethal wild-type WNV infection. Furthermore, TLR7 and MyD88 expression is required for host protection from NS4B P38G infection. Here, we have examined innate cytokine response in THP-1 cells (human monocytic cells) and THP-1 derived macrophages following with NS4B P38G infection. We have found that NS4B P38G induced stronger type 1 IFN and pro-inflammatory cytokine responses in THP-1 cells than those infected by wild-type WNV. Interestingly, we noted that there was a higher production of pro-inflammatory cytokines, but not IFN-b in NS4B P38G infected THP-1 derived macrophages. Our results also showed an upregulation of TLR4, TLR7 and RIG-I expression in THP-1 cells following NS4B P38G infection, whereas TLR2 expression was significantly increased in mutant infected THP-1 derived macrophages. Similar as our findings in mice, NS4B P38G mutant had a very low replication rate in both types of human cells. Overall, these results suggest that NS4B P38G induced a differential innate cytokine response in human monocytic cells and macrophages, possibly via different pathogen recognition receptor signaling pathways in these cells.
Manipulating TLR pathways activated by oncolytic vaccinia virus elicits a potent immune response against tumors (VAC8P.996)
Authors: Rojas Juan , Sampath Padmavathi , Hou Weizhou , Stephen Thorne
Published: January 2023

The immune response raised by oncolytic Vaccinia Virus after the infection and destruction of cancer cells is believed to be a key factor in the promising recent clinical results seen with these viruses. However, Vaccinia Virus primordially activates TLR2, which is associated with Th2 conversion and strong humoral responses. For cancer therapy, elicitation of cellular responses is thought to be more effective for the direct destruction of tumor cells. In this work, we switched the TLR response that a modified oncolytic Vaccinia Virus elicits. Firstly, we looked to activate the TLR3 pathway after infection with Vaccinia Virus, which is more associated with cellular responses. For this purpose, we constructed an oncolytic Vaccinia Virus that codifies for the TRIF protein, a key adaptor of the TLR3 pathway. The resulting virus increased the level of cytokines, chemokines and DAMPs released after infection, resulting in improved cytotoxicity in cancer cells and significantly enhanced therapeutic effects in mouse tumor models. Concurrently, we demonstrated that enzymatic deglycosylation of the surface of the virus is able to ablate the activation of cell surface TLRs, increasing the level of delivery and early viral gene expression in tumors in vivo. Finally, combination of both strategies demonstrated increased amounts of CTLs targeting the tumor, reduced levels of anti-viral neutralizing antibody and a further increase in antitumor activity in orthotopic mouse models.
A Non-lytic form of listeriolysin o induces human dendritic cells to produce IL-12 and IL-23. (VAC8P.1000)
Authors: Hernández Flores Karina , Thomas Dupont Pablo , Vivanco Cid Hector
Published: January 2023

Introduction. Listeriolysin O (LLO), a pore-forming citolysin is a key virulence factor of Listeria monocytogenes (LM). LLO induces a wide range of biological effects during the infection by LM or by itself as recombinant antigen. Here we studied the effects of a non hemolytic form of LLO on the production of two key cytokines for coordinating innate and adaptive immune responses against intracellular pathogens such as IL-12 and IL-23. Objective. We aimed to test the innate immune response of Human Monocyte-derived Dendritic cells (hDCs) to a non-hemolytic form of LLO named as dtLLO (mutated at C484A, W491A, W492A positions). Materials and methods. hDCs were stimulated with dtLLO ( 1μg/mL), in the presence or absence of 50 μg/mL of polymyxin B; 24 hours after stimulation, sups were tested to measure the concentration of TNF-α, IL-6, IL-10, IL-12 and IL-23 by ELISA technique. Results: dtLLO induced the production of IL-6 (1,660.86 pg/mL), TNF α (2,732.36 pg/mL), IL-12 (2,242.92 pg/mL) IL-10 (930.66 pg/mL) and IL-23 (349.47 pg/mL). Cytotoxic effects were undetected. Conclusion: We have identified a potent innate immune response when dtLLO is used as an exogenous antigen on hDCs. The production of IL-12 and IL-23 by human DCs after the recognition of LLO could be very important for the immune response during the human infection by Listeria. As a potential adjuvant for human vaccination purpose, dtLLO could be use to polarized the immune responses toward a Th1 or Th17 responses.
miR-155 overexpression augments anti-viral CD8+ T cell responses (VIR4P.1007)
Authors: Hope Jennifer , Stairiker Christoper , Gracias Donald , Stelekati Erietta , Carey Alison , Mueller Yvonne , Katsikis Peter
Published: January 2023

MicroRNAs (miRNAs) are highly conserved, small, single-stranded non-coding RNAs that play an essential role in regulating cellular processes. One miRNA in particular, miR-155, has been well characterized in many immune cell subsets. Recently, our lab has demonstrated an essential role for miR-155 in CD8+ T cell responses. Using murine models of influenza virus and Listeria monocytogenes (LM) infection, we have shown that CD8+T cells deficient in miR-155 demonstrate an intrinsic defect in anti-viral and anti-bacterial responses. Currently, we are investigating and characterizing the effect of miR-155 overexpression in CD8+ T cells. Adoptively transferred miR-155-overexpressing OT-I CD8+ T cells demonstrate a greater than 2-fold increase in the absolute number of anti-viral CD8+ T cells in the lungs of infected animals when compared to control OT-I CD8+ T cells at days 8 and 10 post-influenza infection. Interestingly, a significant number of miR-155 overexpressing OT-I CD8+ T cells demonstrate IFNγ- and TNFα-production and LAMP1-expression ex vivo. Overexpression of miR-155 in CD8+ T cells does not affect memory formation. These data suggest that overexpression of miR-155 augments effector CD8+ T cell responses.
CD27high/KLRG1low CD8+ T cells that persist throughout MCMV infection are highly expansive and have the ability to reestablish MCMV immunity (VIR4P.1011)
Authors: Quinn Michael , Turula Holly , Snyder Christopher
Published: January 2023

Cytomegalovirus (CMV) is a herpesvirus that establishes life-long latency in 60-80% of Americans. Constant immune surveillance is necessary to prevent viral reactivation from latency and results in the accumulation of functional CMV-specific CD8+ T cells (CD8s) over time, a process termed memory inflation. As such, CMV reactivations remain a clinical concern for immunosuppressed patients and reconstituting CMV immunity is critical for the long-term prevention of CMV disease. Understanding the maintenance of memory inflation may reveal novel approaches to restore CMV immunity. Previous work has shown that the majority of inflationary CD8s express a terminally-differentiated “effector” (TEFF) phenotype, have a short half-life and appear unable to sustain long-term CMV immunity. Interestingly, inflationary populations also include a minor subset of CD8s that express a “memory” phenotype (TM). Utilizing the murine model, MCMV, our data showed that the minor TM population was responsible for the majority of the inflationary population’s expansive capacity following transfer into naive mice and MCMV challenge. In addition, transferred TM cells produced both TM and TEFF progeny, which inflated and persisted in the recipients. Critically, when TM cells were transferred into chronically-infected mice, they survived and produced TEFF CD8s if host immunity was lost. Thus, TM CD8s appear to be a crucial component for the establishment and maintenance of CMV immunity.
Decreased frequency of regulatory T cells related to liver injury in acute viral hepatitis (VIR5P.1032)
Authors: Lee Jeewon , Choi Yoon Seok , Shin Eui-Cheol
Published: January 2023

FoxP3+ regulatory T cells (Tregs) are essential for maintaining self-tolerance and homeostasis in the immune system. Their role and character are already shown in many disease model, however, still concealed in acute viral hepatitis. In this study, we investigated alteration in the peripheral blood Treg population during acute hepatitis A (AHA) and its implication in the immune-mediated liver injury. Using isolated peripheral blood mononuclear cells (PBMCs) of 62 AHA patients, the decreased frequency and attenuated function of Tregs were observed; those results were inversely correlated with the severity of liver damage, represented by serum alanine aminotransferase (ALT) level. In addition, the frequency and suppressive function of Tregs were recovered at the convalescence phase when serum ALT level was completely normalized. We found a cause of the reduced frequency of Tregs from elevated expression level of Fas receptor, related to apoptosis, on Tregs in AHA patients. Indeed, the Tregs of AHA patients were more susceptible to Fas-mediated apoptosis than those of healthy controls in vitro experiments. In present study, we demonstrate that the frequency and suppressive function of Tregs were attenuated during acute HAV infection, resulting from apoptosis of Tregs induced by Fas-mediated mechanism.
Live attenuated rubella vectors express HIV Env and SIV Gag antigens in the context of an acute infection and elicit high antibody titers, memory B cells, and Gag specific CD8+ T cells (VIR5P.1033)
Authors: Berkower Ira , Virnik Konstantin , Hockenbury Max , Rosati Margherita , Alicea Candido , Valentin Antonio , Pavlakis George , Felber Barbara
Published: January 2023

Live attenuated viral strains are among our most potent vaccines. We have used the live attenuated rubella vaccine strain RA27/3 as a vector to express HIV MPER and SIV Gag antigens. These vectors combine the potency, durability and safety of rubella vaccine with the antigenicity of the inserts. We have recently completed the first successful trial of live rubella vectors in rhesus macaques. Rubella vectors grew robustly in macaques, and the vaccine inserts were strongly immunogenic. Simultaneous immunization with two vectors elicited antibodies to both inserts. Anti-Gag antibodies after immunization reached titers equal to natural SIV infection. Antibodies persisted for at least one year and declined at the same rate as anti-rubella antibodies, which protect for life. Re-exposure to the vector boosted the immune response, indicating the induction of memory B cells. Priming with DNA vaccine and boosting with the vector produced high levels of Gag specific CD8+ T cells. The vector can stably express larger inserts, up to 320 amino acids for SIV Gag proteins, including part of MA and all of CA and P2 proteins. They also express engineered HIV Env outer domains up to 180 amino acids that include the CD4 binding site and bind monoclonal VRC01. Rhesus macaques will provide the ideal animal model for evaluating novel vaccine inserts for immunogenicity, induction of neutralizing antibodies, and protection against SIV or SHIV challenge.
SHP-1 regulates neuroinvasion of neurotropic virus into the CNS (VIR5P.1035)
Authors: Schneider Karin , Massa Paul
Published: January 2023

Viral infections in the central nervous system (CNS) may have devastating clinical consequences to the host. Neurotropic virus infections often begin in peripheral tissues, which then spread to the CNS via peripheral nerves following entry into nerve terminals by receptor-mediated endocytosis and subsequent retrograde axonal transport into the CNS compartment. In our lab, a possible novel role for the protein tyrosine phosphatase, SHP-1, in regulating neuroinvasion via peripheral nerves of a neurotropic virus (Theiler’s Murine Encephalomyelitis Virus, TMEV) is being investigated. Preliminary data using immunohistochemistry of TMEV showed extensive staining of spinal cord neurons after intraperitoneal inoculation in mice with a null mutation in the SHP-1 gene (me/me mice) compared to wild type mice. Further, intramuscular (IM) inoculations of TMEV caused virus infection in spinal cord neurons and subsequent paralysis of SHP-1-deficient mice but not of wild type mice. IM and intraperitoneal injections (IP) of retrograde axonal transport tracers fluorogold and horseradish peroxidase showed increased tracer levels in the spinal cords of SHP-1-deficient mice compared to wild type mice. Therefore, we propose SHP-1 may play a role in regulating viral neuroinvasion by controlling retrograde axonal transport from peripheral sites of infection. In this sense, SHP-1 may play novel roles in antiviral responses including inhibition of virus invasion and spreading in the CNS.
Indoleamine 2,3-dioxygenase 2, a novel mediator of pathogenic autoantibodies in autoimmune arthritis (BA12P.110)
Authors: Merlo Lauren , Pigott Elizabeth , DuHadaway James , Grabler Samantha , Metz Richard , Prendergast George , Mandik-Nayak Laura
Published: January 2023

Several autoimmune disorders, including rheumatoid arthritis (RA), are associated with altered activity of the immunomodulatory enzyme indoleamine-2,3-dioxygenase (IDO). However, the precise contributions of IDO function to autoimmunity remain unclear. We examined the effect of two different IDO enzymes, IDO1 and IDO2, on the development of autoimmune arthritis in the KRN preclinical model of autoimmune arthritis by comparing the effects of both genetic knockout models and chemical inhibitors of IDO on joint inflammation. We find that IDO2, but not IDO1, is critical for arthritis development, providing the first direct evidence of separate in vivo functions for IDO1 and IDO2. Specifically, IDO2 knockout mice display decreased joint inflammation relative to wild-type mice due to a reduction in pathogenic autoantibodies and antibody secreting cells. Notably, IDO2 appears to specifically mediate autoreactive, but not normal B cell responses, as total serum Ig levels are not altered and IDO2 ko mice are able to mount productive antibody responses to model antigens in vitro and in vivo. Reciprocal adoptive transfer studies confirmed that autoantibody production and arthritis are modulated by IDO2 expression in a cell type extrinsic to the T cell. Future work will define the relevant cell types for IDO2 activity. Our results provide the first insights into the previously unknown function of IDO2 by defining its pathogenic contributions to autoantibody-mediated autoimmunity.
Superoxide-deficient macrophages exhibit an M2 phenotype to prevent autoimmune diabetes. (BA12P.105)
Authors: Padgett Lindsey , Burg Ashley , Lei Weiqi , Tse Hubert
Published: January 2023

Type 1 diabetes (T1D) is an autoimmune disease culminating in β-cell destruction by inducing reactive oxygen species (ROS), pro-inflammatory cytokines, and islet-infiltrating leukocytes. We recently demonstrated the importance of NADPH oxidase (NOX)-derived ROS synthesis in T1D, as Non-Obese Diabetic (NOD) mice unable to generate superoxide (NOD.Ncf1m1J) were highly T1D resistant. Therefore, we hypothesized that dampened ROS synthesis within NOD.Ncf1m1J mice contributes to blunted pro-inflammatory M1 and/or enhanced anti-inflammatory M2 macrophage responses. During spontaneous T1D progression, islet-resident NOD.Ncf1m1J macrophages exhibited an enhanced M2 phenotype in contrast to age-matched NOD with a significant (p < 0.01) increase in M2 macrophage transcripts (Ccl17, Retnla, Cd206, Arg1), and a significant (p < 0.01) decrease in M1 transcripts (Cxcl10, Ccl5, Nos2, and Tnfa). To confirm that redox status influenced macrophage differentiation, pre-activated diabetogenic BDC-2.5 CD4 T cells, which recruit pro-inflammatory M1 macrophages to destroy β-cells, were transferred into NOD.Rag.Ncf1m1J recipients possessing superoxide-deficient antigen presenting cells. We observed a decrease in IL-1β-expressing, islet-infiltrating M1 macrophages and enhanced Arginase-1-expressing M2 macrophages compared to NOD.Rag. Our results demonstrate the importance of ROS in M1 macrophage differentiation; thus, targeting macrophage redox status may represent a promising therapy in halting T1D.
Toll-like receptor 4 agonistic monoclonal antibody reverses new onset type 1 diabetes (BA12P.106)
Authors: Bednar Kyle , Tsukamoto Hiroki , Wu Yuehong , KImoto Masao , Ridgway William
Published: January 2023

Viral, parasitic or bacterial infections can prevent T1D in NOD mice. The “hygiene hypothesis” proposes that the incidence of autoimmunity is increasing due to decreased immune stimulation in childhood, offering a rationale for stimulation of innate immunity as a treatment for T1D. We demonstrate that a TLR4/MD-2 specific agonistic monoclonal antibody, UT18, can not only prevent T1D, but also reverse new onset T1D (diagnosed by polyuria, weight loss, and elevated blood glucose). UT15, a control TLR4/MD-2 non-agonistic monoclonal antibody, neither prevented nor reversed T1D. In the prevention studies, UT18 treatment resulted in decreased insulitis vs. UT15 treatment. UT18 treatment permanently reversed new onset T1D in 9/13 mice, while UT15 treated mice rapidly progressed to end-stage diabetes (>600 mg/dl). Mice successfully treated with UT18 had less severe islet infiltration and increased insulin positive beta-cell area compared to UT15 controls. Transfer of CD4+ and CD8+ T-cells (from mice successfully treated with UT18) did not cause diabetes for up to 75 days post transfer, whereas cells transferred from UT15 and non-diabetic NOD mice rapidly caused disease. The lack of T cell mediated diabetes transfer suggests that T cell tolerance was restored in new onset diabetic mice treated with UT18. Restoration of T cell tolerance via TLR-4/MD-2 mediated innate immune stimulation is a promising new pathway for treatment of T1D and other T cell mediated autoimmune diseases.
Anti-viral responses to diabetogenic viruses are mediated by superoxide production (BA12P.107)
Authors: Tse Hubert , Padgett Lindsey , Burg Ashley , Lei Weiqi
Published: January 2023

Viral infections may directly trigger the onset of Type 1 diabetes (T1D) by β-cell lysis and/or indirectly via pro-inflammatory cytokines and reactive oxygen species (ROS) by islet-infiltrating leukocytes. We recently demonstrated a crucial role for NADPH oxidase (NOX)-dependent superoxide production in T1D, as NOX-deficient Non-Obese Diabetic (NOD.Ncf1m1J) mice were resistant to autoimmune diabetes, which was partly mediated by diminished pro-inflammatory cytokine and Type I interferon synthesis via dampened macrophage-derived Toll-like receptor (TLR) signaling in response to poly(I:C), a viral RNA mimic. Therefore, we hypothesized that ROS deficiency would diminish anti-viral pro-inflammatory responses and inhibit T1D in response to viral triggers. To define the interplay between redox status and anti-viral responses, we performed infections with Coxsackie B4 (CB4) virus a putative diabetogenic enterovirus associated with T1D induction in both humans and rodents. In the absence of superoxide, NOD.Ncf1m1J macrophages exhibited a 2-fold decrease in TNF-α and IFN-α upon infection due to dampened TLR3 and RIG-I signaling. Corroborating the in vitro results, CB4 infection of NOD.Ncf1m1J mice elicited a 2-fold decrease in TNF-α and IL-1β by pancreas-infiltrating macrophages in contrast to NOD. Future studies will determine if oxidative stress can influence anti-viral responses and uncover novel insights into T1D pathogenesis in response to diabetogenic viruses.
The transcription factor CREMalpha regulates inflammatory T cell subsets in juvenile idiopathic arthritis (BA13P.115)
Authors: Moncrieffe Halima , Ohl Kim , Nickel Helge , Wedderburn Lucy , Tenbrock Klaus
Published: January 2023

The cAMP response element (CRE) modulator (CREM)α binds to genes promoters with CREs and regulates transcription via a chromatin-dependent mechanism. CREMα is important for the T cell pathophysiology of SLE suppressing IL-2 and CD3ζ transcription, but enhances IL-17. Juvenile idiopathic arthritis (JIA) is an autoimmune disease of unknown origin. Th17 cells have a pathogenic role in arthritis and are not controlled by local FoxP3+ regulatory T cells (Tregs). Pathogenic T cells in the inflamed joints of JIA patients have enhanced expression of IL-17, IFNgamma and CD161. CD161+ CD4+ cells also contain FoxP3+ cells that produce proinflammatory cytokines. We observed enhanced CREM expression in JIA patient synovial fluid (SF) T cells and also after culture of healthy donor PBMCs with JIA SF. We furthermore found enhanced expression of CREM in CD4+CD161+ and in CD4+FoxP3+CD161+ cells, which produce inflammatory cytokines. Vice versa Helios+FoxP3+ cells, which are characteristically stable Tregs and do not express inflammatory cytokines, had lower levels of CREM. JIA SF induced IFNgamma, IL-17 and FoxP3 in T cells during incubation, which could be abrogated by CREM siRNA transfection. Within the FoxP3+ population, CREM siRNA reduced CD161+FoxP3+ cells but not Helios+FoxP3+ cells. We thus suggest CREMα overexpression in T cells contributes to T cell pathophysiology in JIA by regulating percentages of inflammatory CD4+ IL-17+ cells, as well as inflammatory CD161+FoxP3+ cells.