bluuebunny/crossref_metadata_2025 huggingface dataset:
Monocyte-derived dendritic cells are critical for the induction of anti-tumor immune responses (TUM4P.932)
Authors: Ronchese Franca , Kuhn Sabine , Yang Jianping , Gilfillan Connie
Published: January 2023

The DC subsets that are required for the induction of effective anti-tumor immune responses and the success of immunotherapy are unknown. We used mouse models to compare different immune-activating agents for their impact on tumor growth, tumor-specific T cells, cytokine production and DC activation. Treatments with poly I:C or a combination of monososdium-urate crystals (MSU) + Mycobacterium smegmatis delayed growth of B16 melanoma and 4T1 mammary carcinoma tumors, whereas LPS, M. smegmatis or MSU alone were ineffective. Effective treatments required both CD8+ and NK1.1+ cells, and increased CD8+ T cell priming, tumor-infiltration by effector cells and effector cytokine production. In addition, only the successful treatments induced the appearance of a population of monocyte-derived DC in tumor-draining lymph nodes. Recent experiments revealed that immunotherapy was required for the differentiation of blood monocytes into DC in the lymph node, and that these monocyte-derived DC were both necessary and sufficient for successful immunotherapy of B16 melanoma tumors. Further experiments are required to clarify the precise function of this DC population. We conclude that the activation of existing DC subsets is not sufficient for the induction of anti-tumor immunity, whereas newly recruited inflammatory DC are critical mediators of the successful activation of anti-tumour immune responses.
Semaphorin7A is up-regulated in breast cancer and promotes macrophage production of angiogenic molecules (TUM4P.933)
Authors: Garcia Areas Ramon , Libreros Stephania , Amat Samantha , Iragavarapu Vijaya
Published: January 2023

Semaphorins are a large family of molecules involved in the axonal guidance and development of the nervous system. In addition, they also play important roles in immunomodulation. Semaphorin 7A (SEMA7A) has been reported to be a modulator of monocytes and macrophages via α1β1integrins. SEMA7A promotes chemotaxis of monocytes and induces them to produce proinflammatory mediators. In this study we explored the role of SEMA7A in breast cancer. We first show that SEMA7A is highly expressed by DA-3, EO771 and 4T1 mammary tumor cells in comparison to normal mammary cells (EpH4). Furthermore, peritoneal elicited macrophages from mammary tumor-bearing mice express higher levels of SEMA7A compared to those from normal mice. We also show that murine macrophages treated with recombinant murine SEMA7A significantly increased their expression of angiogenic chemokine CXCL2 via activation of the MAPK pathway. Peritoneal elicited macrophages from mice bearing SEMA7A-silenced tumors produce significantly lower levels of CXCL2, CXCL1 and MMP-9, compared to macrophages from control tumor bearers. Gene silencing of Sema7A in macrophages from tumor-bearers decreased in their angiogenic potential. We postulate that SEMA7A in mammary carcinomas may skew monocytes into a pro-tumorigenic phenotype to support tumor growth.
Natural cytotoxic antibodies against tumor N-glycolilated ganglioside decrease with age and are absent in NSCLC patients (TUM4P.906)
Authors: Hernandez Ana , Zhurbenko Nely , Martinez Darel , Rondon Teresa , Ramirez Tania
Published: January 2023

N-glycolylated (NeuGc) gangliosides are not naturally expressed in normal human tissues but are overexpressed in several tumors and have immunosuppressive capacity, contributing to cancer progression. The present work shows the existence of a natural antibody response against NeuGcGM3 in healthy donors. These antibodies specifically recognized NeuGcGM3 and killed tumor cells expressing the antigen by a complement mediated mechanism. Interestingly, even after complement inactivation 17% of the positive sera showed a direct cytotoxic effect over the tumor cells. This cytotoxicity was dependent on the presence of the antigen on the tumor cell and resembles an oncotic necrosis kind of cell death. The levels of anti-NeuGcGM3 antibodies in the sera of healthy donors and the percentage of donors with this natural immunity decreases with age. Furthermore we could not detect this reactivity in the sera of 51 non-small cell lung cancer (NSCLC) patients that matched in sex and age the assessed healthy donors. Interestingly the level of sera IgM, but not IgG, was significantly lower in cancer patients in comparison with healthy donors. Also, the capacity of patients B1 cells to spontaneously secrete IgM and to respond to DAMPs, like CpG, seems to be affected. Defects in the natural surveillance against tumor antigens could increase the risk of elderly donors to develop cancer and affect the capacity of cancer patients to effectively fight against them.
The role of danger signals in tumor elicited inflammation (TUM5P.934)
Authors: Francescone Ralph , Hou Vivi , Dmitrieva Oxana , Posocco David , Grivennikov Sergei
Published: January 2023

Sporadic colorectal cancer (CRC) has traditionally been considered a genetic disease, with inactivation of classic tumor suppressors, such as APC and P53, driving tumorigenesis. Recently, however, it has come to light that inflammation plays a major role in the progression of CRC, and that tumor cells can induce a massive infiltration of immune cells into the tumor. Danger Associated Molecular Patterns (DAMPs) are proteins that are released from dying or stressed cells, which promotes an immune response. While these so called “danger signals” are often up-regulated in cancer, the mechanisms behind their role in tumor progression and inflammation require further exploration. Using a number of mouse models of spontaneous CRC, we are beginning to uncover which inflammatory cues induce danger signals, and which immune cells are the major producers. We have found that S100A8/A9 and HMGB-1 are significantly up-regulated in tumor tissue and mesenteric lymph nodes (MLN) of mice with CRC, compared to adjacent normal colon tissue. Additionally, using FACS to sort individual immune populations within the mouse tissue, intraepithelial monocytes and neutrophils seem to be the major immune cell producers of danger signals. Finally, in a kRas inducible model, to simulate a more aggressive cancer, danger signals are elevated even further in tumor tissue and MLN. Future work will focus on elucidating the precise signaling between immune modulators and danger signals.
A B cell riddle: a new tool to study the link between malaria and Burkitt’s Lymphoma (TUM5P.935)
Authors: Hart Geoffrey , Wei Chungwen , Chida Asiya , Crompton Peter , Sanz Ignacio , Pierce Susan
Published: January 2023

Burkitt’s lymphomas (BLs) are B cell lineage tumors that are the most common childhood cancer in the P. falciparum malaria endemic areas of Africa. The molecular basis of malaria infection causing endemic BLs (eBLs) remains unknown. Recent data has confirmed that remarkably 30% of eBLs are enriched for one particular heavy chain V region, VH4-34. The US frequency in normal individuals is 4%, however it was unknown in endemic regions of Africa. This high frequency of VH4-34 in eBLs initiated the use of the anti-VH4-34 antibody, ‘9G4’, to study VH4-34 at the cellular level in Africa. Here we report that in adults and children living in a malaria endemic area of Mali, VH4-34 expressing B cells are present in frequencies similar to those in individuals in the U.S, however more B cells are found in memory B cell subpopulations. We have also found that VH4.34 circulating antibody levels increase when infected with malaria but do not significantly increase with age unlike other malaria specific antibodies. This suggests the VH4-34 heavy chain is possibly important for malaria, but is decreased from the repertoire after infection is controlled. We are initiating studies to determine: the frequency of VH4-34 expressing B cells latently infected with EBV to see if it enriched, whether VH4-34 Abs are protective in malaria, and elucidating a molecular mechanism for transformation. This data will start an avenue to better understand the complex mechanisms behind this childhood cancer.
Combined B cell specific deletion of the deubiquitinating enzymes CYLD and A20 leads to a dramatic accumulation of CD5+ B cells (TUM5P.936)
Authors: Hoevelmeyer Nadine , Hahn Matthias , Waisman Ari
Published: January 2023

Mutations in NFκB signaling molecules and in negative regulators of NFκB, such as CYLD and A20, have been identified in B cell lymphomas. These deubiquitinating (DUB) enzymes remove activating lysine-63 linked ubiquitin chains from numerous NFκB signaling molecules, including oncogene Bcl-3, thus controlling their activity. We found that mice lacking CYLD specifically in B cells develop with age a marked population of CD5+ B cells in the blood and the body, a population that resembles cells found in CLL patients. Importantly, we found that doubleBKO mice, that lack both A20 and CYLD in B cells, develop CD5+ B cells in an enhanced fashion, already at two months of age, with more than 95% of all B cells expressing CD5 before one year of age. Apart of CD5 expression, these B cells also increasingly express CLL markers, such as ZAP70 and Bcl-2. Starting with 14 month of age, these mouse mutants start to die most probably from CLL pathology. Thus, we could assign a separate, yet essential role for each of these two DUB enzymes in the control of B cell function, and have shown that loss of both together resulted in the initiation of a previously not identified chain of events probably leading to lymphoma/leukemia. In addition, we were able to identify a splice variant of CYLD that we previously found in mice, also in human CLL patients, thus indicating that this molecule, we termed sCYLD, may have a tumor-associated role not only in mice but also in human.
Targeting gender specific mechanisms to enhance anti-tumor immune responses against hepatocellular carcinoma (TUM5P.939)
Authors: Barrios Kelly , Vidrine Amy , Watkins Stephanie
Published: January 2023

In cancer, gender disparity has been observed in disease aggressiveness, incidence and prognosis without a clear understanding of the role the immune system plays into this sexual dimorphism. Hepatocellular carcinoma (HCC) occurs much more frequently in men than in women, however mechanisms associated with antitumor immunity in each gender is unclear. We introduce the use of a novel and clinically relevant mouse model of HCC to extricate the role of hormone signaling on immune cell function and the impact on immunotherapy. This model mirrors human disease since it’s driven by the overexpression of the oncogenes c-met and β−catenin. We used hydrodynamic delivery of these oncogenes to induce mutations and HCC. Male and female mice had comparable rates of tumor development; however in the absence of Foxo3, female mice were protected from HCC development. This protection was dependent upon immune cells lacking Foxo3 expression. Our data suggest that in females, silencing of Foxo3 permits the activation and migration of effector cells to the liver and that Foxo3 can control DC function by altering hormone receptor expression. Our results show that immune response and inhibition of tumor growth is differentially regulated by gender. A better understanding of the mechanisms regulating immune mediated tumor protection in females, will aid in devising ways to improve antitumor immune responses in males, and identifying proper targets to design or enhance treatment for all HCC patients
Examination of the role of dendritic cells, macrophages and B cells in the function of vaccine adjuvants. (VAC6P.944)
Authors: Mosaheb Munir , Reiser Michael , Toussi Deana , Liu Xiuping , Masari Paola , Wetzler Lee
Published: January 2023

Vaccines are vital in the fight against infectious diseases. Many vaccines are successful because of adjuvants added to their formulations or in the case of live attenuated vaccine because the adjuvant is inherent, however they have been used with little understanding of their mechanisms. PorB, a TLR2 agonist, is the major outer membrane protein of Neisseria meningitidis with potent adjuvant activity. In this study we investigated the role of individual antigen-presenting cell (APC) types in the adjuvant activity of PorB with comparisons to other vaccine adjuvants. WT mice and MyD88 APC floxed mice were immunized three times two weeks apart with ovalbumin (OVA) and either PorB, Alum, MF-59, MPL or CpG. We confirmed MyD88 deletion by qPCR on the purified cells and by measuring cytokines after stimulation with TLR ligand. Analysis of serum indicated that MyD88 in B cells, macrophages and dendritic cells is important for the production of PorB/OVA induced anti-OVA IgG. Alum, a MyD88 independent adjuvant, was able to induce strong anti-OVA IgG in mice that lacked MyD88 in any of these APCs. Interestingly the adjuvant activity of MF-59, which may be MyD88 dependent, were not affected by deletion of MyD88 in the different APC cell types, suggesting that if MyD88 is involved in its adjuvant activity, it must be due to stimulation of other cell types. These insights will aid in vaccine development by allowing more intelligent and judicious use of these and other vaccine adjuvants.
Biological activity of dialyzable leukocyte extracts in a mouse cutaneous herpes model. (VAC6P.946)
Authors: Pérez-Tapia Sonia , Salinas-Jazmín Nohemí , Estrada-Parra Sergio , Limón-Flores Alberto , Vázquez-Leyva Said , Becerril-García Miguel , Medina-Rivero Emilio , Pavon Lenin , Velasco-Velázquez Marco
Published: January 2023

Human dialyzable leukocyte extracts (DLE) are heterogeneous mixtures of low-molecular-weight peptides. DLE improve the clinical responses in infections, allergies, cancer, and immunodeficiencies. TransferonTM is a human DLE registered as a drug by Mexican health authorities and commercialized nationally. In order to establish a model that could be used routinely to assess the activity of TransferonTM, we standardized and validated a mouse model of HSV-1 cutaneous infection. In such model, we evaluated the biological activity of 27 TransferonTM batches that complied with other quality standards. All batches improved survival of HSV-1-infected mice; average survival increased from 20.9% in control mice to 59.6% in TransferonTM-treated mice. Biological activity of TransferonTM correlated with increased serum levels of IFN-γ and reduced IL-6 and TNF-α concentrations at 7-9 days post-infection (high mice mortality period). Our results show that: a) the mouse cutaneous herpes model allows the evaluation of the biological activity of TransferonTM; b) the assay can be used as a routine tests for batch release; c) TransferonTM is produced with high homogeneity between batches; and d) the protective effect of TransferonTM in this model correlates with differential changes in cytokines related to innate or adaptive immunity, suggesting that immunomodulation is associated to the reported therapeutic effects on herpes infections. Supported by FTU/IB/012/010/PRO.
Adoptive B cell immunotherapy accelerates control of chronic viral infection (VAC6P.947)
Authors: Moseman E. , Heydari Sara , Zinselmeyer Bernd , McGavern Dorian
Published: January 2023

Many viruses can establish persistence and cause disease in humans. Studies have established a role for T cells in resolving persistent viral infections; however, the importance of a humoral response is less clear. In this study, we sought novel insights into the importance of humoral immunity in resolving a persistent infection by utilizing the lymphocytic choriomeningitis virus (LCMV) clone 13 model. Infection with aggressive LCMV variants results in a chronic viral infection during which antiviral T cells pass through a state of functional exhaustion, but the infection is nevertheless controlled over time. We postulated that humoral immunity was required for LCMV control and could be harnessed as an immunotherapy. We observed dramatic increases in splenic plasma cells as early as one week following LCMV infection, which coincided with the emergence of non-neutralizing antibodies. B cell depletion starting at the peak of T cell exhaustion (day 15) resulted in a failure to control virus. Moreover, adoptive transfer of LCMV immune B cells into mice with an active infection dramatically accelerated viral clearance. We found using two-photon microscopy that therapeutic B cells home to the infected recipient spleen where donor antibody secreting cells proliferate to amplify antiviral humoral responses. These data demonstrate that humoral immunity can contribute to the resolution of a persistent viral infection and that B cells can be harnessed for adoptive immunotherapy.
Type A-derived genetically modified live attenuated Francisella tularensis is immunogenic and efficacious in rabbits (VAC7P.965)
Authors: Reed Douglas , Caroline Amy , Stinson Elizabeth , Cole Kelly , Santiago Araceli , Mann Barbara , Barry Eileen
Published: January 2023

Francisella tularensis is a gram negative non-motile coccobacillus that is the causative agent of tularemia. There is concern that F. tularensis could be used as a biological weapon and development of vaccines is a high priority. Type A strains are highly virulent but the current live vaccine strain (LVS) is based on a type B strain and is only partially protective against aerosol challenge with SCHU S4, a type A strain. A series of live attenuated vaccine candidates were derived from the SCHU S4 strain containing specific deletion mutations in target genes. After initial studies in mice, these attenuated strains were evaluated in rabbits. Rabbits were vaccinated by scarification which induced a mild fever and eschar formation at the inoculation site but no other clinical signs of disease. Serum IgG responses could be detected in all vaccinated rabbits as early as day 7 and continued to increase through day 21 before declining slightly on day 28. CD4+ T cells but not CD8+ T cells proliferated when cultured in vitro with heat-killed SCHU S4. Three of the vaccine strains conferred partial protection against death (ranging from 27-40%) and prolonged time to death in animals that did succumb. Vaccination with LVS prolonged survival by 1-2 days but did not protect against death. Type A-derived vaccines are immunogenic and provide better protection than LVS in the rabbit model against a robust aerosol challenge. These candidates warrant further study and evaluation.
ManLAM-aptamer as an immunoadjuvant improves immune-protective effects of BCG (VAC7P.968)
Authors: Zhang Xiao-Lian , Sun Xiao-ming , Pan Qin
Published: January 2023

Tuberculosis (TB) remains an important worldwide health threat. However vaccination with Mycobacterium bovis bacille Calmette-Guerin (BCG), the only available licensed vaccine of TB, provides limited protection against adult pulmonary tuberculosis. Mannosylated lipoarabinomannan (ManLAM) is a kind of glycolipid on the cell wall surface of BCG and has been implicated with remarkable immunosuppressive effect on the host immune cells. In the present study, we used Systematic Evolution of Ligands by EXponential enrichment (SELEX) to generate a single-stranded DNA aptamer that specifically bound to ManLAM of BCG with high affinity. We found that vaccination with BCG combined with BM2 recruited dendritic (DC) cells into peripheral lymph nodes and decreased the numbers of regulatory B cells and regulatory T cells. It could also significantly enhanced the Th1-type cellular response and induced conversion of IgG2a isoforms in mice. We demonstrated that vaccination with BCG plus BM2 significantly reduced the progression of Mycobacterial tuberculosis (M. tb) H37Rv infection, bacterial loads in lungs and spleens of mice via aerosol infection with M. tb H37Rv, and provided much better protective immune response against M. tb than vaccination with BCG alone. Our data provide a new strategy for TB control and the aptamer BM2 holds great promise for developing a new potential immunoadjuvant of BCG.
Increased T and B cell responses in M. bovis BCG vaccinated pigs by co-immunization with pDNA encoding a prototype tuberculosis antigen (VAC7P.973)
Authors: Huygen Kris , Bruffaerts Nicolas , Vandermeulen Gaëlle , Romano Marta , Préat Véronique , Stockhofe-Zurwieden Norbert
Published: January 2023

The only vaccine currently available to prevent tuberculosis, M. bovis BCG, has limited efficacy. BCG is a poor inducer of CD8+ T cells, which are particularly important for the control of latent tuberculosis and protection against reactivation TB. The induction of strong CD8+ T cell responses is a hallmark of DNA vaccines. We postulated that combining the existing BCG vaccine with plasmid DNA encoding immunodominant antigens of M. tuberculosis, could improve the vaccine by generating stronger CD8+ (and CD4+) T cell responses. As a prototype tuberculosis antigen, the mycolyl-transferase Ag85A was selected. Three groups of six pigs each, were vaccinated intradermally with BCG alone, BCG combined with empty vector or BCG combined with codon-optimized V1j.ns-tPA-Ag85A vector. The BCG-pAg85A DNA combination induced strongly increased and sustained humoral and Th1 type cellular responses against Ag85A and against the purified protein derivative of tuberculin. Increased IFN-γ producing CD8 T cells were detected by intracellular cytokine staining and an Ag85A T cell epitope with predicted MHC class I motif, was promiscuously recognized by 5/6 pigs vaccinated with the combination. In summary, our study provides the first proof of concept in a large mammalian species, for a new TB vaccine, based on a BCG-pDNA combination.
Oral immunization with Escherichia coli-expressed enterovirus 71 VP1 and chitosan adjuvant protect mice against lethal challenge (VAC7P.984)
Authors: Li Aqian , Zhang FuShun , Zhang Shuo , Hao Chunsheng , Zhang QuanFu , Wu Wei , Liu Lin , Liu Chuan , Liang Mifang , Li Xiuling , Li Dexin
Published: January 2023

Enterovirus 71 (EV71) is identified as the etiologic agent of hand-foot-and- mouth disease (HFMD) in the Asia-Pacific region while there are currently no vaccines or effective antiviral drugs available. VP1, one of the major immunogenic capsid proteins of EV71, has shown considerable promise for preventing EV71 infection by intramuscular inoculation in mice. In this study, we found that oral gavage vaccination (O.V.) of ICR mice with recombinant VP1 (rVP1) and chitosan adjuvant induced VP1-specific sIgA antibodies in intestine, feces, vagina, respiratory tract and virus-specific neutralization IgG antibodies. The splenocytes collected from the rVP1-immunized mice exhibited significant cell proliferation and induced high levels of Th1 immune responses cytokine IFN-γ and Th2 immune responses cytokine IL-4 after stimulation. Moreover, the rVP1 immunized mother mice conferred protection (survival rate up to 30%) to neonatal mice against the lethal (1000 plaque form unit, PFU) EV71 challenge. All these data indicate that oral immunization with rVP1 is safe and effective in inducing broad-spectrum immune responses and being a promising subunit vaccine candidate for prevention of EV71 infection. Corresponding author: Prof. Dr. Dexin Li Email: lidx@chinacdc.cn
An optimized, synthetic DNA vaccine encoding the toxin A and toxin B receptor binding domains of Clostridium difficile induces protective antibody responses in vivo (VAC7P.958)
Authors: Baliban Scott , Baliban Scott , Shammassian Berje , Michael Amanda , Zetner Isaac , Latimer Brian , Cocklin Simon , Welles Seth , Bouillaut Laurent , Sonenshein Abraham , Kahn Amir , Sardesai Niranjan , Jacobson Jeffrey , Weiner David , Kutzler Michele
Published: January 2023

Clostridium difficile-associated disease (CDAD) constitutes a large majority of nosocomial diarrhea cases within industrialized nations and is mediated by the effects of two secreted toxins, toxin A (TcdA) and toxin B (TcdB). Key toxin-neutralizing epitopes have been discovered within the carboxy-terminal receptor-binding domain (RBD) of the toxins, which has generated interest in developing the RBD as a vaccine target. While numerous platforms have been studied, very few data describe the potential of DNA vaccination against CDAD. Therefore, we created highly optimized plasmids encoding the RBD from TcdA and TcdB and immunized C57BL/6 mice and rhesus macaques intramuscularly followed by in vivo electroporation. In these animal models, vaccination induced significant levels of anti-RBD antibodies within the serum and feces that could neutralize toxins in an in vitro cytotoxicity assay. Moreover, mice that were either immunized with plasmids or given non-human primate immune sera were protected from a lethal intraperitoneal challenge of purified C. difficile toxins. Finally, immunized mice were significantly protected when challenged with strains of C. difficile spores that were homologous (VPI 10463; n=10/10) and heterologous (UK1; n=4/8) to our vaccine antigens. These data demonstrate the robust immunogenicity and efficacy of a TcdA/B RBD-based DNA vaccine in preclinical models of acute toxin-associated disease.
HIV broadly neutralizing antibodies and immunological tolerance (VAC7P.988)
Authors: Yang Guang , Liu Mengfei , Wiehe Kevin , Nicely Nathan , Haynes Barton , Mascola John , Bjorkman Pamela , Kelsoe Garnett
Published: January 2023

It is generally thought that many human antibodies (Ab) that neutralize multiple clades of HIV-1 (bnAbs) are self-reactive. We previously identified kynureninase as the primary self-antigen recognized by 2F5, suggesting that generation of Ab to the 2F5 epitope of HIV-1 may be proscribed by immune tolerance, a notion supported by impaired B-cell development in mice expressing the 2F5 VH and VL regions. However, there is a lack of quantitative and systemic assessment of polyreactivity among HIV bnAbs. In addition, it is unknown whether other classes of HIV-1 bnAbs and non-neutralizing antibodies are also influenced by immunological tolerance. Here, we used protein microarrays to assess bnAb binding to over 9,600 human proteins and compared bnAb binding profiles to HIV non-neutralizing antibodies. We found that bnAbs as a class are more autoreactive and more polyreactive than non-neutralizers. Interestingly, 4 of 7 CD4bs bnAbs screened from 2 distinct lineages (VRC01, VRC02, CH103 and CH106) bind human protein ubiquitin ligase E3A (UBE3A). We confirmed this crossreactivity in ELISA, and demonstrated that UBE3A competitively inhibits gp120 binding to VRC01. Our results demonstrate that HIV-1 bnAbs are significantly more polyreactive and self-reactive than non-neutralizers, which may subject them to immunological tolerance control in vivo. Our identification of UBE3A as a self-antigen of bnAbs provides a mechanism for the rarity and delayed development of certain CD4bs bnAbs.
Evaluation of human cell mediated immune responses to VZV antigens (VAC7P.993)
Authors: Cox Kara , Prokop Michael , Dubey Sheri , Vora Kalpit
Published: January 2023

Zostavax© is a safe and effective vaccine against Herpes Zoster. The precise immune correlate of protection and the corresponding VZV antigens responsible for protective immunity are not currently defined. A few key pieces of data suggest that cell mediated immune responses are more important than humoral responses in the immune control over viral re-activation from latent infection. Primarily, the majority of Herpes Zoster outbreaks occur in the aging population in which antibody titers against VZV are maintained but cell mediated immune responses wane. Additionally, individuals who are immunocompromised as a result of disease or drug treatment have difficulty controlling the virus. A group of ten viral proteins of Varicella were chosen for evaluating human cell mediated immune responses. Donor samples were recruited in the following cohorts: i; Herpes Zoster outbreak within the last 5 years, ii; Subjects age 35+ with no history of Shingles or Zostavax© vaccination, and iii subjects pre and post Zostavax© vaccination. Blood samples from these donors were stimulated with the set of VZV antigens and a panel of cytokine responses from the plasma was measured. Here we show a ranking of VZV viral protein immunogenicity from these cohorts, as well as the cytokine profiles for each. These studies will help to define the viral proteins involved in generating protective cell mediated immunity correlated with protection.
TLR3 preconditioning enhances the therapeutic efficacy of umbilical cord mesenchymal stem cells in DSS-induced colitis (VAC8P.995)
Authors: Carrion Flavio , Fuenzalida Patricia , Fernandez Catalina , Ibañez Cristina , Vega Ana Maria , Fuentealba Rodrigo , Kurte Monica
Published: January 2023

Background and Aim: Mesenchymal stem cells (MSCs) have attracted much attention since exert anti-inflammatory and immunomodulatory effects. Recently, diverse studies have been showed that the stimulation of specific toll-like receptors affects the immunomodulatory properties of MSCs. The aim of this work was to evaluate the effects of TLR preconditioning on therapeutic efficacy of umbilical cord MSCs (UC-MSCs) in a murine model of acute colitis. Methods: C57BL/6 mice were exposed to 2,5 % dextran sulfate sodium (DSS) in drinking water. At days 1 and 3 were injected intraperitoneally with 1 x 106 of UC-MSCs or TLR3 and TLR4-preconditioning UC-MSCs. We evaluated Clinical signs of disease, body weights daily, plasma cytokines levels, colon length and histological changes. Results: The infusion of TLR3-preconditioned UC-MSCs significantly ameliorated the clinical and histopathologic severity of DSS-induced colitis compared with wild type UC-MSCs or TLR4-preconditioned UC-MSCs. In contrast, infusion of TLR4-preconditioned UC-MSCs significantly increased the clinical signs of disease, colon shortening and histological disease index. Conclusions: These results suggest that the immunomodulatory properties of UC-MSCs can be differentially modulated by TLR agonists. Also, these data showed that the short in-vitro TLR3-preconditioning enhances the therapeutic efficacy of UC-MSCs, which can be very useful for treatment or amelioration of proinflammatory diseases.
Developing bacteriophage Qβ as versatile carrier for anti-carbohydrate cancer vaccine: influence of antigen design on antibody responses (VAC8P.998)
Authors: Yin Zhaojun , Gildersleeve Jeffrey , Finn M. g. , Huang Xuefei
Published: January 2023

Vaccines that aim to stimulate the immune system to eradicate tumor cells are very attractive method for cancer therapy. One such strategy, active vaccination against tumor associated carbohydrate antigens (TACAs), which are overexpressed on malignant cells and strongly correlated with tumor progression and metastasis, has shown high potential in clinical studies. However, two major challenges need be circumvented to develop effective TACA anticancer vaccines. First, TACAs are self-antigens and tolerated by the immune system; Second, TACAs are weak T cell independent antigens and cannot generate long lasting immune responses when administered alone. We are interested in exploration of bacteriophage Qβ as versatile platforms for the development of carbohydrate based cancer vaccine. The highly repetitive array of subunit in Qβ permits the organized display of antigens on the surface of the particle. The Qβ also contains natural T-helper epitopes and pathogen associated molecular patterns, which can engage innate immunity and activate the immune system. Herein, we present the vaccination studies with Tn antigen conjugated to bacteriophage Qβ. The effect of antigen density, nature of linker, and immunization routes on the quality and diversity of antibody responses was analyzed. With the optimal construct, significant preventive and therapeutic effects were achieved on highly aggressive TA3Ha tumor model.
Liposomal vaccine formulations incorporating CpG enhance multiple parameters within the lymphatic system that improve immunity (VAC8P.999)
Authors: Neeland Melanie , Meeusen Els , de Veer Michael
Published: January 2023

Liposomal vaccine formulations incorporating stimulants that target innate immune receptors have been shown to significantly increase vaccine induced immunity. Following vaccination, innate cell populations carry antigen from the injection site, via the afferent lymphatic vessels, into the local lymph node. Antigen-specific and activated lymphocytes then leave the lymph node via the efferent lymphatics to perform their effector function. Using unique in vivo models of ovine lymphatic cannulation, this study investigates the immune response occurring in afferent and efferent lymph after vaccination of liposomal delivery systems incorporating the innate stimulator CpG. We show that the addition of CpG to liposomal vaccine formulations enhances DC associated antigen transport and uniquely increases MHC II expression on antigen carrying monocytes. RNA sequencing experiments revealed that 72h after injection of CpG, signatures associated with inflammation, interferon signaling and Th17 cell differentiation are significantly increased in cells migrating to the local lymph node. CpG also promotes the persistence of antigen specific antibodies and significantly increases IFNγ production by CD8 T cells. The results of this study demonstrate the real time in vivo kinetics of the peripheral contribution to liposome based delivery systems and add to our understanding of the immunogenic function of poly(I:C) and CpG when employed as adjuvants within liposomal vaccine formulations
Intranasal vaccination with live attenuated influenza vaccine increases diversity of the human nasal microbiome (VAC8P.1001)
Authors: Lambert Nathaniel , Salk Hannah , Kennedy Rick , Chia Nicholas , Grill Diane , Jacobson Robert , Poland Gregory
Published: January 2023

Pre and post-licensure studies have supported the safety of the intranasally administered live, attenuated influenza vaccine (LAIV). The objective of this study was to measure changes to the nasal microbiome in subjects who received LAIV. We hypothesized that administration would result in only a transient alteration of the nasal microbiome. To test this hypothesis, we swabbed the anterior nares just before vaccination (Day 0), and on Day 7 and Day 28. We performed next-generation sequencing on amplified 16s rRNA genes. There was a significant increase in alpha diversity (Observed, CHAO, and ACE) from Days 0 to 7 (p = 0.02, 0.00, 0.01; respectively) and Days 0 to 28 (p = 0.05, 0.03, 0.05; respectively). When measuring differences in specific observed taxanomic units (OTUs), we identified a significant increase in Flavobacteriaceae and Enterobacteriaceae after vaccination. These data suggest that LAIV may increase the ability of these less abundant OTUs to establish a community niche and, therefore, could be clinically relevant. We have begun to investigate the second objective of this study, which is to correlate microbiome composition with mucosal immunity to influenza virus. To our knowledge, this is the first study to investigate LAIV-induced alterations to the human microbiome. These data may lead to a better understanding of the influences of the host microbiome on the response to vaccine, and perhaps infection.
Enhanced anti-retroviral cytotoxic T cell response by PD-1 blockade (VIR4P.1015)
Authors: Li Wen , Green William
Published: January 2023

LP-BM5 retrovirus induces a complex disease featuring an acquired immunodeficiency syndrome termed murine AIDS (MAIDS) in susceptible strains of mice, such as C57BL/6 (B6). Our lab has previously shown that a cytotoxicT lymphocyte (CTL) response to MAIDS-associated epitopes that are shared by MAIDS-associated terminal B-cell lymphomas can be detected in uninfected, MAIDS-associated tumor cell primed B6 mice. We further confirmed that B6 mice infected with LP-BM5 retrovirus can generate equally robust anti-MAIDS tumor CTLs compared to the B6 mice primed with tumor cells. To determine if the anti-tumor CD8 T-cell responses can be enhanced by interrupting the down-regulatory PD-1 pathway specifically in the CD8 T-cell compartment, thus to treat MAIDS-associated B-cell lymphomas, we determined whether adoptive transfer of PD-1KO CD8 T cells with or without total CD4 T cells, was able to protect Rag-1KO mice from B6-1710 tumor challenge. Rag-1KO recipients that received B6.PD-1KO.CD8 T cells showed delayed tumor growth following B6-1710 tumor challenge compared to Rag-1KO mice that received equal numbers of w.t B6.CD8 T cells. This enhanced anti-tumor activity was similar to what was observed in mice that received combined PD-1 blockade in CD8 T cell compartment and depletion of CD4 T regulatory cells. Our data indicate that anti-tumor CD8 T cell response can be increased by interrupting the down-regulatory, CD8 T-cell mediated PD-1 pathway.
CNS stromal cells modulate antiviral CD8 T cell immunity (VIR4P.1009)
Authors: Swanson Phillip , McGavern Dorian
Published: January 2023

Lymphocytes are essential for control of neurotropic viruses such as HSV, HIV, and JC virus. Understanding the mechanisms involved in the regulation of these cells is essential for the development of therapeutic interventions to treat central nervous system (CNS) infections. Using two-photon microscopy, we observed that CD8 T cells responding to a viral infection interact with meningeal stromal cells (MSCs), a population of specialized fibroblasts that comprise a dense, collagenous fiber network within the lining of the brain. Little is known about the immunological functions of MSCs. To gain novel insights into these cells, we conducted genomic analyses of purified MSCs at different time points following CNS infection with lymphocytic choriomeningitis virus (LCMV). Our analyses revealed that MSCs responded to virus infection through induction of a robust immunological program that included genes associated with antigen presentation, chemotaxis, immune regulation, among others. We also observed in vivo that MSCs were able to present viral-peptide MHC I complexes, which corroborated our intravital imaging studies showing direct, stable interactions between MSCs and anti-viral T cells. MSCs possess the capacity to regulate these interactions through expression of multiple immune regulators such as PD-L1, galectin-9, and PVR. Our data demonstrate that MSCs are a novel CNS-resident antigen presenting cell with the ability to modulate antiviral T cells following infection.
Anti-viral effects of CGRP in mixed glial cells infected with LP-BM5 virus (VIR5P.1022)
Authors: Grlickova-Duzevik Eliza , Malon Jennifer , Cao Ling
Published: January 2023

LP-BM5 retrovirus infection is an established murine model used to study HIV infection in humans due to that susceptible strains of mice (including C57BL/6(B6) mice) develop immunodeficiency syndrome (termed as MAIDS) following LP-BM5 infection. Previously, we observed that calcitonin gene related protein (CGRP), a neuropeptide produced primarily by sensory neurons, reduced LP-BM5 viral loads in infected mixed glial cells (primarily astrocytes and microglia). The mechanism through which CGRP decreases the viral loads is still not known. To elucidate the role of CGRP in glial LP-BM5 infection, we first determined that CGRP did not induce an increased cell death post-infection or directly decrease the viral infectivity. Then, we investigated whether CGRP could affect viral infectivity by enhancing the glycosylation of the cell receptor for viral attachment, murine cationic amino acid transporter 1 (mCAT-1). Our data indicate that blocking glycosylation by Tunicamycin did not affect CGRP’s anti-viral effects. Further, we tested the involvement of p38 MAPK pathway in CGRP-induced inhibitory effects. Preliminary experiment using a p38 inhibitor, SB203580, suggests a p38 mediated mechanism. Future studies will further elucidate the role of p38 and examine the role of other CGRP downstream pathways on LPBM5 infection.
Macrophage depletion ameliorates nephritis induced by pathogenic antibodies (BA12P.111)
Authors: Chalmers Samantha , Chitu Violeta , Stanley E. Richard , Putterman Chaim
Published: January 2023

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by the production of autoantibodies and inflammatory damage in target organs. Kidney involvement affects up to 60% of lupus patients, and is responsible for significant morbidity and mortality. Previous studies using methods to reduce macrophage number have reached conflicting conclusions regarding the role of macrophages in lupus nephritis (LN). Moreover, “off target” effects occur in mice congenitally deficient in macrophages. In this study, we investigated the role of macrophages in mice receiving anti-glomerular antibodies, or nephrotoxic serum (NTS), an experimental model which closely mimics the immune complex mediated disease seen in LN. GW2580, an oral kinase inhibitor monospecific for the CSF-1 receptor, was used as a novel and highly selective method for macrophage depletion. We found that GW2580-treated NTS challenged mice did not develop the significant increases in levels of proteinuria, serum creatinine or serum urea, seen in control treated mice. Further, GW2580-treated mice were protected from the robust kidney expression of inflammatory cytokines associated with LN seen in control treated mice, including RANTES, IP-10, VCAM1, MCP-1 and IL-6. IBA-1 staining confirmed profound depletion of macrophages in glomeruli of GW2580-treated mice. Our results support an important role of macrophages in LN, and suggest targeting this cell type as a promising approach to the treatment of LN.
Pathogenesis of cell death in lupus bone marrow (BA12P.112)
Authors: Zhuang Haoyang , Han Shuhong , Xu Yuan , Li Yi , Yang Lijun , Reeves Westley
Published: January 2023

Pristane-treated mice develop lupus mediated by TLR7-driven production of IFNα (autoantibodies, nephritis) and TNFα (anemia). Anemia is IFN-independent, is caused by TNFα produced by bone marrow (BM) neutrophils, and is associated with a striking accumulation of dead BM niche cells and erythroid precursors in the BM. TNFα and dead cells also were found in BM from anemic SLE patients. The dead cells may drive TNFα/IFNα production by releasing TLR7 ligands. Fas-deficient (B6/lpr) mice were completely refractory to induction of pristane-lupus implicating cell death in its pathogenesis. Interestingly, accumulation of dead cells was abolished in BM from B6 TNFα-/- mice, suggesting that they were killed by TNFα. Signaling through both Fas and the TNF receptor I can activate caspase-3 and processing of the BCL-2 family member BID, which is involved in a mitochondrial amplification loop for Fas-mediated apoptosis. However, pristane injection in BID-/- mice induced TNFα and IFNα production as well as autoantibody production comparable to wild type B6 mice. We conclude that the requirement for Fas signaling in pristane-lupus involves the death of “Type I” cells, which undergo Fas-mediated apoptosis without a requirement for truncated BID and its crosstalk with the mitochondrial cell death pathway.
Autoreactive B cell tetramer analysis reveals a type I interferon-mediated marginal zone precursor B cell defect in autoimmune BXD2 mice (BA12P.102)
Authors: Hamilton Jennie , Li Jun , Wu Qi , Yang PingAr , Luo Bao , Bradley John , Taylor Justin , Randall Troy , Mountz John , Hsu Hui-Chen
Published: January 2023

In human systemic lupus erythematosus and rheumatoid arthritis, development of self-reactive B cells has been associated with defective B-cell checkpoints prior to somatic hypermutation and class-switch recombination in germinal centers (GCs). To identify mechanisms leading to checkpoint defects in the periphery, sera from BXD2 autoimmune mice were screened on a PEPperPRINT Microarray. Highly specific autoepitopes were narrowed down via Discotope epitope analysis and high-throughput ELISA and ELISPOT analyses. Three peptides La, snRNP, and fibromodulin-citrulline (fib-cit) were used to make tetramers (tet) for characterization of B cell subsets in the spleen. Analysis of fib-cit tet+ B cells revealed an age-dependent 5 fold increase in tet+ B cells, especially within the marginal zone-precursor (MZ-P) (10.6% vs 4.85%) and GC (7.3% vs 0%) subsets, in BXD2 vs B6 mice. While the total frequency of La and snRNP tet+ B cells was not different in BXD2 vs B6, there was also an increase in the MZ-P (7.66% vs 1.51%) phenotype. Sorted tet+ B cells exhibited a >100-fold enhanced ability to secrete specific autoAb compared to tet- B cells. Additionally, there was significantly higher CD69 expression (75.5% vs 5.71%), a type I interferon (IFN)-induced protein in MZ-P B cells in BXD2 vs B6 mice. Together, these results establish a general method for characterization of autoAg specific B cells and suggest a checkpoint defect at the MZ-P stage as a result of type I IFN stimulation.
Redox regulation of a new autoimmune mouse model, glucose-6-phosphate isomerase peptide induced arthritis in mice (BA12P.108)
Authors: Yang Min , Holmdahl Rikard
Published: January 2023

Human glucose-6-phosphate isomerase (hG6PI) peptide-induced arthritis is established by immunizing C57Bl/6N.Q (B6N.Q) mice with single hGPI325-339 peptide emulsified in complete freund’s adjuvant. Immunized B6N.Q mice start to develop arthritis on day 10-12, reach to the peak value of severity on day 14-20 and then recover during the next 7-10 days. This peptide-induced arthritis mouse model represents an acute arthritic disease progression. By taking advantage of μMT mice (B cell deficient) and TCRβ-/- (T cell deficient) mice, we have demonstrated that this peptide-induced arthritis is T cell- and B cell- dependent. In addition, by using Ncf1 mutant mice, which are lack of oxidative burst, we have found that reduced reactive oxygen specifics aggravate and prolong the peptide-induced arthritis. Phenotypic analysis results show that increased frequency and absolute numbers of Th17 cells, accompanied by significant increased CD11b+cells, are observed in Ncf1 mutant mice comparing with wild-type mice; moreover, IL-17 levels were dramatically increased in isolated splenic cells or lymph nodes cells from immunized Ncf1 mutant mice when restimulated with hGPI325-339 peptide. Moreover, in vitro experiments suggest that APCs from Ncf1 mutant mice show higher capability to present hGPI325-339 peptide to stimulate hGPI325-339 peptide specific T cell hybridoma, which suggest that enhanced antigen-presentation capability may aggravate the arthritic disease in Ncf1 mutant mice.
TNF receptor superfamily costimulation couples TCR signal strength to thymic Treg differentiation (BA13P.128)
Authors: Farrar Michael , Mahmud Shawn , Manlove Luke , Schmitz Heather , Xing Yan , Wang Yanyan , Owen David , Schenkel Jason , Boomer Jonathan , Green Jonathan , Yagita Hideo , Chi Hongbo , Hogquist Kristin
Published: January 2023

Regulatory T cells express several TNF receptor superfamily (TNFRSF) members, but their role in thymic Treg development is undefined. We demonstrate that Treg progenitors express high levels of the TNFRSF members GITR, OX40, and TNFR2. Expression of these receptors requires TAK1 and CD28 and correlates directly with TCR signal strength. Neutralizing TNFSF ligands markedly reduced Treg development. Conversely, TNFRSF agonists enhanced Treg differentiation by augmenting IL2R/STAT5 responsiveness. Importantly, GITR-L led to a dose-dependent enrichment of lower-affinity cells within the Treg repertoire. In vivo, GITR- or OX40-deficiency modestly attenuated Treg development; in contrast, combined inhibition of GITR, OX40 and TNFR2 abrogated Treg development. Thus TNFRSF expression on Treg progenitors translates strong TCR signals into molecular parameters that specifically promote Treg differentiation and shape the Treg repertoire.