bluuebunny/crossref_metadata_2025 huggingface dataset:
Bacterial metabolites reduce intestinal permeability to protect against food allergen sensitization (MUC8P.724)
Authors: Feehley Taylor , Aitoro Rosita , Stefka Andrew , Nagler Cathryn
Published: January 2023

Food allergies are a growing public health concern. Our laboratory has recently identified a consortium of Firmicutes from the Clostridia class that can protect germ free (GF) or antibiotic (Abx)-treated mice against sensitization to peanut. This Clostridia consortium induces an early innate cytokine response in the colonic lamina propria that is characterized by the production of the barrier protective cytokine IL-22. By measuring the concentrations of two immunodominant peanut allergens, Ara h 2 and Ara h 6, in serum post gavage we demonstrated that Clostridia-induced IL-22 is necessary and sufficient to reduce intestinal permeability in neonatal Abx-treated mice. Administration of FITC-labeled dextran molecules of different sizes has further interrogated the effect of Clostridia colonization on intestinal permeability to food allergens. Treatment of GF mice with heat-killed Clostridia did not induce IL-22 or reduce intestinal permeability, suggesting a role for bacterial metabolites in mediating protection. Metabolomic analysis revealed that our Clostridia consortium produces high levels of the short chain fatty acids acetate and butyrate. Administration of acetate or butyrate to GF mice showed that only butyrate induced IL-22 and reduced the concentration of peanut allergen in the blood after challenge. We propose that production of butyrate is one mechanism by which Clostridia can influence intestinal permeability and host susceptibility to allergic sensitization.
Postnatal colonization shapes the immunologic development of the neonatal intestinal barrier. (MUC9P.751)
Authors: Singer Jeffrey , Blosser Emily , Randolph David , Weaver Casey
Published: January 2023

Microbial colonization of the newborn intestines begins immediately following birth and continues gradually over a period of weeks in both mice and humans. Many preterm infants are unable to secure their intestinal barrier against opportunistic pathogens, resulting in Late-Onset Sepsis (LOS). Here we present a novel murine model of Gram-negative LOS that mimics the natural disease course seen in premies-colonization absent tissue destruction, translocation across the intestines, and bacteremia resulting in sepsis and death. We find that maternal exposure to broad-spectrum Gram-negative (Gentamicin), but not Gram-positive (Vancomycin) antibiotics profoundly affects survival following infection with Klebsiella pneumoniae. Concordantly, neonates from Gentamicin-exposed dams show marked decrease in IL-22 production from Innate Lymphocytes and decreased Tumor Necrosis Factor-alpha (TNF-a) from both CD4+ and CD8+ T cells. These results offer a novel model to study bacterial translocation in the neonatal gut and suggest possible mechanisms through which the intestinal barrier is maintained during the physiologic colonization of the mammalian intestines.
Commensal bacteria-induced MyD88 signaling regulates intestinal permeability to food allergen via anti-microbial peptide and mucin production (MUC9P.741)
Authors: Pawar Sandeep , Feehley Taylor , Nagler Cathryn
Published: January 2023

Intestinal epithelial cells (IEC) play a critical role in maintaining the integrity of the mucosal barrier. We have recently shown that germ free mice exhibit increased intestinal permeability to food allergens, suggesting a role for microbiota-derived signals in protecting the barrier. Decreased permeability to the peanut allergen Ara h 6 in mice selectively colonized with a consortium of Firmicutes in the Clostridia class is associated with upregulation of antimicrobial peptide expression (RegIIIβ/γ) and the production of mucins (MUC2/13). RegIIIβ/γ are well known targets of MyD88 signaling. We hypothesized that Clostridia signals through MyD88 in IECs to enhance barrier protection and generated mice lacking MyD88 in IECs (Myd88ΔIEC). Myd88-/- and Myd88ΔIEC mice exhibit increased permeability to Ara h 6, downregulated expression of RegIIIβ/γ in IECs, and reduced production of MUC2/13 in goblet cells. Antibiotic mediated depletion of commensal bacteria exacerbated the permeability defect in Myd88ΔIEC mice and also led to a barrier defect in MyD88-sufficient littermate controls. Colonization with Clostridia rescues this defect in control mice but not in the Myd88ΔIEC mice indicating that signaling through MyD88 in the intestinal epithelium is necessary for bacteria-induced barrier protection. This restricted permeability of the gut limits the access of dietary antigens to the systemic circulation and contributes to protection against allergic sensitization in these mice.
Peptide microarrays for coverage of sequence diversity in monitoring the B cell immune response (TECH2P.911)
Authors: Castro Aaron , Stephenson Kathryn , Neubauer George , Korber Bette , Barouch Dan , Pawlowski Nikolaus , Zerweck Johannes , Reimer Ulf
Published: January 2023

Pathogens such as HIV or other viruses exhibit considerable sequence diversity as a strategy to escape the host immune system. However, sequence diversity is not limited to infectious agents. The increasing application of DNA sequencing indicates diversity between individuals (germ line mutations) and within individuals (somatic mutations) for many proteins. Such mutations can be connected to diseases such as cancer or autoimmune disease. The knowledge of antibody reactivities within individuals against self and foreign antigens is important for diagnosis, patient stratification, prevention and treatment of disease. Here we describe the application of high content peptide microarrays presenting thousands of peptides to be assayed with ~1µl serum sample of a donor to asses the reactivity pattern of antibodies towards a number of given antigens under consideration of sequence diversity using HIV as an example. A library of 6564 overlapping peptides represents the immunogenic fraction of the HIV proteome and covers the majority of the available sequences from the LANL global HIV-1 database. This high content library enables the assessment of antibody reactivity within different individuals independent on the serotype of the infecting HIV-viruses as well as the breadth and depth of antibody binding after boosting the immune system by vaccination. This concept can be easily adapted to other pathogens as well as self antigens such as tumor-associated antigens.
Development of a RANKL ELISPOT assay as a tool for periodontal disease research (TECH2P.913)
Authors: Zhang Jinrong , Cole Leah , Brown Anna , Ma Bing , Anosova Natalie , McCluskey Jacqueline
Published: January 2023

Periodontal disease (PD) results from the immune response to the bacterial components of the sub-gingival plaque. PD progresses from gingival inflammation, to bone resorption and ultimately tooth loss. Receptor activator of nuclear factor-kappa B ligand (RANKL) is critical for regulating inflammatory bone resorption. Studies have shown that activated T and B cells are one of the main sources of RANKL in PD. The ELISPOT assay is widely used tool that can measure antigen specific cellular immune responses. We developed a RANKL specific ELISPOT assay for quantifying the number of RANKL secreting cells in the murine Porphyromonas gingivalis induced bone loss model. Various potential positive controls were tested for their ability to induce RANKL secretion including IL-7, ionomycin, LPS, and α-CD3 combined with α-CD28. We found that the combination of α-CD3 and α-CD28 was the most effective positive control for cells collected from either murine lymph nodes or spleens. Gingipains are trypsin-like cysteine proteases and have been implicated as important virulence factors for Porphyromonas gingivalis. When we performed the RANKL ELISPOT using the purified native gingipain complex as the recall antigen, we were able to detect more RANKL secreting cells from the mandibular lymph nodes of Porphyromonas gingivalis challenged animals than in the buffer challenged control animals.
Isolation of broadly neutralizing antibodies from single human memory B cell against human influenza A viruses (TECH2P.922)
Authors: Fu Ying , Zhang Zhen , Sun Jiusong , Zhu Quan
Published: January 2023

During the past five years, broadly neutralizing antibodies (BnAbs) represent promising prophylactic and therapeutic treatments against influenza. Here, we have developed a novel method to isolate broadly neutralizing antibodies against influenza A virus from human memory B cells. Antigen-specific memory B cells were isolated from human PBMCs using tetramerized H3 (A/Brisbane/10/2007) hemagglutinin (HA) trimers. The H3-reactive single memory B cells were sorted into plates and stimulated in vitro. More than 40% sorted B cells produced on average 200 ng/ml IgG in the supernatant after 14 days. Supernatants from the expanded B cells were measured for their heterosubtypic binding specificity and neutralizing activity by MSD or a highly sensitive neutralization assay. The selective clones were recovered by single cell RT-PCR. Through screening 2688 memory B clones from 7 individuals, 11% clonable memory B cells were reactive with H3 hemagglutinin. Among them, H3/H7, H3/H7/H1 and H3/H7/H1/influenza B heterosubtypic binding population were 16%, 6.9% and 0.35%, respectively. A new broadly neutralizing Ab, 3I14 encoded by the IGHV3-30 germline gene, was further characterized and shown to possess cross-reactive binding and neutralization activity against both group 1 and group 2 influenza A viruses. Epitope mapping and competition assay revealed a highly conserved epitope located in the HA stalk. Passive transfer of this antibody also conferred in vivo protection.
An innovative systems perspective of protein-protein interactions in the physiologic T cell antigen receptor signaling network (TECH2P.929)
Authors: Neier Steven , Smith Stephen , Davis Tessa , Sinnwell Jason , Nevala Wendy , Enninga Elizabeth , Markovic Svetomir , Eckel-Passow Jeanette , Sciallis Gabriel , Wieland Carilyn , Torgerson Rochelle , Neuhauser Claudia , Schrum Adam
Published: January 2023

Signaling networks and the protein interactions that define them are of great interest, but techniques to assess these networks in unmanipulated primary human cells are limited. We have developed and applied a novel technological and analytical platform to measure proteins in shared complexes detected by exposed surface epitopes (PiSCES). Immunoprecipitation reactions are performed in multiplex using microsphere beads to measure 20+ proteins and 210+ distinct pair-wise protein associations that are important for T cell antigen receptor signaling. Jurkat T cells stimulated with superantigen generated an extensive PiSCES signature using this method and provided a dataset from which we optimized an analytical package. The system is currently being utilized to address the hypothesis that T cells isolated from skin lesions of autoimmune alopecia areta (AA) patients will have a unique PiSCES signature compared to those isolated from non-AA donors. To investigate this, we have isolated non-engineered primary cells from AA patient skin lesions and healthy controls, and have begun to identify differences in their PiSCES signatures, either directly after isolation or after stimulation with anti-CD3 and anti-CD28. We conclude that this novel PiSCES approach is capable of generating protein-protein interaction signatures that provide a unique view of TCR signalosome activity solicited from physiologic non-engineered primary human patient samples.
MIF inhibition as novel treatment for autoimmune myocarditis and dilated cardiomyopathy (THER7P.956)
Authors: Nalawade Saisha , Jamison Braxton , Casabar Julian , Maldonado Daniel , Forsthuber Thomas
Published: January 2023

Myocarditis is an inflammatory disease of the myocardium and a major cause of sudden death in young adults. It is characterized by the presence of immune infiltrates in the myocardium and often progresses to dilated cardiomyopathy (DCM). Despite the inflammatory nature of this autoimmune disease, immunosuppressive treatments with glucocorticoids (GCs) such as dexamethasone (Dex) have not been very effective in preventing myocarditis and progression to DCM. In addition, some patients develop resistance to GCs. We hypothesized that macrophage migration inhibitory factor (MIF) may play a role in resistance to GCs, as it is the only known pro-inflammatory cytokine to be induced by GCs. Importantly, MIF counter-regulates GC-mediated immunosuppression. Using the experimental autoimmune myocarditis (EAM) model, we observed that MIF-/- mice treated with Dex were highly resistant to disease and progression to DCM. In addition, we observe lower expression of CCL3 mRNA in MIF-/- mice treated with Dex compared with wild-type mice during the onset of EAM, which indicates that MIF promotes recruitment of inflammatory cells to the myocardium. Our results suggest that therapeutic inhibition of MIF may increase the efficacy of GC treatment. This study will allow us to better understand the mechanism by which MIF affects treatment of myocarditis by inhibiting the therapeutic effects of GCs.
The spleen is the primary site for Breg induction and is required for anti-TIM-1 mediated tolerance (TRAN2P.963)
Authors: Mohib Kanishka , Ding Qing , Cherukuri Aravind , Rothstein David
Published: January 2023

IL-10+ B cells (Bregs) have potent regulatory activity and can transfer tolerance in auto/allo immune models. We showed that: TIM-1 (T1) is an inclusive Breg marker; T1+ B cells transfer allograft tolerance; and anti-T1 induces IL-10+ T1+ Bregs. However, little is known about where Bregs are induced or act in vivo. We found that most Bregs reside in the spleen (85%) vs LN (1%) or marrow (15%), and few are found in islet allografts. Anti-T1 increased Bregs in the spleen from 2% to 7% of B cells, but had no effect in LN (0.5%) or marrow (1%). In splenectomized (spl-X) mice, anti-T1 did not increase Bregs in LN or marrow. Apoptotic cells (ACs) can induce Bregs in vivo. ACs increased Bregs from 2% to 5% in spleen and from 0.5% to 2% in LN. However, after spl-X, ACs did not increase Bregs in LN. Hence, Bregs are induced in the spleen, but then may migrate. Next, we examined the importance of the spleen for Breg-mediated allograft survival (GS). Anti-T1 was unable to induce long-term GS in spl-X islet recipients vs. controls (MST 24d vs. 100d; p=0.002). To confirm that anti-T1 acts through Bregs, we used inducible hCD20ERT2-CreXIL-10fl/fl mice. In such “B cell-IL-10KO” mice, anti-T1 was unable to prolong GS compared to controls (MST 20d vs. >50d). Moreover, LTβR-KO mice (lack LNs) that received alloreactive T cells, exhibited longer islet GS (MST 18d) vs spl-X LTβR-KO recipients (MST 7d p=0.02). Thus, our data elucidate a vital role for the spleen in Breg induction and function.
Effect of selective CD28 blockade with FR104 alone or combined with CD154 blockade in monkey cardiac allograft transplantation. (TRAN2P.967)
Authors: Azimzadeh Agnes , Zhang Tianshu , Braileanu Gheorghe , Sun Wenji , Kronfli Anthony , Sievert Evelyn , Harris Donald , Parsell Dawn , Dahi Siamak , Kubicki Natalia , Woodall Jhade , Uluer Mehmet , Cheng Xiangfei , Schwartz Evan , Quinn Kevin , Vanhove Bernard , Pierson Richard
Published: January 2023

Selective CD28 blockade, which preserves CTLA-4-dependent immune regulation, is a promising approach to induce tolerance. FR104, a novel CD28 antagonist, was evaluated in monkey heart transplant recipients that received αCD28 alone (n=3, FR104) or in combination with αCD154 (n=5, hu5C8), dosed to achieve receptor coverage for 84 days; results were compared to αCD154 alone (n=4). Acute (AR) and chronic (CAV) rejection scores were quantified histologically. Median graft survival time (MST) with FR104 (167d), hu5C8 (133d), and FR104+hu5C8 (173d) was not statistically different between groups. Rejection was seen in 1 of 3 animals with FR104 monotherapy despite CD28 receptor saturation, but not with additional CD154 blockade. CAV was decreased with FR104 (0.7±0.3) and FR104+hu5C8 (0.5±0.3) compared to hu5C8 (1.6±0.7 p<0.05). AR tended to be lower with FR104 (0.5±0.4, p=0.047) or combined therapy (0.8±0.4, p=0.07) on biopsy/explanted functional grafts relative to hu5C8 (1.4±0.6). Blood Tregs frequency on d7-21 was increased (fold change vs. baseline) with FR104 (1.5±0.2), hu5C8 (1.4±0.3), and FR104+hu5C8 (2.3±0.1). CD28 saturation became undetectable by d150 in blood and LN; subsequent graft failure was associated with alloantibodies. We conclude that selective non-activating CD28 blockade protects primate cardiac allografts from pathogenic alloimmunity, an effect enhanced by simultaneous CD154 blockade, supporting the hypothesis that CD28 blockade is potentially tolerogenic.
Histamine receptor 2 blockade reduces breast tumor development and metastasis (TUM9P.1010)
Authors: Vila-Leahey Ava , Oldford Sharon , Marignani Paola , Marshall Jean
Published: January 2023

Histamine has multiple immunomodulatory roles that impact innate and acquired immunity. Histamine receptor antagonists are widely used clinically to treat allergic disease and gastrointestinal disorders, but their ability to alter effective immune function to breast tumors is unknown. We hypothesized that histamine receptor antagonist treatment could alter immune function in mouse models of breast cancer, therefore altering the growth and spread of breast cancer. Two injectable breast tumor models (the 4T1-BALB/c and E0771-C57BL/6 model) and a spontaneous breast cancer model (STK-/-/NIC model) were utilized. Histamine receptor antagonists were administered in the drinking water prior to tumor cell injection or at time of weaning, respectively. The immune cell populations in the spleen and bone marrow of tumor-bearing mice were analyzed. Ranitidine and famotidine (Hrh2 antagonists) but not selective Hrh1 or Hrh4 antagonists significantly inhibited metastasis in the 4T1 model. Ranitidine treatment also caused significant primary tumor regression in the E0771 breast cancer model. In the STK-/-/NIC model, ranitidine increased latency and decreased the number of tumors per mouse at week 26. Bone marrow and spleen analysis revealed that oral ranitidine treatment significantly and selectively decreased monocyte populations. These studies suggest that HRH2 antagonists may have potential as a beneficial immunomodulatory agent in the prevention or treatment of breast cancer.
NK cell effector functions are differentially regulated by hypoxic conditions (TUM9P.1014)
Authors: Lim Seon Ah , Moon Yunwon , Park Hyunsung , Lee Kyung-Mi
Published: January 2023

NK cells provide the ﬁrst line of defense against tumor and viruses, without the requirement for prolonged pre-activation. However, anti-tumor function of NK cells is progressively dampened in the terminal stages of cancer patients, largely associated with the tumor-induced immune evasion mechanism. Hypoxic tumor environment provides once such mechanism providing NK cell tolerance, and become a potential therapeutic problem to overcome in the patients of solid cancer for NK immunotherapy. To better understand the effect of hypoxia on NK cells, we assessed the anti-tumor function of NK cells in response to decreasing concentration of O2 concentration. As expected, NK cells cultured in severe hypoxia, 0.5% O2, demonstrated diminished cytotoxicity and IFNg against A375 melanoma targets and failed to expand to a larger number ex vivo. However, to our surprise, NK cells cultured in mild hypoxia, 1.5% O2, were expanded to a much higher level and mounted increased anti-tumor effector function, both in normoxic and hypoxic conditions. NK cells grown in mild hypoxia demonstrate upregulation of major activating NK cell receptors, NCRs, while inhibiting apoptosis during ex-vivo expansion. Thus, NK cells respond differentially to O2 concentration, and mild hypoxic conditions can indeed facilitate the expansion and function of NK cells that can overcome NK cell intolerance in the patients undergoing NK cell immunotherapy.
Regulatory B cells accumulate in tumor-draining lymph nodes and promote tumor growth (TUM9P.1015)
Authors: Ganti Sheila , Albershardt Tina , Iritani Brian , Ruddell Alanna
Published: January 2023

Tumor metastasis to regional lymph nodes is a reliable marker for determining cancer progression and is associated with poor prognosis. However, mechanisms utilized by the tumor to modify the tumor-draining lymph node (TDLN) to facilitate metastasis remain to be identified. TDLNs from B16-F10 melanoma-bearing mice exhibit considerable B cell accumulation and lymphatic sinus growth (lymphangiogenesis) which precede and predict metastasis of solid tumors. Phenotypic characterization of B cells in mice bearing B16-F10 melanoma in the rear footpad identified preferential accumulation of T2-MZP B cells in the TDLN. Comparison of TDLNs and non-draining LNs of tumor-bearing mice with LNs and spleens from naïve mice determined that this pattern of B cell accumulation was restricted to the TDLN. B cell-deficient and immunocompetent mice reconstituted with T2-MZP B cells but not with other B cell subsets displayed accelerated tumor growth, demonstrating that T2-MZP B cells possess regulatory activity. Unlike splenic regulatory B cells, however, TDLN T2-MZP B cells did not exhibit increased IL-10 secretion, nor did they promote the conversion of naïve CD4+ T cells to CD4+Foxp3+ Tregs in the TDLN. These findings demonstrate that tumors initially signal via the lymphatic drainage to stimulate the preferential accumulation of T2-MZP regulatory B cells. This local response may be an early and critical step in generating an immunosuppressive environment to permit tumor growth and metastasis.
Enterotoxigenic Bacteroides fragilis induces oncogenic regulatory T cells (TUM9P.1000)
Authors: Geis Abby , Dejea Christine , Fan Hongni , Wu Xinqun , Wu Shaoguang , Huso David , Sears Cynthia , Housseau Franck , Pardoll Drew
Published: January 2023

Enterotoxigenic Bacteroides fragilis (ETBF) is an occasional inducer of inflammatory diarrhea and acute colitis, and preliminary studies have recently demonstrated an association with inflammatory bowel disease and colorectal cancer in humans. The pathogenesis of ETBF can largely be attributed to the secretion of a toxin, BFT (B. fragilis toxin), which has a profound effect on the colonic microenvironment. BFT promotes cleavage of E-cadherin, activates NF-kB and Wnt/b-catenin signaling, and stimulates the secretion of pro-inflammatory cytokines, such as IL-6, IL-8, and TNF-a. The inflammation associated with ETBF is characterized by robust IL-17 cytokine secretion, and in MinAPC/+ mice, ETBF induces IL-17-dependent distal colon tumorigenesis. In an attempt to elucidate the immunologic differences between the distal and proximal colon, we found ETBF induced a greater density of Foxp3+ Tregs in the distal colon. Furthermore, those Tregs initiate the oncogenic IL-17 response to ETBF. While investigating the Treg and IL-17 responses to ETBF, we found that depletion of Tregs in ETBF-colonized C57BL/6 Foxp3DTR-GFP MinAPC/+ mice mitigated intestinal IL-17 production as well as colonic neoplasia. Antibody blockade of IL-2, but not genetic ablation of IFN-γ, restored ETBF-induced microadenoma formation. These findings unveil a new mechanism of cancer-induced inflammation whereby an overt Treg response to intestinal bacteria promotes colon tumorigenesis.
Oral rapamycin (eRapa) requires IFN-γ and promotes γδ T cell cytotoxicity to prevent carcinogen and inflammation-induced dermal cancer (TUM9P.1003)
Authors: Dao Vinh , Pandeswara Srilakshmi , Liu Yang , Hurez Vincent , Liu Aijie , Curiel Tyler
Published: January 2023

Rapamycin, a mammalian target of rapamycin (mTOR) inhibitor, prevents distinct cancers in mice and prolongs life, making it a candidate cancer prevention (and life extension) agent. We hypothesized that eRapa cancer prevention includes immune effects, as mTOR is pivotal in immunity. We tested immune effects in carcinogen (DMBA) and inflammation (TPA)-induced dermal carcinogenesis with eRapa daily at 14 ppm, which protected 100% from skin cancer in wild type (WT) mice. Tumor mTORC1 was not suppressed, supporting eRapa immune effects. γδ T cells and IFN-γ are protective in this model. In δ TCR KO (no γδ T cells) and IFN-γ KO mice, eRapa cancer protection was lost, confirming immune mechanisms. IFN-γ KO mice had significantly reduced CD69+Lamp1+GzB+CXCR3+ epidermal γδ T cells and CXCL10, suggesting IFN-γ is required for epidermal γδ T cell migration (via CXCR3/CXCL10), activation and cytolysis. Bone marrow (BM) chimeras showed eRapa skin cancer protection and maximum γδ T cell epidermal infiltration requires IFN-γ signals in BM (e.g., γδ T cells) and non-BM (e.g., epidermal) cells. Intratumor injections of WT but not Prf1 (perforin) KO γδ T cells regressed DMBA/TPA tumors in δ TCR KO mice on eRapa but not control, showing eRapa boosts anti-tumor γδ T cell cytotoxicity. Our data confirm eRapa cancer prevention through immune mechanisms, challenging the paradigm of mTOR effects solely on cancer cells for prevention, and suggesting novel immune-mediated life extension mechanisms.
Epigenetic modulation of CD1d-mediated antigen presentation by B cell lymphomas (TUM10P.1054)
Authors: Tiper Irina , Webb Tonya
Published: January 2023

Histone deacetylases (HDACs) are a family of enzymes that regulate diverse cellular events such as gene expression, cell proliferation, and immune pathways through deacetylation of their protein targets. We hypothesize that tumors use epigenetic mechanisms to dysregulate CD1d-mediated antigen processing and presentation, which leads to a functional impairment in the ability of natural killer T (NKT) cells to recognize and destroy cancerous cells. We examined CD1d-mediated antigen presentation to NKT cells following treatment with HDAC inhibitors (HDACi). Consistent with previous studies, we found that preventing deacetylation by treatment with Trichostatin A, a pan-HDACi, enhanced both CD1d and MHC class II-mediated antigen presentation. Similarly, treatment of B cell lymphomas with Panobinostat resulted in increased CD1d-dependent NKT cell responses, due at least in part to a dose-dependent increase in CD1d-cell surface expression. In addition, treatment with HDCAi results in a decrease in STAT3 expression as well as IL-10 secretion by B cell lymphomas. Overall, our studies demonstrate the efficacy of HDACi in restoring anti-tumor responses to B cell lymphomas through both cell-intrinsic and extrinsic factors. Collectively, these results suggest that HDACi may work to enhance the immune response and increases the immunogenicity of the tumor itself.
Humoral dysregulation among injection drug users: implications for HIV vaccine strategies (VAC11P.1103)
Authors: Kobie James , Samuel Memorie , Zheng Bo , Piepenbrink Michael , Bunce Catherine , Kiebala Michelle , Maggirwar Sanjay , Morse Diane , Keefer Michael
Published: January 2023

The primary objective of a preventative HIV vaccine is the induction of a persistent humoral response that mediates sterilizing immunity. Although modest, yet encouraging, a reduction in infections in low-risk individuals was obtained in the RV144 HIV vaccine efficacy trial; for high-risk individuals, including injection drug users (IDU) no substantial reduction in infection has been observed in efficacy trials. The impact of injection drug use on humoral responses is poorly defined, particularly in the context of HIV prevention. Through a cross-sectional assessment of peripheral B cells and plasma antibodies, we have identified substantial alterations in the HIV-/HepC- active heroin IDU population. IDU exhibited significantly increased total B cells compared to healthy subjects, that were associated with significantly increased memory B cells and plasmablasts. Additionally, although plasma total IgG levels were similar between IDU and healthy subjects, IDU had significantly higher titers of IgG3 and IgG4 subclass plasma antibodies. Total IgM plasma antibodies were also increased in IDU. This humoral dysregulation may be the consequence of a pro-inflammatory environment as evident by significantly increased plasma sCD40L in IDU. These results demonstrate extensive differences in the steady-state humoral profile of IDU, which could have the potential to severely influence the quality of the B cell and antibody response, and ultimate efficacy of future HIV vaccine strategies.
Polyguanine-conjugated antigens for scavenger receptor targeting and self-adjuvanting vaccines (VAC13P.1125)
Authors: Ciccotelli Jo Erika , Toussaint Helene , Erdos Geza , Donahue Cara , Watkins Simon , Falo Louis
Published: January 2023

We have recently developed a novel vaccine strategy that involves conjugating antigens to polyguanine sequences to fabricate aggregated particulates with exposed scavenger receptor ligands for in vivo targeting to antigen presenting cells. We have previously demonstrated that polyguanine-conjugated ovalbumin (Poly(dG)-OVA) leads to enhanced antigen-specific T cell and antibody responses. In this study, we show that exposing dendritic cells (DCs) to Poly(dG)-OVA results in increased antigen internalization that is inhibited by scavenger receptor ligands. Further, DCs internalizing Poly(dG)-OVA complexes exhibit increased cytokine secretion and inflammatory marker expression. In vivo, intradermal injection of Poly(dG)-OVA results in complex persistence in the skin for up to 7 days. Intradermally immunized mice demonstrated increased immune infiltrates in the skin and a persistent inflammatory cytokine profile. In these immunized animals, Poly(dG)-OVA particles reach the draining lymph nodes directly and in association with migratory DCs. We further demonstrate that Poly(dG)-OVA immunization of mice with established OVA-expressing melanoma tumors exhibited delayed tumor growth and improved survival. Taken together, these results suggest that immunization with polyguanine-conjugated antigens can induce more effective antitumor immunity by targeting DCs through scavenger receptors and inducing proinflammatory DC activation.
Heat shock proteins initiate DNA methylome remodeling to drive dichotomous immune responses. (VAC13P.1127)
Authors: Kinner Lauren , Binder Robert
Published: January 2023

Immunization with heat shock proteins (HSPs) can elicit dichotomous immune responses that are dependent on the dose administered. These responses are characterized by Th1 and Treg cells with low and high dose HSP respectively. The immune responses in either situation are dependent on dendritic cells (DCs) expressing HSP receptor CD91, but the exact mechanism controlling them are unresolved. Here, we focus on the molecular events which control gene transcription and drive this dose-dependent dichotomy. Our data demonstrates that HSP-CD91 signaling results GSK3β phosphorylation which stabilizes DNA methyltransferase (DNMT) levels. DNMTs control the epigenetic state of cells and thus regulate gene transcription and splicing. Thus, we hypothesize that HSP-CD91 signaling induces epigenetic remodeling in a dose-dependent manner, which drives cellular responses. We have mapped the DNA methylome of CD91+ cells following low and high dose HSP immunization. Differential methylation was observed across 229 gene regions in isolated CD91+ cells. These regions include genes involved in chemotaxis, signaling, and metabolism. We also show that neuropilin-1 (Nrp1) is differentially regulated in immunized mice. These studies reveal an early requirement for epigenetic changes and a role for Nrp1 in maintaining a Treg response to HSP.
Ebola virus matrix protein trafficking and cellular innate immune response (VIR9P.1149)
Authors: Subbiah Elankumaran , Sooryanarain Harini
Published: January 2023

Ebola virus (EBoV), a negative sense ssRNA virus (NSV) of family Filoviridae, causes severe hemorrhagic fever in humans, with case mortality rates up to 90%. Recent outbreak in 2014 of Ebola virus disease resulted in over 7500 deaths in West Africa. The viral matrix protein (VP40), is a structural protein involved in virus assembly and budding. Several studies have shown NSV matrix proteins are capable of nucleo-cytoplasmic shuttling (Newcastle disease, Nipah and Influenza) and modulate host innate immune response (Influenza and Marburg). Role of EBoV VP40 in disease pathogenesis remains elusive. Here, we analyzed the trafficking of VP40 and cellular innate immune response using wild type and mutant VP40 fused to EGFP. Our data shows that VP40 gets transiently localized to the nucleus at 6-10 h post-transfection with subsequent transport to the plasma membrane by 16-24 h. Blocking tyrosine phosphorylation by inhibiting phosphokinases or mutating tyrosine phosphorylation sites or blocking ubiquitination by a proteasome inhibitor prevent VP40 nuclear export, transport to the membrane and virus-like particle formation. We also found that VP40 expression lead to activation and nuclear translocation of pIRF3, STAT1 and NFkB (p50), and induced increased expression of LC3b at 24 h post-transfection. Our results suggest EBoV VP40 does not inhibit type I interferon pathway, and may have a potential role in disease pathogenesis by enhancing host inflammatory response and autophagy.
Pulmonary macrophage alterations associated with aging and SIV infection in rhesus macaques (VIR9P.1155)
Authors: Didier Elizabeth , Cai Y , Sugimoto C. , Liu D. , Midkiff C. , Walker E , Arainga M , Haupt E , Alvarez X , Fahlberg M , Kuroda Marcelo
Published: January 2023

Accelerated aging occurs in HIV-infected persons HIV, despite ART, as seen by earlier onset of chronic inflammatory diseases and HIV-associated non-AIDS (HANA) conditions than in non-HIV-infected individuals. In the rhesus macaque SIV model, we reported earlier that increasing monocyte turnover associated with tissue macrophage death predicted onset of terminal disease progression. Here, we hypothesize that macrophage dysregulation contributes to accelerated aging that occurs during SIV infection. We compared macrophage populations of the lung in older and younger uninfected and SIV-infected rhesus macaques. We observed that the ratio of alveolar macrophages (AM) to interstitial macrophages (IM) was significantly lower in chronic SIV-infected younger macaques and older uninfected monkeys compared to uninfected young macaques. The mechanisms differed, however, because the lower ratio in the older uninfected monkeys was due to lower numbers of AM while the shift during SIV infection of younger adults was due to increasing numbers of IM. Functionally, AM of older vs younger uninfected animals exhibited higher levels of pro-inflammatory cytokine secretion (TNFα, IL12) during incubation in medium yet produced lower levels of cytokines after induction with LPS ex vivo. During the acute phase of SIV infection, we observed a higher turnover of monocytes and IM in older compared to younger macaques. This suggests that AM become dysregulated during aging and SIV/HIV infection.
Toll-like receptor 8 signaling facilitates West Nile virus infection in mice (VIR9P.1161)
Authors: Paul Amber , Acharya Dhiraj , Le Linda , Stokic Dobrivoje , Leis A , Bai Fengwei
Published: January 2023

West Nile virus (WNV) is a neurotropic virus that can cause encephalitis, meningitis or possible death in humans. Human and mouse Toll-like receptor 7 (h/mTLR7) and human Toll-like receptor 8 (hTLR8) detect viral single-stranded (ss)RNA motifs. Importantly, mTLR7 recognizes WNV, as TLR7-/- mice are susceptible to lethal WNV infection. However, the role of TLR8 in WNV pathogenesis is not clear. In this study, we found that mice deficient in TLR8 (TLR8-/-) infected with WNV (2,000 plaque forming units) had improved percentage survival (65%) compared to wild-type (WT) controls (25%), which was coupled to reduced viral burden in the brains of TLR8-/- mice. These data suggest that TLR8 signaling facilities WNV infection in mice. Further mechanistic studies showed that expression of TLR7 and the interferon-induced anti-viral gene Isg-56 (IFIT-1) were significantly increased in TLR8-/- mice whole-brain samples and mixed neuronal cultures following WNV infection, compared to WT controls. These results indicate that TLR8 signaling may down-regulate TLR7 and Isg-56 expression, thus promoting WNV-induced neuroinvasive disease.
Activation of cellular innate immunity during primary hepacivirus infection (VIR9P.1137)
Authors: Manickam Cordelia , Reeves R.
Published: January 2023

Hepatitis C virus (HCV) is estimated to infect 170 million people worldwide causing global morbidity and mortality. The course of infection can either lead to natural resolution or chronicity, but the factors dictating these two phenotypes are poorly understood. In this study we utilized an understudied model of HCV infection, GBV-B infection of common marmosets, to characterize acute and chronic immune responses. Plasma viremia was detectable by day-3 post-infection (p.i.) and greater than 50% of animals remained viremic beyond day-28 p.i. In general, plasma viremia was correlated with elevated serum ALT, AST, ALP and GGT. Primary GBV-B infection was also associated with mobilization of innate natural killer (NK) and myeloid dendritic cells (mDCs), as evidenced by increased surface CXCR3 ­— suggesting trafficking to tissues, and increased intracellular Ki67 expression. Indeed, NK cell and mDC frequencies increased 2- to 5-fold in the liver associated with high viral loads at day-14 p.i. Additional cell accumulation was observed in mesenteric lymph nodes and spleen. Functionally, NK cells demonstrated increased expression of intracellular perforin during acute infection, but had no obvious changes in IFN-g or TNF-a secretion. These data indicate the accumulation of innate cells occurs well before development of adaptive T and B cell responses and is associated with virus clearance, suggesting innate cells could be targeted in future vaccine and immunotherapeutic modalities.
How filovirus innate immune antagonists abrogates maturation of human monocyte-derived dendritic cells (VIR9P.1141)
Authors: Yen Benjamin , Basler Christopher
Published: January 2023

Filoviruses, which includes both Ebolavirus (EBOV) and Marburgvirus (MARV), are negative-sense, single-stranded RNA viruses that cause severe hemorrhagic fever in humans with fatality rates that have approach 90% in some outbreaks. Antigen presenting cells such as dendritic cells (DCs) are early and sustained targets of filovirus infection in vivo, and in vitro filovirus infection potently blocks DC maturation and function. Both EBOV-VP35 and MARV-VP35 proteins block IFN-β production, in part, by inhibiting of the viral RNA sensor RIG-I. Further, EBOV-VP24 can inhibit the production of interferon-stimulated genes (ISGs) by blocking type I IFN signaling. We used a lentivirus-based expression system to determine the impact of these filoviral innate immune inhibitors on DCs. Our data demonstrate that EBOV-VP35 and MARV-VP35 can each suppress not only IFN-α/β production but also proinflammatory responses following stimulation of MDDCs with Sendai virus (SeV) or encephalomyocarditis virus (EMCV). Furthermore, either EBOV-VP35 or MARV-VP35 alone is sufficient to inhibit human DC maturation upon SeV infection. EBOV-VP24, despite having the ability to suppress ISG production, fails to block DC maturation induced by SeV or EMCV. Our work suggest that suppression of innate immune response by filoviral VP35s is sufficient to explain the potent blockade of DC function seen in filovirus infection and could be a potential the immune blockade during infection.
Increased activated platelet-T cell conjugates in patients with HIV infection: relationship between coagulation/inflammation and T cells. (VIR10P.1171)
Authors: Green Samantha , Smith Mindy , Hasley Rebecca , Stephany David , Harned Adam , Nagashima Kunio , Imamichi Tomozumi , Qin Jing , Rupert Adam , Ober Alexander , Lane H. , Catalfamo Marta
Published: January 2023

While antiretroviral therapies can successfully suppress HIV replication, there remains evidence of persistent immune activation, which is correlated with increased risks of all-cause mortality. Patients with elevated biomarkers of coagulation/inflammation such as IL-6, sCD14 and D-dimer (a fibrin bioproduct) are at an increased risk to develop non-AIDS defining illnesses such as cardiovascular disease. The interaction between the coagulation system and the immune system is critical for host defense; however, in HIV infection, the mechanisms underlying the ongoing activation of these pathways remains poorly understood. We hypothesized that interaction between platelets and T cells may play a role. We found that platelets from healthy controls and HIV infected patients bind to CD4 and CD8 T cells, preferentially to those with memory phenotypes. HIV infected patients showed a higher proportion of activated platelet (CD42b+CD62P+)-T cell conjugates and significantly correlated to the D-dimer levels. In vitro thrombin stimulation of PBMCs enhanced formation of platelet-T cell conjugates mediated in part by CD62P released by platelets. These data suggest that platelets can promote trafficking of T cells into injured tissues by upregulation of CD62P leading to enhanced rolling of leukocytes along vascular endothelium. In HIV infected patients, recruitment of T cells by this mechanism may promote a state of inflammation/coagulation and contribute to the pathology of the disease.
Gene expression profiling of citrulline-specific B cells in rheumatoid arthritis (BA3P.118)
Authors: Mahendra Ankit , Agarwal Sandeep , Varadarajan Navin
Published: January 2023

Rheumatoid arthritis (RA) affects ~1% of the world’s population and is the most common autoimmune disease afflicting humans. The elicitation of anti-citrulinated protein antibody (ACPA) responses, has been a major milestone for both the detection and treatment of RA. Given the significance of ACPA in RA, B cells have a central role in the progression of the disease. The identification of the differentiation state and phenotype of autoreactive B cells (ABC) is significant from both a fundamental standpoint in understanding autoimmune biology and from a clinical standpoint in providing new avenues to targeting ABCs. In order to identify novel biomarkers targeting ABCs, we have undertaken comprehensive gene expression profiling of citrulline-specific autoreactive B cells isolated from the peripheral blood of RA patients. We demonstrate the ability to selectively isolate autoreactive B cells for single-cell gene expression profiling. The frequency of citrulline-specific B cells was 0.012 % of the PBMCs. The specificity of the mAb isolated using our approach was confirmed using ELISA. We have undertaken whole transcriptome amplification and single-cell gene expression, and by comparison to pathogen-specific B cells, describe sets of genes that may be used to uniquely define ABCs. We anticipate that the key biomarkers identified from these genes will further improve our understanding of autoimmune pathology and will be evaluated for their clinical relevance.
B cell antigen presentation is sufficient to drive neuro-inflammation in an animal model of multiple sclerosis (BA3P.120)
Authors: Parker Chelsea , Archambault Angela , Sim Julia , Ferris Stephen , Mikesell Robert , Linington Christopher , Russell John , Wu Gregory
Published: January 2023

B cells are increasingly regarded as integral to the pathogenesis of multiple sclerosis (MS). Several B cell-dependent mechanisms contributing to inflammatory demyelination of the central nervous system (CNS) have been explored using the CD4 T cell-dependent animal model for MS, experimental autoimmune encephalomyelitis (EAE). While B cell antigen presentation has been suggested to regulate CNS inflammation during EAE, no direct evidence that B cells independently support antigen-specific autoimmune responses by CD4 T cells in EAE exists. Using a newly generated murine model of in vivo conditional MHCII expression, we previously reported that encephalitogenic CD4 T cells are incapable of inducing EAE when B cells are the only antigen presenting cell expressing MHCII. Using this system we now find that B cells cooperate with dendritic cells to enhance active EAE severity resulting from myelin oligodendrocyte glycoprotein (MOG) immunization. Further, increasing the frequency of MOG-specific B cells, but not supplementation with soluble MOG-specific antibody, is sufficient to drive EAE in mice expressing MHCII solely by B cells. By temporally regulating MHCII expression in MOG-specific B cells, we find induction of EAE reliably occurs upon expression of MHCII. These data support a model in which the expansion of antigen-specific B cells during CNS autoimmunity amplifies cognate interactions between B and CD4 T cells and drives neuro-inflammation at later stages of disease.
BANK1 transgenic mice exhibit lupus-associated B cell phenotypes (BA3P.105)
Authors: Pathak Simanta , Guo Yuyuan , Ding Huihua , Du Yong , Min Soyoun , Vanarsa Kamala , Li Quanzhen , Kozyrev Sergey , Alarcón-Riquelme Marta , Mohan Chandra
Published: January 2023

BANK1 is a B-cell adaptor protein that has been implicated in SLE and systemic sclerosis. Previous studies have interpreted that the overexpression of the full length BANK1 isoform plays a pathogenic and the truncated (∆BANK1) version plays a protective role in lupus. However, there is no mechanistic evidence showing that these 2 isoforms can impact lupus development differentially. We have recently generated 2 transgenic strains on the B6 background, BANK1Tg and ∆BANK1Tg, which will be used to address this knowledge gap. The BANK1Tg mice overexpressing the full length Bank1 under the control of CD19 promoter exhibited a dramatic skewing in the splenic B cell population to MZ and B1a cells with a reduction in FO B cells. These mice also exhibited increased plasma cells along with an increased level of total IgM and anti-dsDNA IgG antibodies. Microarray analysis revealed increased transcript levels of several class switched IgG antibodies with a concomitant increase in the level of activation induced deaminase (AID), a master regulator of class switch recombination (CSR). qPCR confirmed higher level of AID and CSR. Interestingly, these phenotypes are observed only in female Tg mice. In contrast, the ∆BANK1Tg mice don’t exhibit the above phenotypes, supporting the hypothesis that the truncated isoform may play a protective role. These studies indicate that the different isoforms of BANK1 observed in humans contribute differentially to the development of B-cell autoimmunity.
Identifying the cellular interactions that result in autoantibodies to insulin in type 1 diabetic autoimmunity (BA3P.112)
Authors: Wan Xiaoxiao , Thomas James , Unanue Emil
Published: January 2023

The presence of insulin autoantibodies (IAAs) is a consistent feature of patients with type 1 diabetes and non-obese diabetic mice. The mechanisms underlying their production and pathogenic role are poorly understood. IAA production is unexpected because insulin-reactive B (INS-B) cells are maintained in a tolerant state. Furthermore, the source of T cell help required for IAA production is limited because T cells recognizing insulin are deleted in the thymus, and those that escape thymic selection only recognize insulin peptides but not native insulin. Increased titers of IAAs to native insulin were found in a T cell receptor transgenic strain, 8F10, whose T cell reactivity is restricted to insulin peptides. The 8F10 T cell only recognizes peptides from insulin that are found in abnormal granules of beta cells. Furthermore, when an insulin B cell receptor heavy chain transgene was introduced into the 8F10 mice, a marked increase in IAAs specific for native insulin was observed. We found that 8F10 T cells differentiated into follicular helper T cells and selected the insulin specific B cells, helping them to differentiate in germinal centers (GC). These changes took place in lymph nodes at various sites. The results suggest that blood insulin was being taken up by the high affinity BCR for insulin and processed there for presentation to the T cells. Thus, pathogenic insulin-reactive T cells are able to reinforce B cell autoreactivity through unusual T-B cell interactions.