bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhancing adoptively transferred T cells by stimulating with dendritic cell stimulation: exploring novel therapies (P4405)
Authors: Murakami Kiichi , Kawamura Kim , Chan Olivia , Tran Michael , Elford Alisha , Nguyen Linh , Ohashi Pamela
Published: January 2023

Adoptive Cell Transfer (ACT) of tumor infiltrating lymphocytes (TILs) is a promising strategy for cancer immunotherapy. The current therapy relies on non-myeloablative lymphodepletion and high dose IL-2 therapy, which has serious side effects for patients. Optimization of the current ACT therapy, using animal models, may help reduce or eliminate these additional treatments and enhance the response rate. ACT has been evaluated in the autoimmune model RIP-GP mice, which express the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) on the β-islet cells of the pancreas. Our findings showed that LCMV memory T cells stimulated with matured dendritic cells (DCs) and pulsed with peptides derived from LCMV, successfully induced diabetes. These stimulated T cells expanded well in vivo and showed strong killing activity after transfer. However, unstimulated memory tissue specific T cells rarely expanded after transfer and showed limited cytolytic activity. To evaluate this method in the tumor model, RIP-Tag2/GP double transgenic mice were used, which express the large T antigen under the control of RIP and spontaneously develop insulinomas. Islet specific T cells cocultured with DCs and LCMV-GP peptides displayed strong anti-tumor activity after transfer in vivo. Surprisingly, mice bearing insulinomas survived longer after ACT than mice treated with LCMV vaccination. Therefore, stimulating TILs before infusion has high potential for enhancing ACT therapy.
Characterization of a human CD27 agonistic monoclonal antibody for use as an agent in glioblastoma therapy (P4411)
Authors: Riccione Katherine , Sanchez-Perez Luis , Flores Catherine , Sampson John
Published: January 2023

CD27 is a costimulatory molecule constitutively expressed on naïve T-cells, B-cells, and subsets of NK cells; signaling through CD27 has been shown to promote Th1 polarization, clonal expansion, enhanced survival of effector and memory cells, upregulation of effector molecules, and enhanced cytolytic activity. We are characterizing the use of an agonistic anti-CD27 antibody, CDX1127, as a treatment in a preclinical glioblastoma model in human CD27 transgenic mice. We have shown that in vitro stimulation of isolated T-cells from hCD27+/- mice with CDX1127, in the context of TCR signaling, leads to increased proliferation, decreased apoptosis, and increased secretion of IFN-γ and TNF-α, corroborating its agonistic activity. Furthermore, we have analyzed the efficacy of CDX1127 as a monotherapy in a mouse glioblastoma model as well as an adjuvant in a total tumor RNA dendritic cell vaccine.
A VCG-based chlamydial vaccine engenders broad-based protective immunity (P4391)
Authors: Eko Francis , He Qing , Black Carolyn , Igietseme Joseph
Published: January 2023

Genital chlamydial infections pose a considerable public health challenge worldwide and a prophylactic vaccine is required for control. Since immunity against natural infection is serovar specific, an efficacious chlamydial vaccine should induce broad-based immunity against the different chlamydial serovars. Intramuscular (IM) immunization with recombinant Vibrio cholerae ghosts (rVCG) expressing chlamydial antigens has shown promise in development of immunity against genital chlamydial infection. We evaluated the ability of a VCG-based multisubunit vaccine expressing the porin B (PorB) and polymorphic membrane protein D (PmpD) proteins of C. trachomatis to induce cross-protective immunity in a mouse model of genital chlamydial infection. IM immunization with the vaccine candidate elicited high levels of antigen-specific genital mucosal and systemic Th1 cell-mediated and humoral immune responses against heterologous serovars and strains, including serovars E-H and L. Also, the multisubunit vaccine conferred significant cross protection that was superior to that provided by VCG expressing the single antigens against the heterologous mouse strain, C. muridarum. These results demonstrate the ability of the serovar D-derived multisubunit vaccine to induce cross protective chlamydial immunity using the VCG platform.
Altered distribution patterns of TLR5 and TLR7 in the central airways of severe asthmatics (P4427)
Authors: Shikhagaie Medya , Bergqvist Anders , Andersson Cecilia , Mori Michiko , Krohn Kortekaas , Dahl Ronald , Hoffman Hans-Jurgen , Bjermer Leif , Erjefält Jonas
Published: January 2023

Toll-like receptors are a major class of pattern and damage recognition receptors controlling innate immune responses. TLR5 recognizes bacterial flagellin and TLR7 identifies single stranded viral RNA. The present study characterizes TLR5 and TLR7 expression pattern in central airways of asthmatics. Methods: Bronchial biopsies were obtained from healthy controls, uncontrolled asthmatics on inhaled glucocorticoids, and severe asthmatics on oral glucocorticoids. Subjects were classified according to GINA guidelines and asthma control score. Tissue sections were processed for immunohistochemical identification of TLR positive cells. Results: TLR5 immunoreactivity was identified in the airway epithelium, glands, smooth muscle, alveolar macrophages and plasma cells. In the airway epithelium, TLR5 had mainly a basolateral distribution. In severe asthmatics the overall and epithelial TLR5 immunoreactivity were significantly decreased. TLR7 displayed a distinct peri-nuclear expression. Quantification of the epithelial TLR7 immunoreactivity showed a significantly decreased expression in severe asthmatics compared to healthy controls and uncontrolled asthma. TLR7 expression was also detected in nerve cells, NK cells and mast cells. Conclusions: Our data suggest that patients with severe asthma have decreased expression of TLR5 and TLR7 compared to healthy controls and uncontrolled asthmatics. This altered expression may reflect a dysfunctional immune-regulation in severe asthmatics.
Hepcidin protects sepsis through regulating iron homeostasis to inhibit immunocyte apoptosis in mice (P4435)
Authors: Zeng CongLi , Zhang Kai , Chen QiXing , Fang XiangMing
Published: January 2023

Background: Hepcidin is a key regulator of iron metabolism and may have a critical role in modulating LPS-induced acute inflammatory responses. The role of hepcidin in cecal ligation and puncture (CLP) induced sepsis model was not clear. Methods: Adenovirus-mediated shRNA to inhibit the expression of hepcidin and a relative negative control were constructed and were administrated to mice through hydrodynamic injection via tail vein. Thirteen days later, the mice were subjected to CLP. Bacterial colony counts in blood and organs, reactive oxygen species (ROS) production in liver and immunocyte apoptosis in spleen were analyzed. Survival rate was estimated after 7 days. Results: The survival rate of hepcidin-knock down (KD) mice in CLP-induced sepsis was significantly decreased. Hepcidin-KD mice exposed to CLP showed more severe lung injury and increased inflammation cytokines. The serum iron concentration in hepcidin-KD mice after CLP was significantly higher, which resulted in more ROS production in liver and increased bacterial counts in blood and organs. In the spleen, there were significantly more apoptotic immunocytes in hepcidin-KD mice than those in controls. However, no significant differences in SOCS3 and phosphorylation of STAT3 level between the two groups were observed. Conclusions: Hepcidin may have a protective role in sepsis through regulating iron balance to inhibit bacterial infection and oxidative stress, which then protected immunocyte from apoptosis.
Monitoring epithelial cell responses to an IBD-associated bacterial pathotype using quantitative proteomics (P1352)
Authors: Lijek Rebeccah , Chan Eric , Fleming Mark , Starnbach Michael
Published: January 2023

Many lines of evidence suggest that the initiation of inflammatory bowel disease (IBD) involves abnormal host-microbial interactions at the intestinal epithelium. One microorganism that is commonly associated with the intestinal mucosa of IBD patients but not from healthy subjects is called adherent invasive E. coli (AIEC). AIEC are a new pathotype defined by their ability to adhere to and invade epithelial cells in the absence of known virulence factors associated with other pathogenic E. coli. Previous work has demonstrated that AIEC infection of epithelial cells induces cell-intrinsic responses such as the activation of autophagy markers, reduction of barrier function and secretion of IL-8. However, a comprehensive analysis of the cellular response to AIEC has not been performed. Here, we take a global approach to define how protein levels are altered in epithelial cells exposed to AIEC using quantitative mass spectrometry. This experimental strategy allows us to determine how the endogenous abundance of known and novel cellular proteins is altered by AIEC infection. Our preliminary results reveal specific differences between the epithelial cell response to adherent invasive E. coli compared to commensal E. coli not associated with IBD. Ongoing work examines whether any of the changes in protein abundance observed in response to AIEC infection are necessary for AIEC attachment, invasion, or intracellular growth.
The role of microRNA in T Helper cell plasticity (P1307)
Authors: Siede Julia , Baumann Claudia , Haftmann Claudia , Mashreghi Mir-Farzin , Löhning Max
Published: January 2023

Upon antigen encounter naive CD4+ T helper (Th) cells differentiate into distinct T helper cell subsets characterized by specific cytokine and transcription factor expression. It is currently debated whether these Th subsets have a fixed or flexible phenotype. Previously we showed that in the course of viral infection Th2 cells can be reprogrammed into a hybrid Th2+1 phenotype co-expressing Th1 and Th2 key transcription factors and cytokines. However, the T cell intrinsic control of subset-specific gene expression is only partially understood. Studies have indicated that post-transcriptional regulation via microRNA contributes to several aspects of T cell gene expression. Nevertheless, only few relevant miRNAs have been described so far. The aim of this project is to identify miRNA specifically expressed in novel hybrid T cell phenotypes as well as to analyze their impact on T cell plasticity and function. We used microarray analysis to identify 22 differentially expressed miRNAs in hybrid Th2+1 cells compared to both Th1 and Th2 cells. Within this subset 8 miRNAs were up and 11 miRNAs were down regulated. We found that type I IFNs are potent inducers of some of these miRNAs. Additionally, we observed an inverse correlation in expression of some miRNAs with their predicted targets that are known for their role in Th cell activation and differentiation. Overall, these miRNAs could prove to be important players in the induction and maintenance of Th2+1 cells.
Regulation of intrathymic γδ T effector subset differentiation by high mobility group box transcription factors (P1342)
Authors: Cho Ok Hyun , Kang Joonsoo
Published: January 2023

Innate-like γδ T effector subsets are exported from the thymus as “memory-like” cells prewired for rapid, specialized function. Previously, we showed that emergent γδ cell subsets distinguished by TCRγ and TCRδ usage in the fetal and adult thymus possess distinct global transcriptomes. In particular, the subset-specific combinatorial expression of High Mobility Group box transcription factors (HMG TFs) was shown to be a primary determinant of γδ effector differentiation. We showed that a quartet of HMG TFs, SOX4, SOX13, TCF1 and LEF1, are critical for the development and function of IL-17 producing γδ T effectors (Tγδ17). The TFs are found co-localized near the transcriptional regulatory sites of Rorc, Blk, Il17a and Gata3 in a subset-specific manner, suggesting that they directly regulate the expression of key proteins that control effector cytokine production. We also observed a positive correlation between active epigenetic modifications and subset-specific gene networks governing effector function, suggesting that the initial programming of γδ effector diversification involves selective chromatin modifications. While the detailed mechanism of HMG TFs cooperativity and counter-regulations is not fully understood, a key feature is predicted to be context-dependent interactions between SOX13 and TCF1 that result in diversified target gene regulation.
Acute-phase serum amyloid A causes Th1 differentiation and proliferation (P1325)
Authors: He Rong , Lui Asona , Hinton Michael
Published: January 2023

Host response to injury and infection is accompanied by a rapid rise of acute-phase proteins in the blood. Serum amyloid A (SAA) is a major acute-phase protein, and has been used as a marker for inflammatory diseases. However, its precise role in inflammation has not been defined. In our previous studies, we found that SAA can induce IL-8 and TNF-a secretion from peripheral blood neutrophils, and can be an endogenous ligand that induces the expression of interleukin-23 (IL-23), a hetero-dimeric cytokine that promotes autoimmune inflammation. SAA is found in very high levels in the arthritic joints of patients, along with a high concentration of certain subsets of T lymphocytes, notably helper T cell type 1 (Th1). In this study, we discovered that SAA can also stimulate CD4+ T cells to produce the cytokine interferon gamma (IFNg) and interleukin-2 (IL-2), and induce Th1 cell differentiation and proliferation. Our data showed that SAA-induced IL-2 production and cell proliferation can be blocked by the inhibitor of MAP kinase p38, and the phosphorylation of p38 indeed can be increased after SAA stimulation. These results suggest that SAA, which is found in arthritic joints, may play a role to induce Th1 cell production, and is perhaps a missing link in our understanding of Rheumatoid Arthritis.
Hypoxia inducible factor-1 alpha links reduced oxygen availability to the generation of anti-inflammatory IL-10 expressing T cells (P1328)
Authors: Clambey Eric , Westrich Joseph , Eltzschig Holger
Published: January 2023

Hypoxia, the state of limited oxygen availability, is a frequent metabolic constraint in physiologic and pathologic states. The impact of hypoxia and hypoxic signaling on T cell differentiation remains incompletely defined, especially regarding inhibitory pathways that may be induced to limit hypoxia-associated tissue damage. Here we identified CD4 and CD8 T cells expressing IL-10 in the context of inflammatory hypoxia in vivo. To test the consequence of limited oxygen availability on the generation of IL-10 expressing T cells, we analyzed T cell differentiation under hypoxic conditions. Hypoxic culture significantly induced IL-10 expression in both CD4 and CD8 T cells. Hypoxia further increased IL-10 expression across diverse differentiation conditions, including conditions known to induce IL-10 expression. The hypoxic enhancement of IL-10 producing T cells was independent of TGF-β and IL-27, but required signals from multiple cell types, as well as T cell-intrinsic expression of the transcription factor hypoxia inducible factor (HIF)-1α. HIF-1α was further required for IL-10 expression in regulatory T cells even in normoxia, consistent with impaired suppressive function of HIF-1α deficient Tregs in vitro and in vivo. In total, these data demonstrate that reduced oxygen availability, interpreted through the HIF-1α transcription factor, is a potent physiologic cue that can drive T cell differentiation towards IL-10 production, thereby limiting immune-mediated pathology.
SNAP-23-dependent surface trafficking of NOX2 regulates ROS-dependent surface up-regulation of BLT1 during exocytotic degranulation induced by Trichomonas vaginalis-derived LTB4 (P3351)
Authors: Shin Myeong Heon , Min Arim
Published: January 2023

Trichomonas vaginalis is a sexually transmitted parasite that causes vaginitis in women. T. vaginalis itself secretes lipid mediator LTB4. Mast cells are important effector cells for tissue inflammation during infection with parasites. In this study, we investigated signaling mechanisms of mast cell degranulation induced by T. vaginalis secretory products (TvSP). Herein, we found that SNAP-23-dependent surface trafficking of NOX2 is required for BLT1-dependent exocytotic degranulation of mast cells induced by LTB4 in the TvSP. Stimulation with TvSP induced p47phox phosphorylation, ROS generation, surface up-regulation of CD63 that is a marker for exocytosis in human mast cell cells. TvSP-induced ROS generation and exocytosis were strongly inhibited by treatment with BLT1 siRNA or NOX2 siRNA. In addition, LTB4 in the TvSP was found to induce trafficking of NOX2, LTB4 receptor BLT1, and CD63 to the cell surface. Moreover, we found that t-SNARE SNAP23 siRNA almost completely inhibited TvSP-induced surface trafficking of NOX2 and ROS generation, which down-regulated surface upregulation of BLT1 and CD63 in HMC-1 cells. In conclusion, our results suggest that SNAP23-dependent surface trafficking of NOX2 are essential for ROS-dependent BLT1 upregulation during exocytotic degranulation in human mast cells induced by T. vaginalis-secreted LTB4. These results can help to explain the signaling talk mechanism between T. vaginalis and mast cells during tissue inflammatory responses.
Entamoeba histolytica induces cell death in HepG2 cells via the change of protein O-GlcNAcylation (P3355)
Authors: Lee Young Ah , Min Arim , Kim Kyeong Ah , Cho Jin Won , Shin Myeong Heon
Published: January 2023

Entamoeba histolytica is an enteric tissue-invasion protozoan parasite that causes amoebic colitis and liver abscess in humans. E. histolytica can induce host cell apoptosis to evade host immune responses. Protein O-GlcNAcyation has been thought to contribute to the various cellular signal processes including apoptosis and proliferation. O-GlcNAc addition and removal are regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. However, it is unknown whether the alteration of O-GlcNAcylated protein level in host cells can affect E. histolytican-induced cell death. In this study, co-incubation of HepG2 cells with virulent amoeba HM-1 strain remarkably increased DNA fragmentation and LDH release compared to cells incubated with medium alone. In addition, Entamoeba HM-1 induced activation of caspases-3 and calpain in HepG2 cells. Amoebic adherence to the HepG2 cells showed the decrease of O-GlcNAcylated protein levels in HepG2 cells within 2 min, while non-virulent Rahman strain did not. Indeed, pretreatment of host cells with O-GlcNAcase inhibitors (PUGNAc or Thi-Met G) or OGA siRNA prevented the E. histolytica-induced protein deGlcNAcylation and cell death in HepG2 cells. These results suggest that decrease of protein O-GlcNAcylation is an important process required for hepatocyte cell death induced by adherence of E. histolytica.
Role of SERPINB3, SERPINB4, and their mouse homolog Serpinb3a in allergen-induced cutaneous inflammation. (P3347)
Authors: Sivaprasad Umasundari , Kinker Kayla , Gibson Aaron , Bass Stacey , Hershey Nicolas , Biagini Myers Jocelyn , Butsch Kovacic Melinda , Martin Lisa , Khurana Hershey Gurjit
Published: January 2023

The serine protease inhibitors SERPINB3 and SERPINB4 have been implicated in allergic disorders. We have recently demonstrated a role for the serine protease inhibitor Serpinb3a (the mouse homolog of SERPINB3 and SERPINB4) in a mouse model of asthma. Elevated levels of SERPINB3 and SERPINB4 are detected in the serum of patients with atopic dermatitis (AD) and they have been proposed as possible biomarkers for AD. Serine proteases are critical for epidermal barrier homeostasis and aberrant expression and/or activity of serine proteases have been associated with AD in human studies. We also observe a significant induction in the expression of Serpinb3a in a mouse model of cutaneous inflammation that has many similarities to AD. However the role of Serpinb3a in the skin is unknown. Using our mouse model of skin inflammation, we observed attenuation in trans-epidermal water loss, skin inflammation scores, decreased levels of total IgE, and pro-inflammatory markers like S100A8 in mice lacking Serpinb3a following exposure to Aspergillus fumigatus extract. In humans, we have identified a single nucleotide polymorphism (SNP) in the locus that encodes SERPINB3 and SERPINB4 that is associated with trans-epidermal water loss (p-val=0.031). In conclusion, we demonstrate that SERPINB3 and SERPINB4 contribute to cutaneous inflammation and work is ongoing to elucidate the role of this protein in regulating the skin barrier.
Anti-tumor activity of obinutuzumab and rituximab in a follicular lymphoma 3D model (P3287)
Authors: Bezombes Christine , Decaup Emilie , Jean Christine , Laurent Camille , Gravelle Pauline , Laurent Guy , Klein Christian , Varoqueaux Nathalie , Fournié Jean-Jacques
Published: January 2023

Follicular lymphomas (FL) account for 35-40% of all adult lymphomas. Treatment typically involves chemotherapy combined with the anti-CD20 monoclonal antibody rituximab (RTX). The development of type II anti-CD20 MAb obinutuzumab (GA101) aims to further improve treatment. Here we show using FL cells that RTX and GA101 displayed similar activity on RL cells cultured in 2D. As 2D culture cannot mimic tumor spatial organization, conventional 2D models may not reflect the effects of antibodies as they occur in vivo. Thus, we created a NHL 3D culture system, termed multicellular aggregates of lymphoma cells (MALC), and compared RTX and GA101 activity. Our results show that both antibodies display greater activity towards FL cells in 3D culture compared to 2D culture. Moreover, we observed that in the MALC model GA101 was more effective than RTX in inhibiting tumor growth through induction of (lysosomal) cell death and senescence; and in inhibiting intracellular signaling pathways such as mTOR, Akt, PLCgamma and Syk. Altogether, our study demonstrates that spatial organization strongly influences response to antibody treatment supporting the use of 3D models for the testing of therapeutic agents in NHL.
Identification of sulfatide reactive type II NKT cells using CD1d dimers (P3291)
Authors: Parish Stanley , Izhak Liat , Xia Zheng , Suzuki Motoshi , Terabe Masaki , Berzofsky Jay
Published: January 2023

Natural killer T cells (NKT) are a unique subset of lymphocytes that bridge the gap between innate and adaptive immunity by recognizing lipid antigen presented by CD1d. While type I NKT cells have been extensively studied by using CD1d multimers loaded with α-galactosylceramide (α-GalCer), a type I specific antigen, studying type II NKT cells has lagged behind, since loading sulfatide, a type II-specific lipid antigen, to the tetramer has been difficult to accomplish reliably. In this study, we have developed sulfatide loaded CD1d dimers that accurately identify type II NKT cells by flow cytometry. Using saposin C, a lipid loading chaperone protein, and acidic conditions, as found in the natural loading environment, we could create sulfatide-loaded CD1d dimers to identify this subset. Sulfatide-loaded CD1d dimers specifically identified type II NKT cells in both WT and type I NKT deficient mice, and were not observed in NKT cell-deficient CD1d-/- mice. The specificity of the binding through TCRβ was confirmed by TCRβ blockade. Consistent with previous findings, in the liver, the sulfatide-reactive type II NKT cells were a distinct population that encompasses about 2% of liver lymphocytes. These data suggest that the method we developed can create sulfatide-loaded CD1d dimers to identify type II NKT cells, and has enabled us to identify other markers of this subset. This method can be used to load other lipids onto CD1d to identify other NKT subsets across multiple models.
FPLC in a pipette tip (P3297)
Authors: Mundodi Suparna , Rishi Porecha , Murray Anderson , Nicole Rapicavoli
Published: January 2023

Molecular characterization of the immune response and how this process is implemented requires the purification of native proteins and antibodies. There are numerous purification methods to isolate proteins of interest, but these methods usually require multiple steps to attain the level of purity required for most studies. The Rainin PureSpeed Protein Purification System utilizes pipette tips containing purification resin at their distal end interfaced with an E4 XLS pipette to simplify enrichment procedures for virtually all proteins. The E4 XLS pipette, when set up within a 96-deepwell plate, is able to carry out semi-automated purification of antibodies and other native or recombinant proteins. The pipette is able to drive up-and-down sample flow over the immobilized resin matrix for as many as 12 samples in parallel. The ability of the pipette to carry out liquid handling steps enhances laboratory productivity by allowing an investigator to step away from the apparatus until the next step in the protocol. This new, innovative technology from Rainin Instrument is available with pipette tips containing protein A, protein G, Ni-IMAC, or one of four different ion exchange resins, which allow for multiple options for protein purification. Finally, the PureSpeed System functions well in advanced applications such as protein and chromatin immunoprecipitation, offering speed and versatility relative to agarose and magnetic bead technologies.
ES-62 prevents development of lupus-like pathology in the MRL/Lpr mouse (P5210)
Authors: Rodgers David , McGrath Mairi , Pineda Miguel , Al-Riyami Lamyaa , Harnett William , Harnett Margaret
Published: January 2023

The prevalence of autoimmunity in developing countries is lower than that in the developed world and this inverse relationship correlates with the level of chronic parasitic infections. Co-evolution of parasites with the human immune system has resulted in evasion strategies that prevent the host clearing the parasite yet limit pathology. One mechanism involves the release of immunomodulators like ES-62, secreted by the filarial nematode Acanthocheilonema viteae: ES-62 exhibits broad anti-inflammatory activity and therapeutic potential in autoimmune diseases such as rheumatoid arthritis. Here we show that ES-62 protects against nephritis, as indicated by proteinuria levels, in the murine MRL/Lpr model of systemic lupus erythematosus (SLE). It has been proposed that the pro-inflammatory cytokine IL-17 is a primary driver of disease, in mouse models as well as in SLE patients, but our data do not support targeting of IL-17 as the major protective mechanism underlying ES-62 efficacy. Indeed, and consistent with this, we have shown that neutralising antibodies specific for IL-17 do not block development of proteinuria. ES-62 is a large immunogenic glycoprotein and therefore not suitable for therapeutic use. Thus, since the active moiety of ES-62 is phosphorylcholine (PC), we have recently developed small molecular analogues based around PC that mimic the immunomodulatory effects of ES-62 in autoimmune disorders, including SLE.
The filarial nematode product, ES-62 targets IL-22 to mediate protection in collagen-induced arthritis (P5212)
Authors: Pineda Miguel , Harnett William , Al-Riyami Lamyaa , Harnett Margarett
Published: January 2023

Parasitic worms skew immune responses to a Th2-like phenotype, inhibiting Th1/Th17 pathways and expanding regulatory cells such as Tregs. Worms can achieve such immunoregulation by secreting specific products like ES-62, a glycoprotein secreted by the filarial nematode A. viteae. We have previously shown ES-62 to be protective in Collagen-Induced Arthritis, a murine model of rheumatoid arthritis, and that the mechanism associated with protection involves a down-regulation of the cytokine IL-17. We have now analyzed the effects of ES-62 on other Th17-related cytokines focusing on IL-22, a cytokine that has been reported to exhibit both pro and anti-inflammatory actions in animal models of inflammatory disease. In CIA, administration of ES-62 appears to target the IL-17/IL-22 balance in lymph nodes and joints of animals resulting in modulation of the number and inflammatory phenotype of infiltrating and resident cells in the joint, and consequent attenuation of inflammation. Interestingly, using a combination of recombinant IL-22 and blocking antibody in in vivo studies, the precise type of effect of IL-22 on CIA progression was shown to depend largely both on the location of IL-22 action and also the stage at which the cytokine is produced during disease progression. These findings have allowed us to identify a novel mechanism used by the helminth product ES-62 to reduce autoimmune arthritis and they also contribute to our understanding of the complex biology of IL-22
Therapeutic effect of mesenchymal stem cells in combination with oncolytic adenoviruses for the treatment of solid tumors in an immunocompetent mouse model (P2020)
Authors: Rincon Esther , Kanojia Deepak , Auffinger Brenda , UIlya Ilya , Han Yu , Alemany Ramon , Ramírez Manuel , García-Castro Javier , Lesniak Maciej
Published: January 2023

Oncolytic adenoviruses (Ad) constitute a promising alternative for patients with solid tumors. Unfortunately, clinical trials have shown limited efficacy due to insufficient viral delivery to tumor and the ability of Ad to trigger antiviral immune response. We have previously documented enhanced Ad delivery in vivo using human mesenchymal stem cells (MSCs). However, a major limitation in the field is the lack of immune- and replication-competent models. In this study, we used syngeneic CMT64 cells as an immunocompetent model to analyze the antitumoral response followed by treatment with MSCs and oncolytic Ad ICOVIR5. First, we demonstrated that ICOVIR5 replicates and has cytopatic effect in CMT64 cells. To evaluate the in vivo efficacy of ICOVIR5 using MSCs as carriers, CMT64 cells were grown in mouse flanks. Once the tumors were established, mice were divided into 4 groups (n=5/group). Groups 1/2 were treated with saline and groups 3/4 with MSCs. Given the fact that mouse MSCs do not support the replication of human Ad, tumors were injected with saline (groups 1/2) or ICOVIR5 (groups 3/4) to mimic the carrier role of human MSCs. This therapy was performed weekly for 3 weeks and the tumor volumes were measured. A significant decrease in the tumor volume (p<0.05) was found in the group treated with MSCs and ICOVIR5. These results establish a potential permissive model to study immune mechanisms involved in the antitumoral effects of MSCs as carriers for oncolytic Ad.
Viral infection alters subnuclear localization of PD-1 and CD62L loci in LCMV-specific CD8 T-cells (P6369)
Authors: Sacristan Catarina , Youngblood Ben , Xu Jean , Lu Peiyuan , McGary Katelyn , Hewitt Susannah , Skok Jane , Boss Jerry , Ahmed Rafi , Dustin Michael
Published: January 2023

During chronic viral infection, CD8 T-cell differentiation yields a unique transcriptional profile: T-cell exhaustion. The suboptimal effector function of exhausted cells is partly due to sustained expression of PD-1. Using the LCMV mouse model of infection, we examined pdcd1 (PD-1) and sell (CD62L) gene regulation at different stages of T-cell differentiation. We hypothesized that epigenetic programming and nuclear localization of these genes were coupled to viral persistence and T-cell differentiation. Changes in gene expression are linked to locus positioning in specific compartments such as nuclear lamina (silencing). Using DNA-FISH, we report a significant decrease in pdcd1 alleles associated to nuclear lamina in exhausted cells relative to other CD8 subsets. Dissociation from lamin was consistent with upregulated expression of PD-1 mRNA and protein, and with loss of PD-1 DNA methylation in exhausted cells. In contrast, we observed a significant increase in sell recruitment to lamin with exhaustion, mirroring changes in sell gene expression. CD8 T-cells from Blimp-1-deficient chronically infected mice exhibited an impaired ability to regulate pdcd1 and sell association to lamin, thus implicating Blimp-1 in modulating expression of both genes during chronic infection. Our findings contribute to the understanding of the molecular pathways mediating T-cell memory and exhaustion, and may provide insight into implementing therapeutic strategies to resolve chronic infections.
Longitudinal analyses of human sera reveal expansions of antibody titers specific to previously-encountered pandemic and seasonal influenza viruses (P6366)
Authors: Miller Matthew , Gardner Thomas , Krammer Florian , Aguado Lauren , Tortorella Domenico , Basler Christopher , Palese Peter
Published: January 2023

It is known that early exposures with influenza viruses impact the outcome of subsequent encounters. However, how sequential exposures to antigenically variant viruses shape the humoral immune response remains very poorly understood. To address this, a longitudinal analysis of antibody titers against pandemic (Japan/57 H2N2, Hong Kong/68 H3N2 and USSR/77 H1N1) and seasonal (Alabama/81 H3N2 and Texas/91 H1N1) strains of influenza virus was performed on matched serological samples collected over a 20 year period (1987-2008). These samples were obtained from the Framingham Heart Study Offspring Cohort. Sustained increases in neutralizing antibody titers were observed against both the pandemic and seasonal isolates. Hemagglutinin stalk-reactive antibody titers rose modestly over the 20 year study period, even in the absence of major antigenic shifts which are known to boost these antibodies most effectively. Neutralizing antibody titers against cytomegalovirus were also investigated. In contrast to influenza virus, no sustained increase in neutralizing antibody titers specific to cytomegalovirus was observed. These findings represent the first detailed longitudinal analysis of the humoral immune response against influenza virus in humans, and suggest a unique role for sequential exposures to antigenic variants in the maintenance of neutralizing antibodies.
The tapasin-related protein TAPBPR is a new component of the MHC class I presentation pathway (P5000)
Authors: Boyle Louise , Hermann Clemens , Boname Jessica , Rhodes David , Lehner Paul , Trowsdale John
Published: January 2023

Tapasin is an integral component of the peptide-loading complex (PLC) important for efficient peptide loading onto MHC class I molecules. We investigated the function of the novel tapasin-related protein, TAPBPR. Like tapasin, TAPBPR is widely expressed, interferon-γ inducible and binds to MHC class I coupled with β2-microglobulin (β2m) in the endoplasmic reticulum. In contrast to tapasin, TAPBPR does not bind ERp57 or calreticulin and is not an integral component of the PLC. β2m is essential for the association between TAPBPR and MHC class I. However, the association between TAPBPR and MHC class I occurs in the absence of a functional PLC, suggesting peptide is not required. Expression of TAPBPR decreases the rate of MHC class I maturation through the secretory pathway and prolongs the association of MHC class I on the PLC. The TAPBPR:MHC class I complex trafficked through the Golgi apparatus, demonstrating a function of TAPBPR beyond the ER/cis-Golgi. The identification of TAPBPR as a novel component of the MHC class I antigen presentation pathway demonstrates that mechanisms controlling MHC class I expression remain incompletely understood.
Dipeptides enhance folding and peptide binding of “empty” MHC class I molecules (P5002)
Authors: Saini Sunil Kumar , Ostermeir Katja , Ramnarayan Venkat Raman , Zacharias Martin , Springer Sebastian
Published: January 2023

MHC (Major Histocompatibility Complex) class I molecules play a central role in the mammalian antiviral immune response by presenting the viral proteome at the cell surface for recognition by cytotoxic T lymphocytes. The folding of recombinant MHC class I molecules from denatured bacterial inclusion bodies and their assembly with the light chain beta-2 microglobulin largely depend on specific high-affinity peptides of usually eight or nine amino acids. To find the minimum peptide requirement for folding and stabilization of class I molecules, we have investigated the influence of dipeptides on the folding and peptide binding of recombinant HLA-A*02:01 (A2) and H-2Kb (Kb). In the presence of the dipeptide Gly-Leu (GL), both A2 and Kb folded well, even without a high-affinity peptide present, as measured by a tryptophan fluorescence assay. When folded in the presence of GL, they also bound much faster to their respective specific high-affinity peptides as when folded without GL. Kinetic analysis by fluorescence anisotropy revealed that the presence of GL enhances the on-rate of peptide binding to empty A2 and Kb by six to eight fold. Molecular dynamics simulation indicates that the dipeptide GL interacts with the C terminus of the peptide binding groove. In conclusion, our data demonstrate that the dipeptide GL improves the folding of A2 and Kb and induces a peptide-receptive conformation for enhanced peptide binding.
The role of B cells in neuropsychiatric disease in murine systemic lupus erythematosis (P4000)
Authors: Wen Jing , Stock Ariel , Gulinello Maria , Putterman Chaim , Wang Haowei , Shlomchik Mark
Published: January 2023

Neuropsychiatric disease in systemic lupus erythematosus (NPSLE) is a common and dangerous disease manifestation. However, the mechanisms underlying NPSLE are not fully understood, and the optimal treatment is not known. One postulated mechanism involves lupus autoantibodies with affinity for brain tissue, which due to a compromised blood brain barrier that allows for the passage from the serum into the brain can deposit in the CNS to induce cell damage and neuropsychiatric abnormalities. Therefore, B-cell depletion would be expected to be an effective intervention for NPSLE. To address this question experimentally, we studied B-cell depletion in a murine lupus model with prominent neuropsychiatric manifestations, the MRL-lpr/lpr strain (JHD-MRL/lpr). Comprehensive neurobehavioral tests including forced swim, anhedonia, open field, object recognition, object placement, and social preference were employed to evaluate the neuropsychiatric manifestations in B cell depleted JHD-MRL/lpr as compared to B cell sufficient MRL/lpr mice. Unexpectedly, we found that complete B cell depletion did not ameliorate major NPSLE features, including depression-like behavior and cognitive dysfunction. However, improved motor activity was observed in JHD-MRL/lpr mice. Thus, the absence of B cells and autoantibodies is not sufficient to ameliorate the expression of NPSLE in MRL/lpr mice. Whether therapeutic depletion or conditional B cell knockout would display similar effects is not yet known.
Autoantibody profiling for Systemic Lupus Erythematosus diagnosis using the Immunarray CHIP™ (P4018)
Authors: Putterman Chaim , Blanco Irene , Jordan Nicole , Daniel-Carmi Vered , Belanis-Meirovich Liron , Sorek Rachel , Cohen-Gindi Ornit , Dromi Nir , Lerner Miriam , Sahar Eli , Batty D. Scott , Tamir Idan , Cohen Irun
Published: January 2023

SLE is a chronic, recurrent, potentially fatal multisystem inflammatory disorder mainly affecting women. SLE patients produce antibodies to many different self-antigens. Yet, currently used serological markers for SLE are lacking in specificity and/or sensitivity. The aim of this study was to develop an improved diagnostic test by measuring and multiplexing specific autoantibody reactivities in SLE patients. Autoantibody reactivity profiles in serum samples collected from 97 SLE patients within three years of the diagnosis were compared with those of 56 healthy controls. Autoantibody profiles were determined using the ImmunArray iCHIP™ - a proprietary protein microarray technology that allows the display of antigens representing a wide range of SLE-associated biochemical pathways on a single chip. Using this novel platform, SLE patients could be differentiated from healthy subjects by a relatively small subset of auto-antigens and Epstein Barr Virus (EBV) antigens. The autoantibody reactivity profile that allowed SLE diagnosis with high sensitivity and specificity displayed differential response to known SLE-specific antigens, such as single-stranded DNA and EBV, and to several novel ones. Conclusion: An antibody profile for SLE diagnosis using a single multiplexed chip was successfully developed. This profile may differentiate between SLE patients and healthy individuals. Validation of this profile in additional patient samples is ongoing.
Modulation of autoimmune diabetes by antibodies specific for N-acetyl-D-glucosamine (P4026)
Authors: New James , Dizon Brian , Kearney John
Published: January 2023

The increasing incidence of autoimmune disease in developed societies has been linked with decreased exposure to environmental antigens, implying that antigen exposure modifies immune system development. Vaccination with Group A Streptococci (GAS) produces a strong antibody response to N-acetyl-D-glucosamine (GlcNAc). This GlcNAc moiety is conserved in mammals, and GlcNAcylated proteins are enriched in pancreatic β-cells. Anti-GlcNAc antibodies generated against GAS bind GlcNAc epitopes in human and murine β-cells. Developmental remodeling of the pancreas is accompanied by significant β-cell apoptosis, which may serve as an initial source of autoantigen priming in Type 1 Diabetes (T1D). We therefore hypothesize that anti-GlcNAc IgM generated during GAS infection mediates non-inflammatory clearance of apoptotic β-cell antigens. Using the Min6 insulinoma cells, we demonstrate that β-cell apoptosis results in surface exposure of GlcNAc residues reactive with anti-GAS Abs. During DC priming with irradiated β-cells anti-GlcNAc Abs suppressed CD4 T cell activation and cytokine production. We show that neonatal immunization with GAS, but not Group C Streptococci, reduces the incidence of diabetes in female NOD-mice. Furthermore, passive transfer of anti-GlcNAc Abs protects NOD.Rag1ko mice from diabetes onset following adoptive transfer of diabetogenic BDC2.5 T-cells. Our data suggest that anti-GlcNAc Abs generated by vaccination with GAS can suppress development of T1D.
The IgM receptor, Toso/FAIM3, is critical for inflammation and diabetes induction. (P4010)
Authors: Lin Gloria Hoi Ying , Marques Ana , Tusche Michael , Brustle Anne , Brenner Dirk , Luk Cynthia , Schroer Stephanie , Winer Daniel , Woo Minna , Mak Tak
Published: January 2023

The mechanisms of type-1 and type-2 diabetes induction are different, yet the two diseases are tightly linked to excessive inflammation. Studies with mice lacking soluble IgM have implicated a protective role of IgM against inflammation and autoimmune progression. Recently, both human and mouse studies have demonstrated Toso/FAIM3, a type one transmembrane protein, to be the receptor for IgM. In our study, we investigated the role of Toso in both type I and type 2 diabetes models in mice. Toso has only been reported to be expressed on B cells and activated T cells. In a type 1 diabetes model, we found that Toso on dendritic cells is required for breaking T cell tolerance and diabetes induction. In a type 2 diabetes model which is induced by 12-14 weeks of high fat diet feeding, WT mice became obese and developed glucose intolerance and insulin resistance. Interestingly, although Toso-deficient mice had similar weight gain, they had improved glucose tolerance and insulin sensitivity compared to the WT mice. Furthermore, Toso-/- mice were found to have decreased B cell number and proinflammatory macrophages in the adipose tissues. In contrast to the protective role of IgM in autoimmune disease progressions, our findings demonstrate a novel and pathogenic role of Toso in both type 1 and type 2 diabetes induction, and suggest that Toso respresents a potential therapeutic target against both autoimmune and type 2 diabetes.
Phospholipid-reactive T cells negatively regulate the function and expansion of glycolipid-reactive T cells (P4012)
Authors: Halder Ramesh , Tran Cynthia , Okorogu Rita , Valera Isela , Prasad Priti , Kim Peter , Singh Ram
Published: January 2023

Glycolipid antigens such as αGalCer and phospholipid (PL) antigens such as phosphatidyl choline bind CD1d and activate T cells. Extensive work on αGalCer-reactive T cells has identified their pathogenic or protective roles in inflammation, infection and cancer. Little is known about the biology and functions of CD1d-restricted PL-reactive T (PL-T) cells. Here, we identified T cells that recognize a battery of PL antigens loaded onto mouse CD1d in various lymphoid organs. While CD1d/PL-T cells were fewer than CD1d/αGalCer-reactive T cells in the liver and spleen, PL-T cells were relatively more abundant than glycolipid-reactive T cells in organs such as mouse decidua. The CD1d/PL-T cells secreted IFN-γ upon in vivo priming with PL antigens in an antigen-specific manner. The PL-T cells did not stain with CD1d/αGalCer tetramers and did not respond to glycolipid antigen αGalCer. Intriguingly, activation of CD1d/PL-T cells markedly reduced the proliferation of αGalCer-reactive T cells in vitro, ex vivo, and in vivo. Thus, PL-T cells are a subset of CD1d-restricted T cells that are phenotypically and functionally distinct from glycolipid-reactive T cells. PL-T cells also negatively regulate the function of glycolipid-reactive T cells. Ongoing studies will investigate whether PL-T cells will modulate the pathogenic and protective roles of glycolipid-reactive T cells in various immune-mediated conditions.
Development of novel Sjogren's Syndrome susceptible mouse lines expressing floxed COX-2 within the major Aec2 disease susceptibility region in chromosome 1 (P4016)
Authors: Mongini Patricia , Haque Shabirul , Chiorazzi Nicholas , Ishikawa Tomo-o , Herschman Harvey , Esposito Donna
Published: January 2023

SS patients exhibit impaired exocrine gland function and high risk for B cell lymphoma. IgG autoAbs to acetylcholine receptors, detectable in humans, impair salivation in SS-afflicted mice with NOD Aec2 and Aec1 susceptibility loci. COX-2 is a potential target for reducing SS pathogenic IgG Ab and B cell lymphoma, since (a) within in vitro cultures B cell-expressed COX-2 and PGE2 augment DNA-modifying AID and downstream IgG switching and (b) mice deficient in COX-2 have reduced IgG. If B cell specific COX-2 function is critical for SS disease, cell-targeted delivery of inhibitors might circumvent adverse side effects. With a downstream goal of examining whether B cell COX-2 contributes to autoimmunity, we introduced a floxed COX-2 gene and a CD19Cre gene independently onto the NOD.B10-H2b SS-susceptible background. To our surprise, early congenic lines HET for either CD19Cre or COX-2flox failed to show aging-related hyposalivation (xerostomia), in contrast to wild-type NOD.B10. Reduced disease in CD19cre HET mice might reflect haplodiploid CD19 or flanking non-NOD genes. Extended backcrossing of COX-2flox onto NOD.B10 yielded two congenic lines that are comparable to wild-type NOD.B10 in developing xerostomia upon aging. Fine genetic mapping of the chr 1 Aec2 region within the novel COX-2flox lines showed that (a) variations in COX-2 do not explain the observed diversity and (b) unidentified genes within a presently more narrowed Aec2 interval are important for xerostomia.