bluuebunny/crossref_metadata_2025 huggingface dataset:
Adoptive T cell immunotherapy using chimeric antigen receptor against a novel cancer target Axl (VAC11P.1007)
Authors: Intisar Ejaj , Walsh Anthony , Weinberg Benjamin , Chakravarti Deboki , Wong Wilson
Published: January 2023

The Axl protein belongs to a subfamily of receptor tyrosine kinase and has been implicated in a large variety of cancers like colorectal, prostate, mesothelioma, breast, etc., beginning from its overexpression initially detected in chronic myeloid leukemia. Hence Axl presents as a novel and promising target for multiple cancer models. Meanwhile chimeric antigen receptor (CAR) is already a developed platform technology used in genetically modified T cells in order to make them recognize and elicit an immune response against cancer cells. Here we designed 3 single-chain variable fragments from existing monoclonal antibodies against Axl and constructed them into third-generation CARs. For testing, we transduced these CARs into Jurkat T cells and primary CD4 T cells. We cultured the cells in different doses of Axl and assayed the level of their activation using flow cytometry and ELISA. All 3 CARs demonstrated functional efficacy in being able to recognize the antigen and produced successful activation. This was detected by increase in GFP expression, cloned downstream of the NFAT promoter in the Jurkat T cell line. Furthermore the primary T cell experiment demonstrated increased secretion of IFNγ for all 3 CARs and IL-2 for 2 CARs. Our study confirmed the successful construction of 3 new therapeutic CARs against the novel cancer antigen, Axl, with varying levels of activation. This can now be utilized in a switchable CAR system to deliver a tunable therapeutic immune response.
Potential of immune biomarkers for predicting survival in glioblastoma patients receiving dendritic cell cancer immunotherapy (VAC12P.1012)
Authors: Visus Carmen , Fischhuber Katrin , Klingenbrunner Simone , Reitermaier Rene , Knausz Heike , Erhart Friedrich , Moricz Sandra , Barresi Caterina , Felzmann Thomas
Published: January 2023

Activartis is developing AV0113, an autologous dendritic cell (DC)-based cancer vaccine for glioblastoma multiforme (GBM). AV0113 is characterized by high release of IL-12, Th1 polarization and a potent anti-tumor immune response. A total of 78 GBM patients have been recruited into a randomized Phase II clinical trial. All patients received first line standard therapy for GBM and patients in the treatment group received AV0113 as an add-on treatment. A recent evaluation revealed a clear trend towards survival benefit for patients receiving AV0113 compared to the control group. In the treatment group 3 of 19 patients had died (16%), whereas in the control group 9 of 20 patients had died (45%). Patients’ chances to survive the first year are close to 3 times greater if they receive AV0113. Based on the hypothesis that there are immune parameters that could predict response to cancer immunotherapy (CIT), we investigated a total of 90 variables in the peripheral blood of GBM patients before, during and after CIT. We detected pre-existing anti-tumor immune responses and found that these responses were higher in patients that survived longer after CIT. Furthermore, a high increase in the anti-tumor responses after CIT was observed in long-term survivor patients and was associated with high percentage of CD3, CD8, NK and Th1 phenotypes as well as lower T regulatory cells in the peripheral blood. These Immune Parameters might be used as prognostic biomarkers and guidance for CIT.
Somatic hypermutation and use of immunoglobulin variable regions at shark T cell receptors (VET2P.1038)
Authors: Criscitiello Michael , Eubanks Jeanine , Chen Pat , Castro Caitlin , Wallace Andrea , Ota Yuko , Flajnik Martin
Published: January 2023

The nurse shark (Ginglymostoma cirratum) is part of the earliest clade found to possess an adaptive immune system employing immunoglobulin superfamily lymphocyte antigen receptors. The hallmarks of adaptive immunity, memory and specificity, are accomplished through B or T cells employing immunoglobulin (Ig) or T-cell receptors (TCR) respectively. Both lineages undergo somatic gene rearrangement for receptor diversification, a process mediated by RAG proteins, in which V, D, and J gene segments are joined together. Surprisingly, we found somatic hypermutation (SHM, normally a B cell process) to occur extensively at shark TCRα loci. Unlike B cell SHM that follows antigen exposure in sharks and most other vertebrates, SHM at TCRα loci occurs during thymic development. TCRα SHM is supported with isolation of clusters of mutated clones having the same CDR3 rearrangement.We suggest that such SHM, in addition to TCRα receptor editing, permits developing T cells with evolving receptors to scan cortical epithelial cells for positive selection on self-peptide/self MHC. Additionally, we have found that shark TCRδ can employ variable gene segments of IgM and IgW in rearrangements, a situation that may be similar to the IgHV use by TCRδ noted in higher vertebrates. Future studies must further study the lineage commitment of shark B and T cells and the receptor plasticity of these lineages in all vertebrates.
Bovine monocyte-derived dendritic cell populations from animals in late gestation show altered response to E. coli (VET2P.1039)
Authors: Pomeroy Brianna , Sipka Anja , Klaessig Suzanne , Schukken Ynte
Published: January 2023

During late gestation the bovine immune system is less capable of eliminating pathogens. Most new intra-mammary infections occur in late gestation, and the majority of these infections are caused by E. coli. Unlike non-pregnant, lactating cows, late gestation cows are unable to elicit an effective inflammatory response to intra-mammary infection caused by E. coli. During pregnancy, the immune system appears to shift to a humoral, anti-inflammatory, so called T helper 2 bias with increased tolerance. Dendritic cells are antigen presenting cells that orchestrate activation and differentiation of naive T cells, and they are hypothesized to play a key role in directing the T helper 2 bias and immune tolerance observed in gestation. We stimulated monocyte-derived dendritic cells with E. coli and compared responses from late gestation cows to those of non-pregnant cows. Results show monocyte-derived dendritic cells from late gestation cows have altered surface molecule expression and cytokine production compared to those from non-pregnant cows. Our findings support the hypothesis that late gestation bovine dendritic cells are likely to produce suboptimal T cell activation in response to E. coli antigens, and play a role in observed immune-bias in pregnancy. In addition, altered dendritic cell function in gestation may impede current, routine vaccination regiments in late gestation. Future work will identify likely pregnancy-factors that modulate bovine dendritic cell responses.
Suppression of B cell responsiveness by LP-BM5 retrovirus-induced myeloid derived suppressor cells generated during a murine acquired immunodeficiency syndrome: a role for negative checkpoint regulator expression on the MDSCs (VIR7P.1059)
Authors: Green Kathy , Wang Li , Green William
Published: January 2023

The inhibitory activities of myeloid derived suppressor cells (MDSC) against T cell responses are well established in tumor microenvironments. We published (2013, J. Virol. 87:2058-2071) on the induction of monocytic MDSCs during infection of susceptible B6 mice by LP-BM5 retrovirus, which causes a profound immunodeficiency. These MDSCs inhibited not only T, but also B cell, responsiveness to polyclonal stimulation in ex vivo suppression assays. Whereas MDSC inhibition of stimulated T cell proliferation and IFN-gamma production was largely iNOS/NO dependent, MDSC suppression of B cell responses was only partially (~50%) due to iNOS/NO - as shown by using iNOS inhibitors and LP-BM5 infected iNOS k.o. mice as a source of MDSCs. Here, we further study additional suppressive mechanism(s) in the MDSC inhibition of B cell responsiveness. Using MDSCs from LP-BM5 infected mice of negative checkpoint regulator knock-out origin, and specific blocking antibody, MDSC suppression of B cell responses was partly dependent on MDSC expression of certain checkpoint regulators. Combining the use of reagents to interrupt both iNOS/NO and checkpoint regulation lead to a synergistic blocking of MDSC suppression of B cell responsiveness. These results were compatible with a role for MDSC in LP-BM5 induced immunodeficiency and highlight the involvement of multiple and unique suppressive pathways in the under-studied area of MDSC suppression of B cell responses.
T and B cell memory responses in 2012 filovirus outbreak survivors (VIR7P.1060)
Authors: Stonier Spencer , Herbert Andrew , Sobarzo Ariel , Kuehne Ana , Eskira Yael , Lutwaama Julius , Lobel Leslie , Dye John
Published: January 2023

From 2011 to the present, there have been four confirmed filovirus outbreaks with a fifth case involving a single patient. Four outbreaks/cases were attributed to the Ebolavirus genus, with Marburgvirus responsible for one. With the high lethality rates seen in these outbreaks (generally ~40-90%), and the potentially dramatic disease sequelae, filovirus outbreaks can devastate local communities and present great health risks for affected regions. These issues obviate the need for effective vaccines and therapeutics to limit and manage future outbreaks. In 2012 alone, three outbreaks occurred in Uganda - two of Sudan virus and one Marburg virus. We collected PBMC and serum samples from survivors from each outbreak to evaluate the presence of immune memory and its composition. Serum antibodies displayed broad specificity to Marburg virus and Sudan virus glycoprotein in addition to other proteins. Virtually all serum samples displayed neutralizing activity. PBMCs stimulated with viral and recombinant antigen were evaluated by flow cytometry for functional parameters as well as level of antigen-responsive T cells. Cytokines in the supernatants of these cultures were determined by multiplex ELISA. Cytokine responses were largely pro-inflammatory produced predominantly by CD4 T cells. Establishing immune memory parameters and the nature thereof gives us targets for vaccine development and opens avenues for development of immune-based therapeutics.
Provirus activation plus CD59 blockage triggers antibody-dependent complement-mediated lysis of latently HIV-1-infected cells (VIR7P.1050)
Authors: Yu Qigui , Lan Jie , Byrd Daniel , Hu Ningjie , Amet Tohti , Shepherd Nicole , Desai Mona , Yang Kai , Gupta Samir
Published: January 2023

Latently HIV-1-infected cells are recognized as the last barrier towards viral eradication and cure. To purge these cells, we combined a provirus stimulant with a blocker of human CD59, a key member of the regulators of complement activation (RCA), to trigger antibody-dependent complement-mediated lysis (ADCML). Provirus stimulants including romidepsin (RMD), suberoylanilide hydroxamic acid (SAHA) and prostratin activated proviruses in the latently infected T cell line ACH-2 as virion production and viral protein expression on the cell surface were induced. These results would lead to cytolytic clearance by viral cytopathic effects (CPEs), host immune responses or both. Immune attack against provirus-activated cells is critical as antiretroviral drugs did not affect the percentage of cells expressing viral proteins, but markedly suppressed virus production, thereby attenuating CPEs. RMD was the most attractive provirus stimulant as it not only effectively activated proviruses at nanomolar concentrations, but also enhanced cell surface expression of MHC-I and NK cell-activating receptors, potentially rendering these cells sensitive to cell-mediated immunity. Provirus-activated ACH-2 cells became sensitive to ADCML in the presence of a CD59 blocker plus anti-HIV-1 antibodies or plasma from HIV-1-infected patients. Therefore, a combination of provirus stimulants with RCA blockers represents a novel approach to eliminate HIV-1.
A first look at the protective immunity elicited by the yellow fever virus 17D live-attenuated vaccine (VIR7P.1051)
Authors: Watson Alan , Lam LK Metthew , Gardner Christina , Klimstra William , Ryman Kate
Published: January 2023

A hallmark of anti-viral vaccination for 77 years has been the safe and effective live-attenuated 17D yellow fever virus (YFV) vaccines. Among more than 500 million individuals vaccinated, only five cases of yellow fever have been reported. This efficacy is presumed to be the result of both neutralizing antibodies and a robust CD4+ and CD8+ T cell response. However, the importance of these immune components for protection following YFV challenge has never been evaluated. For the first time, we have addressed this question using a murine model in which YFV infection is lethal and 17D immunization protects 100 percent of animals. Similar to humans, vaccination in mice elicits life-long protection against challenge characterized by high neutralizing antibody titers and a robust polyfunctional T cell response. YFV-specific T cells express markers congruent with the differentiation of long-term memory. Additionally, T cell mediated cytotoxicity is detected for greater than one year post vaccination against MHC-I and MHC-II restricted targets. Adoptive transfer of immune sera or CD4+ T cells alone provides partial protection against YFV, but CD8+ T cells do not contribute to disease mitigation. Complete protection from disease requires both immune sera and CD4+ T cells. Further understanding the immune mechanisms that make 17D an effective vaccine will aid in the improvement of 17D-chimeric vaccines targeting flaviviruses and other pathogens.
Differentiation and function of murine gammaherpesvirus specific CD4+ T cells (VIR7P.1052)
Authors: Hu Zhuting , Blackman Marcia , Usherwood Edward
Published: January 2023

Gammaherpesvirus infection in AIDS patients is associated with severe disease. CD4+ T cells are essential for immune control of viral infection and prevention of malignancy. Progressive loss of CD4+ T cells results in an erosion of immune surveillance, which may lead to gammaherpesvirus associated tumors. A recently generated murine gammaherpesvirus-68 gp150-specific TCR transgenic mouse provides a model for studying CD4+ T cells more precisely. We aimed to study differentiation of MHV-68-specific CD4+ T cells and the contribution of the CD4+ T cells in immune responses independent of CD8+ T cells and B cells. The gp150-specific CD4+ T cells differentiated into two interconvertible populations based on Ly6C expression. The majority of these cells differentiated into Th1 cells, producing IFN-γ and expressing T-bet but not eomes, GATA3, RORγ or Bcl6. Ly6C expression was positively correlated with T-bet expression, IFN-γ, TNF-α and granzyme B production. The gp150-specific CD4+ T cells prolonged survival of MHV-68-infected mice and controlled growth of MHV-68-immortalized SL1 cells in gp150-Tg mice without CD8+ T cells and B cells. Their antiviral functions were dependent on IFN-γ, and the CD4+ T cells also had cytolytic activity. In summary, CD4+ T cells that arise during MHV-68 infection are mostly Th1 and display antiviral ability in the absence of CD8+ T cells and B cells through IFN-γ.
Contribution of DCs and monocytes/macrophages to disease control or progression in SIVmac251-infected Mamu-B008+ rhesus macaques (VIR8P.1065)
Authors: Bouwer Anthea , Smith Amanda , Swan Zachary , Barratt-Boyes Simon
Published: January 2023

Dendritic cells (DC), monocytes and macrophages are known to play a critical role in the induction of antiviral immune responses. However in the case of HIV/SIV, it remains unclear if this response is beneficial or detrimental in the progression of the disease. To examine the host-virus relationship we are longitudinally following the viral load and assessing the DC and monocyte/macrophage responses in the blood, lymph nodes and ileum of SIVmac251-infected Mamu-B*008+ rhesus macaques. Mamu-B*008 is an MHC class I allele highly correlated with control of SIV replication and a model for controlled and progressive HIV-1 disease. Previous studies have shown that approximately 50% of Mamu-B*008+ rhesus macaques control SIVmac239 replication in the chronic phase in a manner resembling elite control that has been observed in a small subset of HIV-infected humans. Utilizing intracellular cell staining, we are currently investigating the inflammatory cytokine contributions of pDC, mDC and monocytes/macrophages during disease progression. Preliminary findings suggest that pDC may not solely be responsible for the persistent interferon response, characteristic of HIV/SIV infection. As our study continues, we hope to gain a more complete understanding of the roles that DC and monocytes/macrophages play in the progression or control of SIV infection.
Integrin α4β7 is an immunological marker for HIV preference in activated peripheral and cervical CD4+ T cells (VIR8P.1066)
Authors: Chang Theresa , Ding Jian , Tasker Carley , Lu Wuyuan , Lespinasse Pierre , Heller Debra
Published: January 2023

CD4+ T cells, the principal target in acute SIV and HIV infection, are crucial for the establishment and dissemination of HIV infection in mucosal tissues. Studies indicate that α4β7 CD4+ T cells are preferentially infected by HIV in vitro and during acute SIV infection. Integrin α4β7 is thought to promote HIV capture by target cells; however, the role of integrin α4β7 in HIV transmission remains controversial. In this study, we characterized immunological profiles of human cervical T cells and examined HIV preference in α4β7+ CD4+ T cells. In vitro all-trans retinoic acid differentiated peripheral CD4+ T cells were included as a comparison. In both peripheral and cervical cells, HIV preferentially infected CD38+ activated cells. Although integrin α4β7 was not the major determinant for HIV preference, the majority of HIV p24+ cells were found in activated CD4+ T cells expressing α4β7. Interestingly, CCR5 was not a marker for HIV preference. The cyclic hexapeptide CWLDVC and monoclonal antibody against integrin α4β7 did not block HIV attachment or gp120 binding to target cells regardless the presence of CD4, indicating that integrin α4β7 did not contribute to facilitating HIV capture by target cells. Our results suggest that integrin α4β7 may play a role in HIV susceptibility but not through promoting HIV binding to target cells.
Relative resistance to HLA-DM editing is a distinguishing characteristic of Type-1 diabetes associated HLA-DQ molecules (APP2P.100)
Authors: Zhou Zemin , Reyes-Vargas Eduardo , Escobar Hernando , Rudd Brant , Rockwood Alan , Delgado Julio , He Xiao , Jensen Peter
Published: January 2023

Homozygous HLA-DQ2, 8 or heterozygous 2/8 are highly associated with developing Type-1 diabetes (T1D), but the underlining molecular mechanism is not clear. HLA-DM is essential for editing peptides bound to MHC-II, thus influencing the repertoire of peptides presented by APCs, with potential impact on selection/activation of CD4+ T cells. We hypothesized that unique properties of T1D associated DQs, including different binding affinity for CLIP or other peptides & different sensitivity to DM editing, might confer high risk of T1D. Higher CLIP levels were found on the surface of DQ2+, 8+ or 2/8+ cells, compared to T1D protective DQ6+ cells, indicating a relative resistance to DM editing. HPLC-MS/MS analysis of eluted peptides from DQs showed that CLIPs containing longer N-terminal tails & more long peptides (>20 aa) were loaded on DQ2, 8 or 2/8, compared to DQ6. Among eluted peptides, several were derived from known auto-Ag identified in T1D. Peptide binding motifs of DQ2, 8 & 2/8 were different from DQ6, but similar to that of DQ8 as previously reported. Furthermore, DQ peptide binding assay demonstrated that CLIP2 had higher relative binding affinity to DQ2, 8 or 2/8, compared to DQ6. Disruption of hydrogen bonds near P1 pocket in DQ8 reversed its DM editing resistance. Collectively, our results showed that the DM sensitivity of DQ2, 8 & 2/8 is lower than that of DQ6. The relative resistance to DM editing of the T1D associated DQs may contribute to the pathogenesis of T1D.
Eight novel H-2Kb and H-2Db restricted epitopes of ovalbumin (APP2P.109)
Authors: Karandikar Sukrut , Srivastava Pramod
Published: January 2023

CD8+ T cells recognize epitopes, which are generally eight to ten amino acid long peptides presented by MHC class I molecules. Ovalbumin (OVA) has two well documented H-2 Kb restricted epitopes: SIINFEKL- the immunodominant epitope and KVVRFDKL- the sub-dominant epitope. Analyzing the sequence of OVA with NetMHC, a peptide-MHC binding affinity prediction tool, revealed 17 additional putative epitopes presentable by H-2Kb or H-2Db. Eight of these 17 elicit antigen-specific CD8+ T cell responses in naïve C57BL/6 mice. We are currently testing the direct presentation and the immunodominance hierarchy of these epitopes.
Bacterial amyloids promote type I interferon production and accelerate autoimmunity (BA3P.206)
Authors: Gallo Paul , Rapsinski Glenn , Tukel Cagla , Gallucci Stefania
Published: January 2023

Infection is a major cause of morbidity and mortality in patients with systemic lupus erythematosus (SLE). Curli is a bacterial amyloid that serves as a component of enteric bacterial biofilms. Patients are exposed to curli during urinary tract infections, gastrointestinal infections, and sepsis. Here we studied the immune response to curli amyloids in the context of murine SLE. We found that curli amyloids from bacterial cultures contain abundant nucleic acids and that exogenous DNA enhances amyloid fibrillization. Bone marrow-derived myeloid DCs strongly respond to curli-DNA composites, producing large quantities of IL-12, IL-6, and IL-10. Curli potently induces expression of IFN-β and IFN-stimulated genes. Using both natural and synthetic curli, we found that curli and nucleic acids synergize to activate DCs. Young, pre-disease lupus-prone NZBW-F1 mice injected i.p. with nucleic acid-containing curli produce high titers of anti-dsDNA and anti-chromatin autoantibodies within 2 weeks of the first injection, much earlier than mice treated with PBS. In conclusion, the bacterial amyloid curli contains nucleic acids which are integral to the immune response to bacterial biofilms. Curli is a potent activator of dendritic cells and can rapidly accelerate autoimmunity in lupus-prone mice. This work suggests that nucleic acid-containing bacterial amyloids are an important environmental trigger for lupus and that curli injection may be a novel method to accelerate murine lupus.
Molecular aspects of IL-13 up-regulation by CD8+ T cells from systemic sclerosis patients (BA3P.132)
Authors: Fuschiotti Patrizia , Cascio Sandra , Moss Alissa , Ivanco Dana , Domsic Robyn , Milcarek Christine , Medsger Thomas
Published: January 2023

Systemic sclerosis (SSc), an incurable connective tissue disease, is characterized by vascular damage, inflammation and fibrosis. T cells are important in pathogenesis and produce cytokines that contribute to the induction of fibrosis. We found that dysregulated production of the profibrotic cytokine IL-13 by effector CD8+ T cells is associated with more severe skin thickening in SSc and defects in the molecular control of IL-13 production, such as increased expression of the Th2-specific transcription factor GATA-3. Silencing of GATA-3 by siRNA blocks IL-13 production in CD8+ T cells, demonstrating a causal relation¬ship between GATA-3 and IL-13. GATA-3 is also highly expressed by CD8+ T cells in lesions of patients where it may be associated with overproduction of IL-13. Th1-specific T-bet induces IFN-γ production and inhibits Th2 cytokines, including IL-13, by antagonizing GATA-3 expression and/or function. Here we show that CD8+ T cells from patient blood express high levels of IL-13 and GATA-3 but levels of IFN-γ and T-bet similar to controls. We also found that the interaction between T-bet and GATA-3 in SSc CD8+ T cells is weaker allowing more GATA-3 to bind to the IL-13 promoter and induce its expression. We conclude that increased IL-13 expression by SSc CD8+ T cells results, at least in part, from reduced down-regulation of GATA-3 by T-bet. Our new insights will establish novel biomarkers of immune dysfunction in SSc patients that can be used as therapeutic targets.
Memory B cells are critical in gamma herpes virus driven disease enhancement of a mouse model for MS (BA2P.124)
Authors: Marquez Ana , Casiraghi Costanza , Shanina Iryna , Horwitz Marc
Published: January 2023

Genetic and environmental factors are known to be involved in the development of Multiple Sclerosis. However, the mechanism describing how these factors are responsible for the onset of MS has not been elucidated. One of the environmental factors believed to play an important role in the development of MS is previous infection with the Epstein Barr Virus (EBV). The study of the relationship between MS and EBV remains elusive since EBV only infects humans. We hypothesized that latent EBV infection alters the immune system’s response in genetically predisposed individuals, thereby exacerbating autoimmunity. To address this question our lab previously developed a model where latent infection with murine gamma herpesvirus 68 (γHV-68), the murine homolog to EBV, enhances the symptoms of experimental autoimmune encephalomyelitis (EAE), which closely resembles MS pathogenesis. These enhanced symptoms are driven by the upregulation of CD40 on dendritic cells (DCs) of latently infected mice which drives the priming of a strong TH1 response and a decrease in Tregs frequencies. Latency of γHV-68 mainly occurs in memory B cells and the virus is not found in the DCs driving the TH1 response. Here we demonstrate that latently infected memory B cells are the cells responsible for the upregulation of CD40 in DCs, most likely through the upregulation of type I interferons in DCs. This represents a novel mechanism by which infection with EBV can influence MS and other autoimmune diseases.
Genetic variant at ETS1 locus increases lupus risk and affects Stat1 binding (BA2P.115)
Authors: Lu Xiaoming , Zoller Erin , Weirauch Matt , Namjou Bahram , International Consortium on the Genetics of SLE The , Greis Kenneth , Shen Nan , Kaufman Kenneth , Harley John , Kottyan Leah
Published: January 2023

Over the past 10 years, genetic association studies have identified over 55 systemic lupus erythematous (SLE)-risk loci. Genetic variants in ETS1 have been associated with SLE in several independent studies in populations of Asian ancestry. Several recent studies have implicated Ets1 as a critical driver of B and T cell function; indeed, mice deficient in Ets1 develop an SLE-like autoimmunity. rs1128334, in the 3’ UTR of ETS1, has been associated with the decreased expression of Ets1. We performed a fine-mapping study of over 20,000 subjects of two multi-ancestral cohorts using genotyped and imputed variants spanning the ETS1 locus. By constructing genetic models using frequentist and Bayesian association methods, we identified a set of variants that are most likely to be causal. Of the three variants that we tested, only rs6590330 differentially binds lysate from B cell lines as assessed by electrophoretic mobility shift assays and DNA affinity precipitation assays. We found that the active form of the transcription factor Stat1 binds the risk allele of rs6590330 but not the non-risk allele using mass spectrometry, a finding confirmed by Western blot analysis. rs6590330 is in strong linkage disequilibrium with rs1128334 and is predicted to also be associated with decreased Ets1 expression. We propose a model in which the risk allele of rs6590330 increases SLE risk by binding Stat1 and depressing the expression of Ets1.
Shared VH1-46 gene usage by anti-desmoglein 3 antibodies in pemphigus vulgaris suggest a common mechanism for developing autoimmunity (BA2P.116)
Authors: Cho Michael Jeffrey , Lo Agnes , Mao Xuming , Nagler Arielle , Choi Eun Jung , Sharma Preety , Uduman Mohamed , Li Hong , Rux Ann , Farber Sara , Rubin Courtney , Kleinstein Steven , Sachais Bruce , Posner Marshall , Cavacini Lisa , Payne Aimee
Published: January 2023

Pemphigus vulgaris (PV) is a potentially fatal blistering disease caused by autoantibodies (autoAbs) to desmoglein 3 (Dsg3). To better understand the mechanism by which PV Abs develop, we cloned human anti-Dsg3 Abs from four PV patients using phage display (n=2) or heterohybridoma (n=2). We identified VH1-46 anti-Dsg3 Abs from all 4 patients, comprising 5 unique heavy chain complementarity determining region (CDR) 3 sequences. All 5 Abs caused suprabasal blisters in human skin explants. Surprisingly, most VH1-46 Abs had few replacement mutations and did not show evidence of antigen-driven positive selection by focused binomial testing of mutation patterns. To determine if somatic mutations are required for Dsg3 binding, we reverted mutated Abs to their germline (GL) VDJ sequences. Three GL Abs bound Dsg3 by ELISA, with a 2- to 80-fold decrease in affinity as measured by surface plasmon resonance. Analysis of mutation patterns in 2 other Abs which lost Dsg3 binding after mutation reversion revealed homologous acidic amino acid CDR mutations. Site-directed mutagenesis indicated that these acidic residues are necessary and sufficient for Dsg3 binding. Our data indicate that VH1-46 autoAb gene usage is shared among PV patients which may be due to naive autoreactivity of some VH1-46 Abs or the requirement for very few acidic amino acid mutations in the CDRs to confer Dsg3 binding. Common VH1-46 autoAb gene usage may suggest a common mechanism for developing autoimmunity in PV.
Defective lysosomal degradation of immune complexes drives activation of cytosolic sensors in lupus-prone mice. (BA4P.210)
Authors: Monteith Andrew , Ah Kang Sun , Scott Eric , Costello Joe , Clarke Stephen , Vilen Barbara
Published: January 2023

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by heightened production of autoantibodies to nuclear antigen resulting in immune complex (IC) formation. While the loss of tolerance in SLE has been connected with the formation of ICs, little is known about the mechanism of immune dysregulation. Recently, we found that low levels of ICs are displayed via FcγRs on dendritic cells, macrophages (MFs) and B cells from C57BL/6 mice. In contrast, cells from lupus-prone (MRL/lpr) mice show a 30-50-fold increase in surface bound ICs. We’ve demonstrated that MRL/lpr MFs fail to properly mature their lysosomes following phagocytosis of ICs, causing them to be recycled back to the cell surface 48-72 hours later. Impaired lysosomal degradation has been demonstrated to form frustrated phagosomes, which can leak vesicular cargo into the cytosol. Internalized ICs contain both nuclear antigen and IgG, both of which can bind to intracellular sensors. Here we demonstrate that impaired lysosomal degradation in MRL/lpr MFs results in dsDNA from ICs activating the cytosolic sensors Aim2 and IFI204. In addition, the IgG activates the cytosolic sensor TRIM21. The combined activation of these cytosolic sensors leads to increased cell death from pyroptosis and the secretion of the IFNα. Overall, we have defined a novel defect associated with SLE where the inability to degrade ICs drives the activation of cytosolic sensors that contribute to SLE related pathologies.
TLR9 restricts survival of anergic anti-DNA B cells in systemic lupus erythematosus yet is also required for their activation (BA4P.219)
Authors: Nickerson Kevin , Christensen Sean , Cullen Jaime , Meng Wenzhou , Luning Prak Eline , Shlomchik Mark
Published: January 2023

Anti-DNA autoantibodies are a clinical hallmark of systemic lupus erythematosus. Nucleic acid reactive B cells arise in the bone marrow even in healthy individuals, but are tolerized by mechanisms including receptor editing, functional anergy, and/or deletion. TLR9, a sensor of endosomal dsDNA, both promotes and regulates systemic autoimmunity in vivo, but the mechanisms of its apparently contradictory roles in autoimmunity remained unclear. Here, using the 3H9 anti-DNA B cell receptor (BCR) transgene in the autoimmune-prone MRL.Fas-lpr mouse model of lupus, we identify the stages at which TLR9 contributes to establishing and breaking B cell tolerance. Although TLR9 is dispensable for editing and establishment of anergy, TLR9 limits anti-DNA B cell lifespan in the periphery and is thus tolerogenic. In the absence of TLR9, anti-DNA B cells have much longer lifespans and accumulate in the follicle, neither activated nor deleted. These cells retain some characteristics of anergic cells, in that they have elevated basal BCR signaling but impaired induced responses and downregulate their cell surface BCR expression. Nonetheless, in older autoimmune-prone animals, TLR9 is required for spontaneous peripheral activation of anti-DNA B cells and their differentiation into AFCs via an extrafollicular pathway. Thus, TLR9 has paradoxical roles in regulating anti-DNA B cells: it helps purge the peripheral repertoire of autoreactive cells yet is also required for their activation.
Lymphotoxin-expressing marginal zone B cells support marginal zone integrity by maintaining the megakaryoblastic leukemia-1 expression (BA4P.221)
Authors: Li Hao , Hsu Hui-Chen , Wu Qi , Yang PingAr , Li Jun , Luo Bao , Fu Yang-Xin , Zhou Yong , Mountz John
Published: January 2023

Marginal zone macrophages (MZMs) engulf apoptotic debris and play a key role in preventing autoimmunity. We previously reported that in autoimmune BXD2 mice, there is a progressive reduction in the function and numbers of MZMs associated with a gradual decline in MZ B cells. MZ B cells, compared with follicular B cells, expressed higher levels of membrane bound lymphotoxin (mLT). A mixed bone marrow reconstitution(Ly5.1+Ltβr+ : Ly5.2+Ltβr-=1:1) in Rag1-/- mice with an biased repopulation of MZMs (88:12), demonstrated that LT was essential for the maintenance of MZMs. To identify the mechanism of mLT on the maintenance of MZMs, MZM gene expression of BXD2 mice treated with PBS or soluble LTβ receptor (LTβR-Ig) was analyzed by the Illumina MouseWG-6 chips. Pathway analysis revealed that megakaryoblastic leukemia-1(MKL1) mediated genes were the most significantly down-regulated by LTβR-Ig. MKL1 is a Rho kinase mechanosensing signal that mediates cell elasticity. There was dramatic reduction of MKL1 in MZMs of BXD2 and CD19.Cre LTβf/f mice, compared to normal B6 mice. Similarly, there were reduced MZMs as well as spontaneous development of germinal centers and autoantibodies in B6-Mkl1+/- mice, compared to Mkl1+/+ mice. Administration of CCG1423 (0.15 mg/kg), a Rho/MKL1inhibitor, to BXD2 mice exacerbated the loss of MZMs. Together, our results suggest a novel immune tolerance mechanism that LT+ B cells in the MZ support MZM integrity by maintaining cell elastic signal, MKL1
The role of a Stat4 deficiency in Atherosclerosis (CAM1P.237)
Authors: Taghavie-Moghadam Paresa , Butcher Matthew , Kaplan Mark , Nadler Jerry , Galkina Elena
Published: January 2023

Atherosclerosis is a chronic inflammatory process that leads to plaque formation in large and medium size vessels. Th1 cells constitute a majority of plaque T cells and play a pro-atherogenic role. Signal transducer and activator of transcription 4 (Stat4) is a transcription factor involved in Th1 cell differentiation. To test a role of Stat4 in atherogenesis, we bred Stat4-deficient (Stat4-/-) and atherosclerosis-prone Apolipoprotein E (Apoe-/-) mice. Stat4 deficiency resulted in a 70% reduction in plaque burden in Stat4-/-Apoe-/- vs Apoe-/- mice (p<0.01). Surprisingly, reduced atherogenesis was not due to attenuated IFNγ production suggesting an IFNγ-independent pro-atherogenic role of Stat4. Stat4 is highly expressed in T cells, but also detected in macrophages (MΦ). To further test an impact of Stat4 deficiency in myeloid cells, we analyzed activation status of MΦ. Stat4-/-Apoe-/- CD11b+ MΦ expressed low levels of CD69, MHCII, and CD86 when pulsed with LPS vs Apoe-/- MΦ. Differentiated in vitro Stat4-/-Apoe-/- M1 or M2 MΦ also had reduced expression of CCL2 and CXCL10 vs Apoe-/- M1 and M2 MΦ. Importantly, the percentage of aortic Ly6C+ (p<0.02) and CD68+ (p<0.06) cells was reduced in Stat4-/-Apoe-/- vs Apoe-/- mice. The data provides evidence for a functional role of Stat4 in atherosclerosis by elevating levels of MΦ activation and aortic MΦ content, and thus promoting the localized aortic pro-inflammatory immune response. Supported by R01 HL112605 and Suppl.Grant 02S1.
Expression of CCL20 in hapten challenged skin induces recruitment of hapten-primed CD8 T cells producing IL-17 into the skin (CAM1P.229)
Authors: Kish Danielle , Fairchild Robert
Published: January 2023

Contact hypersensitivity (CHS) is a CD8 T cell-mediated response to skin sensitization and challenge with hapten. Hapten-presenting endothelial cell (EC) activate hapten-primed CD8 T cells to produce IFN-γ and IL-17 in the challenge site. These cytokines stimulate the EC to produce CXCL1 and CXCL2 that direct required CXCR2+ leukocyte recruitment into the skin challenge site for elicitation of CHS. This study investigated mechanisms directing hapten-primed CD8 cell localization to the hapten presenting EC in the challenge site. In the draining lymph nodes of hapten sensitized mice, CD8 T cells expressing CCR6 produce IL-17 in response to hapten-presenting cells and CD8 T cells expressing CXCR3 produce IFN-γ. Hapten skin challenge induces CXCL9/10 and CCL20 expression within 1 h and hapten labeling of EC cultures induces CXCL9/10 and CCL20. Anti-CCL20 antibody given at the time sensitized mice are challenged with hapten reduces IL-17 and CXCL1, but not IFN-γ, expression in the skin challenge site. IL-17 and CXCL1, but not IFN-γ, expression is also reduced in the skin challenge site of sensitized TLR4-/- mice, that have low CHS responses despite wild-type levels of hapten-reactive IFN-γ and IL-17 producing CD8 T cells in the skin draining lymph nodes. These results suggest that TLR4 activation of hapten-presenting EC is required to induce the CCL20 expression directing hapten-primed CCR6+ CD8 T cells to the challenged skin site to mediate CXCL1 production and elicit CHS.
Sounding the alarm: studying the role of signal cascades in the immune response (EDU1P.250)
Authors: Trice Mark
Published: January 2023

The purpose of this research opportunity was to investigate the methods and roles of cell signaling via protein cascades and their connections to the functionality of the immune system. During my summer internship, I worked with investigators at the United States Army Medical Research Institute for Infectious Diseases (USAMRIID) to develop and test the activity of man-made protease cascades that could someday mirror the complement system in its aggressive response to specific signal molecules. My research experience inspired me to develop a unit for a second year Biology/AP Biology course. Students will examine the effects of several factors, including protease concentration, substrate concentration, and a non-cascade reaction, on reaction rates through construction of both physical models and simulated protease cascades. In this unit, students first work with a hands-on physical manipulative model to familiarize them with how a protein cascade works. Then, all of the students will work in tandem on a kinesthetic activity modeling exactly how protein cascades behave. This activity allows for a cost-effective method of demonstrating protease cascades without expensive lab equipment and reagents. Students translate their knowledge into the application stage when they examine how protease cascades work in the human body and discuss various uses and effects of cascades, such as protein kinases, on the immune system.
Loss of human B cells bearing highly charged and highly hydrophobic amino acids in CDR-H3 at the pre B cell receptor stage (HEM2P.267)
Authors: Khass Mohamed , Blackburn Tessa , Saad Ayman , Lamb Lawrence , Burrows Peter , Schroeder Harry
Published: January 2023

Successive checkpoints in B cell development including differentiation in the bone marrow and the peripheral lymphoid organs control the composition and reactivity of the antibody repertoire. One of the first key checkpoints is testing of the efficiency of the heavy chain (HC) by forming a pre B cell receptor (pre BCR) composed of the newly generated μHCs and invariant surrogate light chains. Similar to mouse, human B cells with highly charged or highly hydrophobic antigen binding sites are rare in the mature B cell repertoire. Suppression of highly hydrophobic antigen binding sites in mouse reflects, in part, reduced use of RF2 due to Dμ protein containing preBCRs, but the mechanism(s) by which these clones are suppressed in human is unknown. We cloned and sequenced HC transcripts from early to late pre B cells in bone marrow samples obtained from healthy human donors and then used our bioinformatics approach to dissect and analyze the diversity of the third complementary determining region of the HC (CDR-H3). The prevalence of both highly hydrophobic and highly charged CDR-H3s dropped in the transition from early to late pre B cells. This suggests a novel mechanism of selection in human, where the pre BCR controls the passage of B cells based on their CDR-H3 amino acid content. These findings can help explain the diminished production of HIV neutralizing antibodies with abnormal highly charged or highly hydrophobic CDR-H3s due to their loss at the pre BCR stage.
Age-associated B cells (ABC) cause reduced surrogate light chain expression and loss of B cell precursors in old age (HEM2P.269)
Authors: Riley Richard , Ratliff Michelle , Alter Sarah , Frasca Daniela , Wright Jacqueline , Khan Wasif , Blomberg Bonnie
Published: January 2023

Age-associated B cells (ABC, CD21/35- CD23-) accumulate in spleen and bone marrow of ~2 yrs. old mice and induce TNF-α-dependent apoptosis of pro-B cells. The remaining B cell precursors in aged mice exhibit reductions in surrogate light chain (SLC) and compromise of the preBCR checkpoint. Notably, the residual pro-B cells in aged bone marrow are resistant to ABC/TNF-α-mediated apoptosis. SLC is associated with cadherin 17 on the surface of pro-B cells. Like SLC-low aged pro-B cells, pro-B cells from λ5 SLC-deficient young adult mice were also resistant to TNF-α-induced apoptosis. This implies that expression of the SLC/cadherin 17 complex is required for normal susceptibility to apoptosis in pro-B cells. Resistance to apoptosis in aged pro-B cells and λ5-deficient young pro-B cells coincided with increased phosphorylation of the pro-apoptotic protein Bim, a modification that tags Bim for degradation. We hypothesize that, in old mice, expanded pro-inflammatory ABC within the bone marrow cause apoptosis of pro-B cells. However, this cell death occurs preferentially in pro-B cells which express relatively high levels of SLC. The remaining pro-B cells in aged mice are consequently skewed to lower SLC expression. We suggest that ABC, by inducing the loss of “high SLC” pro-B cells, deviate B lymphopoiesis into an ”SLC low pathway” with inefficient B cell production and altered B cell antibody repertoire.
Chronic TLR4 stimulation selectively cripples human lymphopoiesis (HEM3P.286)
Authors: Wang Yujuan , Liu Ailing , Ding Ying , Payne Kimberly , Borghesi Lisa
Published: January 2023

Long-term hematopoietic stem cells (LT-HSCs) must replenish all immune cells lost due to natural turnover, inflammation or injury throughout lifetime. Our recently published data demonstrate that following chronic, low-dose TLR4 agonist stimulation, murine HSCs hyperproliferate, preferentially undergo myeloid-specific differentiation and exhibit compromised self-renewal. However, the impact to human LT-HSC function in vivo has not been established. Here we reconstituted NSG mice with human CD34+ HSCs derived from 3 different donors, and tracked hematopoiesis. Following chronic, low-dose exposure to the TLR4 agonist lipopolysaccharide (LPS), human bone marrow CD34+ HSCs were decreased ~2-fold while downstream CLPs and pro-B cells were dramatically reduced about 90%. In spleen and peripheral blood the percentage of CD19+ B cells was decreased 2-4-fold while CD56+ NK and CD3+ T were proportionately increased. Myeloid cells were largely unaffected. These findings are potentially important because TLR4 on HSCs is activated by two ligands, LPS and dietary fatty acids that are commonly elevated in diseases of chronic inflammation as well as in diet-induced obesity. Our studies may provide a previously unrecognized mechanism of human HSC exhaustion and/or compromised hematopoiesis in patients suffering from these chronic conditions.
Plasma gelsolin as a novel biomarker for cardiac surgery associated acute kidney injury in infants and young children (HUM1P.311)
Authors: Shu Qiang , Shi Shanshan , Fan Jiajie , Chen Chi , Zhao Dongyan , Liu Xiwang , Cheng Baoli , Wu Shuijing , Lin Ru , Tan Linhua , Fang XiangMing
Published: January 2023

Objective Cardiac surgery associated acute kidney injury (CS-AKI) strongly correlated with postoperative morbidity and mortality, especially in pediatric population. The purpose of this study was to investigate the role of plasma gelsolin (pGSN) in patients ≤3 years old, and determine whether pGSN was associated with the occurrence and severity of CS-AKI. Methods 77 patients ≤3 years of age performed on cardiac surgery were enrolled. Plasma gelsolin, creatinine, cystatin C and total protein were measured at 7 perioperative time points. Results Normalized pGSN (pGSNN) of AKI group dropped immediately after CPB (p=0.029), and still remained at lower level at 48 hours after CPB (p=0.036); PGSNN of AKI group at 2 hours after CPB was significantly lower than that of non-AKI group (p=0.002). The earliest increase of normalized pCys C (pCys CN) in AKI patients was 24 hours after CPB (p=0.008); PCys CN of AKI group at 24 hours after CPB was much higher than that of non-AKI group (p=0.002). PGSNN at 2 hours CPB was the only independent predictor of CS-AKI (p=0.032), and negatively correlated with the severity of CS-AKI (r=-0.488, p=0.002). Conclusions This study suggested that CS-AKI patients underwent a rapid and persistent drop of pGSNN after surgery; Compared with pCys CN, pGSNN appeared to be a more sensitive predictor of CS-AKI in infant and young children; PGSNN at 2 hours CPB was the only independent predictor of CS-AKI, and negatively correlated with the severity of CS-AKI.
Defining pathogenic T cells in experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS) (HUM1P.318)
Authors: Wagner David , Waid Dan , Vaitaitis Gisela , Carter Jessica , Corboy John
Published: January 2023

The etiology of MS is unclear, but autoimmunity plays a significant role. The autoimmune components are defined in the EAE model yet defining a biomarker remains problematic. We described a cell subset, Th40 cells, which proved highly auto-aggressive in diabetes. We compared Th40 levels in 90 MS subjects including clinically isolated syndrome (CIS), relapsing/remitting (RRMS) and secondary progressive (SPMS) patients to non-autoimmune controls. Diagnosed MS subjects have significantly increased percentages of Th40 cells in PBL compared to controls. Th40 cells produce pro-inflammatory cytokines. HLA haplotypes of MS patients were diverse although many carry DR2 (DR15). Th40 cells were elevated in MS patients regardless of HLA haplotype. Th40 cells responded to CNS antigens in an HLA dependent fashion. TRAV 8.1.3+ cells were expanded within the Th40 population in MS subjects. We used the EAE model to better define Th40 cells. EAE induction drastically expanded Th40 cell levels prior to symptoms. Th40 cells produce IFNg and IL-17 and infiltrate both brain and spinal cord. Th40 cells transfer EAE but surprisingly CD4+CD40- T cells also did but significantly (p < 0.001) more slowly and with less severity. CD4+CD40- cells became CD40+; suggesting the disease process induces CD40 on T cells. CD40flox.CD4.Cre mice did not develop EAE even after multiple disease induction attempts. These data suggest an important role for CD40 in T cell pathogenesis of MS and EAE.