bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of the human CD8 T cell response to exogenous antigen using MIMIC® technology (52.5)
Authors: Byers Anthony , Haupfear Kelly , Wittman Vaughan
Published: January 2023

Vaccines typically achieve immune protection by induction of humoral immunity. Recently, interest has increased in vaccines capable of eliciting both humoral and cellular immunity. Since the majority of vaccines are subunit or protein-based and CD8 T cells are typically primed by endogenous proteins expressed within the target cell, effective induction of CD8 T cell (CTL) responses by vaccines has been erratic. Exogenous antigen can be presented on MHC class I to CD8 T cells via an inefficient and poorly defined process termed cross-priming. We have used our in vitro MIMIC® technology to optimize CTL cross-priming immune responses to subunit vaccines. We have shown in vitro CTL responses to soluble influenza nucleoprotein (NP) by activating, or licensing, of dendritic cells (DC) by CD4 T cells. Further optimization has involved modification of various assay parameters: cell ratios/kinetics, antigen load, DC types used, addition of DC maturation/activation factors. Generation of the cross-priming response has been evaluated by the ability of CTL to make combinations of multiple cell factors (CD107a, IFN-γ, IL-2 and TNF-α) via intracellular cytokine staining and the ability of the CTL to eliminate targets in a fluorescent-based cytotoxic assay. The MIMIC® system can be used to evaluate CTL response to various protein/adjuvant combinations and identify corresponding vaccine candidates capable of efficiently inducing cellular immunity.
Promotion of the neutralization-competent structure of the HIV-1 gp41 membrane proximal external region when tethered to its native transmembrane domain, and expressed in the context of the plasma membrane: implications for vaccine design (53.24)
Authors: Gulzar Naveed , Montero Marinieve , Klaric Kristina-Ana , Donald Jason , Wang Shixia , Lepik Christa , Tsai Sue , Wu Sampson , Julien Jean-Philippe , Hessell Ann , Lu Shan , Burton Dennis , Pai Emil , DeGrado William , Scott Jamie
Published: January 2023

The limited success of vaccines targeting the MPER, a target of three broadly neutralizing (bNt) monoclonal antibodies (MAbs), we hypothesize, reflects the difficulty of mimicking the neutralization-competent structure (NCS) of the MPER. We determined the contribution of the amino-acid sequence and the transmembrane domain (TM), to the antigenicity of the MPER in the context of the plasma membrane. DNA constructs encoding various gp41 ectodomain fragments, and the TM of either the platelet-derived growth factor receptor (PGDFR), or that of gp41, were produced and transiently expressed in COS-7 cells. Constructs expressing the MPER tethered to the gp41 TM followed by a 27-residue cytoplasmic tail fragment, MPER-TM1, produced optimal binding of MAbs 2F5, 4E10 and Z13e1. A series of 24 single amino-acid substitutions in the MPER-TM1 revealed critical binding residues for the three MAbs; similar substitutions were previously shown to ablate Ab-mediated viral neutralization. Neutralization-incompetent 2F5 Fab and 4E10 IgG mutant Abs failed to bind MPER-TM1, yet retained the ability to bind to peptide epitopes, indicating the plasma-membrane expressed MPER-TM1 closely approaches the NCS of the MPER. Substitution of the TM of gp41 with that from the PGDFR reduced binding by MAb 4E10, but not MAbs 2F5 or Z13e1. Our studies reveal that the gp41 TM appears to play a pivotal role both in orienting the 4E10 epitope, and affecting exposure of the MPER epitopes for all three bNtMAbs.
Induction of memory responses following transcutaneous immunization requires presence of immunoadjuvantative cholera holotoxin (53.4)
Authors: Kalsy Anuj , Bhuiyan Md , Charles Richelle , Arifuzzaman Mohammad , Clemens John D. , Larocque Regina C. , Harris Jason B. , Calderwood Stephen B. , Czerkinsky Cecil , Qadri Firdausi , Ryan Edward
Published: January 2023

Vaccination induces both mucosal & systemic immune response. Polysaccharide antigen (PAg) which is T cell independent antigen does not stimulate production of memory responses. Cholera holotoxin (CT) is shown to be a potent immunoadjuvant. Transcutaneous immunization (TCI) is recently shown as an important alternative route of immunization to induce both systemic & mucosal immune responses. Induction of memory responses upon TCI with PAg is unkown. We evaluated if TCI with Vi & Vi-conjugate vaccine (Vi-DT)could induce anti-Vi specific memory responses in mice. Parenteral & TCI of Vi can not produce memory response. Splenic memory B cell IgG responses were induced by parenteral immunization with Vi-DT but not Vi, but were induced following TCI with Vi-DT/CT and Vi/CT. TC & parenteral immunization with Vi & Vi-DT induced serum anti-Vi IgG & IgA responses but more prominently along with CT & induced T cell independent PAg specific memory response in mice. Stool & lamina proprial lymphocyte (LPL) anti-Vi IgA responses were highest following TCI. Splenic antibody-secreting-cell (ASC) anti-Vi IgG responses were induced by parenteral immunization with Vi or Vi-DT, and by TCI with Vi/CT or Vi-DT/CT. Our results show that TCI induces both systemic & mucosal immune responses with PAg & use of CT as adjuvant induces this response more prominently. Memory responses only found when CT was used TC. This observation could assist in developing of vaccines providing long-term protection.
Virus induced airway hyperreactivity in allergen sensitized animals is mediated by immunological memory and is ablated by dexamethasone (55.14)
Authors: Skappak Christopher , Illarraza Ramses , Wu Ying-qi , Adamko Darryl
Published: January 2023

Common cold viruses are the leading cause of asthma exacerbations. Why people with asthma develop such severe reactions is unclear. We hypothesize that it is not the severity of virus infection but the activation of memory T-cells and eosinophils, which cause the airway hyperreactivity (AHR). We have a model with virus memory T cells in allergen-sensitized guinea pigs (GP). GP are sensitized (i.p.) to ovalbumin (OVA) or not. All receive an inhaled OVA challenge. Some of each group are exposed once to parainfluenza virus (PIV) then, 6 weeks later, re-exposed to either live virus or UV-inactivated virus. Measurements of AHR (histamine i.v.), inflammation (BAL/histology), and T cell memory (BrdU incorporation ex vivo) are measured. Additionally some animals from each group were pre-treated with dexamethasone (DEX) (i.p.) before re-exposure to PIV. Both non-sensitized and OVA-sensitized GPs develop T cell memory to virus based on BrdU incorporation data from lymph node T cells. Unlike non-sensitized GP, OVA-sensitized GP show elevated numbers of eosinophils in the airways, except those pre-treated with DEX. Unlike non-sensitized GP, OVA-sensitized GP develop AHR upon re-exposure to UV-inactivated virus, which is prevented by DEX. GP can develop T cell memory to PIV. Upon re-exposure to PIV antigens, this T cell memory can cause AHR but only sensitized animals, which we believe is mediated by eosinophils. DEX prevents AHR in sensitized animals re-exposed to viral antigens.
A novel composition from herbal extract (SCAP) alleviates skin severity and serum IgE level in NA/Nga mice, an atopic dermatitis model (55.17)
Authors: Lee Geum Seon , Oh Se Koon , Cheong Jae Hoon , Kim Kyungjae , Kang Tae Jin
Published: January 2023

Pimecrolimus (Elidel cream®) is an immunomodulating agent and is used for the treatment of atopic dermatitis (AD). However, it is reported that this treatment has side effects regarding the potential increased risk of lymph node or skin malignancy and is not sufficiently effective. SCAP is a composition of herb extracts from Sonchus oleraceus, Chelidonium majus var. asiaticum, Arctii Fructus, Portulacae Herba. which were traditionally used in oriental medicine as an analgesic, diuretic, and anti-inflammatory agents. The anti-atopic effect of SCAP was investigated in an atopic dermatitis animal model, NC/Nga mice. AD was induced by repeated application of 1-chloro 2, 4-dinitrobenzene (DNCB) in NC/Nga mice. And then mice were treated with SCAP or Pimecrolimus cream by dermal application. Scratching behavior, clinical skin severity, and the levels of total serum IgE were measured. After AD induction, scores of scratching behavior and clinical skin severity, and the levels of total serum IgE were increased. Treatment with SCAP significantly attenuated the scratching behavior and improved skin severity in a dose dependent manner in NC/Nga mice. Especially, treatment of SCAP 0.5 % reduced more rapidly scratching behavior, clinical skin severity, and the level of total serum IgE, compared to that of pimecrolimus cream. These results suggest that the SCAP may be an alternative substance for the treatment of AD.
Non-apoptotic Fas (CD95) signaling on T cells regulate the resolution of airway inflammation in a murine model of asthma (55.9)
Authors: Williams Jesse , Ferreira Caroline , Blaine Kelly , Rayon Crystal , Velazquez Francisco , Peter Marcus , Sperling Anne
Published: January 2023

Asthma is a Th2-mediated airway disease associated with chronic lung inflammation and mucus production. The mechanism by which inflammation resolves in the lung remains unidentified. We have previously shown that Fas deficiency on T cells in an adoptive transfer model of experimental asthma, results in a delay in resolution of inflammation compared with control. Surprisingly, using T cells with survival advantages independent of Fas on T cells (Bim-/- and BcLxL-tg), we found that inflammation resolution was not delayed, thus suggesting a dependency of T cells on a Fas-specific mechanism for normal resolution of inflammation. Fas has long been known to function as a death initiator by activating caspase-mediated apoptosis. However, recently it has also become clear that Fas has non-apoptotic functions including activation of NF-κB via MAPK pathways. Using an adoptive transfer model, Fas mutant T cells capable of non-apoptotic Fas signaling, but not apoptotic Fas signaling, were sufficient to produce normal resolution. Further, we have found that in vitro skewed Th2 cells that are resistant to Fas-mediated apoptosis, still signal through the non-apoptotic Fas pathway. Together these findings demonstrate that non-apoptotic Fas signaling, rather than apoptotic signaling, in T cells is important for normal resolution of airway inflammation in a murine model of experimental asthma.
A Toll/IL-1R/resistance domain-containing thioredoxin regulates phagocytosis in Entamoeba histolytica (56.23)
Authors: Mancilla Ismael , Mendez Alfonso , Wong Isabel , Alcántara Marcela , Arriaga Lourdes , López Constantino , González Jorge , Isibasi Armando
Published: January 2023

Entamoeba histolytica is a parasite protozoan that infects humans and causes amebiasis in developing countries. Phagocytosis of epithelial cells, erythrocytes, leucocytes, and commensal microbiota bacteria is a major pathogenic mechanism used by this parasite. A Toll/IL-1R/Resistance (TIR) domain-containing protein is required for bacterial phagocytosis in the social ameba Dyctiostelium discoideum, both for nutrition and for protection against infection. In insects and vertebrates, TIR domain-containing proteins regulate phagocytic cell activation. Therefore, we investigated whether E. hystolytica expresses TIR domain-containing molecules that may be involved in the phagocytosis of erythrocytes and bacteria. Using Hidden Markov models (HMM), we identified an E. histolytica thioredoxin containing a TIR-like domain. The primary structure of this putative TIR domain exhibited low similitude with the TIR domain of human Toll-like Receptor (TLR)-2; however, the predicted tertiary structures of these two TIR domains were remarkably similar. Interestingly, the phagocytosis of erythrocytes and E. coli by E. histolytica trophozoites increased after the inhibition of the expression of thioredoxin using a specific siRNA. This suggests that TIR domain-containing thioredoxin is a negative regulator of phagocytosis in E. histolytica.
Toxoplasma gondii infection of human plasmacytoid dendritic cells inactivates TLR7 and -9 mediated induction of IFN-α and TNF-α in response to HIV and HSV (56.26)
Authors: Pierog Piotr , Zhao Yanlin , Singh Sukhwinder , Yap George , Fitzgerald-Bocarsly Patricia
Published: January 2023

pDC are the major producers of IFN-α, an antiviral cytokine involved in control of HIV replication, while T.gondii is a life-threatening opportunistic infection in AIDS patients. In this study, we investigated pDC responses in the context of virus and T.gondii co-infection. T.gondii invaded but did not induce IFN-α or TNF-α in human pDC. However, Class I, II and III strains of T.gondii inhibited IFN-α and TNF-α in response to HSV and HIV. Within the T. gondii exposed pDC population, IFN-α production was inhibited only in cells infected by T. gondii, whereas TNF-α was inhibited in both the infected and to a lesser extent, uninfected pDC. T. gondii inhibited neither uptake of GFP-HSV nor recruitment of TLR9 to EEA1+ endosomes. Using imaging flow cytometry we found that virus-induced nuclear translocation of IRF-7 was abolished by the parasite. Taken together, these data indicate that the block of the intracellular signaling cascade by T.gondii occurs downstream of TLR9 recruitment but upstream of IRF-7 translocation. Unexpectedly, T.gondii did not inhibit HSV-induced nuclear translocation of NFκB. We identified that Rop16, a parasite-derived kinase, is partially responsible for IFN-α and TNF-α inhibition. Additionally, T.gondii altered the virus-induced maturation profile of pDC. These findings suggest a novel mechanism of inhibition of TLR signaling by T.gondii and suggest potential negative consequences of HIV/T. gondii co-infection.
Ccr7 is critical for resistance against experimental Trypanosoma cruzi infection (56.4)
Authors: Roffe Ester , Santiago Helton , Lionakis Michail , Barber Daniel , Murphy Philip
Published: January 2023

The protozoan parasite Trypanosoma cruzi is the etiological agent of Chagas’ Disease, which affects ~12 million people in Latin America despite coordinated international control efforts. Cardiomyopathy due to chronic inflammation and fibrosis is the most common and serious presentation of Chagas’ Disease, but its pathogenesis is poorly understood. Several inflammatory chemokines and chemokine receptors have been implicated in mouse models of T. cruzi infection; however, roles for homeostatic chemokines have not previously been reported. Here, we found that mRNA for the homeostatic chemokine Ccl19 and its receptor Ccr7 were both increased in the heart during the acute phase after infection of WT C57BL/6 mice. Consistent with an important functional role, infection was uniformly fatal during the acute phase in infected Ccr7-/- mice, whereas mortality was only 10% in WT control mice. Death in infected KO mice was associated with uncontrolled parasitemia and increased parasite burden in both heart and liver, whereas infected WT mice controlled parasite burden in all 3 compartments. Leukocyte infiltration was also greater in the hearts of infected KO mice compared to infected WT controls, and was characterized by a lower frequency of CD8+ T cells and a greater frequency of Gr-1hiCD11b+IA- cells. Thus Ccr7 is a critical factor for survival and control of T. cruzi infection in C57BL/6 mice. The mechanisms of resistance conferred by Ccr7 are currently under investigation.
A novel mouse model of Schistosoma haematobium infection to study inflammatory mechanisms of bladder fibrosis (56.5)
Authors: Fu Chi-Ling , Knudsen Giselle , Thathireddy Anuradha , Caffrey Conor Caffrey , McKerrow James , Herbert De’Broski , Thai Kim , Xie Mingqian , Hsieh Michael
Published: January 2023

Chronic inflammatory diseases often result in tissue fibrosis through poorly understand mechanisms. Over 112 million people worldwide are infected with the parasitic worm Schistosoma haematobium. Long-term infection with S. haematobium is associated with parasite egg-induced bladder inflammation and bladder fibrosis. Studying S. haematobium infection may provide clues regarding mechanisms of tissue fibrosis in general. We sought to develop and characterize a more reliable mouse model of S. haematobium infection to study inflammatory mechanisms of tissue fibrosis. BALB/c mice underwent bladder wall injection with S. haematobium eggs and exhibited synchronous bladder granuloma development by 4 days post-injection. Egg-injected bladders began showing fibrosis by 14 days post-injection. Urine was serially sampled to profile the proteome by mass spectrometry and revealed that distinct blood, coagulation, and immune proteins were increased in the urine of egg-injected mice relative to controls. Bladder granulomas grew over time and contained many eosinophils and macrophages. Serum cytokines were analyzed by Luminex analysis and showed early and extended upregulation of IL5, IL1A, and VEGF; and late upregulation of multiple chemokines (KC, MCP1, MCP3, and IP10). We have successfully recapitulated inflammation-associated schistosomal bladder fibrosis in mice. We are now poised to employ genetically modified mice to dissect the inflammatory mechanisms which drive bladder fibrosis.
Characterization of sepsis pathogenesis using molecular biomarkers and drug treatments in the mouse CLP model (56.6)
Authors: Bender Andrew , Wu Yin , Burgess Laurette , Teceno Tyler , Skokanova Eva , Cao Qiongfang , Wilcoxen Keith , Ishizaka Sally , Shirota Hiroshi
Published: January 2023

Sepsis is a severe systemic disease caused by an uncontrolled immune response to an inflammatory stimulus. Sepsis has a high mortality rate and few successful treatments are available. Furthermore, one of the confounding aspects of sepsis treatment is the highly variable and dynamic manifestation of the disease. Thus, there is a need not only for new sepsis treatments but also for biomarkers of sepsis that may guide physicians in patient treatment. The cecal ligation and puncture model (CLP) is a clinically relevant model that replicates the nature of human sepsis after trauma as it has a focus of infection rather than a systemic initiation. Thus, we have chosen to characterize the CLP model and use it to evaluate candidate sepsis biomarkers and therapeutics. We evaluated a variety of potential biomarkers and correlated them with body temperature to determine which ones best reflect the health of the animal. Interestingly, some cytokines and blood chemistry markers highly correlated with health and bacterial load while others did not. To gain further insight into sepsis pathogenesis we tested drugs with differing mechanisms of action. A significant observation was that an antibiotic was highly efficacious while the immunosuppressive dexamethasone was not beneficial; suggesting that bacterial elimination is crucial for survival. In summary, our results suggest potential biomarkers for guiding sepsis treatment and provide insight into sepsis pathogenesis.
Single-cell heterogeneity analysis provides rapid validation of biophysical parameters in living cells and quantifies heterogeneous sensitivity to interleukin-2, -7, and -15. (57.12)
Authors: Coward Jesse , Hathorn Matthew , Karabacak Volkan , Even Dar Orly , Altan-Bonnet Grégoire
Published: January 2023

The single-cell resolution of flow cytometry provides a quantitative measurement of the heterogeneity of protein expression within clonal populations of cells. As the biological significance of this variability is becoming more widely appreciated, there is a need for new tools and methodology to analyze and leverage the effects of endogenous variation. These tools can provide alternatives to genetic methods, such as siRNA knockdown or germline knockout, that are commonly used to manipulate protein expression. We introduce such a new tool, named ScatterSlice, that enables rapid quantification of the correlation between heterogeneity in cellular responses (measured by staining of phospho-proteins) and varied expression of signaling molecules. We demonstrate its application to the validation of biochemical models of cellular responses to IL-2, IL-7, and IL-15 in living, unperturbed cells. In addition, we demonstrate that heterogeneous distribution of receptor subunits within a population of lymphocytes results in gradations of sensitivity to each cytokine. Finally, using experimental measurements of error and uncertainty in combination with biochemical models, we demonstrate the robustness of this analysis to experimental noise, and consider both limits and extensions to its use.
Engineering cell-type selective immune responses using mechanism-based designer IL-4 cytokines. (57.8)
Authors: Creusot Remi , Junttila Ilkka , Bates Darren , Moraga Ignacio , Lupardus Patrick , Fathman C. Garrison , Paul William , Garcia K. Christopher
Published: January 2023

IL-4 supports the differentiation and function of B and T cells, but also induces a response in virtually all cell types. Lymphocytes express mostly the IL-4 type I receptor (IL-4Ra and common gamma (CG) chains), and non-hematopoietic cells express primarily the type II receptor (IL-4Ra and IL-13Ra1 chains; also receptor for IL-13), while other leukocytes express both. We have engineered human IL-4 variants with up to 3-logs higher affinity for CG, or up to 2-logs increased binding to IL-13Ra1. Binding of these variants did not perturb receptor orientation. Selected variants were tested on various human cell lines or peripheral blood cells for their ability to differentially induce phospho-STAT6. The RGA variant, which has the highest affinity for CG and lost 1-log binding affinity to IL-13Ra1, induced significantly more pSTAT6 in the Ramos B cell line and in primary lymphocytes, and less pSTAT6 in epithelial and fibroblastic cells, compared to IL-4. Conversely, the KFR variant, which had the highest affinity to IL-13Ra1, had the opposite effect on these cells. Relative abundance (or scarcity) of CG and IL-13Ra1 on the cell surface dictated the potency of each variant: cells expressing high levels of both receptors were not affected, whereas cells expressing limited CG benefited most from the increased affinity afforded by RGA. These new variants have potential therapeutic application in targeting selective cell types, and thereby reducing toxic effects in vivo.
Lack of direct antigenic processing results in delayed CD8+ T cell response, which fails to control Salmonella typhimurium infection (58.12)
Authors: Tzelepis Fanny , Alcon Valeria , Russmann Holger , Krishnan Lakshmi , Sad Subash
Published: January 2023

CD8+ T cells are activated rapidly to control prolific pathogens, however this is not the case during Salmonella typhimurium (ST) infection. In resistant mice, recombinant ST expressing ovalbumin (ST-OVA) induces a delayed CD8 T cells response and a chronic infection is established. As ST-OVA resides in the phagosomes of infected cells, CD8+ T cells are considered to be activated by the alternative cross-presentation pathway. We wanted to address if the CD8 T cells can be activated through the classical MHC-I antigen presentation in this model, and whether that would improve the control of virulent ST-OVA infection. We modified ST to secrete and translocate the immunodominant antigen OVA directly to the cytosol of infected cells through the bacterial type III secretion system. Our results reveal that this modification induces rapid antigen-presentation, which results in profound CD8+ T cell response and differentiation into memory cells. More importantly, this modification makes the targets susceptible to CD8+ T cell-mediated death, which results in abridgment of ST persistence even in susceptible hosts. Thus, lack of direct antigen-processing in ST infected cells facilitates the virulence of ST and perpetuation of infection.
Innate and adaptive Type 2 cytokines drive mucosal eosinophilia (58.2)
Authors: Nussbaum Jesse , Liang Hong-Erh , Sullivan Brandon , Price April , Locksley Richard
Published: January 2023

Helminth infection, asthma, and allergy share classical hallmarks of Type 2 immunity, in which Type 2 helper T (Th2) cells coordinate antibody production, mucus secretion, and innate effector recruitment via IL-4, IL-5, and IL-13 in affected tissues. Identifying the cells and interactions that drive Type 2 immunity may lead to improved treatments of human diseases. IL-5 is the specific survival and maturation factor for eosinophils and is essential for eosinophilia. Although eosinophils are a key Type 2 effector cell, their precise physiologic role in immunity is controversial. We generated an IL-5 reporter mouse and crossed it to our previously published IL-13 reporter strain to study eosinophil recruitment and tissue maintenance. IL-5-producing cells were found in resting lung, small intestine, and adipose tissue. After helminth infection, lung, liver, and intestinal IL-5-producing cells increased in number and in fluorescence intensity, corresponding with local eosinophilia. Intriguingly, much of the IL-5 signal arose not from Th2 cells, but instead from an “innate lymphocyte” population lacking T cell, B cell, and natural killer markers. Expression of IL-5 and IL-13 was similar but not identical. Innate lymphocytes did not express IL-4, and their distribution in mucosal tissues and their cytokine pattern suggest they may help maintain barrier and/or metabolic homeostasis. Studies are ongoing to define the relationship of these cells to Th2 cells and eosinophils.
Toll-like receptor 2 signaling regulates the tubercle granuloma (58.6)
Authors: McBride Amanda , Salgame Padmini
Published: January 2023

The granuloma developed in the host in response to Mycobacterium tuberculosis (Mtb) is a protective structure that curtails the spread of the pathogen and controls reactivation of latent/persistent Mtb. Despite this knowledge, the molecular pathways that regulate the structural reorganization of immune cells in the lung remains poorly understood. We have found that TLR2KO mice can resist acute infection following low dose aerosol infection with Mtb, but are unable to maintain the granulomatous response and control chronic infection. Consequently, the goal of this study was to identify molecular pathways downstream of TLR2 signaling that may regulate granuloma maintenance in the chronic phase. We therefore examined the transcriptional signature of WT and TLR2KO lungs obtained at several time points following Mtb infection using Affymetrix GeneChip Mouse Genome 430A 2.0 Array. Gene set enrichment analysis (GSEA) indicated that enriched upregulated gene sets in the TLR2KO mice corresponded to Cell cycle, cell proliferation and cell activation related gene sets, while enriched down-regulated gene sets in the TLR2KO mice corresponded to extracellular matrix related gene sets, specifically matrix metalloproteinases (MMPs). Together with earlier reports that MMPs regulate the developing tubercle granuloma, our finding of reduced MMP expression in the lungs of TLR2KO mice indicates a novel role for TLR2 in host defense against Mtb through the control of MMP secretion.
Characterization of B cell responses to Salmonella infection (58.7)
Authors: Nanton Minelva , Lee Seung-Joo , Jackson Amy , Davies D. Huw , McSorley Stephen
Published: January 2023

Infection of susceptible mice with Salmonella typhimurium provides a model for studying immunity to typhoid or disseminated non-typhoidal bacteremia. Although B cells are required for adaptive immunity to Salmonella infection the mechanism of protection is unclear. We immunized IgA-, and polymeric Ig receptor-deficient mice with attenuated Salmonella and examined resistance to secondary infection. Neither of these strains displayed any deficiency in acquired immunity to secondary infection. We also used proteomic microarrays to examine the specificity of B cell responses in mice vaccinated with attenuated Salmonella. These studies have revealed new protein targets that can be used to quantify the antibody response and track Salmonella-specific B cells. Our data suggest that B cells primarily contribute to anti-Salmonella immunity at systemic sites and recognize a diverse array of protein targets in vivo.
Innate stimulation of bacterial-specific Th1 CD4 T cells (58.8)
Authors: O'Donnell Hope , Srinivasan Aparna , McSorley Stephen
Published: January 2023

CD4 T cells are activated in secondary lymphoid organs upon T cell receptor (TCR) ligation with MHC-II bound peptide on antigen presenting cells (APCs). Effector Th1 CD4 T cells traffic to sites of infection, where subsequent interactions with infected APCs can stimulate production of IFNγ. Our laboratory has recently described an alternative mechanism of bacterial-specific Th1 re-activation via innate stimulation in the absence of MHC-II interaction. Using mixed bone marrow chimeric mice we extend these observations and show that CD4 T cell stimulation is dependent upon intrinsic MyD88 and/or TRIF expression. Current work attempts to identify whether innate stimulation occurs directly on CD4 T cells through TLR4 or indirectly by MyD88-dependent cytokine signaling. Understanding innate stimulation of bacterial-specific CD4 T cells may lead to the development of more effective vaccines and therapeutics.
Immunization of heat-killed Klebsiella pneumoniae promotes strong mucosal Th17 responses and protects mice from live infection (59.4)
Authors: Chen Kong , Fisher Candice , Zheng Michael , Weaver Casey , Kolls Jay
Published: January 2023

Bacterial pneumonia is a leading cause of mortality and one major pathogen associated with this disease is Klebsiella pneumoniae (KP). Thus, a vaccine against KP is in great need especially for immunocompromised patients. Here we show that intranasal immunization of mice with heat-killed KP strongly induces IL-17 production in the lung. Using an Il17f reporter mouse, we were able to sort IL-17F positive cells after immunization by FACS. Further analysis reveals that these cells are conventional CD4+TCR-beta+ Th17 cells and they express Il17a, Il17f, Il22, Rora and Rorc. These Th17 cells are antigen specific as they proliferate and produce IL-17 upon re-stimulation with heat-killed KP but not heat-killed Escherichia coli or Staphylococcus aureus. These responses ultimately lead to the protection of immunized mice from live KP infection. This immunization strategy also confers protection in B cell-deficient (mu-MT) mice, demonstrating the vaccination can be antibody independent. We also show that other mucosal routes of vaccination such as intragastric immunization also reduces bacterial burden during pulmonary infection, suggesting that Th17 cells can be primed or recruited from other mucosal sites distal to the lung and oral vaccine can be effective in pulmonary infection. These data suggest that the Th17 pathway can be a target of pulmonary vaccine development through intranasal or intragastric immunization.
The role of Pax5 and E47 on AID expression in B lymphocytes of Aged BALB/c Mice (61.1)
Authors: Landin Ana Marie , Frasca Daniela , Scallan Martina , Harrison Patrick , Hagman James , Blomberg Bonnie
Published: January 2023

Previous results from our laboratory demonstrated that stimulated enriched (95%) B cells from senescent mice (19 to 24 months) have an intrinsic defect in class switch recombination (CSR), and a decrease seen in both Pax5 and E47. The decrease in both Pax5 and E47 is proportional to the decrease in activation induced cytidine deaminase (AID) with aging. Binding sites for both Pax5 and E47 have been identified in the gene for AID, Aicda, and stimulation of B cells results in the recruitment of Pax5 and E47 to the Aicda gene and its expression. Our current results show that overexpression of both E47 and Pax5 rescues levels of AID and CSR in old B lymphocytes to young-like levels. Furthermore, exogenous retroviral E47 mRNA is more stable than the endogenous E47 due to the removal of the 3’UTR. Overexpression of Pax5 leads to up-regulation of E47 in both primary B cells and 293T cells; however, overexpression of E47 up-regulates Pax5 in B cells but not in 293T cells. Nuclear extracts from B cells of senescent mice show lower levels of binding consistent with reduced levels of E47 and Pax5 in aging. Using a DsRed lentiviral vector driven by the Aicda promoter and containing the E47 and Pax5 enhancer region, we show that in 293T cells DsRed is only expressed when the cells are co-transduced with E47 and/or Pax5.
B cell extrinsic MyD88 and FcRγ control contraction of the extrafollicular B cell response (61.12)
Authors: Sweet Rebecca , Cullen Jaime , Shlomchik Mark
Published: January 2023

The extrafollicular (EF) B cell reaction is emerging as a lynchpin of systemic autoimmune responses. External factors that limit this response remain undefined. AM14 transgenic mice have been useful in elucidating how autoreactive Rheumatoid Factor B cells are activated. On a lupus-prone genetic background, AM14 B cells undergo an isotype-switched EF plasmablast response. A similar response ensues when these cells are exposed in vivo to IgG2aa anti-chromatin antibodies, which presumably form immune complexes (ICs) with chromatin shed from dying cells. ICs have Fc that could also stimulate myeloid cells via FcRγ and TLRs, which in turn could influence the EF response. To investigate this, we transferred AM14 B cells into mice lacking FcRγ, MyD88 or both. Initial expansion and differentiation of AM14 B cells was equivalent in all hosts. However, by day 7, whereas the AM14 B cell response contracted in the WT environment, dysregulated expansion continued in the deficient environments. Furthermore, MyD88 and FcRγ are non-redundant, as responses in mice lacking both proteins were larger and more prolonged compared with mice deficient in either factor alone. By d10, the AFC frequency was 40-fold larger in double-deficient compared to WT recipients. These results reveal novel regulatory roles in the EF B cell response for two receptors that are typically considered proinflammatory. Targeting these pathways may provide a way to curtail dysregulated autoreactive B cell activation.
Follicular dendritic cell activation during vaccination correlates with antibody response and germinal center formation (61.14)
Authors: Cantisani Rocco
Published: January 2023

FDCs are stromal cell of the lymphoid organs that play a central role in the induction of humoral immune response. FDCs deliver signal to B cells by trapping intact antigen onto their surface as antigen-antibodies or antigen-complement fragments immune complexes. Thus cognate B cells recognize the antigen presented by FDCs via BCR, while surface proteins or soluble factors expressed by FDCs serve as co-stimulation. FDC activation is pivotal for the origin of germinal center (GC) reaction, and TLR4 engagement appears critical for this purpose. Nevertheless, no studies analyzing the commitment of FDC during vaccination with licensed adjuvants have been performed. Here we show for the first time, that the intramuscular injection of adjuvants approved for human use leads to FDC activation, which occurs late after injection. In addition, we observe that the activation of FDCs upon injection of an adjuvanted antigen takes place only in the follicles where a GC reaction occurs and the number of follicles with activated FDCs strictly correlates with the antigen-specific antibody titers. Moreover, retention of the injected antigen is observed only in the follicles that contain activated FDCs. Thus the extent of the humoral immune response against an antigen correlates with the activation state of FDCs, which can be modulated by the presence of the adjuvants, maybe through an indirect effect. The elucidation of the adjuvant affect on FDCs can help to design more efficacious vaccines.
PARP-14 mediates B cell memory response through the regulation of energy metabolism and gene transcription (61.5)
Authors: Cho Sung Hoon , Trochtenberg Alyssa , McGuinness Owen , Boothby Mark
Published: January 2023

Immunological memory, including both persistent and recall antibody (Ab) responses, enables more efficient and effective protection against recurrent or chronic infection. Although many basic features of memory responses are understood, key molecular mechanisms regulating emergence and persistence of memory B cells and plasma cells remain unknown. Recent evidence suggests that the regulation of energy metabolism might mediate memory T cell differentiation and function. PARP-14, a Stat6-interacting protein is highly expressed in lymphoid organs, influences B cell subset ratios as well as the initial IgA response to antigen, and has intrinsic ADP-ribosyltransferase activity. We now show that PARP-14 is essential for the regulation of energy metabolism in B cells and has been significant role in the increased Ag-specific Ab levels that follow booster immunization. Investigation of the mechanism(s) revealed that PARP-14 mediates at least in part to the formation of long-lived plasma cell formation and regulates IL-4 induction of PRDM1 (Blimp-1), a master regulator of plasma cell differentiation. These results reveal a novel function of a mammalian ADP-ribosyltransferase in memory of B lineage cells and may shed a light on a new target for vaccine development.
B lymphocytes secrete FasL+ exosomes capable of inducing apoptosis in CD4+ T cells (61.6)
Authors: Klinker Matthew , Reed Tamra , Shaw Campbell , Fox David , Holoshitz Joseph , Lundy Steven
Published: January 2023

B lymphocytes possess immunosuppressive properties such as secretion of the anti-inflammatory cytokine interleukin-10 and the expression of apoptosis-inducing molecules. We have previously identified a murine splenic B cell population which expresses the death-inducing molecule Fas Ligand (FasL) and showed that these cells can directly kill antigen-specific T cells. Upon screening a panel of human B cell-derived lymphoblastoid cell lines (LCLs) for expression of FasL, we found that a high frequency of LCLs constitutively produce intracellular FasL as determined by immunoblot and intracellular staining. Secretory exosomes (<100nm diameter microvesicles) were isolated from LCL culture supernatants and it was found that all lines tested secreted exosomes containing MHCII and FasL. The presence of MHCII+FasL+ exosomes was confirmed by incubating exosomes with anti-MHCII-coated beads and showing the presence of FasL on MHCII+ exosome-coated beads by flow cytometry. Exosomes derived from LCLs were capable of inducing significant levels of apoptosis in autologous CD4+ T cells in the presence of superantigen. Finally, preliminary data suggest that splenic murine exosomes bound to anti-IgM-coated beads stain positive for the presence of FasL. These data indicate a novel role for B cells as producers of immunosuppressive exosomes and suggest that LCL-derived MHCII+FasL+ exosomes may represent a novel therapy for T cell-mediated autoimmunity.
The V(D)J recombination machinery is organized as large complexes anchored on the nuclear matrix (62.1)
Authors: Xie Wanqin , Lange Miles D , Hong Sang Yong , Huang Lin , Xue Weihua , Ding Shi-jian , Jones Dallas , Yu Zhihong , He Ti , Yu Yangsheng , Burrows Peter D. , Lu Runqing , Swanson Patrick C. , Su Kaihong , Zhang Zhixin
Published: January 2023

V(D)J recombination provides the foundation for adaptive immunity. This complicated process involves the cleavage of specific recombination signal sequences (RSS) by the recombination activating gene products, RAG1 and RAG2, followed by repair of the DNA double-stranded breaks (DSBs) by the non-homologous end joining (NHEJ) factors, including DNA dependent protein kinase (DNA-PK), Artemis, Ku86, Ku70, DNA ligase IV, XRCC4, XLF, and HMGB1. Currently, it is not clear how these factors are organized inside of the nuclei for recombination. Accumulating studies indicate that the multiple-component DNA replication and RNA transcription machineries are organized as large complexes on the nuclear matrix (NM). Here we show that RAG1, RAG2, and many NHEJ factors are associated with the NM, where they appear to form large complexes and are distributed closely at discrete foci. Ongoing RSS cleavage mainly occurs on the NM in developing B, T lineage cells and HEK293 cells with an artificial recombination system; Purified NMs containing RAG and NHEJ proteins possess the entire enzymatically active recombination system to cleave RSS substrates and generate coding joints in vitro. These results support a novel model that the V(D)J recombination machinery is organized as large complexes anchored on the NM to conduct recombination.
Profiling the pattern of human immunoglobulin SHM and CSR in B memory cells via high-throughput sequencing (62.11)
Authors: Han Jian , Sanders Catherine , Yang Qunying , Byrne-Steele Miranda , Pan Wenjing , Wang Chunlin
Published: January 2023

One of the defining features of the adaptive immune system is that it adapts its response during an infection to improve its recognition of the pathogen. The adaptive feature is best embodied in the genetic alteration mechanism of somatic hypermutation (SHM) and class-switch recombination (CSR) as they are only activated to increase recognition to a previously encountered pathogen. Due to the limited number of sequence data, the hypermutation pattern such as mutation rate, and the distribution of mutation spots remains poorly understood. Using the combination of arm-PCR and high-throughput sequencing, we have determined about one million cDNA sequences of human immunoglobulin genes in B memory cells isolated from the peripheral blood from two subjects at various time points after administration of the 2009-2010 flu vaccine. We identified the location of the hypermutations, the distribution of mutation rate, mutation patterns of different classes of immunoglobulin, and the impact on primer design for immunoglobulin genes.
Modulation of biallelic TCRα expression in influenza-specific CD8 T cells (62.6)
Authors: Dash Pradyot , McClaren Jennifer , Oguin III Thomas , Morris Melissa , Doherty Peter , Thomas Paul
Published: January 2023

Our understanding of complementary T cell receptor (TCR)α and β chain utilization is very limited for pathogen-specific immunity. This problem has been solved with the development of a novel, multiplex nested RT-PCR method for the simultaneous amplification of TCRα and TCRβ chain transcripts from single cells. Using this technique, we found surprisingly, 25% of influenza-epitope-specific CD8+ cytotoxic T lymphocytes recovered directly from the pneumonic lung expressed a dominant, non-productively rearranged TCRα transcript, an effect that is never seen for TCRβ. T cells with these out-of-frame TCRα mRNAs also express an alternate, in-frame TCRα, while approximately10% have two productive TCRα transcripts. The proportion of cells with bi-allelic transcription increases over the course of a response; stayed high in the memory compartment but decreased in the recall response. However these bi-allelic cells appear to expand with low efficiency. Further, cells with out-of-frame expression appear to be specifically excluded from the recall response, a finding that has implications for immune memory and for autoimmunity.
AID-dependent gene expression in activated B cells (62.9)
Authors: Unniraman Shyam , Arya Rahul , Gregory Briana , Agrawal Anurodh , Carico Zachary , Upton Dana
Published: January 2023

Activation induced deaminase (AID) initiates both somatic hypermutation (SHM) and class switch recombination (CSR) by deaminating cytosines to uracils in the immunoglobulin genes. AID is also mistargeted to the transcribed portion of several other genes in activated B cells. In contrast, during early stages of development, AID appears to play a very different role in some non-B cells. Specifically, it targets methylated cytosines in CpG islands leading to demethlylation and induction of silent genes. Notably, both antibody diversification (SHM and CSR) as well as demethylation rely on the same deamination activity of AID but the choice of co-factors determines the genes and portions within the genes that are deaminated. Therefore, we wanted to test whether AID-mediated demethylation could occur in activated B cells as well. Towards this end, we compared gene expression profiles between wild-type and AID-deficient B cells, and identified several genes that are induced in an AID-dependent manner. Here, we present the role played by some of these genes in regulating antibody diversification and mature B cell development. These results reveal a non-canonical function of AID by activating silent genes in B cells and have implications both in normal B cell function as well as tumorigenesis.
Modulation of T-dependent recall humoral immunity by Staphylococcal enterotoxin B (63.12)
Authors: Lee William , Janik David
Published: January 2023

Some microbial toxins are superantigens (SAg) that target large numbers of CD4 T cells. We previously showed that the SAg SEB can lead to memory CD4 T cell inactivation (anergy). Given that SAgs target TCRs independently of their peptide-binding properties, memory T cell help for unrelated antigens might be impaired. Here we show that SEB prevented T-dependent recall humoral immunity. In a mouse model, ovalbumin (OVA)-specific, Vβ8-expressing memory CD4 T cells provided help to hapten-specific B cells after challenge with the T-dependent antigen, TNP-OVA. Only when specific memory T cells were present did the mice make TNP-specific secondary (IgG) humoral responses; otherwise a primary (IgM) humoral response was observed. Exposure of the immune mice to SEB prior to challenge with TNP-OVA led to a loss of IgG production, indicating impaired memory helper function. Aberrant recall immunity was long-lived, as even 3 weeks after exposure to SEB, immunization with TNP-OVA resulted in IgM, but not IgG, anti-TNP Ab. Comparable numbers of carrier-specific T cells were present in SEB-treated and control mice, suggesting that anergic memory cells were maintained in vivo. Memory cell anergy occurred due to Fyn kinase-dependent negative signaling, and Fyn-deficient memory cells promoted IgG production even after SEB exposure. Hence, bacterial toxins can target memory helper cells to eliminate recall responses to unrelated antigens, suggesting a potential need for re-vaccination.
PD-L1 costimulation of the CD8 T cell response to Listeria monocytogenes infection (63.16)
Authors: Xu Daqi , Fu Han-Hsuan , Obar Joshua , Park Jang-June , Tamada Koji , Lefrancois Leo
Published: January 2023

It is well known that programmed death ligand-1 (PD-L1) transduces a suppressive signal, increasing the activation threshold of T cells via its counterreceptors PD1 and CD80 (B7.1). On the other hand, other studies have suggested a possible role of PD-L1 as costimulator. In this study, we analyzed the role of PD-L1 in the primary CD8 T cell response to Listeria monocytogenes infection. PD-L1 blockade using the monoclonal antibody (mAb) 10F.9G2 impaired antigen-specific CD8 effector T cell expansion, particularly limiting short-lived effector cell differentiation. Simultaneous CD4 T cell depletion and anti-PD-L1 blockade exacerbated the effect of either treatment alone demonstrating that PD-L1 provides important costimulation even in the absence of CD4 T cells. Most importantly, specific blockade of PD-L1 binding to CD80 (43H12 mAb) or PD-1 (RPM1-14 mAb) during Listeria monocytogenes infection did not recapitulate blockade with 10F.9G2. These data suggest a 3rd unknown binding partner for PD-L1 that is responsible for positive costimulation of antigen-specific CD8 effector T cells. We conclude that PD-L1 plays an important role in antigen-specific CD8 effector T cells during Listeria monocytogenes infection through a novel costimulatory pathway.