bluuebunny/crossref_metadata_2025 huggingface dataset:
The Adjacent Flanking Region Plays a Critical Role in Facilitating the Presentation of theListeria monocytogenesProduct lemA to H2 M3wt-Restricted, Peptide-Specific Murine CD8 Cells
Authors: Kurlander Roger J. , Chao Elizabeth , Fields Janet , Nataraj Chandrasekaran
Published: December 2022

Mice infected with Listeria monocytogenes (LM) generate CD8 effectors specific for f-MIGWII, the amino terminus of the bacterial product lemA presented by the class Ib MHC molecule H2 M3wt. lemA has several distinctive properties: 1) it is readily presented as an exogenous Ag in the absence of bacterial infection; 2) it is processed by a TAP-independent pathway, which is sensitive to chloroquine, pepstatin, and brefeldin; and 3) the immunogenic portion of the molecule is extremely resistant to proteolytic degradation even by proteinase K. To assess the structural basis for these findings, we expressed a truncated variant (t-lemA) containing the amino-terminal hexapeptide and the subsequent 27 amino acids linked to a histidine tail in Escherichia coli, and purified the product by affinity chromatography. Purified t-lemA could be presented to f-MIGWII-specific effectors by macrophages and fibroblasts at 1–10 nM. Unlike f-MIGWII, which binds directly to H2 M3wt, t-lemA required processing by a chloroquine-, pepstatin-, and brefeldin-sensitive pathway. Brefeldin sensitivity often implies endogenous processing in the cytoplasm, but several lines of evidence suggest translocation to the cytoplasm and proteosomal degradation are not critical for t-lemA presentation. Unlike f-MIGWII, t-lemA was profoundly resistant to proteinase K, and, using 35S-labeled t-lemA, we could identify the region from position 1 to ∼30 as the protease-resistant element. Thus, the hydrophobic peptide sequence following f-MIGWII can account for the unusual properties of lemA noted above. Analogous modification could be used to alter the properties of other peptide Ags presented by class I MHC products.
A DNA Immunization Model Study with Constructs Expressing the Tick-Borne Encephalitis Virus Envelope Protein E in Different Physical Forms
Authors: Aberle Judith H. , Aberle Stephan W. , Allison Steven L. , Stiasny Karin , Ecker Michael , Mandl Christian W. , Berger Rudolf , Heinz Franz X.
Published: December 2022

We have conducted a DNA immunization study to evaluate how the immune response is influenced by the physical structure and secretion of the expressed Ag. For this purpose, we used a series of plasmid constructs encoding different forms of the envelope glycoprotein E of the flavivirus tick-borne encephalitis virus. These included a secreted recombinant subviral particle, a secreted carboxyl-terminally truncated soluble homodimer, a nonsecreted full-length form, and an inefficiently secreted truncated form. Mice were immunized using both i.m. injection and Gene Gun-mediated application of plasmids. The functional immune response was evaluated by determining specific neutralizing and hemagglutination-inhibiting Ab activities and by challenging the mice with a lethal dose of the virus. As a measure for the induction of a Th1 and/or Th2 response, we determined specific IgG subclasses and examined IFN-γ, Il-4, and Il-5 induction. The plasmid construct encoding a secreted subviral particle, which carries multiple copies of the protective Ag on its surface, was superior to the other constructs in terms of extent and functionality of the Ab response as well as protection against virus challenge. As expected, the type of Th response was largely dependent on the mode of application (i.m. vs Gene Gun), but our data show that it was also strongly influenced by the properties of the Ag. Most significantly, the plasmid encoding the particulate form was able to partially overcome the Th2 bias imposed by the Gene Gun, resulting in a balanced Th1/Th2 response.
Critical Role of CD28 in Protective Immunity AgainstSalmonella typhimurium
Authors: Mittrücker Hans-Willi , Köhler Anne , Mak Tak W. , Kaufmann Stefan H. E.
Published: December 2022

Efficient T cell activation requires both TCR signals and costimulatory signals. CD28 is one of the molecules that provide costimulatory signals for T cells. We used mice deficient in CD28 expression (CD28−/− mice) to analyze the role of CD28 in the immune response against the intracellular bacterium Salmonella typhimurium, the causative agent of murine typhoid fever. CD28−/− mice were highly susceptible to infection with wild-type S. typhimurium and even failed to control infection with attenuated aroA− S. typhimurium. More detailed analysis revealed that CD28−/− animals did not mount a T-dependent Ab response and were highly impaired in the production of IFN-γ. Thus, CD28 cosignaling is crucial for immunity against S. typhimurium. To our knowledge, this is the first report describing an essential role for CD28 in protective immunity against an intracellular microbial pathogen.
Protection Against the Mortality Associated with Disease Models Mediated by TNF and IFN-γ in Mice Lacking IFN Regulatory Factor-1
Authors: Senaldi Giorgio , Shaklee Christine L. , Guo Jane , Martin Laura , Boone Thomas , W. Mak Tak , Ulich Thomas R.
Published: December 2022

Mortality and cytokine production associated with disease models mediated by TNF- and IFN-γ were studied in mice lacking IFN regulatory factor-1 (IRF-1). IRF-1 knockout (KO) mice showed no mortality after the injection of a dose of LPS lethal in intact control mice (LD95). KO mice showed lower circulating levels of TNF and IFN-γ than controls. KO mice also showed lower TNF and IFN-γ mRNA in the spleen or liver than controls. KO mice had smaller spleens than controls, which contained similar percentage but lower absolute count of macrophages and lower percentage and absolute count of NK cells. IRF-1 KO mice survived longer than controls after the coinjection of LPS and galactosamine. IRF-1 KO mice also showed less mortality than controls after the injection of Con A and in a model of cerebral malaria. After the injection of a lethal dose of TNF (LD88), mortality was similar between KO and intact mice. Mortality was also similar after the coinjection of two nonlethal doses of TNF and IFN-γ, a lethal combination (LD100). This study shows that the lack of IRF-1 protects against the mortality associated with disease models mediated by TNF and IFN-γ but has no effect on the mortality directly induced by TNF and IFN-γ. The lack of IRF-1 appears to result in impaired production of TNF and IFN-γ, reflecting a down-regulation of gene expression in the liver and spleen as well as a reduction in the number of splenic cells.
Development of Adenovirus Vectors Encoding Rat Complement Regulators for Use in Therapy in Rodent Models of Inflammatory Diseases
Authors: McGrath Yvonne , Wilkinson Gavin W. G. , Spiller O. Brad , Morgan B. Paul
Published: December 2022

C activation has been implicated in the pathogenesis of numerous inflammatory human diseases and disease models. A therapy based on C inhibition might therefore be of benefit to reduce inflammation and ameliorate disease. C inhibition in vivo can be accomplished by the delivery of soluble recombinant C regulators either systemically or directly to a target site, but effects are transitory. We have developed a strategy for the efficient delivery of the membrane-bound rat C inhibitors, CD59, Crry, and decay-accelerating factor (DAF), using replication-deficient adenovirus vectors with the intention of treating rat models of disease in which C is implicated. The adenovirus recombinants(RAd), RAdCD59, RAdCrry, and RAdDAF, respectively, have been tested for expression and function of the transgene in vitro. Infection of human fetal foreskin fibroblasts resulted in high levels of expression of each of the rat inhibitors. The constructs were also tested for inhibition of rat C-mediated cell lysis and C3b deposition. In a cell lysis assay, each inhibited to varying degrees of efficiency in the order RAdCD59 = RAdDAF > RAdCrry. In a C3b deposition assay, RAdDAF caused a greater reduction in C3b deposition than RAdCrry and RAdCD59 was ineffective. These agents, individually or in combination, provide the tools for testing the effects of prolonged inhibition of C at a target site on the progress of experimental models of disease.
In Vivo Administration of Recombinant IL-2 to Individuals Infected by HIV Down-Modulates the Binding and Expression of the Transcription Factors Ying-Yang-1 and Leader Binding Protein-1/Late Simian Virus 40 Factor
Authors: Bovolenta Chiara , Camorali Laura , Lorini Alessandro L. , Vallanti Giuliana , Ghezzi Silvia , Tambussi Giuseppe , Lazzarin Adriano , Poli Guido
Published: December 2022

Leader binding protein-1 (LBP-1)/late SV40 factor (LSF) and ying yang-1 (YY1) transcription factors are involved in the regulation of HIV expression. In particular, YY1 and LBP-1 have been shown to cooperate in repressing HIV-1-long terminal repeat reporter gene expression by in vitro cotransfection experiments. However, no information is available on the levels of expression and activation of these transcription factors in PBMC of HIV-infected individuals. Therefore, we have evaluated the expression and DNA binding activity of YY1 and LBP-1 (LSF) in PBMC of HIV-infected individuals before, during, and after administration of IL-2 in association with antiretroviral therapy (ART), a regimen under consideration for broad clinical use in this disease based on its ability to stably raise the absolute number of circulating CD4+ T lymphocytes. Both YY1- and LBP-1 (LSF)-DNA binding were profoundly down-modulated during administration of IL-2/ART, and a proteolytic activity probably responsible for the reduced expression of the two cellular transcription factors was found activated in PBMC of individuals receiving the immunotherapeutic regimen. This study is the first evidence of modulation of cellular transcription factors following IL-2/ART administration and provides a potential correlate of the transient raises in plasma viremia early reported in patients receiving IL-2 in the absence of ART, thus underscoring the importance of always administering this cytokine to HIV-infected individuals together with potent antiretrovirals.
I. Isoagglutinins in the Blood of Cattle
Authors: Little Ralph B.
Published: December 2022

Introduction Since the identification by Landsteiner of the four definite human blood groups numerous workers have attempted to group the blood of mammals with varying results. In 1907, Hektoen (1) was unable to find any isoagglutinins in the serum of rabbits, guinea pigs, dogs, horses, and cattle. In each instance 0.1 cc. of 10 or 20 different sera was tested upon 0.1 cc. of 2.5 per cent suspension of washed corpuscles from as many animals. Ottenberg and Friedman (2), in 1911, were able to divide by agglutination the blood of steers into three definite groups, as follows.

Group I. Agglutinative but not agglutinable.

Group II. Agglutinable but not agglutinative.

Group III. Neither agglutinative nor agglutinable.

The authors assume that in the blood of steers there is one isoagglutinin and one isoagglutinable substance. They also thought that the examination of a large number might reveal the existence of a second agglutinin and a second agglutinable substance.
Hypersensitiveness to Diphtheria Bacilli
Authors: Neill James M. , Fleming William L.
Published: December 2022

The present example of diphtheria bacterial hypersensitiveness agrees with the previous examples in that it is manifested by an immediate skin reaction and in that the capacity for the reaction can be passively transferred. It differs, however, in that the individual is particularly reactive with a heat (60°C.) labile product not involved in the reactions of the previous diphtheria-sensitive persons. This product is apparently contained in effective concentration only in the unheated culture filtrates of toxicogenic diphtheria bacilli and not in the filtrates of nontoxicogenic strains nor in solutions of material derived from the washed bacterial cells. If it is not toxin, it is a hitherto unrecognized diphtheria bacterial product that possesses a degree of lability not usually encountered among the bacterial substances associated with hypersensitive reactions.

The results are discussed in respect to the possibility that the described reactions may represent an example of true hypersensitiveness of man to diphtheria toxin.
The Influence of Blockade of the Reticuloendothelial System on the Formation of Antibodies
Authors: Cannon Paul R. , Baer Raymond B. , Sullivan F. L. , Webster J. R.
Published: December 2022

Summary and Conclusions

The liberation into the blood stream of hemolysins to sheep's red corpuscles following blockading procedures depends upon the manner of administration of the blockading material and the amount of antigen injected. Repeated injections of suspensions of India ink, particularly when given intravenously and continued after the injection of a small quantity of “test-antigen,” are followed by a significant decrease in the liberation of antibody into the blood stream, this liberation apparently varying inversely with the extent to which the phagocytic cells are blocked. This decrease in output of antibody is independent of individual variation of animals, loss of weight or of depression by fluids alone. Evidence is presented that small amounts of blockading material stimulate the liberation of antibody whereas large amounts tend to depress such a liberation. These experiments offer additional evidence that antibodies, at least hemolysins, are formed by the phagocytic cells of the mesenchymal tissues.
Development of CD8αβ+ iNKT cells in a humanized mouse model (APP3P.112)
Authors: Yuan Weiming , Wen Xiangshu , Kim Seil , Xiong Ran , Li Michelle , Lawrenczyk Agnieszka , Besra Gurdyal , Delabona Paolo , Casorati Giulia , Akbari Omid , Exley Mark , Rao Ping
Published: December 2022

Invariant NKT (iNKT) cells are innate-like T cells showing potent anti-tumor function in conventional mouse models. In sharp contrast, iNKT cell ligands have had limited efficacy in human anti-tumor clinics, mostly due to the profound differences in the properties and compositions of iNKT cells between human and mice, including the presence of CD8+ iNKT cells only in humans. To build more relevant in vivo models for studying human iNKT cells, we recently developed the first humanized mouse model (hCD1d-KI) with human CD1d knocked in (Proc. Natl. Acad. Aci. USA 110: 2963-8, 2013). To further humanize the mouse model, we introduced human invariant TCRα chain (Vα24Jα18) into the hCD1d-KI mice. Interestingly, we observed a substantial subset of iNKT cells expressing CD8αβ. The presence of these CD8αβ+ cells in the thymus suggested that they are developed in the thymus. Functionally these iNKT cells show a strong Th1-biased cytokine response and potent cytotoxicity upon activation. The low binding of iNKT TCRs to human CD1d/lipid complex and high prevalence of Vβ7 TCRβ among these CD8+ iNKT cells strongly suggested a novel low avidity-based developmental program for the ontogeny of these iNKT cells which includes the suppression of Th-POK and up-regulation of Eomes transcriptional factors. Our establishment of a near fully-humanized mouse model in CD1d/iNKT TCR system will greatly facilitate the future design and optimization of iNKT cell-based immunotherapies.
Effects of immunosenescence on professional antigen presenting cell immunogenicity in the context of Varicella zoster viral infection (APP3P.114)
Authors: Cheney Elizabeth
Published: December 2022

A protracted decline in immune system fitness occurs over one’s lifespan. Both the innate and adaptive immune systems exhibit numerous alterations with age. Despite these alterations, aged people often have robust responses to pathogens they encountered in child and adulthood. Memory lymphocytes that develop during these challenges are protective and remain largely intact. In contrast, protection against Varicella Zoster Virus (VZV) wanes with increasing age and results in viral reactivation known as Herpes Zoster (HZ) or shingles. This increased incidence correlates with a decrease in cell-mediated immunity (CMI) that is critical for protection against VZV. This is perplexing as most people are exposed to VZV when young, and have functional anti-VZV CMI in adulthood. Viral latency of both VZV and a closely related herpes virus, Herpes simplex 1 (HSV-1), is maintained by CMI. Importantly, maintenance of Tm cell responses is dependent on the recruitment of professional APC (pAPC) to the site of reactivation in HSV-1 infection. We hypothesize that increased incidence of HZ in the elderly is due to a deficit in pAPC function or numbers that then results in loss of tissue resident Tm responses. Specifically we predict that aging affects Ag processing and presentation in pAPCs. Data will be presented that examines the capacity of human pAPCs to process and present antigen as a function of age.
Quantitative expression analysis of rhesus macaque MHC alleles (APP3P.102)
Authors: Peng Xinxia , Pyo Chul-Woo , Thomas Matthew , Pyon Yoon Soo , Law Lynn , Wang Ruihan , Geraghty Daniel , Katze Michael
Published: December 2022

The relevance of MHC genes to HIV infection and disease progression is well established, and nonhuman primates are the most important model for AIDS vaccine research and development. However, due to the structural and sequence complexity of the macaque MHC, the expression changes across this complex region have not been determined accurately. We are developing a strategy for accurately quantifying rhesus MHC allele expression. Our strategy leverages a large collection of full-length rhesus MHC genomic sequences and a custom-developed computational pipeline, and only requires regular macaque RNA-seq data from users. We used this technique to analyze RNA-seq data collected from normal rhesus macaque tissue samples and PBMC samples from acutely infected rhesus macaques. We show that the expression of both MHC class I and class II genes was highly tissue specific, and multiple class I alleles were expressed within a single tissue. Compared to animal matched baseline PBMC samples, there was an overall increase in the expression of class I alleles in acutely infected rhesus macaques, as early as 3 days post infection. Both up-regulation and down-regulation of class II gene expression in PBMC samples was also observed during acute SIV infection. This study presents for the first time a systematic quantification of macaque MHC allele expression. The results may reveal limits in our understanding to fully account for the impact of MHC expression on immunological outcomes.
Identification of endogenous murine leukemia retrovirus antigens attributing to exosome-induced autoimmunity in NOD mice (BA6P.122)
Authors: Dai Yang , Bashratyan Roman , Regn Danielle , Binley James , Marquardt Kristi
Published: December 2022

Endogenous autoimmune triggers and antigens are required for initiating the innate and cellular immune responses in the islet of non-obese diabetic (NOD) mice. We found that secreted, nano-sized microvesicles or exosomes are potent inflammatory triggers that, importantly, stimulated autoreactive B and T cells. By performing proteomic analysis, we identified endogenous retrovirus (ERV) proteins, highly homologous to murine leukemia retrovirus (MLV), that are enriched in the exosomes. To study whether these ERV antigens attribute to the autoimmune induction, we produced exosomes lacking the ERV Gag protein and virus-like particles (VLPs), which are nano-sized microvesicles overexpressing Gag and Env proteins. We found that NOD mice spontaneously develop autoantibodies binding to the MLV Env, and also accumulate Env-specific B cells in periphery as compared with diabetes-resistant mice. Autoreactive T cells did not respond to the MLV antigens directly; however, immunization with the exosomes or VLPs induced Gag-specific T cells that inhibited the function of diabetogenic T cell clones in the pancreas. Therefore, ERV antigens may be the targets of autoreactive B and T cells in this T1D mouse model. Consistently, we detected endogenous MLV antigens expressed in the pancreas and by the islet stromal/stem cells in NOD mice. These data support that unique ERV antigens and/or altered formation of exosomes in islet cells may trigger tissue-specific autoimmunity in NOD mice.
Commensal microbiota regulate inflammatory cardiac remodeling (BA6P.126)
Authors: Barin Jobert , Diny Nicola , Gebremariam Elizabeth , Talor Monica , Bedja Djahida , Kass David , Peterson Daniel , Rose Noel , Cihakova Daniela
Published: December 2022

In this study, we sought to understand how the microbiota regulates inflammatory mechanisms underlying cardiac autoimmunity. We employ an autoimmunization model of heart disease in mice, termed experimental autoimmune myocarditis (EAM). This disease model depends on CD4+ T cells for pathogenesis, immediately implying a cytokine product of these cells as a critical mediator of disease pathogenesis, and an attractive target for therapeutic intervention. Our laboratory has previously reported that Th17 cells are critical for the disease process. In order to establish a role for the commensal microbiota in autoimmune heart disease, we treated animals with a comprehensive polyantibiotic cocktail to induce antibiotic dysbiosis. Treating BALB/cJ animals with antibiotic limited the development of myocarditis, and furthermore prevented their progression to later heart failure. Segmented filamentous bacteria (SFB) represent a clade of Clostridia that have been found to elicit intestinal Th17 differentiation upon colonization, and subsequently modulate susceptibility to Th17-associated autoimmune disease. We colonized BALB/cJ mice with SFB and observed that these animals progress to diminished cardiac function following the induction of EAM. Microbiota manipulations were associated with differences in canonical Th17-associated programs, pointing to these pathways as an important mechanism in coupling commensal sensing to the progression of heart disease.
Overexpression of Pbx1-d, a novel splice isoform of Pbx1, in CD4+ T cells promotes follicular helper T cell differentiation (BA6P.127)
Authors: Choi Seung-Chul , Morel Laurence
Published: December 2022

Pbx1 controls chromatin accessibility to a large number of genes. Pbx1-d is a Pbx1 dominant negative isoform lacking the DNA binding domain, and its expression is more frequent in the CD4+ T cells from lupus patients than in healthy controls. Pbx1-d is associated with the production of autoreactive CD4+ T cells in the Sle1a1 lupus susceptibility locus and lupus susceptibility in the B6.Sle1.Sle2.Sle3 mouse model. We have generated Pbx1-d Tg mice that specifically express Pbx1-d in CD4+ T cells. These Tg mice reproduce the phenotypes of Sle1a1 mice, with increased inflammatory functions of CD4+ T cells and impaired regulatory T (Treg) cell homeostasis. Pbx1-d Tg mice also present an increased number of follicular helper T (TFH) cells, which expand with age in naïve mice, and result in enhanced TH1-biased antibody production in response to T-dependent immunization. Interestingly, the relative distribution of TFH and regulatory T (TFR) cell populations is different in both Pbx1-d Tg and Sle1a1 mice as compared with wild-type mice. Adoptive transfers of OT-II.Pbx1-d Tg CD4+ T cells showed that Pbx1-d overexpression expands TFH cell differentiation in a cell-intrinsic and antigen-specific manner. Our results suggest that Pbx1-d impacts lupus development by regulating effector T cell differentiation by promoting TFH to the expense of Treg cell. In addition, our results identify Pbx1-d as a novel regulator of CD4+ T cell effector function.
Innate cells in autoimmune diabetes in mice and man (BA7P.150)
Authors: Bayer Allison , Umland Oliver , Fraker Chris
Published: December 2022

Donor Treg engraftment following immunomodulation is lower in recent diabetic compared to prediabetic NOD mice. This may be related to altered immune compartment that cannot fully support Tregs and represents an obstacle to successful therapy. Interplay of adaptive and innate immune cells is often overlooked and role natural killer cells (NKs) have only recently been appreciated. But NK subsets remain poorly understood in mice. The current study was aimed at identifying NK functional subsets with effector and regulatory properties using NOD mouse model. We found CD335+CD122+ cells among CD45+CD3- cells in lymph nodes (LN), pancreatic LN (pLN), spleen, and blood in young female non-autoimmune B6 and diabetes-prone NOD mice. Among bulk NKs, c-Kit+ and c-Kit+CD127+ were found in LN, pLN, and spleen, but c-Kit+CD127+ cells was considerably lower in NOD mouse. Interestingly, majority of CD127+ NKs were CD25+ and IL-18R++ compared to other NKs. Blood samples of long-standing diabetic and patients at-risk for diabetes in comparison to control subjects revealed majority of human NK (hNK) are NKeff (CD56+CD16+), but ratio NKeff:NKreg (CD56++CD16-) statistically decreases with disease progression resulting in a more regulatory environment. c-Kit+ hNKs are largely found in NKreg and express IL-18R with long-standing and at-risk having highest %c-Kit+ NKreg compared to controls. The challenge is to resolve the inflammatory roles of NK cells with their regulatory roles in autoimmunity.
Modulation of autoimmune diabetes by innate-like B cell derived antibodies specific for N-acetyl-D-glucosamine. (BA7P.148)
Authors: New James , Dizon Brian , King Rodney , Kearney John
Published: December 2022

The immunodominant epitope of Group A Streptococcus (GAS) is the cell wall associated N-acetyl-D-Glucosamine (GlcNAc) of Group A Carbohydrate (GAC). Previous reports in humans and rodent models have suggested a link between GAS exposure and protection from T1D. Through neonatal immunization with GAS and serum-transfer experiments we find that T1D protection elicited by neonatal GAS exposure is conferred by the antibody (Ab) response to GAC, and the differentiation of unique GlcNAc-specific clonotypes into long-lived plasma cells. Glycan microarray analysis of GAC-specific antibodies indicates they recognize aberrantly truncated mammalian glycans. GlcNAc-terminating glycans exist on a subset of insulin-containing granules and are generated as neoepitpoes during beta cell apoptosis. We find GAC-specific Abs directly block recognition of GlcNAc-modified autoantigens by innate lectins, dampen inflammatory complement activation, and reduce the capacity of dendritic cells to present beta cell antigen. Importantly, T1D patients are deficient in GAS-reactive IgM antibodies. These observations suggest that early exogenous antigen exposure in crucial for developing protective GlcNAc-specific natural antibodies and validate a role for the hygiene hypothesis in T1D. Subsets of GlcNAc-specific antibodies may serve as biomarkers for identifying at-risk patients with natural antibody deficits, and boosting these clonotypes may serve to suppress T1D development
Identification of immunogenic epitopes of β-adrenergic receptor 1 that induce heart autoimmunity in A/J mice (BA8P.164)
Authors: Basavalingappa Rakesh H , Massilamany Chandirasegaran , Arumugam Rajasekaran Rajkumar , Steffen David , Reddy Jay
Published: December 2022

Coxsackievirus B3 (CVB3) is one of the important infectious causes of myocarditis that may lead to dilated cardiomyopathy. To study the autoimmune events in this viral infection, various mouse models are commonly employed. In our studies with A/J mice, we recently demonstrated that the animals infected with CVB3 show the appearance of pathogenic T cells specific to cardiac myosin heavy chain (Myhc)-alpha 334-352, leading us to hypothesize that CVB3 infection might accompany with the generation of T cells with specificities for multiple antigens. To test this hypothesis, we sought to identify the immunogenic epitopes of one other putative cardiac antigen namely, β-adrenergic receptor 1 (BAR1) that has been previously implicated in the immune pathogenesis of CVB3 infection. Our data revealed that the peptides spanning the amino acids, 170 to 230 are capable of inducing epi/endocarditis by active immunization. We also noted that the disease induced by BAR1-peptides was associated with the induction of CD4 and/or CD8 T cell responses, with a skewed T helper (Th)1 and Th17 cytokine production. In conjunction with Myhc-alpha 334-352, identification of BAR1-peptides may thus permit us to determine the kinetics of the generation of autoreactive T cells with multiple antigen-specificities, which might be generated as a result of epitope spreading during the course of CVB3 infection.
Heterodimeric IL-15 regulates the balance of effector and regulatory cells, promoting anti-cancer tumor effects (CCR4P.213)
Authors: Felber Barbara , Bergamaschi Cristina , Ng Sin Man , Chen Stephanie , Bear Jenifer , Alicea Candido , Beach Rachel Kelly , Chertova Elena , Sowder Raymond , Pavlakis George
Published: December 2022

Interleukin-15 (IL-15) is a common γ-chain cytokine that plays a significant role in the activation and proliferation of T and NK cells and holds great potential in fighting infection and cancer. We have previously shown that bioactive IL-15 in vivo comprises a complex of the IL-15 chain with the soluble or cell-associated IL-15 receptor alpha chain that are together termed heterodimeric IL-15 (hetIL-15). HetIL-15 produced from engineered human cell lines showed favorable pharmacokinetics and bioactivity compared to monomeric IL-15 in both mice and macaques. We investigated the anti-tumor efficacy of hetIL-15, using the MC38 colon carcinoma mouse model. hetIL-15 resulted in a significant delay in tumor growth, when administered intraperitoneally every 2 days for 2 weeks. In addition, intratumoral delivery of hetIL-15 induced regression of established tumors and cured 50% of mice. Repeated injections of hetIL-15 resulted in an increased CD8+/Treg cells ratio in spleen, lymph nodes and tumor microenvironment. Preclinical cancer studies support the use of hetIL-15 in tumor immunotherapy approaches to promote the development of anti-tumor responses by favoring effector over regulatory cells.
Differential synthesis and release of IL-18 and its antagonist, IL-18 binding protein, from human platelets (CCR4P.209)
Authors: Ahmad Ali , Allam Ossama , Aldbah Zainab , Samarani Suzanne , Sagala Patrick
Published: December 2022

IL-18 is a multifunctional pro-inflammatory cytokine belonging to the IL-1 family. The cytokine is known to be produced from macrophages, dendritic cells, keratinocytes, adrenal cortex etc in the human body. We found that the cytokine as well as its antagonist, IL-18 Binding Protein (IL-18BP), are produced in human platelets. Interestingly, the two of them are synthesized and released differentially from these anuclear cellular elements. Human platelets contain transcripts for the cytokine and synthesize it de novo upon activation as an inactive precursor protein, which is cleaved into mature form by activated caspase 1, whose activation occurs in activated platelets. The platelets continue to release IL-18 over several hours after activation. Freshly isolated resting platelets as well as paraformaldehyde-fixed ones contain very little IL-18. In a sharp contrast to IL-18, its antagonist (IL-18BP) is present pre-made in platelets and is released upon their activation. Platelets contain no transcripts for it and do not synthesize de novo upon activation. Freshly isolated resting platelets as well as paraformaldehyde-fixed platelets contain IL-18BP. Furthermore we found that the two soluble mediators are secreted from platelets via different mechanisms: glyburide inhibits synthesis of active IL-18 but not of IL-18BP from activated human platelets. Our findings bear important implications for a variety human diseases which are accompanied by chronic inflammation.
Identification and characterization of a novel inflammatory DC precursor (HEM5P.235)
Authors: Rogers Peter , Schwartz Elizabeth
Published: December 2022

DC differentiated through culture with GM-CSF are used in many clinically promising applications including vaccines. However, the developmental timeline during which specific stimulatory or immunosuppressive functions are acquired by these cells is poorly understood. Here, we developed a method of isolating progenitor populations at specific stages of development to monitor their phenotype and function. Bone marrow cells were cultured in GM-CSF for three days, then sorted based on Ly6c and CSF-1R (CD115) expression, two hallmarks of myeloid cell development. We then monitored the developmental progression and differentiation of each population based on the expression of these proteins as well as several other hallmarks of the dendritic cell phenotype. We measured their gene expression profiles by qRT-PCR and their response to inflammatory stimuli at each stage of development. Through this analysis, we have identified five distinct cell types representative of Common Myeloid Progenitors (CMP), Granulocyte Macrophage Progenitors (GMP), Monocytes, inflammatory DC (iDC), and a novel dendritic cell precursor which is phenotypically and functionally distinct from previously described myeloid cells. Our findings suggest this novel cell type, Inflammatory DC precursor (IDP), arises from monocytes and serves as an immediate precursor to immature inflammatory DCs. These studies will aid in the refinement of protocols to acquire cells for specific clinical applications.
Pharmacological targeting of luteinizing hormone-releasing hormone receptor promotes survival after lethal irradiation (HEM5P.222)
Authors: Velardi Enrico , Tsai Jennifer , Young Lauren , Lieberman Sophie , Gupta Jyotsna , Dudakov Jarrod , Van Den Brink Marcel
Published: December 2022

Sex steroid inhibition (SSI) is a well-described strategy to promote lymphopoiesis in the bone marrow and thymus of aged and immunodepleted mice. Although the precise mechanisms underlying this regenerative effect are unclear, there is increasing evidence that sex hormones can have a profound impact on hematopoietic stem cell (HSC) function. Here we show that SSI induced by a novel class of Luteinizing Hormone-Releasing Hormone Receptor antagonist (LHRH-Ant) protected both male and female mice from BM failure, increased the recovery of myeloid and lymphoid compartments, and ultimately promoted overall mouse survival when administered 24 hours after lethal total body irradiation (LTBI). Underlying this, we could demonstrate both phenotypically and functionally that LHRH-Ant spared HSCs from radiation-induced depletion and induced HSCs into cell cycle, thereby promoting hematopoiesis. Intriguingly, use of an LHRH agonist, which causes an initial surge in sex steroids, also mediated enhanced survival from LTBI but surgical castration did not, suggesting that the benefit of SSI is not mediated by the sex steroids themselves but rather by upstream regulators such as LH. Consistent with this, irradiated mice receiving LHRH-Ant and treated daily with LH were no longer protected from radiation injury. Taken together these data not only reveal a novel mechanism that regulates HSC function, but also offer a therapeutic approach to regenerate hematopoiesis after radiation exposure.
An essential role for Birc5 in innate lymphocyte development (HEM5P.223)
Authors: Beaulieu Aimee , Sun Joseph
Published: December 2022

NK cells and innate lymphoid cells (ILCs) are two important classes of innate lymphocytes, and both have essential roles in early immune responses. NK cells provide systemic protection against viruses and cancer, while ILCs defend against a broad range of pathogens, including viruses, helminths, and bacteria, particularly at barrier organs such as the lung, gut, and skin. The molecular pathways that regulate innate lymphocyte development and function are an area of active investigation. Here we show that the inhibitor of apoptosis (IAP) protein, Birc5, is essential for the development of NK cells and ILC subsets. We observed high expression of Birc5 in NK cell and ILC precursor populations in the bone marrow, and consistent with a non-redundant role for Birc5 in innate lymphocyte development, steady-state populations of NK cells and ILCs were severely reduced in mice with cell-specific deletions of the Birc5 gene. BrdU-labeling experiments indicated an important role for Birc5 in cell cycle progression in developing innate lymphocytes in the bone marrow. Moreover, Birc5-deficient NK cells in the peripheral organs of these mice exhibited a maturation defect. Together, these findings highlight a new and crucial role for Birc5 in the proliferation and maturation of developing innate lymphocytes.
Regulation of NK cell maturation by the intramembrane protease SPPL3 (HEM5P.224)
Authors: Hamblet Corinne , Makowski Stefanie , Tritapoe Julia , Pomerantz Joel
Published: December 2022

Natural killer (NK) cells play an essential role in tumor surveillance through direct tumor cell lysis. Signal peptide peptidase-like 3 (SPPL3) is a highly conserved intramembrane aspartyl protease, but very little is known about its functions in vivo. We developed an SPPL3 conditional knockout mouse and found that deletion of SPPL3 within the hematopoietic system using Vav1-iCre resulted in a peripheral defect in NK cell number. The reduced NK cell population was hypofunctional in tumor clearance in vivo and direct tumor lysis in vitro. Analysis of SPPL3-deficient NK cells revealed a substantial block in terminal maturation, at the CD27+CD11b- stage. Deletion of SPPL3 in NK cells by NKp46-iCre confirmed a cell-autonomous requirement for SPPL3 in NK development and function. CRISPR/Cas9 genome editing was used to create a knockin mouse expressing a protease-dead SPPL3 knockin allele, D271A. NK cells expressing SPPL3 D271A exhibited the same block in terminal differentiation as SPPL3-deficient NK cells. Our results reveal that the proteolytic function of the SPPL3 intramembrane protease is critical for normal NK cell maturation at a checkpoint that regulates the transition from CD11b- to CD11b+ cells.
Influenzal Antibodies in Lymphocytes of Rabbits Following the Local Injection of Virus
Authors: Harris Susanna , Harris T. N.
Published: December 2022

Following the injection of inactivated influenzal virus into the foot-pad of the rabbit antibodies to the viral protein can be found in the popliteal lymphnode, which drains the site of injection, and in lymph obtained from the efferent lymphatic vessel of that node. These antibodies are found earlier in the local lymphatic system that in the serum and in higher titer until the 4th day after the injection. Thereafter the serum-titer rises above that in the lymphatic tissues.

At the same time there is a burst of activity in the local lymphatic tissue. There is marked enlargement of the lymphnode, almost entirely of cortical tissue. This consists of diffuse lymphocytic hyperplasia which very soon becomes organized into the conventional follicular structure of lymphnodes. The absolute and relative count of lymphocytes in the lymph emerging from the popliteal lymphnode rises sharply.

Injection of serologically distinct strains of influenzal virus into the two legs of individual rabbits give results which corroborate the specificity of the antibodies produced.

On separation of lymph emerging from the popliteal lymphnode into cells and plasma and testing each for antibody-content, it is found that the titer of antibody in the lymph-cells exceeds that in the lymph-plasma, by ratios as high as 16:1. This ratio is found to be highest at the time when the rate of increase of antibody-content of the local lymphatic system is greatest.

These findings are interpreted to mean that the lymphocyte can be a primary source, or site of final synthesis, of antibodies to viral protein. This conclusion is in keeping with those of earlier studies in which a similar role was demonstrated for the lymphocyte in the formation of antibodies to bacterial and cellular antigens.
Immunologic Studies of Japanese Encephalitis Virus in Japan
Authors: Scherer William F. , Kitaoka Masami , Grossberg Sidney E. , Okuno Takeshi , Ogata Takayuki , Chanock Robert M.
Published: December 2022

Following inapparent infection by Japanese encephalitis virus in August and September 1956, 14 children without preexisting JE virus neutralizing antibody, developed a primary hemagglutination-inhibition antibody response by November; 13 of the 14 children formed neutralizing antibody and 11 of 14 children developed complement fixing antibody. Neutralizing antibody usually appeared concurrently with HI antibody, though in some children it appeared before or after HI antibody.

Inapparent “reinfections” by JE virus occurred during August and September 1956 in seven children with preexisting neutralizing antibody in plasma. The HI and neutralizing antibody responses were similar in time of occurrence and frequency to those seen in the 14 children without preexisting neutralizing antibody; however, complement fixing antibody more promptly and uniformly rose to detectable levels in the “reinfected” children than in the group with primary infection. Although the exact mechanism of antibody stimulation following “reinfection” of humans by JE virus is uncertain, this phenomenon may play a role in Japan in maintenance of JE virus antibody levels and immunity in humans and possibly in animals.

Maximal HI antibody titers that develop within two months of inapparent infection were lower than those usually found after overt Japanese encephalitis. Contrariwise CF antibody did not display a difference in maximal titers. Neutralizing, CF and HI antibodies persisted at detectable levels in plasma and serum for different periods of time after inapparent infection (neutralizing for many years, CF for at least 5–6 weeks but less than 9 months, and HI for years in many persons.)
Immunologic Studies of Japanese Encephalitis Virus in Japan
Authors: Buescher Edward L. , Scherer William F. , Rosenberg Murray Z. , Kutner Leon J. , McClure H. Elliott
Published: December 2022

Maternal antibodies reacting with JE virus were found in newly hatched domestic chicks and wild birds near Tokyo, Japan. The neutralization test detected these antibodies more frequently than the hemagglutination-inhibition test; in wild birds of the family Ardeidae, an average of only 25% of birds with maternal neutralizing antibody during the first 15 days of life were positive at 1:10 plasma dilution by HI test. Neutralizing antibody disappeared below detectable levels (log neutralization index less than 1.0) in half the ardeids by 5–15 days after hatching and in all birds by 21–30 days. Maternal neutralizing antibody in chicks prevented viremia upon challenge with JE virus, but did not affect viremia from WEE virus. In ardeids, maternal antibody apparently prevented infection by JE virus in one bird but not in another, suggesting that some ardeid hens have specific antibody to JE virus and others have nonspecific “antibody”. This observation is compatible with summer infection of some ardeids with JE virus in Japan and winter infection of others with immunologically-related Group B arthropod-borne viruses in southeast Asia. Nevertheless after maternal antibody becomes undetectable, ardeids and chicks are fully susceptible to JE virus.
Specific and Cross Reactions by Complement Fixation with Boston Exanthem Disease Virus (ECHO-16)
Authors: Neva Franklin A. , Malone Mary F.
Published: December 2022

Satisfactory complement fixing antigens were prepared from strains of Boston exanthem viruses by the concentration of fluids from infected tissue cultures. CF antibody responses were readily demonstrated in patients infected with these agents. No definite antigenic differences were found between the viruses isolated from several exanthem disease patients, when the agents were studied by the complement fixation reaction. However, cross reactions to these exanthem virus antigens were observed on study of sera from patients with certain Coxsackie and certain heterologous ECHO virus infections.
Immunological Studies of Anthrax
Authors: DeArmon Ira A. , Klein Frederick , Lincoln Ralph E. , Mahlandt Bill G. , Fernelius Albert L.
Published: December 2022

Immunized and nonimmunized guinea pigs, rats and mice given graded challenge doses of Bacillus anthracis spores were compared. The reciprocal of time-to-death was found to be linearly related to log10 challenge dose over a reasonable range of doses, and, therefore, a difference in position of the parallel regression lines represented a difference in response of control and immunized animals. This difference in position of regression lines is the log increase in dose required to cause the immunized group to give the same response as the control. This log increase is called the mean Immunity Index. The mean index of an immunized population of guinea pigs was 3.2. The distribution of individuals in the control and immunized groups overlapped and thus it was concluded the variability of response of immunized guinea pigs was greater than the control animals, and that only 46% of the immunized population had a significant protection against B. anthracis challenge.
Preparation and Properties of Antibodies to Sheep Erythrocytes
Authors: Amiraian Kenneth , Leikhim Edward J.
Published: December 2022

The use of anion-exchange chromatography for purification of rabbit antisheep hemolysin (γ1) is confirmed. The technique is extended to the purification of γ2 hemolytic and agglutinating antibodies. The latter types of antibodies have been digested with papain and some immunologic properties of the fragments so derived are reported.
Purification of Complement-Fixing Antigen of Rickettsia Orientalis
Authors: Kobayashi Yuzuru , Tachibana Nobuyoshi
Published: December 2022

A stable complement-fixing antigen of Rickettsia orientalis, free of anti-complementary substance, was obtained by sodium deoxycholate (DCA) sucrose extraction of yolk sac suspensions previously treated with Amberlite XE-64.

The optimum concentration of DCA, 1.0% in 0.25 M sucrose solution, yielded an antigen containing approximately one tenth of the nitrogen of the material extracted by sucrose alone without any change in the complement fixation titer of antigen.

The DCA-extracted antigen was stable on storage at -70°C for periods of 5 months or more.