bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of X-linked agammaglobulinemia patients in Mexico (B222)
Authors: Lopez-Herrera Gabriela , Berron-Ruiz Laura , Mogica Dolores , Espinosa-Rosales Francisco J , Santos-Argumedo Leopoldo
Published: January 2023

Humoral immunodeficiences are very common in the National Registry of Primary Immunodeficiences in Mexico. Among the humoral defects, X-linked agammaglobulinemia (XLA) is one of the most frequently found. In this work we report Btk mutations for 13 unrelated XLA patients some of wich has not been reported previously, most of the mutations affect Btk kinase domain, and PH and SH2 domain affecting mutations were also detected. Btk protein expression was found to be present in the majority of patients, in some cases we detect low expression levels and it was absent in other patients.
Pinning down Bruton’s tyrosine kinase (Btk) (B26)
Authors: Vargas Leonardo , Yu Liang , Mohamed Abdalla J. , Berglöf Anna , Smith C. I. Edvard
Published: January 2023

Bruton’s tyrosine kinase (Btk) is a nonreceptor tyrosine kinase belonging to the Tec family of Src-related protein tyrosine kinases (PTKs). Btk is predominantly expressed in B-lymphocytes as well as in myeloid and erythroid progenitor cells. It has been shown that Btk plays critical roles in B-lymphocytes development, differentiation, and signaling. Mutations in Btk cause X-linked agammaglobulinema in humans and X-linked immunodeficiency in mice. However, the mechanism of activation and signaling of this enzyme has not been fully investigated.

The peptidyl-prolyl cis/trans isomerase (PPIase) Pin1 is an important regulator of cell proliferation and DNA-replication. Recent report shows that pin1 is overexpressed in diffuse large B cell lymphoma.

In both cell lines and Pin1 knock-out mice experiment, We identified that the Pin1 is a negative regulator of Btk, controlling its expression level by reducing its half-life, and the negative regulatory effect of Pin1 is dependent on a functionally intact Btk. We also found that pin1 interact with Btk in a cell cycle dependent manner, Phosphorylation of Serine 21 leads to Pin1 binding and regulation only during mitosis, whereas phosphorylation of Serinev115 leads to Pin1 binding and regulation specifically in the resting cells. Our results indicate that, a key regulator of Btk activity and half-life, Pin1 prolyl isomerase controls a critical non–receptor tyrosine kinase for normal B cell biology.

This study was supported by the Swedish Cancer Fond, The Magnus Bergvall Foundation, the Wallenberg Foundation and the European Union Grant EURO-POLICY-PID.
Phosphorylation and ubiquitination of the I kappa B kinase complex by two distinct signaling pathways (B29)
Authors: shambharkar prashant
Published: January 2023

The IκB kinase (IKK) complex serves as the master regulator for the activation of NF-κB by various stimuli. It contains two catalytic subunits, IKKα and IKKβ, and a regulatory subunit, IKKγ/NEMO. The activation of IKK complex is dependent on the phosphorylation of IKKα/β at its activation loop, and the K63-linked ubiquitination of NEMO. However, the molecular mechanism by which these inducible modifications occur remains undefined. Here, we demonstrate that CARMA1, a key scaffold molecule, is essential to regulate NEMO ubiquitination upon T cell receptor (TCR) stimulation. However, the phosphorylation of IKKα/β activation loop is independent of CARMA1, or NEMO ubiquitination. Further, we provide evidence that TAK1 is activated and recruited to the synapses in a CARMA1-independent manner and mediate IKKα/β phosphorylation. Thus, our study provides the biochemical and genetic evidence that phosphorylation of IKKα/β and ubiquitination of NEMO are regulated by two distinct pathways upon TCR stimulation.
The protective effect of the NOD.Idd3 genetic interval is associated with enhanced regulatory T cell function and IL-10 production (B37)
Authors: Anderson Ana C , Chandwaskar Rucha , Kuchroo Vijay K
Published: January 2023

The Idd3 genetic interval confers protection against multiple autoimmune diseases, including type 1 diabetes and experimental autoimmune encephalomyelitis (EAE). However, the cellular and molecular mechanisms by which the Idd3 locus mediates suppression of autoimmunity have not been analyzed. The favored candidate gene in this interval is Il2, which has been shown to be polymorphic between susceptible and resistant strains of mice. IL-2 regulates the growth/death of effector T cells as well as the generation/maintenance of regulatory T cells (Tregs). We have examined both Tregs and effector T cells in NOD and NOD.Idd3 congenic mice and find that NOD.Idd3 Tregs are more suppressive and NOD.Idd3 effector T cells produce more IL-10 when compared to NOD-derived T cells. Interestingly, we find that both these effects are driven by NOD.Idd3-derived antigen presenting cells, suggesting that the protective effect of Idd3 is due to alterations in the APC compartment and not T cells themselves.
Sterile protection against malaria infection requires TAP in spite of completely operative TAP-independent vacuolar cross-presentation pathway (B40)
Authors: Liepinsh Dmitry J , Steers Nick , Schwenk Robert J , Krzych Urszula
Published: January 2023

Induction of long-lasting protective immunity to malaria by repetitive immunization with P. berghei γ radiation-attenuated sporozoites (γ-spz) requires MHC class I-restricted CD8+ T cells, implying that γ-spz/liver stage derived antigens have to be presented through one of the cross-presentation pathways. Based on accumulation of proliferating and IFN-γ producing effector/memory CD8+ T cells in the livers of TAP−/− mice, we showed that TAP-independent vacuolar pathway efficiently operates in vivo during priming and subsequent boosts with γ-spz. Likewise, infectious sporozoite challenge increased the number of IFN-γ+ CD8+ T cells in γ-spz immune TAP−/− mice and induced IFN-γ production by wt γ-spz immune CD8+ T cells transferred into TAP−/− environment suggesting that γ-spz/liver stage derived MHC class I specific peptides generated in a TAP-independent manner participate in response to infectious sporozoites. Nevertheless, the pattern of IFN-γ secretion, parasitemia and survival data upon infectious sporozoite challenge clearly indicated that TAP-associated antigen processing is indispensable for sterile protection.
An automatically curated MHC ontology for intelligent immunogenetic information retrieval (B42)
Authors: DeLuca David S. , Beisswanger Elena , Hahn Udo , Blasczyk Rainer
Published: January 2023

Accurate information retrieval of scientific articles is complicated by the intricacies of natural language. Ontologies are useful for formalizing scientific concepts and providing a foundation to mediate between formal, computationally accessible, representations and informal natural language expressions. The terminology associated with the MHC exemplifies the need for a sophisticated translation process between a natural language query and the relevant documents. For example, a researcher searching for “peptides that bind MHC” should be presented with a text containing “peptides were eluted from HLA-A2.” To achieve this goal, we have developed a multi-species MHC ontology which encompasses all human MHC alleles and serologic groups. Because of the rapid rate of new allele discovery, allele incorporation is automated using the IMGT/HLA and IPD-MHC databases. In the context of the StemNet knowledge management project, this MHC ontology contributes to machine-learning-driven text annotation, as well as to search-string interpretation and mapping. In this way, StemNet aims to offer researchers a platform to efficiently access the goldmine of clinically relevant immunogenetic information, which is otherwise inaccessible by standard search engines and databases.

This work was financed by the Federal Ministry of Education and Research, Germany, funding number 01DS001C; the authors are solely responsible for content.
Phenotypic changes induced by IL-12 priming regulate effector and memory CD8 T cell differentiation (B48)
Authors: Lee Jee-Boong , Lee Kyoo-A , Chang Jun
Published: January 2023

Inflammatory cytokines such as IL-12 provide important signals for optimal activation of naïve T cells in addition to TCR and costimulatory signals. In the previous study, we demonstrated that IL-12 provides survival signals to the CD8 T cells and thus increases the generation of effector and memory CD8 T cells. In the present study, to investigate the mechanisms by which IL-12 priming contributes to differentiation and survival of CD8 T cells, we searched the differentially regulated genes and markers by IL-12 during antigenic stimulation. Here, we show that IL-12 priming results in the increased subpopulation of CD127hi cells, which is known to develop into long-lived memory cells. We also found that IL-12 priming dramatically induces IL-10 expression from activated CD8 T cells. Direct IL-10 priming of CD8 T cells resulted in the significant increase of effector and memory CD8 T cell population after adoptive transfer and this priming effect is closely associated with less susceptibility to apoptosis. Although IL-10 is known as a cytokine with important anti-inflammatory and immunosuppressive properties, these results have shown that IL-10 has a direct and positive effect on the survival of CD8 T cells. Together, our results suggest that cytokine environment such as IL-12 and IL-10 play important roles, at the early stage of antigenic stimulation, in regulating differentiation of activated CD8 T cells into effector and memory cells.

*This work was supported by the grant R11-2002-098-05005-0 from Korea Science and Engineering Foundation and by the grant R15-2006-020 from the NCRC program of the Ministry of Science & Technology and the Korea Science and Engineering Foundation
Act1 adaptor protein is an immediate and essential signaling component of interleukin-17 receptor (B52)
Authors: Chang Seon Hee , Park Heon , Dong Chen
Published: January 2023

Interleukin (IL)-17, the founding member of the IL-17 cytokine family, is the hallmark of a novel subset of CD4+ T cells that is regulated by TGFbeta, IL-6, and IL-23. IL-17 plays an important role in promoting tissue inflammation in host defense against infection and in autoimmune diseases. Although IL-17 has been reported to regulate the expression of proinflammatory cytokines, chemokines, and matrix metalloproteinases, the signaling mechanism of IL-17 receptor has not been understood. An earlier study found that IL-17 activates NF-kappaB and MAPK pathways and requires TRAF6 to induce IL-6. However, it is unknown what molecule(s) directly associates with IL-17 receptor to initiate the signaling. We demonstrate here that IL-17 receptor family shares sequence homology in their intracellular region with Toll-IL-1 receptor (TIR) domains and with Act1, a novel adaptor previously reported as an NF-kappaB activator. MyD88 and IRAK4, downstream signaling components of TIR, are not required for IL-17 signaling. On the other hand, Act1 and IL-17 receptor directly associate likely via homotypic interaction. Deficiency of Act1 in fibroblast abrogates IL-17-induced cytokine and chemokine expression, as well as the induction of C/EBPbeta, C/EBPdelta, and IkappaBzeta. Also, absence of Act1 results in a selective defect in IL-17-induced activation of NF-kappaB pathway. These results thus indicate Act1 as a membrane-proximal adaptor of IL-17 receptor with an essential role in induction of inflammatory genes. Our study not only for the first time reveals an immediate signaling mechanism downstream of an IL-17 family receptor but also has implications in therapeutic treatment of various immune diseases
Interleukin-22 Induces Lipopolysaccharide Binding Protein in Hepatocytes: a Potential Systemic Role of Interleukin-22 in Crohn’s Disease (B60)
Authors: Wolk Kerstin , Witte Ellen , Hoffmann Ute , Döcke Wolf-Dietrich , Endesfelder Stefanie , Asadullah Khusru , Sterry Wolfram , Volk Hans-Dieter , Wittig Bianca Maria , Sabat Robert
Published: January 2023

Crohn’s disease (CD) is characterized by intestinal infiltration of activated immune cells and distortion of the gut architecture. Here, we demonstrate that IL-22, a cytokine that is mainly produced by activated Th1 and Th17 cells, was present in high quantities in the blood of CD patients in contrast to IFN-[gamma] and IL-17. In a mouse colitis model, IL-22 mRNA expression was elevated predominantly in the inflamed intestine but also in the mesenteric lymph nodes. Simultaneously, IL-22BP showed strong a constitutive expression in the gut and lymph nodes and was decreased in the inflamed intestine. To investigate the possible role of systemic IL-22 in CD, we administered IL-22 to healthy mice and found an up-regulation of LPS binding protein blood levels reaching concentrations known to neutralize LPS. This systemic up-regulation was associated with increased hepatic but not renal or pulmonary LBP mRNA levels. IL-22 also enhanced the secretion of LBP in hepatocytes in vitro. This increase was mainly transcriptionally regulated and synergistic with that of other LBP inducers. Finally, elevated LBP levels were detected in CD patients and the mouse colitis model. These data suggest that systemic IL-22 may contribute to the prevention of systemic inflammation provoked by LPS present in the blood of CD patients through its induction of hepatic LBP.
Thromboembolic stroke promotes the formation of the inflammasome complex (B70)
Authors: Abulafia Denise P , de Rivero Vaccari J Pablo , Lozano J Diego , Ragan Timothy J , Keane Robert W , Dietrich W Dalton
Published: January 2023

Inflammatory responses contribute to the pathogenesis of cerebral ischemia and stroke. Activated caspase-1 cleaves the pro-inflammatory cytokines IL-1β and IL-18, initiating a cascade of detrimental inflammatory events. Cell culture studies of macrophages have reported that activation of caspase-1 involves the formation of a macromolecular complex termed the inflammasome. Whether this complex is present after cerebral ischemia is unknown. Here, we utilized a clinically relevant model of thromboembolic stroke in mice to investigate the formation of the inflammasome and subsequent activation of inflammatory responses. Our results demonstrate for the first time that cerebral ischemia triggered the formation of a high molecular weight complex comprised of caspase-1, caspase-11, the adaptor protein ASC (apoptosis-associated speck-like protein with CARD) and NALP1 (NACHT/LRR/pyrin domain-containing protein 1) in the cerebral cortex, 24 hr after ischemia. Formation of the inflammasome complex resulted in activation of caspase-1 and cleavage of IL-1β and IL-18. Immunohistochemical analysis revealed the presence of inflammasome proteins in cortical neurons and inflammatory cells. Finally, the therapeutic potential of targeting the inflammasome after ischemia was demonstrated by reduced cytokine activation in mice treated with a neutralizing antibody against NALP1.
IgA plasma cell migration to the lactating mammary gland is mediated by an alpha4 dependent, MAdCAM-1 independent pathway (B71)
Authors: Martino Ben , Wilson Eric
Published: January 2023

It has been previously shown that the accumulation of immunoglobulin A (IgA) antibody secreting cells (ASC) in the lactating mammary gland leads to secretion of antibodies into mothers’ milk and their passive transfer to the suckling newborn. The lactating mammary gland also provides a unique model in understanding the molecular mechanisms governing lymphocyte homing to the common mucosal immune system. Lymphocyte homing is mediated through a multi-step process involving the vascular expression of adhesion molecules and chemokines, as well as lymphocyte expression of cognate adhesion molecule ligands and chemokine receptors. We have previously shown that the accumulation of IgA ASC to the lactating mammary gland is highly dependant on the chemokine CCL28. However, the adhesion molecules governing this homing event have not been defined. Here we show that IgA ASC homing to the lactating mammary gland is blocked by the addition of function blocking anti-alpha4 antibodies but not function blocking anti-MAdCAM-1 antibodies. These results suggest that IgA ASC homing to the lactating mammary gland is mediated in an alpha4 beta7/MAdCAM-1 independent manner.
Hypersensitiveness to Diphtheria Bacterial Products
Authors: Neill James M. , Fleming William L. , Sugg John Y. , Gaspari E. L.
Published: January 2023

Previous evidence indicative that toxin is the substance involved in C's hypersensitiveness was based upon the consistent association of high toxin content of test material with high degree of hypersensitive reactivity and of low or no toxin content with weak or no hypersensitive reactivity. The evidence in this paper was based upon the constant association between the antitoxin content of serum and the capacity of the serum to neutralize the hypersensitive agent when the toxin-containing filtrate is incubated with the serum before injection in individual C. Experiments with similar results were obtained with antitoxin-containing serum of horses, rabbits, and men. With the rabbit sera the use of an “antibacterial” as well as an “antitoxic” serum made it possible to show not only that a serum containing antitoxin was able to neutralize the hypersensitive agent but also that a serum rich in other antidiphtheria antibodies was unable to neutralize it. The most convincing results were obtained with human sera because it was possible by choice from a large number to select for the tests more purely “antitoxic” sera than could be obtained by the laboratory immunization of animals. The selected human sera, although containing no precipitins and no or only traces of “group agglutin,” were able to neutralize the agent of the hypersensitive reaction. Further experiments demonstrated a quantitative relationship between the antitoxin content and the amount of the respective serum required to neutralize the substance hypersensitively reactive with individual C.
Stomatin-like protein 2: a link between T cell activation and mitochondrial function (35.19)
Authors: Christie Darah , Lemke Caitlin , Hatch Grant , Madrenas Joaquín
Published: January 2023

During a study to characterize the proteome of lipid raft microdomains of activated human T cells we identified stomatin-like protein 2 (SLP-2). We have previously shown that T cell activation through the TCR leads to up-regulation of SLP-2 expression, which in turn correlates with enhanced T cell effector responses. Since in human T cells the major pool of SLP-2 is associated with mitochondria, we examined the function of this protein in mitochondria during T cell activation. Here, we report that up-regulation of SLP-2 expression increases mitochondrial mass and count. Such an effect correlates with an increase in the expression of the nuclear transcriptional co-activator PGC-1α, in lipid biosynthesis linked to mitochondrial membrane biogenesis, and in mitochondrial DNA replication. As expected, these changes translate into an increase in both cellular and mitochondrial ATP levels, higher mitochondrial uptake of calcium, and enhanced resistance to apoptosis through the intrinsic pathway. Based on these results we propose that SLP-2 expression is a key regulatory step for mitochondrial biogenesis and function in response to T cell activation.
In vitro PTEN deletion in peripheral T cells enhances activation but does not replace CD28 costimulation or prevent anergy (35.37)
Authors: Locke Frederick L , Zha Yuan-yuan , Gajewski Thomas F
Published: January 2023

PTEN plays a pivotal role in T cell development by negatively regulating the PI3K signaling pathway important for activation, growth, and proliferation. In Cre/loxp mouse models when PTEN is deleted during thymogenesis, peripheral T cells show CD28-independent IL-2 production and are relatively resistant to anergy induction. These animals also develop autoimmunity and T cell lymphomas. However, the consequences of PTEN deletion directly in peripheral T cells are not known, and we hypothesized that much of this phenotype might be explained by the elimination of PTEN during thymic development. The use of CAR Tg-PTENflox/flox mice allowed deletion of PTEN using a Cre adenovirus in vitro. PTEN-deleted splenic T cells or Th1 clones exhibited enhanced IL-2 and IFN-gamma production and proliferation upon TCR/CD28 engagement. Gene expression profiling revealed a subset of induced genes that were augmented upon PTEN deletion. However, PTEN-deficient T cells still required CD28 costimulation for IL-2 production and were not resistant to anti-CD3-induced anergy. Our results indicate that deletion of PTEN can augment the activation of post-thymic T cells but does not mediate CD28-independence or anergy resistance. Ongoing experiments will determine if the absence of PTEN protein in post thymic T cells will lead to improved immune responses in vivo, and if overt autoimmunity or lymphomagenesis will occur.
Trogocytosis leads to sustained signaling and selective survival of CD4+ T cells (35.44)
Authors: Osborne Douglas G. , Wetzel Scott A
Published: January 2023

Antigen recognition by CD4+ T cells leads to large-scale spatial and temporal molecular redistributions, forming the immunological synapse. We have previously shown that upon dissociation, T cells capture large membrane fragments from the antigen presenting cell directly from the immunological synapse. The mechanism and biological significance of this process, termed trogocytosis, is still unclear. In this study we examine the impact that trogocytosis has on the individual T cell after capturing molecules from the antigen presenting cell. We employed murine fibroblast and dendritic cell lines expressing an I-Ek molecule loaded with a covalently attached antigenic peptide (moth cytochrome C 88-103) and containing a GFP-tagged cytoplasmic tail as antigen presenting cells for T cells from a peptide-specific TCR transgenic mouse. Using a combination of high-resolution microscopy, ELISA and flow cytometry, we have shown that the transferred material is retained on the surface of the T cell and is associated with sustained signaling after removal of the antigen presenting cells. The extent of transfer correlates with increased proliferation and sustained survival of the transfer-positive cells in vitro. These findings suggest that trogocytosis may lead to sustained signaling and survival, potentially altering the effector functions of T cells.

Funded By NIH COBRE 2P20RR017670 (Subproject 5)
Identifying ZAP-70 kinase-dependent and -independent functions in T cells using a chemical genetic approach (35.5)
Authors: Au-Yeung Byron B , Levin Susan E , Zhang Chao , Shokat Kevan , Weiss Arthur
Published: January 2023

The tyrosine kinase ZAP-70 activates signals proximal to the TCR and is critical for the activation of downstream signal pathways. To identify which T cell functions require ZAP-70 kinase activity, our lab has generated a novel mouse that expresses an analog-sensitive ZAP-70 mutant that can be inhibited specifically by an analog of the Src family kinase inhibitor PP1. This titratable, chemical genetic approach gives us the ability to rapidly and specifically inhibit ZAP-70 activity in primary mouse T cells. We asked whether naïve and effector responses upon TCR stimulation required ZAP-70 activity. TCR induced calcium mobilization, upregulation of CD69, and proliferation were highly dependent on ZAP-70 activity. Similarly, effector cytokine production by primed CD4+ and CD8+ cells was ZAP-70 kinase-dependent. Inhibition of ZAP-70 activity also severely impaired CD8+ cytolytic activity. In contrast to conventional CD4+ and CD8+ T cells, we observed that CD4+ CD25+ Treg suppress CD4+ CD25- T cell proliferation, even in the presence of the ZAP-70 inhibitor. Thus, while conventional T cell activation and function are ZAP-70 kinase dependent, CD4+ CD25+ Treg suppressive function appears to be ZAP-70 kinase-independent. Our results imply that an inhibitor of the WT form of ZAP-70 could be effective for the attenuation of T cell-mediated pathology.
Ran overexpression leads to a compromised transcription factor nuclear translocation and diminished T-cells response to TCR activation (35.8)
Authors: Pham Thi Ngoc Diep , Qiao Xiaoying , Valderrama-Carvajal Hector , Luo Hongyu , Wu Jianping
Published: January 2023

Ran (Ras-related nuclear protein) is a Ras family GTPase, and its documented functions are the regulation of DNA replication, cell cycle progression, nuclear structure formation, RNA processing and exportation, and nuclear protein importation. In this study, we performed detailed mapping of Ran expression during mouse ontogeny using in situ hybridization. High Ran expression was found various organs and tissues including the thymus cortex and spleen white pulp. Ran was induced in T cells 24 hours after their activation. Ran's function in the immune system was investigated. In transgenic T cells with actin promoter-driven Ran expression, there was compromised activation marker expression, lymphokine secretion, and proliferation upon T cell receptor (TCR) activation in vitro, compared with wild-type T cells. At the molecular level, transgenic T cells were defective in nuclear factor kappa-B (NFκB), activator protein 1 (AP-1) and nuclear factor of activated T cells (NFAT) nuclear translocation. Our experiments showed, for the first time, that Ran has important regulatory function in T-cell activation, and such function is probably related to its role in nuclear import of critical transcription factors.
The Regulation and Function of the TSLP Receptor in the Airway Epithelium (36.15)
Authors: Miazgowicz Michael , Headley Mark , Ziegler Steve
Published: January 2023

Epithelial cells of the airway and lung have been shown to participate actively in the response to both pathogenic and allergenic environmental stimuli. In particular, the airway epithelium is a potent source of thymic stromal lymphopoetin (TSLP), a cytokine that is strongly associated with asthma and other atopic pathogenesis. In the lung, TSLP acting in concert with an antigenic stimulus is capable of inducing hallmarks of the asthmatic response: eosinophilia and inflammatory cell infiltrates, sub-epithelial fibrosis, goblet cell hyperplasia and mucus production, airway hyperresponsiveness, and Th2 cytokine and chemokine production.

TSLP production by airway epithelial cells is known to be NF-κ B dependent. Surprisingly, we have found that factors known to upregulate TSLP is also capable of upregulating the heterodimeric TSLP receptor. Stimulation with either TNF-α or infection with Sendai virus results in the upregulation of both TSLPR and IL7R on a human airway epithelial cell line as well as primary murine airway epithelium. These same stimulatory conditions, in combination with the Th2 cytokine IL-4, furthermore result in the upregulation of TARC and CXCL5, chemokines associated with airway inflammation. Upregulation of these chemokines can be inhibited by the addition of an anti-TSLP monoclonal antibody, indicating a role for TSLP signaling in the expression of these genes by the airway epithelium.
Student use of learning resources to understand antimicrobial actions: use in hybrid vs on-line format (37.6)
Authors: Samuel Lysa , Muirhead Bill , Green-Johnson Julia M
Published: January 2023

Increased demand for online computer-based instructional resources has led to the need for effective utilization of learning objects in health biology courses. Our aim was to examine how well learning objects assist students in understanding challenging concepts in antimicrobial activity against infectious disease. Our specific focus was on learning object use in an introductory microbiology and immunology course in two different settings: a fully on-line section and a hybrid section, which integrates online materials with face-to-face lectures. To collect information on use of the learning objects, an anonymous survey was given to both sections. Survey participation rates varied, with a 56% rate from the online course, and only 28% from the hybrid course. Survey data analysis indicated many similarities between online and hybrid sections in learning object utilization, including high usage as study material for examinations. Differences were observed in the positive impact learning resources have on students learning difficult concepts, with the online course showing 100% agreement, and the hybrid course only 76% agreement, suggesting differences in learning object utilization for learning challenging new concepts. Qualitative responses provided further indications for effective learning object integration into differentiated instruction formats.

Funded by UOIT's Teaching Innovation Fund.
Active learning improves comprehension in large section immunology course (37.7)
Authors: Ersin Özlem H , Bandrick Meggan , Molitor Thomas W
Published: January 2023

Active learning eschews the traditional sage-on-stage model of education in favor of a collaborative learning environment where students share with faculty the responsibility for making learning happen. The approach is especially well suited for students in large lecture classes who might otherwise feel isolated, unengaged or reluctant to interact. At University of Minnesota, we use a variety of active learning techniques in our large section, upper level undergraduate immunology course. Active learning has allowed us to incorporate formative assessment as an ongoing aspect of our class, as well as to encourage active engagement of a wide base of students in discussions. We describe here an active learning module on the complement system and report on the relative efficacy of the approach in fostering student achievement. The complement system is a conceptually trying topic for the typical undergraduate student of immunology. Results of pre- and post-tests over three academic years indicate students in the active learning intervention score higher than those in the control group. However, despite superior knowledge gains, student satisfaction with the learning experience declines.
ENteric Immunity SImulator: A tool for in silico study of gut immunopathologies (166.15)
Authors: Wendelsdorf Katherine , Bassaganya-Riera Josep , Bisset Keith , Eubank Stephen , Hontecillas Raquel , Marathe Madhav
Published: January 2023

Here we present the newly developed ENteric Immunity SImulator (ENISI) that simulates the antagonistic inflammatory and regulatory immune pathways of the gut as individual immune cells interact with and respond to commensal and foreign bacteria. This tool has been used to reproduce a typical inflammatory response to foreign bacteria as well as the immunopathological effects of autoimmunity to commensal bacteria with 10^6 individual cells. ENISI is built on an agent-based model that incorporates spatial effects and randomness of cell-cell and cell-bacteria contact, which can have a significant impact on whether a triggered inflammatory cascade is successfully regulated or spirals out of control. Preliminary simulations indicate the importance of cell density and epithelial cell-mediated recruitment in determining gut immunopathology. In addition, such representation allows emergent properties such as changes in bacterial demographics and evolution as a result of interaction with host immune cells. This tool will be useful to i) test the plausibility of in vitro observed immune cell responses as explanations for tissue-level damage, ii) propose mechanisms not yet tested in vitro that could explain tissue-level phenomenon, iii) conduct low-cost, preliminary tests of proposed interventions/ treatments for inflammatory diseases, iv) identify specific data to be gathered experimentally in order to characterize mechanisms of immune modulation in the gut.
Modeling human rheumatoid arthritis in NHP: Type II collagen induced arthritis in cynomolgus macaques (167.4)
Authors: Zhang Kevin , Liu June , Deng Jilin , Chang Liangtang , Shao Jian , Lu Jun , Bendele Alison , Fu Yunfeng , Duan Jeff
Published: January 2023

Collagen induced arthritis (CIA) rodent models have been extensively used in rheumatoid arthritis (RA) research. An RA model in non-human primate (NHP) is particularly demanded because of the close phylogenesis that provides the cross-reactivity to human for different development compounds using most modern drug technologies. However, NHP RA model has been reported extremely difficult because of the low and inconsistent disease incidence. We studied type II collagen induced arthritis in Cynomolgus monkeys. Following immunization with collagen, the disease progression was monitored for 8 weeks. Overall the arthritic incidence reached 87% and the average arthritic incidence of proximal interphalangeal (PIP) joint reached near 90%, significantly higher than what was previously reported. The average swelling of PIP joint increased approximately by 45%. Radiography, histopathology and histomorphometry analysis of the joint bones well supported the arthritic disease with the similar characteristics of human RA joints. The average arthritic score was significantly reduced with the single agent treatment of Methotrexate or Dexamethasone. Our results demonstrated the successful establishment of an reliable CIA in Cynomolgus monkeys, providing a valuable tool for studies of RA disease in pathogenesis, biomarker, translational research, and most importantly, anti-arthritic therapeutics as well as other relevant diseases, such as anemia of chronic disease and arthritic pain.
Regulation of immunity during viral lung infection (168.1)
Authors: Loebbermann Jens , Thornton Hannah , Sparwasser Tim , Johansson Cecilia , Openshaw Peter
Published: January 2023

Regulatory T cells (Tregs) play a clear role in chronic infections and inflammatory disorders, but their role in acute infections have not been fully elucidated. Respiratory syncytial virus (RSV) is the major cause of serious lower respiratory tract infection in infants, resulting in over-exuberant immune responses. In order to examine the role of Tregs in primary RSV primary infection, we infected “Depletion of regulatory T cells” (DEREG) mice which express the diphteria toxin (DT) receptor enhanced GFP fusion protein under the control of FOXP3 gene locus. Injections of DT ablated Tregs, and depletion prior to RSV primary infection caused increased weight loss and increased influx of cells into the lung and the airways (bronchioalveolar lavage, BAL) with lung and BAL showing elevated numbers of CD4+ and CD8+ T cells on d4, d8 and d14 post RSV infection. Interestingly, Treg depletion also caused an increase in neutrophils and eosinophils in the airways. Therefore, Tregs do not only control the magnitude of the T cell response, but also the quality of the responses that follow. The effects of RSV infection in mice with defective immune regulation closely parallels the observed effects of RSV in children with bronchiolitis, suggesting that the pathogenesis of bronchiolitis may involve an inability to regulate virus-induced inflammation
The miR-17-92 cluster is essential for regulatory T cell function in vivo (168.14)
Authors: Jeker Lukas , De Kouchkovsky Dimitri , Esensten Jonathan , Bluestone Jeffrey
Published: January 2023

microRNAs (miRNAs) are posttranscriptional regulators of the proteome. We have identified CD28-responsive miRNAs in human CD4+ T cells. Among others, the miR-17-92 cluster was of particular interest because abnormal miR-17 levels have been reported in peripheral blood mononuclear cells from patients with multiple sclerosis. When activated with low CD28-costimulation the miR-17-92 cluster was necessary to drive maximal proliferation in conventional mouse CD4+ T cells (Tconv) in vitro. However, the miR-17-92 cluster was largely dispensable for total T cell numbers in secondary lymphoid tissues and for antigen-response in vivo. Since FoxP3+ regulatory T cells (Treg) are strongly co-stimulation dependent we deleted the miR-17-92 cluster specifically in Treg. Preliminary data suggest that as for Tconv the miR-17-92 cluster was dispensable for Treg numbers and function under homeostatic conditions, even in aging mice. By contrast, mice with miR-17-92-deficient Treg develop much more severe experimental autoimmune encephalitis than control mice. Preliminary results suggest that miR-17-92-deficient Treg numbers are significantly reduced in the spinal cord while antigen-specific effector cells are present in normal numbers. We are currently investigating how miR-17-92 mediates this crucial function. Our results demonstrate that miRNAs are critical for optimal T cell co-stimulation via CD28. The miR-17-92 cluster is essential for Treg function under inflammatory conditions in vivo.
Induction of adaptive regulatory T cells by HIV-infected dendritic cells is defective. (168.15)
Authors: Presicce Pietro , Rusie Laura , Moreno-Fernandez Maria , Fichtenbaum Carl , Chougnet Claire
Published: January 2023

Recent studies have shown that dendritic cells (DCs) can induce peripheral conversion of conventional T cells into regulatory T cells (Tregs). DCs are among the first targets of HIV and it is not known whether their infection or exposure to the virus alters their capacity to convert Treg. In our study, 10% of CD3+CD4+CD25-FOXP3- T cells (non-Tregs) expressed FOXP3 5 days after culture with autologous monocyte-derived DCs (moDCs). These converted FOXP3+ cells suppressed proliferation of responder T cells similarly to natural Tregs. In contrast, moDCs from HIV-infected patients were defective at inducing FOXP3 in autologous non-Tregs. MoDCs infected in vitro with a CCR5-using strain were also unable to induce FOXP3. Productive infection of the DC was not required, as DC exposure to inactivated HIV also impaired FOXP3 induction. This defect did not depend on IL-10 or TGF-β, as adding these cytokines did not restore FOXP3 induction. Higher T cell death was observed in infected cocultures than in uninfected ones. Our findings suggest that HIV-infected moDCs kill converted Tregs, leading to a poor induction of adaptive Tregs.
Adenosine signaling mediates the protective effects of regulatory T cells in kidney ischemia reperfusion injury (168.17)
Authors: Kinsey Gilbert , Huang Liping , Lobo Peter , Okusa Mark
Published: January 2023

Regulatory T cells (Tregs) suppress the innate inflammation and injury associated with kidney ischemia-reperfusion injury (IRI). Activation of adenosine 2A receptors (A2ARs) on immune cells also prevents renal injury after IR. Tregs express CD73, which is the rate-limiting enzyme in production of extracellular adenosine. We hypothesized that the production of adenosine by Tregs is required to block innate immune responses in kidney IRI and that the Treg-generated adenosine would signal through A2ARs on inflammatory cells. The ability of adoptively-transferred Tregs, isolated from WT and CD73KO mice, to protect WT mice from kidney IRI was compared in terms of renal function, tubular necrosis and kidney infiltrating leukocytes. WT Tregs protected WT mice from kidney IRI, but in the absence of adenosine generation (CD73KO) the functional capacity of Tregs to block kidney IRI was impaired (plasma creatinine 18hr post ischemia: Sham: 0.3±0.1*; IRI+non-Tregs: 1.4±0.3; IRI+WT Tregs: 0.4±0.1*; IRI+CD73KO Tregs: 1.0±0.3; *p<0.05 vs. IRI+non-Tregs). WT Tregs offered significant, but incomplete, protection to A2ARKO mice. In contrast, WT Tregs completely protected A2BRKO mice, similar to WT mice. Taken together these findings suggest that adenosine production is vital for the protective effects of Tregs in IRI and suggest Treg-generated adenosine acts partially through A2ARs, in the recipient, to inhibit innate immune responses in kidney IRI.
Ex-vivo expanded DCs induce donor-specific central and peripheral tolerance and prolong the acceptance of donor skin allografts (169.12)
Authors: Kishimoto Hidehiro , Yamano Tomoyoshi , Suzuki Toshihiro , Abe Ryo
Published: January 2023

The principal function of Dendritic cells (DCs) is to promote T cell activation and differentiation into effecter T cells by presenting foreign Ags and providing costimulatory signals. Also DCs are known to regulate immune responses by inducing both central and peripheral tolerance. DCs play a vital role in negative selection of developing thymocytes by deleting T cells with high-affinity for self-peptide-MHC complexes. In the periphery, DCs mediate peripheral tolerance by promoting regulatory T cell development, induction of T cell unresponsiveness, and deletion of activated T cells. We studied whether allogeneic DCs obtained from bone marrow cultured either with Flt3L (FLDC) or GM-CSF (GMDC) could induce allo-specific central and peripheral tolerance after i.v. injection; B cells were used as a control. The results showed that only FLDC reached the thymus after injection, and these cells induced both central and peripheral tolerance to donor MHC. For central tolerance, injection of FLDC induced antigen-specific negative selection of both CD8 and CD4 single-positive thymocytes. For peripheral tolerance, injection of FLDC induced donor-specific T cell unresponsiveness and prolonged survival of donor-derived skin grafts. Tolerance induction by adoptive transfer of Flt3L-induced DC could be a useful approach for promoting graft acceptance after organ transplantation.
Liver transplant tolerance: a new look at an old puzzle (169.17)
Authors: Ahearn Aaron , Klein Ingo , Lee Karim , Hayden Tracy , Liu Feng Chun , Tang Qizhi , Kang Sang-Mo
Published: January 2023

We explored the tolerogenicity of liver allografts (Ltx) by characterizing the phenotype and persistence of alloreactive T cells after spontaneously tolerant Ltx in mice. Using adoptive transfer of TCR-tg direct and indirect donor-reactive T cells, we found that LTx (BALB→B10) induced a stronger CD4 T cell response than either skin or heart Tx. Moreover, large numbers of donor-reactive T cells persist 21 days after Ltx and do not upregulate Foxp3, even though tolerance to subsequent allospecific skin grafts is established. In vivo MLR confirmed the persistence of endogenous effector CD4 T cells at 21 and 250 days post-Ltx. Overall, these findings are consistent with an ongoing effector alloresponse rather than immune regulation after Ltx. In contrast, the CD8 donor-reactive T cells were significantly reduced, though still detectable, after Ltx. However, post-Ltx animals selectively eliminate allogenic target cells in an in vivo CTL assay. Thus, deletion of effector CD8 T cells may help protect liver allografts, but cannot completely account for Ltx tolerance. Finally, CD4, CD8, and Total T cells isolated from day 90 post-Ltx mice reject donor skin grafts when transferred into Rag1-/- hosts, indicating that Ltx tolerance cannot be transferred via the immune compartment. These data demonstrate the magnitude and persistence of the anti-donor T cell responses after Ltx and opposes either deletional tolerance or regulatory T cell induction as mechanisms for Ltx tolerance.
Operationally tolerant kidney recipients exhibit higher numbers of suppressive CD19+CD24hiCD38hi b cells in relation to chronic rejection (169.25)
Authors: SILVA Hernandez , Takenaka Maisa , Vieira Pedro , Monteiro Sandra , Lemos Francine , Saitovitch David , Kalil Jorge , Coelho Veronica
Published: January 2023

Introduction: Immunotolerance in human transplantation remains a great challenge. The study of special individuals, called operationally tolerant (OT), that maintain stable graft functions after immunosuppression (IS) withdrawal can shed light into the field of transplantation tolerance. Objective: Investigate if OT involves a differential activation profile in the suppressive B cell population, CD19+CD24hiCD38hi (Bregs). Methods: We evaluated 5 OT, 10 Chronic rejection (CR), 19 stable graft function under standard IS (Sta) and 10 healthy (HI) individuals. We investigated the activation status of CD40/STAT3 signaling pathway in B cell subpopulations, analyzing STAT3 pTyr705 phosphorylation by flow cytometry. Results: We observed higher percentages in OT in relation to CR and STA (p<0.05) and higher absolute numbers in both OT and HI in relation to CR (p<0.05) of circulating Bregs. Despite the contrasting numbers of Bregs, the activation status of the CD40/STAT3 signaling pathway in these B cells was similar among all groups. Nevertheless, the fold of STAT3 phosphorylation increase (STAT3p+ cells post-stimulus/STAT3p+ cells pre-stimulus) was higher in OT than in CR (p<0.05). Conclusions: Despite the normal suppressive B cell CD40/STAT3 pathway activation in all study groups, the significantly lower numbers and a less efficient functional mobilization of Bregs, in CR, suggest that the preservation of this population may be relevant to keep the state of operational tolerance.
Defective B cell memory and autoantibodies in subjects of cardiac transplantation in infancy (169.27)
Authors: Bender Diane , Schlater Amy , West Lori , Chinnock Richard , Platt Jeffrey , Cascalho Marilia
Published: January 2023

Aberrant functions of T cells and B cells may impair B cell memory and are thought to allow development of autoantibodies. Rarely, B cell memory defects and/or autoimmunity can be traced to specific mutations; however, in most cases which properties of T cells and B cells cause deficient B cell responses and/or autoimmunity are unclear. To answer these questions we studied child recipients of cardiac transplantation during infancy who, owing to removal of the thymus and T cell depletion at the time of surgery, have a greatly reduced T cell receptor repertoire diversity. We found these children to have impaired memory IgG antibody responses to polypeptide vaccines compared to controls, even though they appeared to mount IgM responses. These results suggest that the defective T cell compartment in recipients of cardiac transplantation in infancy impairs B cell memory and/or isotype class- switching. Remarkably, despite these defects and despite ongoing treatment with immunosuppressive drugs, 6 of the 9 subjects studied produced anti-ssDNA and rheumatoid factor antibodies as early as of 2 years of age. These findings suggest that the diversity of T cells and/or the availability of recent thymic emigrants are needed to generate B cell memory and prevent autoimmunity.
The role of antibodies in controlling reactivation of mCMV in immunocompromised bone marrow transplantation recipients (169.28)
Authors: Seefried Martina , Schneider Andrea , Mach Michael , Winkler Thomas
Published: January 2023

Reactivation of cytomegalovirus (CMV) is a frequent complication of bone marrow transplantation (BMT) and results in morbidity and mortality in immunocompromised recipients. In a previous study using murine CMV (mCMV) as a model, we have shown that transfer of memory B cells protects the immunodeficient host from the lethal course of mCMV infection. Here we demonstrate the impact of antibodies on the reactivation of mCMV in a murine model of BMT. We find that previous depletion of B cells (anti-CD20 antibody) during primary mCMV infection results in a profound reactivation of mCMV after syngeneic BMT. Our data show a negative correlation between virus-specific serum IgG titer and reactivation of mCMV after BMT. Therefore, our studies suggest that antibodies play a major role in controlling recurrent mCMV infection after BMT.