bluuebunny/crossref_metadata_2025 huggingface dataset:
Studying the cooperativity at the SLP-76 signaling complex critical for the immune response (50.7)
Authors: Barda-Saad Mira , Shirasu Naoto , Pauker Maor , Hasan Nirit , Perl Orly , Balbo Andrea , Yamaguchi Hiroshi , Houtman Jon , Appella Ettore , Schuck Peter , Samelson Lawrence
Published: January 2023

Following T-cell antigen receptor (TCR) engagement, multiprotein signaling complexes essential for regulating T-cell functions are formed. A multi-molecular complex consisting of SLP-76, Nck and VAV is recruited to the T-cell/antigen presenting cell (APC) site during initial T cell activation and is crucial for regulation of the actin machinery. Most of the studies performed to date have concentrated on the role of the signaling molecules that comprise this molecular complex. However, the regulation of the complex formation and the cooperation and competition among its components during formation have not yet been explored. In this study, a multidisciplinary approach including molecular imaging, biochemical and biophysical techniques was utilized in order to define the composition, stoichiometry, and specificity of the SLP-76:Nck:VAV complex. High-resolution imaging techniques, such as fluorescence resonance energy transfer (FRET) analysis, were performed in live activated T cells in order to focus on the binding properties within the trimolecular complex. Our data reveal a direct interaction between Nck and VAV which is mediated by the binding of the C-terminal SH3 domain of Nck to VAV’s N-terminal SH3 domain. Disruption of the VAV:Nck interaction significantly impaired actin polymerization. This research provides novel insights into the regulation of a signaling complex formation critical for immune response.
Identification of a class of small molecules that up-regulate production of anti-inflammatory cytokine IL10 via inhibition of GSK3β (51.10)
Authors: Gururaja Tarikere , Ding Rongxian , Yung Stephanie , Yi Sothy , Zhao Haoran , Singh Rajinder , Kinoshita Taisei , Masuda Esteban
Published: January 2023

Interleukin-10 (IL-10), also known as human cytokine synthesis inhibitory factor (CSIF), is an anti-inflammatory cytokine produced primarily by monocytes and to a lesser extent by lymphocytes. This cytokine exhibits pleiotropic effects in immunoregulation and inflammation and is capable of inhibiting synthesis of various pro-inflammatory cytokines such as IFNγ, IL-2, IL-3, IL-6, TNFα and GM-CSF made by cells such as macrophages and Th-1 cells. During the course of our investigation to find small molecules that are capable of inhibiting the production of a pro-inflammatory cytokine, IL-23, we came across a sub-set of small molecules that exhibited an interesting property of up-regulating expression of IL-10 besides inhibiting IL-23 production. Such observation prompted us to further investigate the mechanism of action and target identification for this class of compounds. Preliminary experiments revealed that enhanced IL-10 production upon LPS stimulation of human THP-1 monocytic cells in the presence of compounds appears to be due to inhibition of GSK3β kinase. Results from these studies were further substantiated by screening the lead candidate against a panel of kinases in-vitro including GSK3β and a cell-based β-cateinin accumulation assay that directly measures GSK3β kinase on-target activity.
Modulation of CD40L expression by polypyrimidine tract binding protein (PTB) (51.8)
Authors: Matus Nicodemos Rodrigo , Vavassori Stefano , Laughlin Jennifer , Covey Lori
Published: January 2023

The expression of CD40L is tightly regulated at the posttranscriptional level throughout a time course of T cell activation, which corresponds to a significant increase in message stability at late times of activation (24-48 hr). Previous work revealed a cytoplasmic polypyrimidine tract binding protein (PTB)-containing-complex binds to the CD40L 3’UTR at late times of T cell activation. We used RNA interference to downregulate PTB in D1.1 Jurkat T cell line to address the functional roles of PTB in CD154 mRNA stability and surface expression. Real-time qPCR measurement of the CD154 mRNA decay revealed that downregulating PTB caused a > 2-fold decrease in the CD40L mRNA half-life. The downregulation of PTB also caused an approximate 2-fold decrease in the mean fluorescence (MFI) of CD40L. Analysis of PTB cellular distribution during a time course of CD4+ T cell activation revealed cytoplasmic and nuclear localization in all resting and activated cells. However, there was an increase in expression at late times of activation. Binding studies revealed that CD40L mRNA is bound by nuclear PTB at all times of activation indicating that the requirements for binding of CD40L message by nuclear versus cytoplasmic PTB is highly distinct. Finally, the binding of CD40L message corresponded to a change in phosphorylation status of cytoplasmic PTB. These findings support a model whereby activation-induced phosphorylation of PTB is required for cytoplasmic binding to CD40L mRNA.
Lymphocyte type determines the effect of single nucleotide polymorphisms on Lymphotoxin Alpha expression. (51.9)
Authors: Yokley Brian , Posch Phillip
Published: January 2023

The lymphotoxin alpha (LTA) gene contains many single nucleotide polymorphisms (SNPs); some influence expression levels and are associated with disease. The impact of SNPs on LTA expression primarily has been analyzed in B cells, yet T cells contribute the most to LTA levels. Thus, we investigated the cell type-specific effects of the LTA +81C/A and +369G/C SNPs on expression. The LTA +81A SNP significantly increased expression relative to +81C in T cells; opposite of its impact in B cells. Conversely, the LTA +369G SNP was found to significantly decrease expression only in T cells under basal conditions. The impact of this SNP was partly due to stronger binding to and repression of LTA +369G by Sp1. ChIP assays showed Sp1 binding to this region of the LTA locus in vivo, but the two Sp1 sites in close proximity could not be distinguished. However, mutation of the LTA +369 SNP-associated Sp1 site did result in an expected increase in expression further implicating that Sp1 binding to this site suppresses expression. Numerous studies associate SNPs with disease and mechanisms are proposed to explain these associations based on the known impact of the SNP on expression. We show that LTA SNPs can differentially impact expression levels depending on stimulation status and cell type. Thus, it is critical to consider the cell type-specific impact of a SNP and the pertinence of different cell types to disease pathogenesis when proposing a mechanism behind a disease association.
Towards the development of a universal T cell influenza (FLU) vaccine (52.10)
Authors: Xu Dong , Popmihajlov Zoran , Arnold Katherine , Sumner Jonathan , Hussell Tracy , Feldmann Marc , Smith Kendall
Published: January 2023

Present day FLU vaccines have not changed since WWII, and depend upon the generation of neutralizing antibodies, which the virus readily escapes via mutations. Therefore, we focused on immunizing T cells with serologically non-cross reactive viral strains in a murine model. C57Bl/6 mice infected intra-nasally (i.n.) with H3N2-FLU were protected against a lethal challenge of H1N1-FLU. Cellular composition of the Broncho-Alveolar Lavage (BAL), lungs and mediastinal lymph nodes monitored by flow-cytometry 8 days after infection with H1N1-FLU yielded a vigorous cellular response, with a 2:1 ratio of CD8+ vs. CD4+ T cells in the BAL, and a high proportion of immunodominant nucleoprotein (NP) peptide-specific tetramer+ cells (~ 40%). By comparison, mice immunized i.n. with replication-incompetent adenovirus containing NP (10e7 pfu) were also protected against lethal challenge. Although the H3N2-FLU-infected control animals mobilized greater numbers of CD8+ T cells in the BAL 14d post H1N1 challenge, the vaccinated animals had 3X as many NP-specific tetramer+ T cells. Therefore, a non-replicating adenovirus vaccine can protect against a lethal challenge with a non-serologically cross-reactive flu strain, so that a “T cell vaccine” is effective in preventing symptomatic disease.
Differential lymphocyte activation induced by mammalian-derived Ebola virus-like particles in a post-exposure murine model. (52.23)
Authors: Anthony Scott , Bradfute Steven , Bavari Sina
Published: January 2023

Ebola virus is a member of filoviradae, a family of negative single stranded RNA viruses which cause hemorrhagic fever with mortality rates of up to 90%. To date there is no approved treatment to combat this deadly disease. During studies in a murine model, it was found that Ebola virus-like particles (eVLP) provided sufficient protection when given 24 hours after Ebola virus exposure. However, a study determining the specific, in vivo eVLP activation of immune cell subsets during the course of infection has not been performed. Utilizing flow cytometry, lymphocytes within the draining lymph node of the eVLP vaccination site were compared to corresponding cells in unvaccinated mice. In preliminary studies, eVLP induced significantly different levels of B cell activation on day 7 of the infection. In day 7 eVLP vaccinated mice, activation was found to be significantly higher in the draining inguinal lymph node compared to the contra-lateral inguinal node. Future studies will attempt to expand upon the functional significance of this lymphocyte response, while also incorporating the role of macrophages and dendritic cells. This study better characterizes the local, in vivo, adaptive immune responses induced by eVLP in this post-exposure model of protection. By understanding the proper adaptive immune response to therapeutically protect against this virus, it may be possible to modulate future therapies to target these specific successful responses for further up regulation.
Immunological evaluation of Recombinant Mycobacterium vaccae expressing Mycobacterium tuberculosis-specific antigens in mice (52.5)
Authors: Amoudy Hanady
Published: January 2023

Tuberculosis (TB) is considered to be one of the leading causes of death from infectious diseases all over the world. The danger is coupled with the emergence of multi-drug resistant strains which renders treatment a real challenge. The Vaccine-BCG- used against TB is very disputable giving wide ranges of protection reaching as low as nil, in some parts of the world. Several M. tuberculosis-specific DNA regions including RD1 were found to be deleted from the vaccine strains. Deleted region RD1 encode proteins that are major targets for T cell recognition in humans and animals infected with M. tuberculosis and M. bovis, respectively. The aim of this study is to express open reading frames (ORFs) from RD1 region in M. vaccae and evaluate their ability to induce specific immune responses in mice. The appropriate ORFs were cloned in M. vaccae using the shuttle vectors pSMT3, and pDE22. Expression of the corresponding proteins was detected by western blotting using anti-hemagglutinin (HA) and/ or antigens- specific antibodies. Recombinant mycobacteria were injected in Balb/C mice, spleens were removed, and specific immunity to the recombinant antigens was assessed using standard spleenocytes culture and cytokines quantitation assays. This system of expression using shuttle vectors proved to be a promising system to identify candidate antigens to be used in diagnosis and to develop improved vaccine strains of BCG.
15d-PGJ2 ameliorates collagen-induced arthritis by convergence of Th17 to induced-Tregs (83.1)
Authors: Napimoga Marcelo , Carregaro Vanessa , Vieira Silvio , Grespan Renata , Silva João , Cunha Fernando
Published: January 2023

Regulatory T cells (Treg) are CD4+T subtype cells with a critical role in inhibiting autoimmunity. Several works reported efficacy therapeutic of 15d-PGJ2 in autoimmune models as well in vitro is able to generate the differentiation of naïve T lymphocytes to the regulatory profile. However, this prostanoid use and its interrelationship with Tregs during the arthritis rheumatoid are unknown. In view of a therapeutic perspective, we investigate the potential role of 15d-PGJ2 in a model of collagen-induced arthritis (CIA) and its interplay with Tregs-induced anti-inflammatory mechanism. CIA was elicited in DBA male mice by collagen-emulsion injection and then treated with 15-PGJ2 daily during 7 days. 15d-PGJ2 administration at the disease onset attenuated the severity of CIA, reducing clinical score, pain and edema. Furthermore, while the mRNA expression of ROR-γt was reduced, FOXP3 expression was up-regulated in draining lymph nodes cells from 15d-PGJ2-treated mice. In vitro, 15-PGJ2 increased the expression of FOXP3, GITR and CTLA-4, Tregs makers, on CD4+CD25- population, suggesting the induction of Tregs on effector cells. Coculture of Th17 cells with 15d-PGJ2-induced Treg reduced significantly the IL-17 production by collagen and α-CD3 stimuli whereas IL-10 production was enhanced. 15d-PGJ2 converge Th17 lymphocytes to regulatory profile, reducing the number of pathogenic T cells and consequently suppressing the inflammatory immune response. FAPEMIG 097/09
IL-12p35 promotes antibody-induced joint inflammation by activating NKT cells and suppressing TGF-β. (83.14)
Authors: Kim Hye sung , Park Yuna , Ahn Ji Ye , Yun Daesun , Cho Mi La , Hong Seokmann , Kim Ho Youn , Chung Doo Hyun
Published: January 2023

The functional role of IL-12 in rheumatoid arthritis is controversial. Moreover, whether IL-12 contributes to regulation of antibody-induced joint inflammation remains unclear. To address these issues, we explored the functional roles of IL-12 in the K/BxN serum transfer model. IL-12p35-/- and IL-12R2-/- mice were resistant to the development of arthritis. Injection of K/BxN serum into IL-12p40 reporter (yet40) mice induced CD11b+,CD11c+ cells and Gr-1+ granulocytes to produce IL-12p40 in the joints. The levels of IFN-γ, IL-4, and IL-6 production were lower in joint tissues of IL-12p35-/- and IL-12Rβ2-/- mice than B6 mice, whereas that of TGF-β expression was higher. Administering IL-12p35-/- mice recombinant IL-12 or IFN-γ restored joint inflammation and suppressed TGF-β production in joint tissues. Moreover, administering neutralizing αTGF-β mAb enhanced joint inflammation. Among the immune cells that infiltrated joint tissues during antibody-induced arthritis, NKT cells expressed IL-12Rβ2 receptors. Furthermore, the adoptive transfer of splenocytes from B6 or Gr-1+ granulocyte-depleted mice restored joint inflammation in IL-12Rβ2-/- mice as much as in B6 mice, whereas splenocytes from Jα18-/- mice did not. These findings indicate that signals via IL-12 receptors on NKT cells play a critical role in the development of antibody-induced arthritis. In conclusion, the IL-12p35/IFN-γ axis promotes antibody-induced joint inflammation by activating NKT cells and suppressing TGF-β.
Autoreactive cytotoxic T lymphocytes require target tissue stimulus to acquire cytotoxic effector capacity. (83.2)
Authors: Graham Kate , Krishnamurthy Balasubramanian , Santamaria Pere , Thomas Helen , Kay Thomas
Published: January 2023

Cytotoxic T lymphocytes (CTL) are required for the development of autoimmune diseases such as type 1 diabetes. CTL are first activated in lymph nodes (LN) draining target tissue and then concentrate as fully differentiated CTL in the target tissue. We have studied the need for further stimulation of CTL after LN activation and the importance of APCs and the target tissue itself in CTL differentiation. Naïve CFSE-labeled pancreatic β cell specific 8.3-TCR transgenic CD8+ T cells were transferred into NOD mice. Autoreactive CTL proliferated and acquired limited expression of GranzymeB and IFNγ, signatures of cytotoxicity, in the pancreatic LN. CTLs in the islets had proliferated further and acquired higher expression of GzmB and IFNγ. This increased differentiation appeared to take place in the islet because expression of GzmB and IFNγ in CFSE-labeled T cells was similar in peripheral blood and PLN and T cells prevented from migrating from the PLN by FTY720 did not have increased cytotoxic differentiation. Reducing pancreatic β cell antigen presentation affected CTL proliferation but had little effect on differentiation. Increasing the density of antigen on APCs also had more impact on proliferation than differentiation. The data provide in vivo support for a signal required for CTL differentiation, in addition to TCR ligation and co-stimulation, which is produced in the inflamed target tissue. This suggests the target tissue is the critical location for CTL differentiation.
Th17 polarized cells from NOD mice following mycobacterial adjuvant immunotherapy delay type 1 diabetes development (83.26)
Authors: Nikoopour Enayat , Schwartz Jordan , Huszarik Katrina , Sandrock Christian , Kroughly Olga , Lee-Chan Edwin , Singh Bhagirath
Published: January 2023

IL-17 producing T cells are regarded as potential pathogenic T cells in the induction of autoimmune diseases. Previously we have shown that injection of Mycobacteria containing products such as Bacille Calmette-Guerin (BCG) or Complete Freund Adjuvant (CFA), can prevent type 1 diabetes (T1D) in nonobese diabetic (NOD) mice. We injected NOD mice with mycobacterial products subcutaneously and analyzed the IL-17 producing cells from the draining lymph nodes and spleen by restimulating whole cell populations in vitro with or without IL-17 polarizing cytokines. Mice receiving CFA had a concomitant rise in the level of IL-17, IL-22, IL-10 and IFN-g in the draining lymph node and spleen. Adoptive transfer of splenocytes from CFA-injected NOD mice polarized with TGF-b plus IL-6 or IL-23 delayed the development of diabetes in recipient mice. IL-17 producing cells induced by CFA maintained their IL-17 producing ability in the recipient mice. Injection of CFA also changed the cytokine profile of cells in pancreatic tissue by increasing IL-17, IL-10 and IFN-g cytokine gene expression. Compared to wild type mice, injection of CFA into MyD88-/- mice drastically reduced but did not abolish the level of IL-17 in the lymphoid organs and pancreas indicating that the MyD88 pathway is a major pathway through which Mycobacteria exerts its effect. We suggest that the rise in the level of IL-17 after adjuvant therapy in NOD mice has a protective effect on T1D development.
Natural regulatory T cells (Treg) determine post vasectomy sequlae: autoimmune orchitis (EAO) versus testis-specific tolerance. (83.3)
Authors: Wheeler Karen , Luu Brian , Tardif Steve , Rival Claudia , Qiao Hui , Bui Elise , Hardy Daniel , Tung Kenneth
Published: January 2023

Vasectomy (Vx), a popular male contraceptive, causes a persistent epididymal granuloma. Using unilateral Vx (uni-Vx) as an inducer of a localized danger signal, we queried whether Treg control the transition to autoimmunity. When Treg were depleted (CD25 Ab) in uni-Vx mice, frequent and bilateral EAO developed. This was accompanied by T cell activation in the regional lymph node and a robust testis antibody response. CD4+ T cells that produced IFNγ predominated in the diseased testes. CD4+ T cells are sufficient for disease induction as in vivo depletion of CD4+ cells blocked the emergence of EAO and CD4+ cells from uni-Vx mice were able to adoptively transfer disease. We also identified sperm Zonadhesin (Zan) as the major orchitogenic autoantigen. By immunoblot, autoantibodies from uni-Vx mice prominently recognized Zan, and immunization with Zan in adjuvant induced EAO. As EAO induction in Vx men would be devastating, we depleted Treg two weeks following uni-Vx and found they were free of EAO. To test whether uni-Vx in fact induces tolerance to testicular antigens, we compared uni-Vx and Sham-Vx mice in their response to EAO induction by immunization with testis homogenate in CFA. Indeed, uni-Vx mice developed significantly lower EAO scores and autoantibody and T cell proliferative responses. Thus, Treg protect Vx mice from post-Vx EAO by blocking propagation of danger signals to pathogenic autoimmunity while granting systemic tolerance to testis antigens.
Critical mouse strain dependency of IL17A and IFNγ requirements in experimental autoimmune orchitis (EAO) (83.5)
Authors: Rival Claudia , Qiao Hui , Paul Alberta , Tung Kenneth
Published: January 2023

Many autoimmune diseases depend critically on the proinflammatory cytokines IFNγ and IL17, but their relative contribution is controversial. This may be due to variations in mouse strain, adjuvant and autoimmune model. For example, IL17 is deemed critical for EAE and collagen-induced arthritis in B6, whereas IFNγ is critical in proteoglycan-induced arthritis in BALB/c. EAO develops in B6, BALB/c, and (BALB/cxB6) F1 (F1) mice after immunization with testis Ag in CFA and pertussis. Herein, we compared their cytokine requirements using gene KO mice (IFNγ, IL17A) and cytokine Ab blockade. In B6, IL17A is required and IFNγ is redundant. In BALB/c, IL17A and IFNγ are redundant; in fact, EAO still occurs in IL17A and IFNγ double KO BALB/c. In contrast, both IL17A and IFNγ are required in the F1. The variable cytokine dependency may reflect mechanistic differences in EAO amongst these wild type strains, since the testis-infiltrating cells are neutrophil dominant in B6, monocyte dominant in BALB/c, and eosinophil dominant in F1 mice. In addition, the severity of EAO in wild type F1 mice far surpasses either parental strain, indicating genetic complementation. Finally, IFNγ deficient BALB/c developed severe epididymitis and vasitis, which are absent in wild type BALB/c. We have therefore documented fundamental mouse strain differences in IL17A and IFNγ requirement in a single autoimmune disease, and this may reflect strain dependent differences in disease mechanisms.
Regulatory B10 cells have the potential to differentiate into Ab-secreting plasma cells (84.16)
Authors: Smith Susan , Maseda Damian , Bryant Jacquie , Weaver Casey , Tedder Thomas
Published: January 2023

B cells with IL10-dependent regulatory functions have been observed in mouse models of autoimmunity, colitis and in T-dependent immune responses such as delayed-type hypersensitivity. The rare regulatory B10 cell subset that is competent to produce IL10 was examined in the current studies using two distinct lines of reporter mice to track IL10 production. Tiger mice expressed both IL10 and GFP proteins from the same transcript, with GFP expression faithfully marking IL10 production by B10 cells. By contrast, 10BiT mice express Thy1.1 as a separate transcript. While Thy1.1 and IL10 transcripts appeared concurrently as determined by real-time RT-PCR, cell surface Thy1.1 expression was delayed by >24 h and persisted on the cell surface even after a decline in IL10 production. Thus, Thy1.1 expression functioned as a marker for B10 cells after IL10 induction in vivo during later stages of differentiation. Following LPS stimulation in vivo, Thy1.1+ B10 cells upregulated CD138 expression and secreted IgM. Likewise, B10 cells from wild type mice were a major source of secreted Ab following LPS-stimulation ex vivo. Thereby, B10 cells are competent to express IL10 following LPS stimulation in vivo, which is followed by their differentiation into IgM-secreting plasma cells.
Regulation of Immunoglobulin Secretion During Adaptive Immunity (84.17)
Authors: Jones Shannon , Vilen Barbara
Published: January 2023

Regulating BCR-mediated adaptive immune responses and subsequent immunoglobulin (Ig) secretion is critical for normal immune function. Defective regulation could lead to chronic inflammation, autoimmunity, or cancer. Several regulatory mechanisms control the threshold for B cell activation and Ig secretion. One mechanism negatively regulates the threshold for BCR activation through coligation of FcγRIIb and subsequent recruitment of SHIP. It was thought that FcγRIIB was the primary mechanism by which humoral responses were terminated. However, mice deficient in FcγRIIb do not exhibit excessive Ig secretion indicating other regulatory mechanisms must exist. We recently identified two additional mechanisms by which immunoglobulin secretion is attenuated during adaptive immunity. First, upon engagement of FcγRIII on dendritic cells by antibody/antigen immune complexes (ICs), dendritic cells secrete soluble mediators that repress BCR-mediated Ig secretion. Second, an autocrine mechanism functions to inhibit Ig secretion wherein B cells stimulated via the BCR with anti-μ or antigen secrete soluble mediators that repress Ig secretion. These data suggest that in addition to FcγRIIb, soluble factors secreted by both dendritic cells and B cells have a critical role in regulating immunoglobulin secretion during BCR-mediated adaptive immune responses. Studies are underway to define the relative importance of these pathways in terminating Ig secretion during adaptive immunity.
Transcriptional control of rapid recall by memory CD4 T cells (85.10)
Authors: Lai Wendy , Yu Minjun , Okoye Francesca , Keegan Achsah , Farber Donna
Published: January 2023

The defining feature of memory T cells is the capacity for rapid effector function upon restimulation through the T cell antigen receptor (TCR), although the signaling mechanisms controlling memory recall remain undefined. We hypothesized that enhanced transcription factor activation regulated the distinct TCR-coupled functional responses in naive and memory CD4 T cells. We generated memory CD4 T cells by in vitro-priming of naive CD4 T cells from DO11.10 transgenic mice, adoptive transfer into mouse hosts, and recovery 4-8 weeks post-transfer. OVA-specific memory CD4 T cells produced high levels of IFN-γ early (6hrs) after antigen stimulation, compared to IFN-γ production after 24-48hrs stimulation of OVA-specific naive CD4 T cells. Early IFN-γ production by antigen-stimulated memory CD4 T cells occurred in the absence of upregulation of the T-bet transcription factor, while IFN-γ production following naive CD4 T cell activation was associated with high T-bet expression. Chromatin immunoprecipitation (ChIP) analysis revealed that NFκB, but not T-bet was bound to the IFN-γ promoter in memory CD4 T cells early after TCR stimulation. In addition, expression of the active nuclear p50 subunit of NFκB was increased in resting and activated memory versus naive CD4 T cells, and inhibition of NFκB activity impaired early memory T cell recall. These results reveal a molecular mechanism for immediate recall responses in memory CD4 T cells through enhanced NFκB activity.
Comprehensive and semi-quantitative TCR repertoire analysis with a novel multiplex PCR method and 454 sequencing (85.3)
Authors: Han Jian , Wang Chunlin , Sanders Catherine , Yang Qunying , Schroeder Harry , Wang Elijah , Babrzadeh Farbod , Gharizadeh Baback , Myers Richard , Hudson James , Davis Ronald
Published: January 2023

CDR3 sequences, composed by V(D)J combination, form the center of the antigen binding site where they often play a critical role in defining the affinity and specificity of the receptor for individual peptide-MHC complexes of both the TCRα and TCRβ chains. The goal of our study was to produce comprehensive, unrestricted profiles of TCR diversity among key developmental and effector subsets of T cells isolated from the blood of a single, healthy donor at sequence-level resolution using a novel multiplex PCR method combined with Roche 454 Life Sciences high-throughput sequencing technology. From the sequence reads, about 1.48 million CDR3 intervals were identified, totaling 169,977 and 113,290 unique CDR3 intervals for TCRα and for TCRβ, respecitively. Our data also show numerous examples of identical CDR3 sequences shared by different T subsets. Using the compound Poisson process model, we estimated that the diversity of the TCRα and β chains expressed by our donor is around 0.47 x 106 and 0.35 x 106 unique CDR3 nucleotide sequences, respectively. Our comprehensive data demonstrates that our new method has overcome past challenges in studying the T cell immune repertoire and is highly sensitive, repeatable, and semi-quantitative. This approach provides a useful tool for assessing immune competence, tracking T cell expansion kinetics, assessing vaccine efficiency, and detecting antigen-specific T cell clones in patients with infection or cancer.
Regulation of IL-13 in mast cells by the Let-7 miRNA family (86.15)
Authors: Takemoto Clifford , Gamper Christopher , Lee Youl-Nam , Mendell Joshua , Brandal Stephanie , Powell Jonathan , McDevitt Michael
Published: January 2023

In mast cells, IL-13 protein is robustly expressed with IgE activation; IL-13 mRNA transcript also increases, but rapidly returns to baseline levels within several hours. Based on these observations, we postulated that miRNAs may regulate IL-13 protein expression at the post-transcriptional level. miRNAs play roles in a number of biologic processes but little is known about their function in mediating allergic disease. To explore if miRNAs contribute to IL-13 regulation and other IgE responses, we performed a microarray screen for miRNAs regulated by IgE in both mouse and human mast cells. We found that IgE stimulation resulted in widespread depression of miRNAs, including downregulation of the Let-7 miRNA family. We looked for proteins expressed in IgE activated mast cells that were potentially targeted by Let-7. We found a highly conserved Let-7 binding site in the 3’UTR of the IL-13 gene. We further demonstrate that the IL-13 UTR is regulated by Let-7; forced expression of Let-7 represses expression of a reporter gene with the IL-13 UTR, and repression is abrogated by mutation of the Let-7 binding site. Conversely, we show that Let-7 inhibition results in increased expression of a reporter gene with the IL-13 UTR, either by transfection of “antagomirs”, or by overexpression of the Let-7 family inhibitor, LIN28B. These results suggest that in mast cells, IL-13 protein expression is regulated by Let-7 miRNAs and support a role for miRNAs mediating IgE responses.
Contrasting requirements for DAP12 in receptor-mediated mast cell activation (86.4)
Authors: Smrz Daniel , Iwaki Shoko , Metcalfe Dean , Gilfillan Alasdair
Published: January 2023

By inducing FcϵRI aggregation, antigen/IgE promotes mast cell cytokine production; a response markedly enhanced by specific TLR agonists. Critical to the ability of FcϵRI to activate mast cells is the recruitment of the tyrosine kinase Syk via the FcϵRIγ chains. Recent reports in other cell types have indicated that TLR’s may alternatively signal via the adaptor molecule DAP12 which also has the ability to bind Syk following tyrosine phosphorylation. We therefore examined whether DAP12 similarly regulates TLR-mediated responses in mast cells. DAP12 was confirmed to be expressed in both human and mouse mast cells (BMMCs) and, following its phosphorylation by pervanadate treatment, DAP12 recruited Syk. Although the TLR agonists LPS, Pam3Cys, and MALP2 induced cytokine production and synergistically enhanced this response mediated by FcϵRI, they failed to increase basal DAP12 phosphorylation. Furthermore, no up-regulation or down-regulation of TLR-mediated responses, nor FcϵRI-mediated responses, was observed in DAP12-/- BMMCs. In contrast, DAP12 phosphorylation and Syk recruitment were observed in mast cells following concanavalin A (Con A)-induced aggregation of mannose-glycosylated receptors, and these responses, together with ConA-induced degranulation, were substantially reduced in the DAP12-/- BMMCs. These data show that in mast cells DAP12 does not regulate TLR- or FcϵRI-mediated responses, but is required for responses elicited by mannose-glycosylated receptors.
Stat5 is Activated by FceRI via Fyn Kinase (86.7)
Authors: Ryan John , Barnstein Brian
Published: January 2023

We have recently found that the transcription factor Stat5 is rapidly activated by IgE crosslinkage, and that its expression is critical to IgE-mediated degranulation, LTB4 production, cytokine secretion, and survival signals. Stat5KO mast cells induced cytokine mRNAs normally following IgE crosslinkage, but these mRNAs were not sustained over time, and were degraded at twice the rate observed in wild type cells. We find that Stat5 is associated with the IgE receptor after antigen-mediated activation. Moreover, cholesterol depletion or Fyn kinase deletion prevents Stat5 tyrosine phosphorylation in response to IgE crosslinkage. In contrast, the Lyn, Syk, MAPK, and PI-3 kinase pathways do not appear to regulate Stat5 tyrosine phosphorylation via the IgE receptor. We also find that Stat5 is serine phosphorylated during IgE signaling. These data suggest that Stat5 is activated through a lipid raft- and Fyn-dependent pathway. Our work demonstrates that Stat5 is a critical factor in IgE-induced mast cell activation that could provide a new clinical target for mast cell-associated inflammatory diseases.
Aryl Hydrocarbon Receptor independent production of IL-17A in a murine model of Hypersensitivity Pneumonitis (87.12)
Authors: Mohinta Sonia , Perdew Gary , August Avery
Published: January 2023

AhR (Aryl hydrocarbon Receptor) is a cytosolic transcription factor functioning as a sensor for environmental toxins. Recently, AhR has been suggested to have a cardinal role in Th17 biology. Although AhR is not a direct regulatory factor for Th17 differentiation its activation is required for the functional differentiation of Th17 cells, and production IL-22. To study the role of AhR in T cell differentiation we have used selective ligands of AhR in an in vitro assay and found that AhR agonist beta-naphthoflavone enhances IL-17 production whereas an AhR inhibitor suppresses it, both at the level of transcription and translation. The Th2 cytokine IL-4 was suppressed by beta-naphthoflavone and induced by the inhibitor and the Th1 cytokine IFNγ was inhibited by both the compounds. To further investigate the role of AhR in Th17 biology we used a well established Th17 dominated murine model of hypersensitivity pneumonitis mediated by a thermophilic actinomycete Staphylopolyspora rectivirgula (SR). Analysis of the cytokine transcripts from the SR exposed lungs revealed that IL-17 was not only expressed in WT mice but was more highly expressed in Ahr-null mice. These data suggest that the AhR may regulate cytokine production by other T cell subsets, and that the requirement for the AhR in IL-17 expression may be restricted to T cells. Further work is required to determine the role of the AhR in the innate immune cells, which are potential sources of IL-17.
Meningeal Immunity, Learning and Memory, and IL-4: Do T cells Make You Smart? (87.35)
Authors: Derecki Noel , Cardani Amber , Quinnies Kayla , Crihfield Anastasia , Lynch Kevin , Kipnis Jonathan
Published: January 2023

T cells were recently shown to support learning and memory, although the underlying mechanisms for this phenomenon are yet unknown. Here, we show that severe combined immune deficient (SCID) mice, shown to be impaired in assays of learning and memory, display a pronounced pro-inflammatory (M1) skew of meningeal myeloid cells. Acute lymphopenia induced by FTY720, a sphingosine-phosphate receptor agonist, or blockade of T cell migration into the meninges using anti-VLA4 antibodies, results in similar cognitive impairment and M1-skewed meningeal myeloid phenotype in wild type mice. The role of IL-4 is critical: we show accumulation of IL-4+ CD4+ T cells in the meninges of wild type mice after MWM training. Moreover, IL-4 knockout mice exhibit profound cognitive impairment and M1 skew. Transfer of IL-4 knockout bone marrow to wild type mice results in meningeal myeloid inflammation and impaired learning, while transfer of wild type T lymphocytes to IL-4 knockout mice is sufficient to rescue both behavioral phenotype and M1 skew. Our results point to a critical role for meningeal T cells, and for IL-4 in particular, in the regulation of meningeal myeloid cells and concomitant cognitive function. These results might lead to development of new immune-based therapies for cognitive conditions associated with immune decline, such as HIV- and age-associated dementia.
Computational modeling of Hsp90 inhibition illustrates a potential therapy to inhibit immune-mediated inflammation in systemic lupus erythematosus. (87.8)
Authors: Shimp Samuel , Reilly Christopher , Rylander Marissa
Published: January 2023

Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disorder that can affect nearly every organ in the body. Mesangial cells from lupus mice are hyper-responsive to immune stimulation and overproduce inflammatory mediators including IL-6 and nitric oxide (NO). Heat Shock Protein 90 (Hsp90) is known to chaperone numerous signaling client proteins including phosphatidylinositol 3-kinase (PI3K) and Akt, which belong to the immune activated PI3K, Akt, and mTOR (PI3K/Akt/mTOR) inflammatory pathway. Geldanamycin (Geld) inhibits Hsp90 function by binding to the carboxyl (C-) terminal domain resulting in deactivation, destabilization and degradation of Hsp90 inflammatory signaling clients, such as Akt, for decreased inflammation. Our long-term goal is to elucidate and mathematically model the cellular mechanisms of the Hsp90 modulated PI3K/Akt/mTOR pathway in SLE. Lupus mouse mesangial cells were cultured and treated with various concentrations of Geld followed by immune stimulation (LPS/IFN-γ) for 12, 18, and 24 hours. Cell cytokine analysis shows a sharp transition from stimulated levels of NO and IL-6 to significantly reduced NO and IL-6 concentrations at the Geld treatment range of 0.01 µM to 0.1 µM. Based on the measured data an empirical model for predicting the extracellular cytokine concentration following treatment with Geld was formulated. The empirical model suggests that N-terminus inhibition of Hsp90 may be a potential therapy for SLE.
IDENTIFICATION OF CIS-REGULATORY ELEMENTS TARGETING AID-MEDIATED SEQUENCE DIVERSIFICATION TO THE CHICKEN IMMUNOGLOBULIN LIGHT CHAIN GENE (88.2)
Authors: Kothapalli Naga Rama , Norton Darrell , Fugmann Sebastian
Published: January 2023

Somatic hypermutation (SHM) and gene conversion (GCV) are two closely related processes that increase the diversity of the primary immunoglobulin (Ig) repertoire. Both processes are initiated by the activation-induced cytidine deaminase (AID) that converts cytosine residues to uracils in a transcription-dependent manner and processed by direct replication and error-prone DNA repair. It is unknown how this mutagenic activity is targeted almost exclusively to Ig loci. To identify cis-acting elements involved in targeting SHM and GCV to the chicken Ig light chain (IgL), we used DT40 cells and systematically deleted non-coding sequences from the IgL locus using standard gene targeting strategies. We identified a novel as of yet uncharacterized regulatory region (named 3′RR) in the chicken IgL gene, containing a classical transcriptional enhancer and also cis-acting DNA elements essential for targeting AID-mediated sequence diversification. We are continuing our systematic deletion strategy to identify the minimal sequence required for targeting and to determine the trans-acting factors that bind to it. To identify similar targeting elements in the murine Igκ locus, we use a cross-complementation approach replacing the 3′RR in DT40 cells with murine Igκ sequences. Previous mouse transgenic studies indicated that both iEκ and 3'Eκ were necessary for the SHM of Igκ transgenes, and we are currently testing the ability of these enhancers to restore SHM/GCV in the chicken locus.
Th1 bias and gender differences in the ex vivo functional capacity of human NKT cells. (89.17)
Authors: Snyder-Cappione Jennifer , Tincati Camilla , Eccles-James Ijeoma , Cappione Amedeo , Koth Laura , Nixon Douglas
Published: January 2023

Human NKT cells are a unique T cell subset capable of producing Th1, Th2, or Th17 cytokines. It is hypothesized that the composite cytokine profile of the NKT cell compartment may determine disease pathogenesis. In order to define the functional signature of individual human NKT cells ex vivo with greater resolution, we FACS-Aria sorted NKT cells from fresh PBMC of 12 healthy donors and assessed their functional profiles, on both a population and single cell level, using Luminex and Elispot platforms, respectively. The purified NKT cell populations produced seven of the 14 analytes measured by Elispot, including IL2, IL4, IFNγ, TNFα, MIP1α, MIP1β, and perforin. Surprisingly, IL5, IL6, IL10, and IL17 were not detected from any subjects, and only one subject’s NKT cells produced IL13. Comparison of Elispot numbers with the distribution of several circulating NKT subsets revealed a significant positive association between the percentage of CD4/CD8 Double Negative cells and the number of perforin-secreting cells. NKT functional profiles of age-matched subjects revealed significantly higher frequencies of Th1-producing cells in male donors. These results suggest that 1) the panoply of human NKT Th2 and Th17 responses may not be as pronounced as previously believed, and 2) NKT cells may contribute to the sex bias found in autoimmune and other diseases.
The role of fibroblasts in neonatal intestinal inflammation (89.46)
Authors: Zawahir Shamila , Okogbule-Wonodi Adora , Li Guanghui , Blanchard Thomas
Published: January 2023

Background: Fetal intestinal fibroblast cell lines (FIF) produce more IL-8 in response to LPS compared to non-neonatal intestinal fibroblast lines (NNIF). Necrotizing Enterocolitis (NEC) is the most common gastrointestinal emergency in neonates and occurs more frequently in premature infants than term infants. We propose that fetal fibroblasts are inherently more responsive to stress and contribute to increased inflammation. Method: Primary human FIF and NNIF cell lines were exposed to E. coli LPS for 24 hours and the amount of IL-8 in the supernatants was determined by quantitative ELISA. A membrane blotting cytokine detection assay was also used to establish the cytokine profiles in the supernatants. Supernatants were used to stimulate neutrophils and activation was measured by increased CD11b expression by flow cytometry. Results: LPS stimulation induced a cytokine profile in FIF distinct from NNIF that included MCP-1, IL-6 and GRO-alpha. The supernatants from LPS stimulated FIF also activated neutrophils significantly more than supernatants from LPS treated NNIF. Conclusions: Fetal fibroblasts are more sensitive to stimulation by bacteria endotoxin and produce cytokines that promote inflammation, including the activation of neutrophils. Lamina propria fibroblasts in the developing gut may drive inflammation and put premature infants at an increased risk for developing NEC.
Automated analysis of flow cytometry data for the identification of cellular markers of allergy therapeutic responses (91.2)
Authors: Qian Yu , Kong Megan , Casale Thomas , Campbell John , Sadat Eva , Thomson Elizabeth , Dunn Patrick , Xiang Diane , Dahlke Carl , Scheuermann Richard
Published: January 2023

Mechanistic studies of biological specimens obtained during the conduct of clinical trials are frequently used to identify markers of therapeutic responses and to understand the underlying physiological processes associated with therapeutic efficacy. The Immunology Database and Analysis Portal (ImmPort; www.immport.org) is a public database resource funded by NIAID to support the management and analysis of clinical and mechanistic data generated by their funded investigators. We demonstrate the use of the ImmPort clinical data management platform and novel data analysis methods in the analysis of the allergen immunotherapy experiments conducted by the Casale group of Immune Tolerance Network. In the Casale study, immune response assays were conducted on samples taken before, during and after a 12-week course of allergen-specific immunotherapy to investigate whether anti-IgE antibody (omalizumab) can enhance the safety and efficacy of rush immunotherapy. Flow cytometry data was re-analyzed with FLOCK, a novel automated flow cytometry analysis pipeline in ImmPort, to determine if the level of any blood cell population correlated with treatment conditions. The analysis has identified a unique cell population expressing CD14, CXCR3 and high levels of the Fc-epsilon receptor (CD23), which is significantly increased in patients undergoing rush immunotherapy. Such data-driven analysis using ImmPort can generate hypotheses for further experimental design and verification.
Immune response profiles after caterpillar exposure (91.3)
Authors: Smith-Norowitz Tamar , Norowitz Kevin , Silverberg Jonathan , Kohlhoff Stephan , Kalra Kaushal , Chice Seto , Nowakowski Maja , Durkin Helen , Bluth Martin
Published: January 2023

Rationale: The role of the immune response to caterpillar exposure is not well described. Methods: Distributions of blood T and B lymphocytes, monocytes (CD1d+), levels of serum Igs(IgM, IgG, IgA, IgE), and cytokines (IFN-γ, IL-4, TNF-α) were studied in an allergic asthmatic serum IgE+ (181 IU/mL) adult who was exposed to the caterpillar, Halysidota harrisii Walsh, 1864. Results: Numbers of CD4+ T cells, CD45 RA+ T cells, CD25 + cells, CD19+ B cells, and CD1d+ monocytes decreased (20-33%) one week post reaction, while CD8+CD60+ T cells and CD23+ cells decreased 48 hrs (33, 74%) post reaction. In contrast, numbers of CD16/56+ NK precursor cells increased (60%) 12 hr, then decreased (65%) 48 hr post reaction; other lymphocyte subsets were unaffected. Serum IgM, IgG and IgA were normal; however, serum IgE levels were elevated 2 hrs post reaction (15%), and returned to pre-exposure levels by 24-48 hrs. High levels of IFN-γ (187-319 pg/mL) and TNF-α (549-749 pg/mL) were detected in serum 24-36 hrs and 3.5-24 hrs post reaction, respectively. In contrast, levels of IL-4 were undetected (<1.0 pg/mL) in serum at all time points. Conclusions. Caterpillar exposure may result in a variety of clinical reactions including increased serum IgE levels, blood CD16/56+ NK precursor cells, and cytokines. Understanding immunomodulation in caterpillar exposure will provide better insight and mechanisms of caterpillar contact and tailor more effective therapeutic intervention.
Roles of TSLP in diverting airway tolerance to Th2 sensitization (91.6)
Authors: Zhou Baohua , Lei Liying , Zhang Yanlu
Published: January 2023

Induction of Foxp3+ regulatory T cells (iTreg) is an important mechanism to generate mucosal tolerance against harmless antigens. On the other hand, mucosal tolerance breakdown is an important step towards allergen sensitization that ultimately leads to allergy and asthma. Recent studies demonstrated that thymic stromal lymphopoietin (TSLP) is essential for the development of allergic inflammation. The precise mechanism by which TSLP induces allergic response remains inadequately understood. Here we report that intranasal treatment of Ovalbumin (OVA) + TSLP was sufficient to induce Th2 sensitization against OVA while inhalation of OVA induced airway tolerance. In fact, elevated airway TSLP was able to suppress the development of antigen-specific iTregs in a dose dependent manner. We further demonstrated that TSLP significantly inhibited the differentiation of Tregs in vitro. This inhibition was TSLP specific as naïve CD4 T cells isolated from TSLP receptor deficient mice showed normal iTreg differentiation in the presence of TSLP. Although TSLP could directly promote Th2 differentiation in vitro, IL-4 or Stat6 deficiency only partially rescued the inhibition of iTreg induction by TSLP. Taken together, our findings suggest that TSLP, in addition to promote Th2 differentiation, can directly suppress antigen-specific iTreg induction in response to inhaled antigens, and thus induces Th2 sensitization and allergic inflammation.