bluuebunny/crossref_metadata_2025 huggingface dataset:
Cre-mediated excision of exon 8 of the Smad4 gene specifically in T cells is not associated with enhanced CD4+ T cell activation and differentiation (63.19)
Authors: Lv Ming , Zhang Jiyan
Published: January 2023

Numerous studies have revealed the critical importance of TGF-beta signaling on T cells. As a central mediator of TGF-beta signaling, Smad4 (homolog 4 of mothers against decapentaplegic, Drosophila) has been demonstrated to play essential roles during vertebrate development, tumorigenesis and the maintenance of gastrointestinal tract homeostasis. Recently, it has been reported that Cre-mediated excision of exon 1 of the Smad4 gene specifically in T cells has little role in the in vivo production of IFN-gamma, IL-10, IL-17, IL-17F, and IL-22 after KLH immunization of 4-week-old mice. Here, we report that Cre-mediated excision of exon 8 of the Smad4 gene specifically in T cells showed no significant effect on T cell proliferation and T cell development. In the peripheral tissues, the number of Treg cells, CD4+ T cell activation and differentiation were similar between aged wild-type and Smad4-deficient mice. Thus, our data are consistent with the previous report and suggest that Smad4 is indispensable for the maintenance of CD4+ T cell homeostasis.
Targeting the PD-1/PD-L1 complex with drug-like small molecules to induce T-cell tolerance (63.26)
Authors: Fuhrman Christopher , Gubernick David , Brusko Todd , Ostrov David
Published: January 2023

The interactions between T-cell costimulatory molecules and cognate receptors expressed on antigen presenting cells result in important signaling events that control T cell homeostasis and tolerance. The costimulatory ligand Protein Cell Death Ligand 1 (PD-L1) binds T-cell expressed Protein Cell Death-1 (PD-1) and promotes tolerogenic signaling and regulates T-cell responsiveness. Blocking PD-1/PD-L1 interactions inhibits tolerogenic signaling thus accelerating the onset of autoimmunity in several in vivo model systems. In contrast, PD-1 agonists are expected to promote T-cell tolerance in autoimmune settings. We developed a novel strategy for the development of tolerogenic PD-1 agonists. A large library of drug-like small molecules were targeted to the PD-1/PD-L1 interface via virtual high throughput screening. We coupled the grid-based energy scoring function with the AMBER molecular mechanics scoring function of Dock 6.4 to prioritize compounds for screening. The highest-ranking compounds are under investigation to characterize their effects on the association and dissociation kinetics of the PD-1/PD-L1 complex. The discovery of small molecules that increase the duration of PD1/PD-L1 contact (dwell time) and promote T-cell tolerance, may offer an invaluable tool in exploring T-cell homeostasis and regulating autoimmunity in vivo.
The generation of a B7 conditional knockout mouse (63.27)
Authors: Watanabe Masashi , Bhatia Sumeena , Hodes Richard
Published: January 2023

B7.1/B7.2 molecules are ligands for CD28 and CTLA-4, major T cell co-stimulatory and co-inhibitory molecules, respectively. B7.1/B7.2 molecules are essential not only for eliciting normal immune responses through CD28 mediated T cell activation, but also for maintaining immune homeostasis via CTLA-4 mediated negative regulation of T cell function. B7/CD28 interaction is critical for development and maintenance of specific immune cell populations, including iNKT cells and Foxp3+ regulatory T cells. Although many cell types (i.e., TEC, DC, MΦ, B cell and T cell) express B7 molecules in vivo, the precise role of B7 on each cell type for immune cell development and immune response is not well defined. To address these questions, we here report the generation of a B7 conditional KO mouse. Since B7.1 and B7.2 have largely redundant roles in vivo, we generated B7.1flox BAC Tg mouse on B7.1/B7.2 DKO background (designated as B7flox). The pattern of B7.1 expression in the B7flox mouse is comparable to that of WT mouse regarding both expression levels and tissue distribution. Cre-dependent deletion of B7.1 expression appears to be efficient in the B7flox mouse, and studies are in progress to evaluate tissue specific roles of B7 in immune system development, homeostasis, and response by tissue-specific Cre-mediated deletion.
Preformed CD40L is stored in an intracellular, secretory compartment in Th1, Th2, Th17, and T follicular helper cells as well as CD4 single positive thymocytes and invariant NKT cells, but not in Treg cells (63.5)
Authors: Parker David , Buenafe Abigail , Thauland Timothy , Gardell Jennifer , Bivins-Smith Elizabeth , Jacoby David , Slifka Mark , Koguchi Yoshinobu
Published: January 2023

CD40L is essential for development of adaptive immune responses. It is generally thought that CD40L is made from new mRNA following antigen recognition like other cytokines. However, we and others have shown that some CD4+ effector and memory cells also store preformed CD40L (pCD40L) in an intracellular secretory compartment and mobilize it onto the plasma membrane immediately upon antigenic stimulation, suggesting that CD4+ T cells may use pCD40L to activate APC during brief encounters in vivo that are too short to allow de novo CD40L synthesis. In this study, to understand relevance of pCD40L in different immune responses, we investigated the presence of pCD40L among different effector CD4+ T cells and Treg cells. We show that pCD40L is present in Th1 and follicular helper T cells that develop during infection with lymphocytic choriomeningitis virus, Th2 cells in the airway of asthmatic mice, and Th17 cells from CNS of experimental autoimmune encephalitis (EAE) animals. pCD40L is absent in both natural and induced Treg cells, even in the presence of intense inflammation such as occurs in EAE. We also found pCD40L expression in CD4 single positive thymocytes and invariant NKT cells. Together, these results suggest that pCD40L may function in T cell development as well as an unexpectedly broad spectrum of innate and adaptive immune responses, but is turned off in Treg cells to avoid compromising their suppressive activity.
PPARγ promotes age-related thymic involution (64.12)
Authors: Youm Yun-Hee , Leff Todd , Dixit Vishwa
Published: January 2023

Aging of the thymus precedes aging of other organs in healthy adults. The decline in naïve T cell production from thymus is related with ectopic adipocyte development within the lympho-stromal thymic zones. The regulators responsible for age-related increase in thymic adiposity and reduced thymopoiesis are poorly understood. Our prior data suggests that age-related elevation of PPARγ expression in aP2 expressing thymic stromal cells in correlated with thymic adiposity. To directly investigate whether PPARγ activation induces thymic involution, we created transgenic mice with constitutive active PPARγ (CA-PPARγ) fusion protein driven by an aP2 promoter. The CA-PPARγ transgene was expressed in thymus and CA-PPARγ fusion protein mimicked the liganded PPARγ receptor. The CA-PPARγ mice had increased thymic adipogenesis, thymic involution, reduced thymopoeisis and restricted T cell receptor (TCR) repertoire diversity. In addition, the ligand activation of PPARγ by insulin sensitizing class of thiazolidinediones drug, rosiglitazone accelerated the PPARγ driven thymic adipogenic cascade and promoted thymic involution andreduced naïve T cells. Our data suggest that activation of PPARγ induces thymic adiposity and accelerates age-related thymic involution.
Activation of the non-classical NF-kB pathway in thymic epithelial cells drives mTEC development in the absence of TCR+ thymocytes (64.14)
Authors: Williams Joy , Jenkinson Rhiannon , Zhang Jingjing , Xie Ping , Bishop Gail , Hollaender Georg , Hodes Richard
Published: January 2023

Activation of the non-classical NF-kB signaling pathway is required for the formation of a normal medullary thymic epithelial cell (mTEC) compartment, critical for tolerizing developing thymocytes. Mice deficient for key signaling molecules in this pathway fail to support normal mTEC expansion and differentiation. Known activators of this signaling pathway include tumor necrosis receptor family members; of these, Rank, CD40 and LTbR are all expressed on mTEC and have been shown to be important at various stages of mTEC organization, expansion and differentiation. The ligands for these receptors: RankL, CD40L and LT are provided by TCR-expressing thymocytes; in the absence of TCR+ thymocytes medullary formation is substantially impaired. To test directly whether activation of the non-classical pathway would be sufficient to support mTEC development independently of receptor signaling we made use of a mouse model in which Traf3, a negative regulator of the non-classical NK-kB pathway, is conditionally deleted only in epithelial cells. In B cells where Traf3 has been deleted, constitutive activation of the non-classical NF-kB pathway has been observed in the absence of receptor signaling.We find that epithelial-cell specific Traf3 null mice develop a robust mTEC compartment even in the absence of TCR+ thymocytes, suggesting that engagement of the non-classical NF-kB pathway in mTEC progenitor cells is the critical signal provided by TCR+ thymocytes to promote mTEC formation.
Use of surface enhanced resonance Raman scattering for 96 well plate based immunodetection of biomolecules (65.24)
Authors: Park Jun , Brewster David , Utzat Christopher , Moskow John , Chernokalskaya Elena
Published: January 2023

We report here a novel detection method in which the intrinsic affinity of proteins toward silver is used to induce Surface Enhanced Resonance Raman scattering (SERRS) of Raman active R6G dye conjugated to detection antibodies. We found a simple method of passive adsorption of silver nanoparticles onto PVDF membrane produces readily useful substrate for inducing the enhancement of Raman signal which could then be used to discern the presence of specific target molecules in quantitative manner. The effect is specific for PVDF membrane, other synthetic membranes such as nitrocellulose fail to produce the same effect. The method can be used for an accurate quantitative measurement of proteins labelled with Raman-active dyes. For example, it can be useful for quantification of detection antibodies eluted off after immunodetection of a target protein and subsequently allowed to adsorb onto silver substrate which can induce enhancement of Raman signal. Using mouse IgG molecules as a model antigen, adsorbed mIgG molecules were detected with limit of detection (3x S/N ratio over blank) down to 0.4ng (3 femtomoles) per well with greater than 1 log linear dynamic range. We also tested this method under sandwich immunodetection targeting c-peptide and show detection limit of 20 picogram (7 femtomoles).
Brighter is better: the introduction of Brilliant Violet™ conjugated antibodies for flow cytometry (65.26)
Authors: Nguyen Dzung , Koh Carolyn , Lundsten Kelly , Jiang Nan , Wormsley Susan , Lay Gene
Published: January 2023

There is always a need for improved and more sensitive fluorophores in research applications. Brilliant Violet 421™ and Brilliant Violet 570™ are novel polymeric, highly sensitive fluorophores excited by the violet laser, for use in flow cytometry. These are ideal for producing antibody conjugates that provide significantly improved signal-to-noise ratios, as much as 12-fold, compared to comparable fluorochromes excited by the violet laser, Pacific Blue™ and Pacific Orange™. These antibodies were fully compatible for use in multi-color flow cytometry applications, maintaining expected phenotypic frequencies, and were also useful for intracellular staining of cytokines. Furthermore, they work well with common commercial cell staining, fixation, and permeabilization solutions, proving their versatility and ease of use. Thus, antibodies conjugated to these novel Brilliant Violet™ fluorophores could prove to be an enabling technology allowing researchers to resolve lowly expressed antigens with greater resolution and accuracy, providing new and expanded capabilities in flow cytometry using the violet laser.
A lamprey monoclonal VLR antibody against human CD5 (65.34)
Authors: Yu Cuiling , Ehrhardt Götz , Cooper Max
Published: January 2023

The recently identified VLRB antibodies of the agnathan sea lamprey use highly variable leucine-rich repeats (LRRs) that assume a solenoid shape to bind antigens. Solved structures of monoclonal VLRB antibodies complexed to the H-trisaccharide or HEL show that antigens bind to variable residues in the beta-sheets located on the concave surface of specific VLRB antibodies. In this study we screened clones of a lamprey VLR cDNA expression library prepared from animals immunized with human CD4+ T cells to identify a monoclonal VLRB antibody (T32) that bound to human T lymphocytes, but not mouse T cells. Modulation of the expression levels of the TCR/CD3 complex by mouse antibodies did not affect VLR T32 binding. Staining of T cells, tonsilar B cells and malignant B-CLL cells with VLRB T32 and a mouse anti-CD5 mAb (clone UCHT2) demonstrated a diagonal co-staining pattern for each population of the test cells. Transfection of the CD5-negative OCI-Ly3 cell line with a human CD5 cDNA construct resulted in VLR T32 binding of transfected but not of untransfected cells. Staining of CD5+ cells by VLR T32 was not blocked by pre-incubation with the mouse UCHT2 anti-CD5 mAb, which suggests that they recognizes different CD5 epitopes. We conclude that lamprey monoclonal VLR antibodies offer useful alternatives to conventional antibodies for biomedical purposes.
Agarose slide elisa for diagnosis of HCV-AB in rural areas (65.5)
Authors: Abouelhag Hussien , Safhi Mohammed
Published: January 2023

Hepatitis C (HCV) is a viral infection of the liver affecting 170 million people around the world. Commercially available tests for HCV today are based on enzyme immunosorbent assay (EIA) for detection of HCV-Ab. Regarding the sophisticated equipments required for diagnosis of HCV Thus this work targets offering a dependable field serological test can be used in urban areas as preliminary screening for HCV patients. 150 serum samples were collected from Jazan clinics in Saudi Arabia for HCV patients and 50 serum samples were collected from healthy volunteers. All samples were tested for HCV-Ab using slides coated with 1.5% agarose saturated with HCV Capside synthetic peptide (BIORAD). 50 µl of each sample were applied in spot of Ag saturate agarose and incubated in inverted position in humidified incubator for 90 min at 37\#959;C. After which the slides were washed gently by Tris NaCl buffer PH 7.4 for 20 min. mouse anti human IgG labeled conjugate was added to the slides and incubated in inverted position in humidified incubator for 30 min at 37C. After washing the slides same as before a substrate (BIORAD) was added for 30 min at room temperature then washed out as before. the slides were examined by necked eye and by light microscope against controls. The obtained results were compared with slandered MONOLISA HCV Ag-Ab kite and both results were match. These results proving that agarose slide EIA can be used for preliminary diagnosis of HCV in field clinics of rural areas.
Near-infrared in vivo imaging of human lymphatic architecture and function in lymphedema and normal subjects (65.7)
Authors: Aldrich Melissa , Adams Kristen , Angermiller Bonnie , Darne Chinmay , Fife Caroline , Guilliod Renie , Hawkins Trina , Marshall Milton , Maus Erik , Rasmussen John , Smith Latisha , Tan I-Chih , Wang XueJuan , Sevick-Muraca Eva
Published: January 2023

Lymphedema (LE) is a disabling condition resulting from genetic predisposition, cancer treatments involving lymph nodes, or nematode infection. Obtaining in vivo images of healthy and diseased lymphatic vessels and lymph nodes, until now, has required using radionucleotide imaging agents, and has resulted in grainy, nondistinct images. We used a custom-designed ICCD camera system and low-level laser excitation of intradermally injected fluorescent dye to obtain live, non-invasive images of lymphatic vessel architecture and function in humans with and without lymphedema. This technique has provided exquisite images and movies of lymphatic abnormalities such as tortuous vessels and patchy “starry night” areas, as well as the ability to measure lymph propulsive velocity and frequency. This non-invasive, live, in vivo imaging of the human lymphatic system in health and disease may provide opportunities to diagnose lymphatic disorders, direct cancer resections, and evaluate therapies.
Dendritic cells support long term survival of plasma cells via CD28-CD80/CD86 interactions (66.11)
Authors: Nair Jayakumar , Carlson Louise , Rozanski Cheryl , Koorella Chandana , Murray Megan , Shaughnessy John Jr , Bergsagel P Leif , Boise Lawrence , Chanan-Khan Asher , Lee Kelvin
Published: January 2023

We have previously shown (AAI2009) that dendritic cells (DC) contribute to the survival of long lived plasma cells in the bone marrow, which are important to maintain persistent antibody titres. What is not clear are the molecular interactions that are involved. Both normal and malignant plasma cells(like myeloma cells) express the T-cell co-stimulatory molecule CD28, and both interact with DCs in the bone marrow microenvironment. CD28 activation on MM triggers similar molecular partners downstream as observed in T-cells. Gene expression analysis of normal and malignant plasma cells shows that CD28 expression correlates with poor prognostic subgroups that are associated with specific genetic lesions. In our data, while activation of CD28 with antibodies or DC-CD80/CD86 (B7) protects myeloma cells against cell death, the ligation of B7 on DCs induces DC production of plasma cell survival factor IL-6, and immunosuppressive factor indoleamine 2,3-dioxygenase (IDO). Use of Lenalidomide (a common myeloma drug) to inhibit DC-B7 expression or anti-CD28(Fab)2 fragments to block CD28-B7 interactions reduced DC ability to protect MM cells. Experiments with αCD28(Fab)2 or inhibitors of notch pathway suggest a cross talk that regulates IL-6 production. IDO activity in DC-MM cocultures, suppressed T-cell proliferation in MLRs while an IDO inhibitor alleviated this. Our data suggests a role for CD28-B7 in plasma cell survival by modulating the bone marrow microenvironment.
A small molecule TGF-b signaling inhibitor synergizes with agonistic OX40 antibody to elicit a potent anti-tumor and anti-metastatic response in a breast cancer model (66.22)
Authors: Garrison Kendra , Zhang Xiamei , Lee Wen-Cherng , Ling Leona , Akporiaye Emmanuel
Published: January 2023

SM16 is an orally available small molecule TGF-b signaling inhibitor that binds to the ATP pocket of TGF-b receptor Type 1 and prevents phosphorylation by TGF-BRII following engagement of the ligand. OX40 (CD134) is a member of the TNF-a receptor superfamily and is upregulated on activated CD4 and CD8 T cells. OX40 ligation by its cognate ligand (OX40L) or agonistic antibody (anti-OX40) enhances effector function, expansion and survival of activated T cells. In this study, we examined the therapeutic efficacy and anti-tumor immune response induced by the combination of SM16 plus anti-OX40 in a poorly immunogenic and highly metastatic 4T1 mammary tumor model. Our data show that SM16 synergizes with OX40 to elicit a potent anti-tumor effect against established primary tumors, with a 73% reduction in tumor size and a 92% reduction in the number of metastatic lung nodules; overall 71% of treated mice were either tumor free or showed stable disease by the end of the study. This positive treatment outcome is associated with a two-fold expansion of tumor-infiltrating effector CD8+ T cells and an accumulation of CD4+ and CD8+ cells overall, as well as a high tumor-specific IFN-g response. Depletion of both CD4+ and CD8+ T cells completely abrogates the effect of this therapy, indicating it is T cell dependent. Taken together, these data suggest that combining a TGF-b signaling inhibitor with anti-OX40 is a viable approach for treating metastatic breast cancer.
Myeloid derived suppressor cells induced thymic iNKT17 cells that promote tumor metastasis (66.36)
Authors: Liu Yuelong , Cheng Ziqiang , Wang Guijun , Sun Dongmei , Liu Cunren , Zhuang Xiaoying , Zhang Huang-Ge
Published: January 2023

A new subset of NKT cells, an IL-17-producing iNKT cell (iNKT17), has been identified recently. The role of this new iNKT17 subset in a tumor bearing host is unknown. We report that the percentages of iNKT17 cells in the thymus are increased dramatically in naïve mice from 0.5 ± 0.1% to 2.1 ± 0.2% in 4T1 breast tumor-bearing mice. Tumor induced iNKT17 cells are marked as TCRβint CD1dTetramerpos CD4neg CD8neg CD44hi CD25hi CD5pos CD24low DX5neg CD54pos. The iNKT17 cells from 4T1 tumor bearing mice are unique in that upon α-Gelcer-CD1d tetramer stimulation, IL-17 but not IL-4, IFN-γ, IL-10, IL-13, IL-6, IL-12 is induced. The CD11b+Gr1+ myeloid-derived suppressor cells (MDSC) accumulate in dramatic fashion in the thymus in 4T1 tumor bearing mice. The induction of iNKT17 subset is MDSC dependent. Addition of antibodies against TGFβ or IL-6 leads to the inhibition of MDSC-mediated induction of iNKT17 cells, suggesting that TGFβ and IL-6 are required for the induction. The MDSC-mediated induction of the iNKT17 cell subset is correlated with reduction of SLAM but not Ly9, CD84, Ly108 and 2B4. Mice injected intravenously with anti-IL17 antibody leads to the attenuation of 4T1 tumor metastasis to lung. This data suggest that MDSC-mediated induction of iNKT17 cells may play a role in promoting tumor metastasis.
Antibody responses to dengue infection (67.9)
Authors: Dejnirattisai Wanwisa , Malasit Prida , Mongkolsapaya Juthathip , Screaton Gavin
Published: January 2023

Dengue virus infection is a major public health problem in tropical and subtropical countries. Around 50 million people are infected per year of which 500,000 cases develop into the severe form, dengue hemorrhagic fever (DHF). Dengue is a flavivirus circulating as 4 serotypes with an average of 30% amino acid difference between each other, and infection with one serotype dose not provide protective immunity to the others, so secondary infection is very common. So far, the mechanisms involved in the pathogenesis of DHF are not well understood, but the key epidemiological studies indicate that it usually occurs in subsequent heterotypic dengue infection. Antibody dependent enhancement (ADE) is one of the hypotheses where preexisting antibodies from primary infection may be unable to neutralise other serotype from secondary infection but may instead form dengue/antibody complexes which will be taken up by Fc-bearing cells such as monocytes and macrophages leading to increase viral production. In this study, we have generated a panel of human monoclonal antibodies against dengue virus from B cells isolated from dengue infected patients. Neutralizing, enhancing and cross-reactive activity of these antibodies have been characterized and shown different patterns of functioanl properties.
Plasmodium chabaudi infection alters B cell homeostasis and disrupts development of heterologous immunity (99.18)
Authors: Wilmore Joel , Maue Alexander , Rochford Rosemary
Published: January 2023

Several studies have shown that the antibody responses to several vaccines are reduced in children following an acute episode of Plasmodium malaria. At present, the factors underlying these reduced responses are not understood. Using the Plasmodium chabaudi mouse model of blood stage malaria, we show that there are dramatic alterations in B cell hematopoiesis in the spleen and bone marrow. Production of immature B cells in the bone marrow was significantly reduced compared to control mice at 12 and 17 days post-infection resulting in significant reductions in transitional, marginal zone and an overall decrease of splenic B cells. To determine if these changes in B cell hematopoiesis impact the development of a heterologous immune response, we immunized P. chabaudi-infected mice during the acute phase of parasitemia with the model vaccine antigen, NP-ovalbumin. At 12 days post immunization, antigen-specific antibody titers of infected mice were significantly lower compared to uninfected immunized mice. Similar numbers of NP-OVA binding B cells could be isolated from infected and control mice. However, P. chabaudi infected mice had significantly reduced levels of BrdU uptake in NP-OVA+ B cells indicating an impaired ability of NP-OVA specific B cells to proliferate. These data suggest that disruption of B cell homeostasis during acute P. chabaudi infection impacts the capacity of the immune system to respond to heterologous infection and immunization.
Variability in murine airway immune responses to clinical and environmental isolates of Aspergillus conidia (99.21)
Authors: Templeton Steven , Buskirk Amanda , Law Brandon , Green Brett , Beezhold Donald
Published: January 2023

Pulmonary exposure to conidia of Aspergillus species has been associated with morbidity and mortality. However, the potential to cause invasive disease varies between species. Results from a previous study in our laboratory suggest that murine airway immune responses are species-dependent, as airway recruitment of CD8+ T effector and memory-phenotype cells were increased in response to aspiration of viable A. fumigatus, but not A. versicolor, conidia. We sought to test the hypothesis that differences in airway responses are strain dependent, by testing additional clinical and environmental Aspergillus isolates in our model. Airway CD4+ or CD8+ T cells produced IFN-γ and/or IL-17A in response to both clinical and environmental strains of A. fumigatus. In A. versicolor aspirated mice, the T-cell response was characterized by lower numbers of both cytokine-producing cells and infiltrating CD8+ T cells. Both A. fumigatus and A. versicolor induced bronchiocentric inflammation and granuloma formation observed in histopathology. The ability of aspirated conidia to germinate in the lung tissue was variable. Airway eosinophil recruitment was increased in response to A. versicolor and a single environmental isolate of A. fumigatus in comparison with other A. fumigatus isolates. These data provide preliminary evidence that airway immune responses to Aspergillus conidia are both species- and strain-dependent.
CCR6+Tc17 cells induced in the absence of CD4 T-cell help upon vaccination with Blastomyces dermatitidis mediate protective immunity (99.8)
Authors: Nanjappa Som , Heninger Erika , Wuthrich Marcel , Klein Bruce
Published: January 2023

The increasing incidence of fungal infections in immune-compromised patients calls for the development of suitable vaccines to target CD8 T cells. Accumulating studies indicate that Th17 cells play an important role in anti-fungal immunity. However, whether IL-17 producing CD8 T cells (Tc17) can be induced and mediate protective immunity against fungal infection in the absence of CD4 help is not known. Here, using mouse model of vaccination against lethal Blastomyces dermatitidis infection, we show that upon vaccination Tc17 cells are induced and re-called at the site of lethal challenge in the absence of CD4 T-cell help. Vaccine immunity by CD8 T cells was severely impaired in both vaccinated- IL-17A KO and IL-17-receptor KO mice in the absence of CD4 T cells. Neutralization of IL-17A during the re-call response also impaired fungal clearance by effector CD8 T cells. Further, we show that protective immunity by Tc17 cells was not influenced by deficient IL-12 signaling, required for Type I immunity, but neutrophils were essential in Tc17 cell action. Tc17 cells preferentially expressed CCR6 over CXCR3 chemokine receptor (distinct from IFNγ producing cells, which were CXCR3+ve) and antibody blocking of CXCR3 did not affect the re-call response of Tc17 cells to the lung. Collectively, our studies show that protective Tc17 cells are induced in the absence of CD4 T cell help and may aid in designing fungal vaccines for immune-compromised hosts.
Type I IFN drives a distinctive dendritic cell maturation phenotype that allows continued MHC-II synthesis and antigen processing (106.21)
Authors: Wearsch Pamela , Simmons Daimon , Canaday David , Liu Yi , Wang Ying , Boom W. , Harding Clifford
Published: January 2023

Microbial molecules or cytokines can stimulate dendritic cell (DC) maturation, which involves DC migration to lymph nodes and enhanced presentation of Ag to launch T cell responses. Toll-like receptor (TLR) agonists are the most studied inducers of DC maturation and promote a burst of Ag processing with enhanced expression of peptide-MHC-II complexes and co-stimulatory molecules. Subsequently, MHC-II localization is shifted from vacuolar compartments to the surface and synthesis is inhibited, thus decreasing Ag processing and limiting presentation to a cohort of Ags kinetically associated with the maturation signal. Type I interferon (IFN-I) also promotes DC maturation, but the phenotypic changes induced by IFN-I are poorly defined. Our studies show that treatment of DC with IFN-I also increases MHC-II Ag presentation, but, in contrast to TLR-induced maturation, allows for continued Ag processing. IFN-I treated DC expressed high levels of MHC-II at the surface as well as in vacuolar compartments, consistent with sustained Ag processing function. Expression of mRNA for MHC-II and CIITA was inhibited in DC treated with TLR agonists, but maintained in DC matured with IFN-I. Similar to TLR agonists, however, IFN-I induced changes in MARCH1 expression and MHC-II ubiquitination. Our findings suggest that IFN-I drives a distinctive DC maturation program that enhances Ag presentation to T cells without a shutdown of Ag processing, allowing continued sampling of Ags for presentation.
Components of the cytoplasmic antigen processing pathway regulates indirect antigen presentation by MHC class II molecules (106.23)
Authors: Joyce Sebastian , Dragovic Srdjan , Hill Timothy , Christianson Gregory , Roopenian Derry , Van Kaer Luc
Published: January 2023

Indirect presentation is a process in which MHC class II-positive antigen-presenting cells (APCs) acquire and present antigens expressed by class II-negative allografts, tumours or infected cells. It is generally assumed that similar cellular biochemical mechanisms underlie direct and indirect antigen presentation. This is because in both cases antigens are delivered to the endo/lysosomes for proteolytic processing for assembly of derived peptides with class II molecules and their subsequent display on the cell surface of antigen presenting cells. Remarkably, upon testing this assumption, we found that indirect presentation involved LMP2-dependent immunoproteasomes, indicating that the antigen had accessed the recipient APC’s cytosol. In this cytosol, TAP and ERAAP, but not tapasin, partially destroyed the antigens slated for indirect presentation. Because autophagy was not involved in returning the donated antigen from the cytosol to the endo/lysosome, a novel mechanism(s) is predicted to underlie cytosol to endo/lysosome antigen transport. How components of the cytosolic antigen-processing pathway regulate indirect class II-restricted antigen presentation and the mechanism(s) by which cytosolic antigens are returned to the endo/lysosomes will be presented.
Classical antigen presentation by HLA-A2 of Mycobacterium tuberculosis derived ligands (106.40)
Authors: Liles Lauren , McMurtrey Curtis , Schafer Fredda , Bardet Wilfried , Swarbrick Gwendolyn , Null Megan , Toren Katelynne , Lewinsohn David , Lewinsohn Deborah , Hildebrand William
Published: January 2023

Mycobacterium tuberculosis (Mtb) remains a world health threat, with 8.8 million cases reported in 2010, 65% of which were new and recurrent cases of tuberculosis . Cellular immunity is critical for controlling Mtb and recent studies identify CD8+ T cells as important players in controlling infection. Cytotoxic T lymphocytes (CTLs) recognize peptide antigen presented by Major Histocompatibility Complex (MHC) Class I molecules and destroy infected cells, therefore discovery of CTL epitopes is of high importance. Using a method of soluble HLA production, we were able to generate soluble HLA-A2 from THP-1 monocyte cell lines transfected with a soluble form of HLA A*02:01. Transfected cells were seeded into a hollow fiber bioreactor and were infected with Mtb or left uninfected. Following production of 25 mgs of HLA-A2 from infected and uninfected cells, peptides were eluted from the supernatants of uninfected and infected cells. Peptides from each group were fractionated using HPLC and analyzed by mass spectrometry. We were able to identify six Mtb derived ligands presented by the MHC Class I molecule HLA-A*0201, two of which reacted well with donor PBMC from patients with both active and latent Mtb infection. Identifying new vaccine candidates is a top priority in the TB field and the reactive Mtb ligands identified here demonstrate that multiple Mtb proteins are processed for presentation by Class I HLA and a fraction of these ligands are immunologically reactive.
Immunization strategies for high affinity immune response (106.48)
Authors: Hawksworth David , Bax Brian , Himmelsbach Richard
Published: January 2023

In the field of hybridoma development immunization strategies involving varied mouse strains, adjuvants and immunogens have been widely reported with diverse results. We describe here studies completed to evaluate two arms of the immunization strategy - adjuvant selection and hapten-to-carrier ratio. First, Titanium Powder Adjuvant (TPA) and 2-Dose Adjuvant (2DA) were evaluated vs. our standard adjuvant, Adjulite Freund’s Adjuvant (AFA), for resulting antibody titer and affinity by immunizing with either insulin or hepatitis B surface antigen (HBsAg). Second, mice were immunized with the hapten neopterin linked to BSA using a 23:1 or 4:1 coupling ratio. CAF1/J and RBF/DnJ mice were employed for both studies. Adjuvant study results showed a higher titer and higher affinity response using AFA vs. TPA. While all mice immunized with HBsAg developed an immune response, those immunized using AFA showed a significantly higher average relative affinity than those immunized using TPA. Additionally, for CAF1/J mice immunized with insulin, only those using AFA developed an immune response. The 2DA mice yielded a relative affinity similar to the AFA mice. In the hapten-to-carrier ratio study mice immunized with hapten coupled using a 4:1 ratio yielded a significantly higher average affinity. We continue to evaluate all arms of the immune response strategy to ensure a high affinity immune response by testing new adjuvants and animal models.
B1 B cell-derived bone marrow plasmacytes are the primary source of natural IgM (109.11)
Authors: Reynolds Alexander , Kuraoka Masayuki , Kelsoe Garnett
Published: January 2023

Natural IgM is a unique and important component of humoral immunity. It provides early protection against microbial pathogens and helps to maintain tissue homeostasis. Though B1 cells are known to be the primary source of natural IgM, the developmental stage and anatomical location of the cells that secrete natural IgM antibody remain controversial. In order to answer this question, we first confirmed earlier observations that the spleen and BM are the primary tissues in which IgM secreting cells reside. We next examined two lines of VDJ knockin mice and found that both exhibited 10-fold reductions in serum IgM levels despite the presence of normal IgG concentrations. Whereas knockin animals had normal numbers of IgM secreting cells in the spleen and numbers of splenic and BM IgG plasma cells that were indistinguishable from normal controls, the numbers of IgM plasmacytes in the BM of knockin mice was ≤20% of normal animals. In addition, the vast majority of peritoneal B1 cells were missing in both knockin strains whereas all B2 populations remained unaffected. By sorting BM cells, we determined that only those with the CD138high IgM+ phenotype secreted IgM in vitro; analyses of the two knockin mouse strains revealed this BM population to be significantly reduced in concordance with the peripheral B1 B-cell populations. We conclude that natural IgM antibody is produced by differentiated BM plasmacytes that are derived from peripheral, B1 B-cell populations.
Diversification of Toxoplasma gondii-specific cytotoxic T lymphocytes by IL-12 (110.15)
Authors: Shah Suhagi , Grotenbreg Gijsbert , Yap George
Published: January 2023

The obligate intracellular parasite Toxoplasma gondii elicits a robust and uniquely IL-12 dependent CD8+ T cell immune response. We have previously shown that IL-12 is required for the differentiation of KLRG1+ CD8+ T effector cells following T. gondii infection. We have also shown that in the absence of IL-12, the frequency of KLRG1- memory precursor effector cell (MPECs) differentiation is increased at the expense of KLRG1+ short-lived effector cells (SLECs). Therefore, IL-12 may be a critical signal for the early phase bifurcation of naïve CD8+ T cells into KLRG1+ SLEC and KLRG1- MPEC-fated lymphocytes. To determine if SLECs and MPECs are generated in the earliest phase of CD8+ T cell activation, we enumerated T. gondii reactive CD8+ T cells by magnetic bead enrichment of Kb-tgd057 tetramer binding cells. We have found that the initial proliferation of tgd057-specific CD8+ T cells in the spleen occurs between day 3 and day 5 following T. gondii infection. Concurrently, KLRG1+ and KLRG1- CD8+ T cells can be observed in both the spleen and the effector site of the peritoneum, suggesting early diversification of tgd057-specific CD8+ T cells. Current efforts are geared towards visualizing the events leading to the initial bifurcation of the CD8+ T cell response and determining how IL-12 regulates this decision-making process.
Genomic analysis of CD8 T cells during the early phases of the immune response (110.25)
Authors: Blair David , Mayya Viveka , Madar Aviv , Best J. Adam , Bonneau Richard , Dustin Michael
Published: January 2023

The generation of long-lived memory CD8 T cells is critical for the development of effective vaccines and adoptive immunotherapy strategies. However, the molecular mechanisms by which a T cell transitions from naïve to memory remains poorly understood. In an effort to better understand the molecular pathways involved in the differentiation of CD8 T cells, we have analyzed ovalbumin-specific OTI CD8 T cells during the very early phases of the immune response at the genomic level. Following infection with Listeria monocytogenes expressing ovalbumin, OTI CD8 T cells from infected mice were sorted and subjected to microarray analysis. We show that while cell surface phenotype of OTI T cells remained largely unchanged shortly after activation, large scale changes occurred at the level of transcription. Expression levels of many families of genes involved in signaling networks in T cells, in addition to multiple immune response factors, were differentially expressed shortly after T cells were activated, but prior to the first division. Notably, expression of IL-21, a cytokine implicated in T cell memory, was transiently expressed with peak expression occurring at 12 hours post-stimulation. This was confirmed by intracellular staining for the IL-21 protein. Similarly, the peak of STAT3 expression coincided with IL-21, a known activator of STAT3. We are currently investigating autocrine IL-21 signaling in CD8 T cells and its role in memory development.
B cell Transcription Factors Define a Novel Tumor Suppressor Gene Network In Acute Lymphoblastic Leukemia (111.11)
Authors: Heltemes Harris Lynn , Kornblau Steven , Farrar Micheal
Published: January 2023

STAT5 is activated in a subset of patients with ALL and high levels of STAT5 activation prior to treatment predicted poor response to therapy. In mice, we identified synergy between constitutive activation of STAT5 (STAT5b-CA) and Ebf1 or Pax5 haploinsufficiency that rapidly induced progenitor B-ALL in 100% of the mice. The leukemic cells displayed 2-fold reduced expression of Ebf1 and Pax5 that affected a small subset of EBF1 or PAX5 target genes, including tumor suppressor genes and oncogenes. To test whether compound haploinsufficiency of Ebf1 and Pax5 is sufficient to cause transformation we generated Ebf1+/- x Pax5+/- mice. Over 80% of these mice developed progenitor B-ALL. Although these mice lack the Stat5b-CA transgene, the leukemic cells exhibit increased STAT5 activation. In preliminary studies we observed similar results in Ebf1+/- x Ikzf1+/- and Pax5+/- x Ikzf1+/- mice. Our findings suggest a model in which small perturbations in a self-reinforcing network of transcription factors critical for B cell development cooperate with STAT5 to initiate ALL and that EBF1, PAX5 and IKZF1 form an interrelated network of transcription such that loss of any two alleles in the network results in a loss of tumor suppressor function. This hypothesis represents a departure from the conventional idea that transformation is a result of deletion at both alleles of one tumor suppressor gene, and may explain why mutations in Pax5, Ebf1 and Ikzf1 are so common in human ALL.
Characterization of human thymocyte behavior in situ (111.46)
Authors: Melichar Heather , Halkias Joanna , Ross Jenny , Taylor Kayleigh , Yen Bonnie , Robey Ellen
Published: January 2023

Thymocyte development occurs through the coordinated movement of developmental intermediates to defined anatomical locations within the thymus during which thymocytes sample surrounding cells and the microenvironment for signals to promote differentiation. The development of an experimental model in which purified, labeled cell populations migrate into a 3-dimensional living thymic slice gives us the first opportunity to visualize and monitor the behavior of human thymic subsets in situ. Double negative (DN) progenitors and CD4+CD8+ double positive (DP) human thymic subsets primarily localize to the cortex, whereas CD4+ and CD8+ single positive (SP) subsets localize appropriately to the medulla. In many respects, the migration of human thymic subsets mirror that of their murine counterparts: immature DP and DN cells move randomly with slow motility rates, DP cells migrate slower than DN, and both SP populations are fast and exhibit some directional movement presumably toward the medulla. These similarities between human and mouse thymocyte motility occur despite significant differences in the development and microenvironment of the thymus between the species. We are also examining cross-talk between mouse and human factors as well as the sensitivity of human thymic subsets to chemokine signals. The suitability of the thymic slice model to study human thymocyte development is a powerful system to study and manipulate processes that are otherwise inaccessible to researchers.
Role of protein tyrosine kinase seven in human recent thymic emigrant cell survival (111.50)
Authors: Acharya Swati , Ramachandran Vasavi , Dionis Kira , Lewis David
Published: January 2023

Recent thymic emigrants (RTEs) are antigenically naïve T cells that have recently completed intrathymic maturation and emigrated from the thymus to the periphery. Our lab has shown that PTK7 is a novel marker for human RTEs of the MHC class II-restricted CD4+CD8- T-cell lineage and is highly expressed during human and murine intrathymic development. PTK7, a ‘pseudokinase’, is a member of the receptor tyrosine kinase family previously implicated in the planar cell polarity pathway of neuroepithelium. While viable CD4+ RTEs can be identified based on PTK7 surface staining to define the phenotype of RTEs in humans, the function of PTK7 in human T-cell development remains unclear. To decipher a functional role of PTK7 in RTEs, we performed lentiviral small hairpin sh RNA or small interfering si RNA-mediated gene silencing in T and non-T lineage cell lines, primary human adult and neonatal peripheral T cells and thymocytes. In all these cellular contexts RNAi-mediated knockdown of PTK7 consistently decreased cell survival alongside caspase 3 activation. Also, T cells with reduced PTK7 levels showed increased susceptibility to apoptosis inducing agents, such as camptothecin and staurosporin, with apoptosis dependent on caspase 3. Survival that was dependent on AKT phosphorylation downstream of PTK7 was also reduced by PTK7 RNAi knockdown. Together, these observations suggest an essential role for PTK7 in survival of RTEs, and most likely, in developing thymocytes.
TSLP expands the production of human B lineage cells in vitro and in a novel human TSLP expressing xenograft model (111.54)
Authors: Payne Kimberly , Su Rui-jun , Baez Ineavely , Francis Olivia , Weldon Abby , Dovat Sinisa , Milford Terry-Ann
Published: January 2023

IL7 and TSLP are important in murine B cell development, however their role in human B lymphopoiesis is unclear. In contrast to other cytokines that act on B cell progenitors, TSLP lacks species cross reactivity and activates different intracellular signaling pathways in mice and humans. Thus, human models are essential for understanding the role of TSLP in human B lymphopoiesis. We developed a novel human-only model of in vitro B cell development that allows selective cytokine stimulation and used this model to show that IL-7 is essential for human B cell production where it increases B cell progenitors by over 60 fold (JI 2009 82:4255). Here we use this model and a novel xenograft model system to study human B lymphopoiesis from CD34+ HSCs in umbilical cord blood (CB). BrdU incorporation shows that TSLP induces proliferation of human B lineage cells to produce in vitro expansion of B cell progenitors similar to that observed for IL7. TSLP acts directly on human B cell precursors to induce STAT5 phosphorylation and upregulate PAX5. To evaluate the role of TSLP in vivo we developed a novel xenograft model system consisting of human TSLP+ (hTSLP+) and hTSLP- immune deficient NSG mice. Using the hTSLP+/- model system we show that hTSLP augments existing IL7 effects to expand human B lineage cells (from 37% to 68% total B lineage chimerism) in NSG mice, a strain that facilitates human B cell production. These data suggest that TSLP is important in human B lymphopoiesis.
Differential thymocyte motility and microenvironmental localization during normal and neoplastic T cell development (111.55)
Authors: Ehrlich Lauren , Cardenas Kim , Jones Jessica , Selden Hilary
Published: January 2023

As thymocytes develop, they modulate expression of receptors, such as chemokine receptors, that promote migration into distinct thymic microenvironments. Within these thymic regions, developing thymocytes have the opportunity to interact with unique stromal cells that provide survival, selection, and/or differentiation signals. Thymocyte: stromal interactions are essential for proper thymocyte development, implicating the molecules involved in regulating intrathymic migration as key regulators of T cell development. Previously, we have used 2-photon microscopy to study real-time migration of thymocytes within live thymic slices. These studies revealed that immature thymocyte progenitors were restricted to the cortex while only post-positive selection thymocytes were able to enter the medulla. The ability to migrate within cortex versus medulla was regulated by multiple mechanisms, including G-protein coupled receptor mediated chemotaxis and substrate accessibility. We hypothesize that the molecular cues that mediate normal intrathymic migration may be disrupted during thymic lymphomagenesis, contributing to the initiation and/or progression of disease. Consistent with this hypothesis, we find that the thymic microenvironment is disorganized in a murine model of thymic lymphoma. Furthermore, using 2-photon microscopy, we find that migration of lymphoma cells within thymic tissue is abnormal, suggesting that aberrant thymocyte: stromal interactions may contribute to disease.