bluuebunny/crossref_metadata_2025 huggingface dataset:
An IL21 reporter mouse reveals a novel population of TFH precursors in young mice. (LYM8P.630)
Authors: Adkins Elisabeth , Wang Xulong , Sproule Thomas , Christianson Gregory , Park Giljun , Lane-Reticker Sarah Kate , Carter Gregory , Morse Herbert , Roopenian Derry
Published: January 2023

T follicular helper cells (TFH) are a specific subset of activated CD4+ T cells that localize to germinal centers (GC) and secrete Interleukin 21 (IL21). IL21 is vital for driving the proliferation of antigen-stimulated B cells within GC and their differentiation into memory B cells and antibody-secreting plasma cells. TFH and IL21 are crucial for normal humoral immunity, but their dysregulation has been implicated in immunodeficiencies and autoimmune diseases. However, the processes that result in the normal development of TFH and their expression of IL21 are still poorly understood. Here we use a novel IL21-venus fluorescent protein (VFP) reporter mouse to investigate the signals and pathways involved in the early differentiation of TFH and expression of IL21. Using this approach, we have identified a naturally occurring population of IL21-expressing CD44hi CD4+ T cells that are detected in the thymus and periphery of naïve C57BL/6J mice by 2 wks of age. RNAseq profiling and adoptive transfer experiments showed that these IL21-expressing cells are the earliest identified precursor (termed nascent TFH, nTFH) to the fully mature TFH state. nTFH display a highly diverse TCR repertoire, but restricting their TCR to a high affinity foreign antigen does not prevent their development. From this, we propose that nTFH arise by tonic self-antigenic stimulation and are primed for efficient differentiation to mature TFH in response to foreign or autoimmune antigen challenges.
Cryptococcus gattii infection dampens pulmonary inflammation by attenuating chemokine expression in the immunocompetent hosts (MPF6P.644)
Authors: Angkasekwinai Pornpimon , Sringkarin Nuntarat , Supasorn Oratai , Fungkrajai Madtika , Wang Yui-Hsi , Chayakulkeeree Methee , Ngamskulrungroj Popchai , Angkasekwinai Nasikarn , Pattanapanyasat Kovit
Published: January 2023

Cryptococcal infections are caused mainly by two fungal species; Cryptococcus neoformans and C. gattii. While C. neoformans primarily affects the immunocompromised hosts, C. gattii causes disease in immunocompetent individuals. Recent studies suggest that C. gattii is capable of infecting immunocompetent hosts because of its ability in dampening inflammatory cell recruitments. We hypothesizes that C. gattii dampens pulmonary inflammation by attenuating chemokine expression. Mice were infected with the highly virulent strains C. neoformans and C. gattii and then analyzed for kinetics of inflammatory cell infiltration and chemokine expression. Inflammatory cells and chemokine transcripts were found to be up-regulated and reached the peak levels at day 7 post-infection. Mice infected with C. gattii recruited much fewer myeloid dendritic cells, neutrophils, and CD4+ T helper cells into the lungs than those in C. neoformans-infected mice. Interestingly, the expression levels of Cxcl1, Cxcl2, Mcp1, Cxcl9, and Ip10 transcripts known to be important for recruiting neutrophil and Th1 cells were significantly higher in C. neoformans-infected mice than in C. gattii-infected mice, whereas the expression levels of Ccl20, Ccl11 and Ccl17 were comparable. Our data suggest that C. gattii infection dampens pulmonary inflammation by down-regulating the pulmonary chemokine expression, thus resulting in the inability to mount protective immunity in immunocompetent hosts.
Immunological and biochemical characterization of an immunogenic antigen (5G8) of Giardia lamblia (MPF6P.648)
Authors: Velazquez Carlos , Quintero Jael , Valdez Alejandra , Samaniego Brenda , Rascón Lucila , Calderon Boris , Rascón Raúl , Domínguez Víctor , Garibay Adriana , Astiazarán Humberto
Published: January 2023

The protozoan parasite G. lamblia is an intestinal parasite of humans, which the World Health Organization estimates to have infected 250 million people worldwide. Clinical manifestations of G. lamblia infections vary from the asymptomatic carrier state to severe diarrhea, abdominal pain, nausea, malabsorption and weight loss. Our knowledge about the immunogenic antigens of G. lamblia is limited. The purpose of this study was immunologically and biochemically characterize an immunogenic antigen (protein 5G8) of G. lamblia. We generated monoclonal antibodies (moAbs) spe­cific to the protein 5g8 of the parasite by using splenocytes derived from in­fected mice. All of these moAbs recognized a protein of 71 kDa by Western blotting. This protein was isolated from a G. lamblia trophozoite lysate by affinity chromatography. The purified 71 kDa protein was analyzed by mass spectrometry. This immunogenic antigen was expressed inside of the G.lamblia trofozoites (>90% of total cell population) and only a small percentage (≈ 2-5%) of those trophozoites expressed high levels of the 5G8 protein on the cell surface. Trophozoite population expressing the 5G8 protein on the cell surface induced stronger antibody response that trophozoite population that does not express this protein on cell surface. The characterization of this antigen will provide a better understanding of the G. lamblia-host interaction, and will be important for developing effective control strategies against giardiasis.
CCR7+ dendritic cells are sufficient to recover a defective population of KLRG1+ T cells in the brain of chronically infected CCR7-/- mice (MPF7P.716)
Authors: Landrith Tyler , Noor Shahani , Wilson Emma
Published: January 2023

Toxoplasma gondii is a protozoan parasite that establishes a chronic infection in the brain. CCR7-/- mice are susceptible to challenge with normally avirulent strains of T. gondii, but if the mice are treated with sulfadiazine, a chronic infection is established. At this stage, equivalent numbers and proportions of parasite-specific T cells are seen in the brain of CCR7-/- and wild type mice suggesting normal priming is possible in the absence of CCR7. However, in contrast to their wild type counterparts, CCR7-/- mice fail to mount a protective response to rechallenge with a virulent strain, implying deficient effector mechanisms despite equivalent priming. Indeed, a profound defect is observed in the number and proportion of T cells expressing the effector marker KLRG1 in the brain of CCR7-/- mice. Here we present data showing that this defective effector population is restored upon transfer of CCR7+ dendritic cells at the chronic stage. We also present data on the role of transferred dendritic cells in survival upon rechallenge with the virulent RH strain. This data suggests a novel role for CCR7 during chronic T. gondii infection in guiding efficient dendritic cells interactions required to maintain effector populations of T cells in the brain.
Eosinophils support Th2 response and control parasite number in brain during murine neurocysticercosis (MPF7P.717)
Authors: Mishra Pramod , Li Qun , Munoz Luis , Mares Chris , Morris Elizabeth , Teale Judy , Cardona Astrid
Published: January 2023

Neurocysticercosis (NCC) is a common parasitic infection of the brain and leading cause of acquired epilepsy worldwide. NCC is caused by metacestode larvae of the tapeworm Taenia solium and characterized by an initial asymptomatic phase followed by a symptomatic phase. While the asymptomatic phase is attributed to the immunosuppressive capabilities of viable T. solium metacestodes, dying parasites induce inflammation including an eosinophil (EO) response causing pathology and symptoms. Previous studies in T. solium-infected pigs have shown that EOs are present around dying parasites. However, the role of EOs in killing of metacestode has not been verified. To determine the parasiticidal role of EOs in brain, we used a murine model of NCC in which wild type (WT) or EO deficient (ΔdblGATA) mice were infected intracranially. We found a strong EO response in WT NCC mice similar to porcine NCC. In comparison to WT NCC mice, EO deficient, ΔdblGATA NCC mice had an increased parasite burden. Analysis of infiltrates from infected brains revealed that ΔdblGATA mice had reduced leukocyte infiltration into the brain in comparison to WT mice. However, αβ T cell frequency was greater in ΔdblGATA mice brain. Further, higher αβ T cell frequency correlated with increased CD8+ T cell response and reduced CD4+ Th2 responses. Thus, our results show that EOs support Th2 response in brain microenvironment and play a critical role in controlling the parasite number in brain during murine NCC.
Inflammatory monocytes orchestrate innate antifungal immunity in the lung (MPF7P.701)
Authors: Espinosa Vanessa , Rivera Amariliz
Published: January 2023

Aspergillus fumigatus is a mold fungal pathogen and the most common cause of invasive aspergillosis (IA), a serious infection that develops in patients with compromised immune function. Although a variety of immune cells can help confer protection against IA, the mechanisms that govern immune cell cooperation culminating in the eradication of conidia and IA prevention are unclear. Using selective cell depletion strategies our recent studies have shown that, in addition to neutrophils, CCR2+ inflammatory monocytes (CCR2+Mo) and their derivative monocyte-derived dendritic cells (Mo-DCs) are required for the prevention of IA. In preliminary studies, we find that IA development in neutrophil-depleted animals is associated with diminished Mo-DC differentiation and lower conidiacidal activity in these cells. We hypothesize that neutrophil and CCR2+Mo functions are interdependent, continuously cross-regulating each other’s antifungal activities. We are currently investigating the mechanisms of CCR2+Mo and neutrophil cross-regulation, and characterizing their overall contributions to anti-fungal immunity.
MicroRNA-146a constrains intestinal immunity and increases susceptibility to DSS colitis (MUC8P.732)
Authors: Runtsch Marah , O'Connell Ryan
Published: January 2023

MicroRNA-146a (miR-146a) regulates multiple immunological processes via downregulation of proinflammatory pathways, and its loss or decreased expression often results in adverse chronic inflammatory phenotypes in mammals. Although miR-146a has been well studied in systemic compartments of the immune system, its role in intestinal immunity is not well understood. We have found that miR-146a is expressed in a variety of gut tissues, including the colon and small intestine. Further, we demonstrate that this miRNA represses a subset of intestinal barrier genes that include members of the Reg3 and Mucin families, adhesion molecules, IFN response genes, immunoregulatory enzymes, and cytokines, all within a network of immune-related signaling pathways that include NF-κB and Stat signaling. Moreover, miR-146a restricts the expansion of Tfh cells and GC B cells, which decreases the production of luminal IgA, in addition to reducing Th17 and Treg populations within the intestines. Consistent with an altered intestinal barrier, we observe shifts in communities of the fecal microbiota upon loss of miR-146a. Finally, we demonstrate that miR-146a-/- mice (which have a heightened barrier) are resistant to DSS-induced colitis, and find that miR-146a is elevated in colons of human IBD patients. Taken together, our data describe a role for miR-146a in constraining intestinal barrier function and find that this is consequential in the setting of intestinal diseases.
Isolation and ex vivo expansion of murine bone marrow-derived group 2 innate lymphoid cells (MUC8P.734)
Authors: Duerr Claudia , McCarthy Connor , Mindt Barbara , Fritz Jorg
Published: January 2023

Group 2 innate lymphoid cells (ILC2) are recently discovered lymphocyte subpopulations that have innate ability to produce large quantities of a specific set of cytokines regulating innate and adaptive immune responses. Importantly, deregulated ILC2 responses are implicated in detrimental type 2 immunopathologies such as allergies, asthma and atopic dermatitis, as well as fibrotic disease, both in rodents and humans. However, the progress in our understanding of ILC2 biology has been hampered by their scarcity at steady state as well as infected and inflamed tissues. Here, a simple, efficient and reliable method is presented to isolate murine ILC2 from the bone marrow by flow cytometric cell sorting and further cultivate them using a defined cytokine cocktail, yielding a 500-fold expansion. The resulting ILC2 constitute a pure cell population and show the same phenotypic and functional characteristics as ILC2 in vivo. Thus, this protocol enables a high yield of ILC2 from murine bone marrow and thereby provides sufficient ILC2 numbers for standard cell and molecular biology assays. An in-depth understanding of the cellular and molecular mechanisms of ILC2 is essential for the development of new strategies and drugs to fight unwanted type 2 immunopathologies and fibrotic disease.
Interleukin-22 directly regulates intestinal stem cell regeneration after tissue damage (MUC8P.722)
Authors: Hanash Alan , Lindemans Caroline , Mertelsmann Anna , van den Brink Marcel
Published: January 2023

Little is known about regulation of the intestinal stem cell (ISC) compartment after injury. We hypothesized that IL-22 could promote intestinal regeneration by either acting on ISCs or by acting on Paneth cells (PCs) making up the ISC niche. Utilizing an in vitro regeneration model, we found that organoids derived from murine small intestine (SI) and large intestine (LI) crypts demonstrated substantially increased size after culture with IL-22. Innate lymphoid cell co-culture also increased organoid size in an IL-22-dependent fashion. IL-22 expanded the Lgr5+ ISC pool within SI organoids, activated STAT3 phosphorylation in Lgr5+ cells, and increased size of organoids cultured from purified SI ISCs. Using a murine allogeneic bone marrow transplant (BMT) model, we found that IL-22 administration led to reduced intestinal graft vs. host disease pathology, increased maintenance of Lgr5+ ISCs, and significantly greater ISC proliferation. ISC protection post-BMT was not due to niche augmentation, as PC numbers, PC-derived growth factors (EGF, Wnt3), and stroma-derived growth factors (R-spondin3) were all unchanged after IL-22 administration. IL-22 receptor (IL-22R) staining further indicated a direct effect of IL-22 on ISCs rather than the niche, as PCs were found to express very little IL-22R and demonstrated no evidence of STAT3 phosphorylation upon in vitro culture. In summary, we found that IL-22 regulates epithelial regeneration after tissue damage by acting directly on ISCs.
Foxp3+ regulatory T cells restrain pathogenic RORγt+ group 3 ILC-mediated colitis (MUC8P.728)
Authors: Joyce-Shaikh Barbara
Published: January 2023

IL-10 receptor beta (IL-10Rb) is a common receptor chain for both IL-10 and IL-22. Since IL-10 is a protective cytokine that restrains inflammatory responses and IL-22 signaling is required for epithelial cell-dependent tissue repair, targeted deletion of Il10rb should greatly exacerbate inflammatory bowel disease (IBD). Unexpectedly, we found that Il10rb-/- mice are completely resistant to development of spontaneous colitis. In contrast, Il10-/- mice develop IBD by 12 weeks of age and harbor a large number of IL-22-producing RORgt+ innate lymphoid cells (ILCs). Using a CD45RBhi T cell transfer model, we found that induction of intestinal inflammation requires the presence of gut resident RORgt+ ILCs. The IL-22-producing ILCs are essential to perpetuate the disease process initiated by the T cells. Furthermore, Foxp3+ Tregs directly inhibit ILC expression of IL-23R and other tissue injury sensors, thereby suppressing innate cell-driven colitis. Our study has uncovered a novel immune network that maintains intestinal homeostasis.
Bacillus subtilis exopolysaccharide suppresses mast cell responses (MUC9P.754)
Authors: Swartzendruber Julie , Knight Katherine
Published: January 2023

Commensal bacteria are being studied for their probiotic, or beneficial effects in a number of diseases. Probiotics have been proposed as a therapy for food allergy but the mechanism and efficacy of how probiotics may be preventing disease are not understood. We previously showed that we can limit diarrheal disease caused by the enteric pathogen Citrobacter rodentium using the probiotic Bacillus subtilis. In addition we isolated the protective molecule, exopolysaccharide (EPS) and shown EPS alone is sufficient for protection of disease. Food allergy and the anaphylactic response following allergen exposure are mediated by mast cell degranulation. We have evidence that EPS can suppress mast cell-dependent degranulation and anaphylaxis. Therefore, we hypothesize that EPS protects against anaphylaxis through the suppression of mast cell activation. Identifying the mechanism by which EPS prevents anaphylaxis will provide the first safe and effective therapeutic use of a probiotic for the treatment of food allergy.
Mechanism by which commensal exopolysaccharides limit inflammation (MUC9P.744)
Authors: Paynich Mallory , Jones Sara , Knight Katherine
Published: January 2023

Trillions of bacteria live within the gastrointestinal tract and are critical for maintaining intestinal homeostasis; however, the mechanisms utilized by specific bacterial molecules to contribute to homeostasis are not well understood. We utilize a mouse model in which a single dose of the probiotic, B. subtilis, protects mice from acute colitis induced by the enteric pathogen C. rodentium. Our goal is to elucidate the mechanism by which B. subtilis prevents inflammation. We identified exopolysaccharides (EPS) to be the active molecule of B. subtilis, and a single dose of EPS protects mice from disease. EPS specifically binds F4/80+CD11b+ peritoneal macrophages (MΦ). Following EPS treatment, these cells increase expression of CD206+Arg1+YM-1+IL-4Rα+ and inhibit T cell activation and proliferation in vitro, indicative of M2 MΦ. CD4+ T cells drive much of the inflammation associated with C. rodentium infection, and we hypothesized that EPS inhibits CD4+ T cell responses in vivo. Accordingly, we measured levels of IFNγ (Th1), IL-17 (Th17), and IL-13 (Th2) in splenic T cells following EPS treatment and found decreased levels of these cytokines. Similarly, mesenteric lymph node from mice orally gavaged with B. subtilis spores produced decreased levels of T cell cytokines in an EPS dependent manner. We hypothesize that EPS-induced M2 MΦ mediate inhibition of CD4+ T cell responses in vivo and further, that these cells mediate protection from acute colitis induced by C. rodentium.
Generation of antigen-specific monoclonal antibodies by in vitro immunization (TECH2P.904)
Authors: Heilmann Katja , Holzlöhner Pamela
Published: January 2023

Monoclonal antibodies are one of the most important tools in biomedicine but the generation by hybridoma technology is very time consuming and laborious. The adaptation of the process to an in vitro approach is therefore a smart alternative to reduce the disadvantages of hybridoma technology. In this study a protocol for in vitro immunization was developed which enables hematopoietic stem cells to differentiate into naïve dendritic cells. These cells were specifically activated with the antigen of interest and cocultivated with naïve B and T lymphocytes in order to generate antigen specific B lymphocytes. After 4 days the cells were fused with myeloma cells and antigen specific hybridomas were selected. The produced antibody was purified and characterised in comparison to a conventional monoclonal antibody reacting against the same antigen. Further this protocol could be adapted to human cells and the generation of antigen specific hybridoma cells secreting human IgG molecules could be established. The developed protocols allowed a generation of antigen specific hybridoma cells of murine and human origin within 3 weeks. Therefore it seems that the immunization in vitro is useful for avoiding the disadvantages of conventional hybridoma technology.
Measuring autophagic flux by assessing LC3, p62 and LAMP1 co-localization using imaging flow cytometry (TECH2P.908)
Authors: Pugsley Haley , Friend Sherree , Kong Raymond , Vaidyanathan Shobana , Morrissey Phil
Published: January 2023

Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via the lysosome; during starvation it is a survival mechanism that reallocates nutrients from unnecessary processes to more vital processes in the cell. In autophagy, cytoplasmic LC3 protein is processed and recruited to the autophagosomal membranes. The autophagosome then fuses with the lysosome to cause the breakdown of the autophagosome vesicle and its contents. The ubiquitin-associated protein p62 which binds to LC3 can also be used to measure autophagy. p62 is an important marker for the induction of autophagy, clearance of protein aggregates and the inhibition of autophagy. In addition, p62 positive cytoplasmic inclusions have been found in several neurodegenerative diseases. Immunofluorescence microscopy has been used to visually identify LC3 puntca, p62 and/or lysosomes on a per-cell basis; however, an objective and statistically rigorous assessment can be difficult to obtain. To overcome these problems, we employed the ImageStreamX Mark II imaging cytometry platform. We collected large numbers of cell images to assess autophagy and co-localization of LC3, p62 and the lysosomal marker LAMP1 in an objective, quantitative, and statistically robust manner. Defects in autophagic flux have been linked to immune-mediated diseases; we believe this technique may bring insight to these diseases.
SimpleStep ELISA™: Innovative immunoassays in less than 90 minutes (TECH2P.918)
Authors: Campbell Adam , Constable John
Published: January 2023

Here we introduce the novel SimpleStep ELISA™ platform which has improved features over the traditional sandwich assay. The SimpleStep ELISA format is a novel, streamlined protocol that can be completed in less than 90 minutes, requires no additional training and uses standard laboratory equipment. In one step, the complete sandwich complex forms in solution and is anchored to the plate through an immunoaffinity tag. Only a single incubation and wash step is required, compared to multiple incubation and wash steps with standard ELISAs, that can take up to five hours. Fewer steps mean less time is needed and there are fewer opportunities for errors with no loss on sensitivity or increased background. While simple in concept, ELISA performance can be affected by many factors, including the sample type, as many antibody pairs and immunoassays that work well on purified proteins fail to perform acceptably when a complex biological sample, such as plasma or serum, is used. The foundation of SimpleStep ELISA performance is an extensive screening program to identify antibody pairs that perform well in a range of biological samples, and the inclusion of specially-designed buffers to ensure accuracy and minimize the effect of non-specific binding, for example by human serum antibodies reactive against mouse antibodies. Supporting data for each SimpleStep ELISA includes sensitivity, range, precision, linearity, recovery and cross-reactivity.
Quantification of 500 human protein biomarkers using antibody arrays and its applications in immunology research (TECH2P.921)
Authors: Huang Ruo-Pan , Mao Yingqing , Huang Ruochun , Yen Haw-Han , Sun Yanni
Published: January 2023

Proteomic approach continues to be the trend for biomarker discovery through profiling as many proteins as possible in a biological sample. Though LC-MS technology can accurately detect medium-to-high abundance proteins (> ng/ml), many cellular modulators such as cytokines are present at pg/ml level. Immunoassays continue to be the most utilized tools for biomarker discovery and validation due to their convenience, sensitivity and specificity. Here we validated the sandwich detection of 500 most analyzed human protein biomarkers with a pair of antigen-specific antibodies. Subsequently, capture antibodies were fixed on glass slides with non-contact arrayer, and multiplex ELISA detection of these protein biomarkers was achieved through planar antibody array technology with laser fluorescence detection. To overcome the potential cross reactivity among different reagents, multiple non-overlapping arrays were developed based upon their cellular abundance, functionality, and cross reactivity. Most of the markers can achieve pg/ml detection - similar to single ELISA detection but with much wider dynamic range. These arrays were further validated with normal human sera, where over two thirds of these proteins are within the detection range. Separate array panels were developed for those proteins with high abundance in sera, where high sample dilutions are needed. In summary, our system provides a powerful, efficient, and low-cost tool for immunology research and biomarker discovery.
Rapid multiparametric analysis of activation and cytotoxicity in lymphocyte subpopulations (TECH2P.928)
Authors: Gillis Katherine , Clor Julie , Tyagarajan Kamala
Published: January 2023

Multiplexing cell health parameters with phenotyping markers provides greater understanding of time and sequence for activation and cellular stress pathways in immune cells. Sample heterogeneity often makes this type of assessment complex to perform and evaluate. The development of the easyCyte™ 12 instruments, which combines 3 lasers and 12 parameters, has greatly facilitated 6-9 color multiparametric analysis of immune cell populations using small amounts of sample without compensation. In this study, we evaluated the impact of activation agents and cytotoxic treatment on PBMCs using a multiparametric assay which identifies lymphocyte subsets in combination with cell health or activation parameters. Our studies demonstrate that overnight treatment with anisomycin resulted in more sensitive impacts on mitochondrial depolarization on all lymphocyte subsets with lower apoptotic and cell death effects being observed. Overnight treatment with celastrol demonstrated almost parallel changes in mitochondrial potential and annexin V based apoptosis effects in lymphocyte subpopulations. The methodology was also utilized to study dose and time course treatments of activation, cellular stress, and cell death caused by PHA or PMA/ionomycin with PBMCs. Which allowed for dissection of sequence of changes in multiple subpopulations. Such studies can greatly enhance our understanding on response of immune cell subpopulations to compounds that cause activation or cytotoxicity.
Study human antibody repertoire in malaria using ultra accurate immune repertoire sequencing (TECH2P.931)
Authors: Jiang Ning , Wu Di , Qu Mingjuan , Wendel Ben , He Chenfeng
Published: January 2023

Recently developed Immune repertoire sequencing (IR-seq) has become a useful tool to quantify the composition of the various antigen receptor repertoires, such as antibody and T cell receptor. However, early versions of IR-seq suffer significantly from high amplification bias and high sequencing errors. In this study, we developed an improved IR-seq method that clusters antibody sequences within each molecular identifier group to further separate different Ig transcripts that have been tagged with the same molecular identifier. We first validated this method by using naïve B cells sorted to different amounts. Analysis showed that our methods has significantly improved the sequence accuracy and diversity coverage. In addition, it enables us to examine small number of cells, such as memory and plasmablasts. We used this method to study antibody repertoire in children who experienced malaria in Africa. Epidemiology studies showed that children under 5 are most vulnerable to malaria with more than 460,000 cases of malaria caused death in children younger than 5 in Africa in 2012. However, little is known on how immunity toward malaria is established. We sequenced children from as young as 3 month to 10 year old and observed many traits in antibody diversity and mutations that correlate with age and confer protection, demonstrating the power of systems immunology approach to study infection.
Enhancements in mass cytometry for multiplexing samples and increasing cell detection rate (TECH3P.938)
Authors: Bandura Dmitry , Ornatsky Olga , Majonis Daniel , Wei Yuhong , Baranov Vladimir , Tanner Scott
Published: January 2023

Mass Cytometry uniquely enables high dimensional single cell proteomic analysis for systems-level discovery and comprehensive functional profiling applications. At the core of the technology are the ICP ion source and a fast elemental analyzer designed for metal-conjugated affinity reagents. We show how recent advancements in reagents, sample introduction system and the ion source have simplified data acquisition and improved data quality with the mass cytometry platform. Up to 20 samples from an experiment are barcoded with palladium-based cell tagging reagents, combined into one tube, and stained and run as one sample. This multiplexing improves data quality by eliminating sample-to-sample staining and running variation, increases experimental throughput by reducing the number of tubes that need to be processed and run, and reduces reagent consumption. Since each barcode consists of a unique set of 3 Pd isotopes, the de-barcoding algorithm also filters out most doublets (with more than 3 isotopes). The multiplexed sample is run on the CyTOF using the novel large volume sample introduction system. We will discuss results of modifications to the sample introduction system and the inductively coupled plasma ion source, which enable analysis of large samples at high cell detection rate. Examples of barcoded PBMCs from multiple donors, stained with high dimensional antibody panels that combine deep phenotyping with multiple cytokine or signaling targets will be demonstrated.
Residual EAE severity following B cell depletion is due to treatment-resistant plasma cells (THER7P.953)
Authors: Monson Nancy , Chen Ding , Blazek Monica , Ireland Sara , Ortega Sterling , kong xiangmei , Stowe Ann , Wang Yue , Herbst Ronald
Published: January 2023

Plasma cells and the autoreactive antibodies they produce are suspected to contribute to the pathogenesis of Multiple Sclerosis, but recent attempts to target these components of humoral immunity have failed. Recently we reported that MEDI551, an anti-CD19 antibody, disrupts development of EAE, the mouse model of MS. Leukocyte infiltration into the spinal cord is significantly reduced as well as short- and long-lived autoreactive CD138+ plasma cells in the spleen and bone marrow. In addition, potentially protective CD1dhiCD5+ regulatory B cells show resistance to depletion, and myelin-specific FoxP3+ regulatory T cells are expanded. Thus, MEDI551 may offer a compelling alternative that reduces pathogenic adaptive immune responses while preserving regulatory mechanisms. The sparing of long-lived plasma cells by anti-CD20 therapies and targeting of this population by MEDI551 prompted us to compare the efficacy of anti-CD19 Ab and anti-CD20 Ab in our EAE model. Our data demonstrated that anti-CD19 depletion is more effective than anti-CD20 depletion to ameliorate EAE. In fact, the frequency of residual antibody-producing cells correlated with residual disease severity in both the anti-CD19 and anti-CD20 treatment groups. No other correlations with residual EAE severity were found. We conclude that CD19+ plasma cells remaining after B cell depletion therapy contribute to residual EAE severity by producing autoreactive antibodies and pro-inflammatory factors.
Identifying biomarkers for monitoring disease progression in experimental autoimmune encephalomyelitis (THER7P.942)
Authors: Ji Niannian , Chase Carol , Saenz-Trevino Erica , Raphael Itay , Forsthuber Thomas
Published: January 2023

Multiple sclerosis (MS) is the most common demyelinating neuroinflammatory disorder which afflicts over 400,000 Americans. Currently, determining clinical or sub-clinical disease progression in MS patients and development of novel treatments has been hampered by the lack of specific and sensitive laboratory tests. Recently, our lab has developed a technique called M2 proteomics, which is a rapid quantitative approach for identifying putative protein biomarkers and therapeutic targets of experimental autoimmune encephalomyelitis (EAE), a commonly used animal model for MS. Notably, we identified several putative-biomarkers which correlate with different stages of monophasic EAE. The objective of this study is to identify protein biomarkers of disease progression in a progressive EAE model using non-obese diabetic (NOD) mice. We hypothesize that during progressive EAE key central nervous system (CNS) disease-specific proteins will be released into blood and changes of these proteins can be used to determine disease progression. Using the progressive NOD EAE model, we determined the expression of putative CNS-specific protein biomarkers by immune assay in brain homogenate and serum longitudinally over the course of disease. We identified several CNS-specific potential biomarkers in serum that correlated with the progression of disease. The results of this study could help development of biomarkers for disease progression and testing of novel treatments for progressive MS.
Infusion of SMCC-conjugated MOG-coupled spleen cells effectively prevents and reverses experimental autoimmune encephalomyelitis: induction of antigen-specific Foxp3+ regulatory CD4+T cells and suppression of antigen-specific Th17 cells (THER7P.949)
Authors: Xia Changqing , Zhang Langfang , Guo Yixian
Published: January 2023

Experimental autoimmune encephalitis (EAE) is an induced autoimmune disorder in rodent animals with the development of autoimmune T cells attacking axonal myelin protein causing demyelination. This model has been widely used to develop preventive or therapeutic approaches for human multiple sclerosis (MS). In current study, we report that infusion of SMCC-conjugated MOG35-55 coupled autologous spleen cells (either live, or ultraviolet B-irradiated apoptotic) significantly prevents and reverses EAE in mice. Further studies show that the protected animals resist EAE re-induction by MOG35-55 antigen challenge. Moreover, adoptive transfer of CD4+ T cells from the protected mice to naïve syngeneic mice renders the recipients resistant to EAE induction, suggesting that MOG35-55-specific regulatory CD4+ T cells may be induced. Unexpectedly, CD4+T cell proliferation is similar upon ex vivo stimulation by MOG35-55 amongst all groups. However, further analysis of those proliferating CD4+ T cells shows remarkable difference in Foxp3+ cells (70% in MOG-SP groups versus 10-25% in control groups, p<0.05), and in IL-17+ cells (2-3% in MOG-SP groups versus 6-9% in control groups,p<0.05). These findings indicate that MOG35-55 coupled spleen cell treatment induces MOG35-55 specific regulatory T cells, and suppresses EAE pathogenic Th17 cells. Our study provides a novel approach for antigen-based immunotherapy for EAE, which has a great potential for developing immunotherapies for MS .
Computational prediction and functional identification of HLA-A2-restricted T lymphocyte epitopes derived from swine leukocyte antigen (TRAN2P.962)
Authors: Lee Chong-Kil , Park Chan-su , Im Sun-A , Park Young-Jun , Lee Jae Hee , Kim Kyungjae
Published: January 2023

Xenotransplantation using porcine organs is a potential solution to the problem of the shortage of allografts. However, immunological rejection of xenografts remains as a major hurdle to success. Little information is available regarding the precise swine leukocyte antigen (SLA)-derived immunogenic peptides that are presented in the context of human HLA molecules. The SLA-derived peptides that bind to HLA-A*0201, a representative of the A2 supertype, were predicted using a computer-assisted algorithm. The candidate peptides were synthesized, and the stabilities of complexes formed between the peptides and HLA-A*0201 were compared using MHC stabilization assays. The cytotoxic T lymphocyte (CTL)-inducing activity of the selected peptides was examined in HLA-A*0201-transgenic mice. Among 15 candidate peptides synthesized, two peptides, peptide-35 (YLGPDGLLL) and peptide-43 (TLICHVDSI), were selected to have high affinity and stability with HLA-A*0201. Examination of the CTL-inducing activity of the two peptides in HLA-A*0201-transgenic mice showed that immunization with peptide-35, but not peptide-43, elicited potent CD8-specific-CTL responses. The Peptide-35 is present in non-polymorphic a2 domains of 34 SLA-1 alleles, 18 SLA-2 alleles, and 1 SLA-3 allele. These results show that Peptide-35 is a HLA-A2-restricted immunogenic peptide derived from SLA.
PD-1 and IL-10: partners in crime against anti-tumor immunity in ovarian cancer. (TUM9P.1011)
Authors: Lamichhane Purushottam , Karyampudi Lavakumar , Shreeder Barath , Knutson Keith
Published: January 2023

PD-1: PD-L1 is a major inhibitory axis that acts to suppress tumor-rejecting effector responses. In addition to its expression and inhibitory functions in T and B cells; PD-1 has also been shown to be expressed on and thereby impact the functions of cells of innate arm such as dendritic cells (DCs) primarily at the tumor site but not in the periphery. We show here that one of the culprits in the tumor microenvironment responsible for enhancing PD-1 expression on the tumor-associated DCs is the cytokine IL-10; which is expressed in abundance in many malignancies including ovarian cancer. Based on inhibition and knockdown studies, we show that IL-10 mediated PD-1 expression depends on STAT3 activation. Using murine model of ovarian carcinoma (ID8), we show that tumor-associated DCs express not only high levels of PD-1 on the surface but also have increased expression of STAT-3 and p-STAT3 compared to splenic DCs. Blockade of PD-1 on DCs led to increased production of IL-10 which correlated with a further increase in PD-1 expression. In in vivo studies, blockade of PD-1 and IL-10 neutralization as combination therapy augmented the anti-tumor response in ID8 ovarian tumor bearing mice; leading to a decrease in tumor mass and a significant increase in survival. These results show that IL-10 and PD-1 pathway intersect in DCs in theTME hence making them attractive targets for therapy of ovarian cancer.
Regulatory rebound in Interleukin-12-treated tumors is driven by uncommitted peripheral Treg (TUM9P.1012)
Authors: Li Qingsheng , Virtuoso Lauren , Egilmez Nejat
Published: January 2023

Interleukin-12 (IL-12) promotes a rapid reversal of immune suppression in the tumor microenvironment. However the adjuvant activity of IL-12 is short-lived due to T-regulatory cell (Treg) re-infiltration. Quantitative analysis of Treg kinetics in IL-12-treated tumors and tumor-draining lymph nodes (TDLN) revealed a transient loss followed by a rapid 4-fold expansion of tumor Treg between days 3 and 10. Subset-specific analysis demonstrated that the post-treatment rebound was driven by the CD4+ CD25+ Foxp3+ Neuropilin-1(Nrp-1)low peripheral Treg (pTreg) resulting in a 5-fold increase in the pTreg to CD4+ CD25+ Foxp3+ Nrp-1high thymic Treg (tTreg) ratio by day 10. The expanding pTreg displayed hypermethylation of the CpG islands in Treg-specific demethylated region (TSDR), CTLA-4 exon 2 and GITR exon 5; were phenotypically unstable; and exhibited diminished suppressive function consistent with an uncommitted in vitro-induced Treg (iTreg)-like phenotype. In vitro culture of post-therapy Treg populations under TH1-promoting conditions resulted in the production of IFNg by pTreg but not by tTreg, confirming their transitional state. Blockade of selected molecular mechanisms that are known to promote Treg expansion identified Indoleamine 2,3 dioxygenase (IDO)-positive Dendritic cells as the primary mediator of post-IL-12 pTreg expansion. Clinical implications of these findings are discussed.
The role of host B7-H1 in the recurrence of B16 melanoma. (TUM9P.1013)
Authors: Goding Stephen , Wilson Kyle , Rosinsky Carolyn , Antony Paul
Published: January 2023

Our B16 melanoma mouse model, in which ~50% of mice receiving adoptive cellular therapy with cytolytic CD4+ T cells recognizing the tumor/self antigen tyrosinase protein 1 (TRP-1), exhibits tumor relapse following initial tumor regression. CD4+ T cells from tumor-bearing TRP-1 antigen knockout (tyrp1BwRAG-/-) transgenic mice, or tumor sensitized (t.s.) T cells, expressed low levels of the exhaustion marker PD-1 and were 15-25% Foxp3+. Due to this presensitization, B16 tumors recurred faster and more predictably in RAG-/- mice with t.s. CD4+ T cells than naïve TRP-1-specific CD4+ T cells. However, in RAG-/- mice lacking B7-H1 (i.e. PD-L1, the ligand for PD-1), relapses occurred more rarely and developed at slower rates following adoptive transfer of t.s. CD4+ T cells. When CD4+ T cells from mice bearing relapsing tumors, expressing high levels of PD-1 and Foxp3+, were transferred into either RAG-/- or B7-H1-/-RAG-/- mice, the treatment effect was completely abrogated. We previously reported that primary tumors required B7-H1 blockade or Treg depletion to be treated, and relapsing tumors required both treatments. Here we show that Treg cell depletion in B7-H1-/-RAG-/- mice partially restored treatment with fully exhausted CD4+ T cells and relapses took longer to occur. These data indicate that although multiple mechanisms are involved in tumor recurrence (Treg cells and PD-1), the expression of B7-H1 in the host rather than the tumor plays a major role in B16 melanoma relapse.
Cancer mediates effector T cell dysfunction by targeting microRNAs and EZH2 via glycolysis restriction (TUM9P.1007)
Authors: Zhao Ende , Maj Tomasz , Kryczek Ilona , Wei Shuang , Crespo Joel , Wan Shanshan , Vatan Linda , Szeliga Wojciech , Chinnaiyan Arul , Welling Theodore , Marquez Victor , Wang Guobin , Zou Weiping
Published: January 2023

Aerobic glycolysis regulates T cell function. However, if and how primary cancer alters effector T cell glycolytic metabolism and affects tumor immunity remains a question in cancer patients. Here we report that ovarian cancer imposes glucose restriction on effector T cells, dampens their function via maintaining high expression of microRNA101 and microRNA26a, which subsequently constrains EZH2 expression. EZH2 activates the Notch pathway by suppressing Notch repressors via H3K27me3, and consequently stimulates T cell polyfunctional cytokine expression and promotes their survival via Bcl-2 signaling. Moreover, EZH2-/- T cells elicit poor anti-tumor immunity and EZH2+CD8+ T cells are associated with improved long-term cancer patient survival. Together, our data unveil a novel metabolic target and mechanism of cancer immune evasion.
Organ specificity of cancer metastasis depends on the adaptive immune surveillance and the neuronal chemokine fractalkine (TUM10P.1030)
Authors: Nwajei Felix , Beceren-Braun Figen , Gabrusiewicz Konrad , Shanmugasundaram Meenakshi , Zal Anna , Wu Wuenye , Heimberger Amy
Published: January 2023

The phenomenon of organ specificity in cancer metastasis has been traditionally interpreted in terms of the “seed and soil” hypothesis. However, the role of the adaptive immune system in this phenomenon is largely unknown and controversial. We found that MCA-fibrosarcoma cancer cells formed lethal tumors in the lungs, but not in the brain thereby representing a model of organ-specificity of cancer metastasis. Using this model, we assessed the role of the adaptive immune system. In immune competent multi-color fluorescent reporter mice, longitudinal intravital imaging via cranial windows revealed initial engraftment and growth of MCA cancer cells that was followed by tumor regression in concordance with T cell infiltration. However, MCA cancer cells formed lethal tumors in the brains of Rag1-KO mice indicating a key role for the adaptive immune system in the organ specificity of MCA cancer cell metastasis. In contrast, T cells were recruited to pulmonary MCA lesions but were ineffective in tumor rejection in that organ. Interestingly, T cell recruitment to MCA micrometastases in the brain was dramatically impeded in mice lacking the receptor for the neuronal chemokine fractalkine, and MCA tumors progressed in the brains of those mice. Our results reveal a role for brain-specific adaptive immunity to cancer metastasis and implicate fractalkine in regulating this process thereby broadening the “seed and soil” concept.
The DH/PH domain of BCR-ABL mediates INKT cell immunosubversion in chronic myeloid leukemia by downregulating cell-surface CD1D expression in myeloid dendritic cells (TUM10P.1038)
Authors: Herbelin Andre , Basbous Sara , Levescot Anais , piccirilli nathalie , GOMBERT JEAN MARC
Published: January 2023

Chronic myeloid leukemia (CML) is associated with a rearrangement between the Abelson (ABL) and the BCR genes, generating a chimeric fusion gene. The constitutive tyrosine-kinase (TK) activity linked to ABL is sufficient to induce malignant transformation while BCR contains a DH/PH domain ensuring activation of the Rho/ROCK pathway that is implicated in the control of mobility of leukemia cells. iNKT cells constitute a distinct population of T cells that recognize glycolipids as α-GC presented by CD1d, and are believed to play a key role in cancer immunosurveillance. CML in chronic phase (CP) is associated with functional deficiencies of iNKT cells. Here, we hypothesized that the anergic status of iNKT cells during CML-CP results from BCR-ABL-dependent dysfunctions in APC. Accordingly, CML mDC downregulated their cell surface expression of CD1d, as compared to mDC from healthy individuals. Since Rho/ROCK pathway is a negative regulator of CD1d expression, we surmised that its enhancement by the DH/PH domain of BCR-ABL is responsible for the impaired cell-surface CD1d expression in CML mDC. In favor of this hypothesis, we showed that treatment of PBMC by Rho/ROCK inhibitors restores in vitro cell-surface expression levels of CD1d on CML-CP mDC as well as expansion of CM-CP iNKT cells in response to α-GC. Altogether, these results indicate that the DH/PH domain of BCR-ABL mediates iNKT-cell immunosubversion in CML patients by downregulating cell-surface CD1d expression in mDC.
Lymphocyte depletion after neoadjuvant chemoradiotherapy for operable gastric cancer. (TUM10P.1039)
Authors: Wydmanski Jerzy , Chwieduk Agata , Kotyla Przemyslaw , Kaletka Agnieszka , Tukiendorf Andrzej
Published: January 2023

The purpose of this study was to assess the effect of preoperative chemoradiotherapy (CRT) for operable gastric cancer on lymphocyte subpopulations counts. A total of 35 patients with adenocarcinoma of the stomach were investigated. The preoperative CRT regime consisted of radiotherapy and chemotherapy. The total dose of 45 Gy was administered in 25 fractions, with 5 fractions per week for 5 weeks. Analysis of lymphocyte subpopulations in peripheral blood was carried out before and during treatment (5 analyses for each patients) using a BD Multitest IMK Kit (Becton Dickinson). The data were analyzed with ANOVA. Before and after CRT, the mean values (MVs) of the subtype counts (SD) were as follow: lymphocytes, 1,788 cells/µl (±694) and 239 cells/µl (±131); CD3+CD4+ cells, 738 cells/µl (353) and 135 cells/µl (55); CD3+CD8+ cells, 562 cells/µl (±398) and 56 cells/µl (±41); CD3-CD19+ cells, 150 cells/µl (±112) and 2 cells/µl (±2); and CD16+CD56+ cells, 329 cells/µl (±155) and 28 cells/µl (±36). Every week, we have observed the gradual depletion of all subset of lymphocytes and those differences were statistically significant (p<0.0000). Before CRT, the MV of the CD4+/CD8+ ratio was 1.58 (SD±0.97 ). This ratio has gradually increased during CRT. At the end of treatment, the MV of the CD4+/CD8+ ratio was 2,96 (SD±2,04), p<0.0000. Preoperative chemoradiotherapy induces depletion of all lymphocyte subpopulations.