bluuebunny/crossref_metadata_2025 huggingface dataset:
Early complement activation through alternative pathway significantly correlates to myocardial injury in patients undergoing cardiac surgery (142.6)
Authors: Zhang Ming , Hou Yunfang , Gulaya Karan , Lee Daniel , Lee Jeanie , Rushbrook Julie , Ko Wilson , Shevde Ketan
Published: January 2023

Global heart ischemia occurs in the normal course of cardiac surgery during aortic cross-clamping (AXCL), and plasma level of cardiac troponin, a specific myocardial injury marker, increases during reperfusion after releasing the clamp. Earlier clinical studies also found C3, the common factor in three complement pathways, is activated during cardiac surgery. In this prospective study, we investigated three complement pathways in the involvement of myocardial injury during cardiac surgery. Fifty patients undergoing open heart surgery were enrolled. Coronary sinus and peripheral blood were collected at different peri-operative time points. Plasma levels of the initial factors in the complement pathways were analyzed. Our results showed that the level of complement factor B, the initial component in the alternative pathway, increased significantly in both coronary sinus and peripheral blood after release of AXCL compared to samples taken prior to AXCL. Spearman correlation analysis showed that this post-AXCL increase in factor B was significantly correlated with the cardiac troponin level sampled immediately after the operation. In contrast, there was no significant correlation between the initial factors in the classical or the lectin complement pathways and post-operation troponin levels. Thus, alternative complement pathway is activated during open heart surgery and may play a role in human myocardial ischemic injury.
NKG2D/ligands-mediated immune activation promotes atherosclerosis (142.7)
Authors: Xia Mingcan , Yang Kangkang , Guerra Nadia , Sukhova Glina , Miller Carla , Shi Guo-Ping , Raulet David , Xiong Na
Published: January 2023

Atherosclerosis, a chronic vascular disease, is characterized by deposition of fatty substances and accumulation of immune cells in susceptible regions of arteries. Increasing evidence show that abnormal metabolic conditions, such as dyslipdemia and diabetes, induce immune cell activation and inflammation, which play crucial roles in progression of atherosclerosis. However, molecular linkers of metabolic disorder, immune activation and atherosclerosis are not well understood. Here we report that ligands for NKG2D, an immune-activating receptor, were upregulated in multiple tissues and organs under atherosclerotic disease condition, particularly in atherosclerotic lesion and liver. Preventing NKG2D/ligands interaction significantly reduced atherosclerotic lesion formation and systemic inflammation. In addition, preventing NKG2D/ligand interaction alleviated abnormal metabolic conditions by suppressing liver inflammation. Our data clearly show that NKG2D/ligand interaction plays critical roles in the progression of atherosclerosis and might be potential targets against atherosclerosis.
Regulation of the Receptor Activator of NF-κB Ligand (RANKL)-induced activation of the alternative NF-κB pathways by interleukin-4 (IL-4) (142.8)
Authors: Yu Minjun , Moreno Jose , Keegan Achsah
Published: January 2023

NF-κB signaling is essential for RANKL-induced osteoclast (OC) formation. IL-4 inhibited RANKL-induced OC differentiation, while at the same time promoted macrophage fusion to form multinucleated giant cells (MNG). Several groups have proposed that IL-4 acted by suppressing the RANKL-induced activation of NF-κB. However, we found that IL-4 did not block canonical NF-κB signaling. Instead, we found that IL-4 inhibited alternative NF-κB signaling and induced p105/50 expression. Interestingly, in p105/50 deficient bone marrow macrophages (BMM), the formation of both multinucleated OC and MNG induced by RANKL or IL-4 respectively was impaired. This suggests that NF-κB signaling also plays an important role during macrophage fusion and MNG formation. To confirm this, the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) was used; PDTC blocked both RANKL-induced OC and IL-4-induced MNG formation. RT-PCR and western blot analyses showed that E-cadherin, and DC-STAMP, proteins important for macrophage fusion, were downregulated by PDTC. Furthermore, overexpression of p52 or RelB in p105/50 deficient BMM significantly enhanced both OC and MNG formation. DC-STAMP was upregulated in p52 and RelB transduced BMMs. These results suggest that the influence of IL-4 on NF-κB activation pathways is complex, and that NF-κB pathways positively regulate the IL-4-induced formation of MNG by MCSF-dependent macrophages.
Nuclear Factor-κB is a Potential Therapeutic Target in Cystitis (142.9)
Authors: Vykhovanets Eugene , Vykhovanets Olena , Ponsky Lee , Cherullo Edward , Gupta Sanjay
Published: January 2023

NF-κB has been shown to be activated in various inflammatory disorders or by physical and chemical stress. NF-κB regulates the transcriptional of genes important for chronic inflammation and drug resistance. Interstitial cystitis (IC) affects millions of women and is characterized by severe pain and chronic inflammation. Etiology and the mechanisms involved in pathogenesis of IC are still a matter of obscure. Animal models of IC and role of NF-κB as a therapeutic target in the IC have not been elucidated. Using transgenic NF-κB-Luciferase Tag female mice, we performed serial, noninvasive in vivo and ex vivo molecular imaging of NF-κB activation in the mouse body after administration of arsenic trioxide (5 mg/kg), LPS (2 mg/kg), or cyclophosphamide (CYC, 200 mg/kg) to initiate transient cystitis. Pretreatment with dexamethasone (DEX) (10 mg/kg) was used to modulate the chemical-induced NF-κB-dependent luminescence. Treatment of NF-κB-Luc mice with chemicals increased luminescence in a time- and organ-specific manner. Highest levels of bladder NF-κB-dependent luminescence were observed 4 hr after CYC administration. Pretreatment with DEX 1 hr before injection of CYC significantly down-regulated CYC-induce bladder NF-κB-dependent luminescence and gross anatomy signs of cystitis. The molecular imaging of NF-κB activity might be an attractive approach to distinguish the role of NF-κB signaling in transient cystitis and select potential candidate drugs for treatment of the IC.
Intricate regulation of IL-17 response by IFN-γ and IL-27 determines the outcome of the pathogenetic events in experimental autoimmune arthritis (143.18)
Authors: Rajaiah Rajesh , Puttabyatappa Muraly , Moudgil Kamal
Published: January 2023

Recent advances in defining the new subsets of T cells and the related newer cytokines have stimulated new undertakings in examining the pathogenesis of autoimmunity beyond the traditional Th1-Th2 paradigm. We report here novel findings regarding the roles of IFN-γ, IL-17 and IL-27 in the adjuvant arthritis (AA) model of human rheumatoid arthritis that challenge the traditional viewpoints. We hypothesized that the temporal kinetics and the relative levels of antigen-induced cytokine responses determined both the propagation of AA as well as resistance to the disease. We determined the cytokine responses of arthritic Lewis rats at different phases of the disease and that of the arthritis-resistant, MHC-compatible Wistar Kyoto (WKY) rats given a potentially arthritogenic challenge. In Lewis rats, IL-17 dominated the early (incubation phase) response, whereas IFN-γ and IL-27 appeared late (peak phase). Intriguingly, in WKY rats, all these 3 cytokines appeared concomitantly and early, showing that IFN-γ and IL-27 successfully pre-empted the pathogenicity of IL-17. This cross-regulation, along with the novel finding of upregulation of IL-27 by IFN-γ, was validated through mechanistic experiments, including the AA-protective effect of IFN-γ injected in vivo, and the differential modulation of specific signaling biomolecules and inflammatory mediators in the joints. These results provide a novel IL-17-IL-27-centered perspective on the pathogenesis of autoimmune arthritis.
IgD BCR-mediated signals do not have protective roles in negative selection of autoreactive B cells in lpr mice (143.29)
Authors: Guo Linjie , Guo Zhuxiu , Zheng Biao , Han Shuhua
Published: January 2023

IgD is the major antigen receptor isotype coexpressed with IgM on the surface of most peripheral B cells in mice and humans. However, the biological role of IgD as B cell receptor (BCR) has remained enigmatic and controversial. Previous studies have indicated that IgD may play a role in B cell tolerance and the expression of IgD may rescue cells undergo programmed cell death. To understand the role of IgD in B cell tolerance and autoimmunity, we have examined the development of autoimmune syndrome in lpr mice deficient for IgD. The present study showed that IgD deficiency did not alter many aspects of the autoimmunity in lpr mice such as splenomegaly and significant expansion of activated T cells. Interestingly, lpr mice with IgD deficiency exhibited increased levels of autoantibodies and more severe nephritis compared to their IgD-competent littermates. In addition, the absence of IgD did not result in any increase in cell death of B cell subsets including transitional B cells. Thus, our results indicated that IgD BCR-mediated signals do not have a protective role in negative selection of autoreactive B cell clones. In contrast, the absence of IgD leads to heightened autoantibody production and more deposition of immune complex in the kidney, resulting in more severe nephritis.
Loss of P2X7 receptor membrane expression in B220+ double-negative T lymphocytes of autoimmune MRL/lpr mice (143.32)
Authors: Bobé Pierre , Le Gall Sylvain , Legrand Julie , Benbijja Mohcine , Benihoud Karim , Kanellopoulos Jean
Published: January 2023

Lupus is an autoimmune disease influenced by multiple genetic loci. Fas-deficient MRL/lpr mice exhibit lupus and lymphoproliferative syndromes with massive accumulation of B220+CD4-CD8- (DN) T lymphocytes. Pathogenic B220+ DN T lymphocytes are derived from normal activated T lymphocytes which have not undergone activation-induced cell death due to inactivation of Fas. Normal activated peripheral T lymphocytes up-regulate the membrane phosphatase B220 before undergoing apoptosis. In Fas-deficient mice, activated T cells down-regulate CD4 or CD8 molecules but retain the expression of B220. P2X7R is an extracellular ATP-gated cell membrane receptor involved in the release of inflammatory cytokines and cell death. We show herein that MRL/lpr mice carry a P2X7R allele, which confers a high sensitivity to ATP. However, during aging, the MRL/lpr T-cell population exhibits a drastically reduced sensitivity to ATP-mediated stimulation of P2X7R. This decrease in P2X7R activity parallels the increase in B220+ DN T-cell numbers in lymphoid organs. The rare B220+ T cells observed in normal mice are also resistant to ATP treatment. Moreover, B cells, which express B220 as a developmental marker, are also dramatically less sensitive to ATP treatment. Using rabbit anti-P2X7R sera, we found a strong reduction in P2X7R levels on B220+ cells. Our results prompt the conclusion that B220 expression strongly correlates with the negative regulation of ATP/P2X7R pathway on both T and B cells.
REGULATORY CD4-CD8- T CELLS PREVENT AUTOIMMUNE DIABETES (143.4)
Authors: Lesage Sylvie , Dugas Véronique , Hillhouse Erin , Beauchamp Claudine , Chabot-Roy Geneviève
Published: January 2023

Regulatory T cells show great potential for use in cellular therapy. In particular, CD4-CD8- (DN) T cells, which compose 1 to 3% of total T lymphocytes, exhibit prominent antigen-specific immune tolerance properties in models of allografts and xenografts, as well as in an induced model of autoimmune diabetes. Here, we evaluate the immunoregulatory properties of DN T cells in the autoimmune-prone NOD genetic background. Using the 3A9 TCR transgenic mice, we demonstrate that DN T cells from both autoimmune-resistant and -prone mice are equally effective at eliminating potentially autoreactive B cells in vitro. However, autoimmune-prone mice carry at least three fold fewer DN T cells than autoimmune-resistant mice, in both TCR transgenic and non-transgenic setting. Interestingly, a single transfer of DN T cells is sufficient to prevent autoimmune diabetes onset in autoimmune-prone mice. These results suggest that increasing DN T cell number is sufficient to confer protection from autoimmune diabetes onset. Further functional and genetic characterization of DN T cells in NOD mice highlighted a potential role for both CD172a and IL-10 in the regulation of DN T cell homeostasis. Importantly, genetic polymorphisms in both CD172a and IL-10 are associated with diabetes susceptibility in humans. Taken together, our results strongly suggest a role for DN T cell in peripheral tolerance and the association of genetic defects in DN T cell homeostasis with the development of diabetes.
IL-13 Mediated Regulation of Islet-Reactive T Cells (143.44)
Authors: Rasche Sarah , Phillips Michele , Quinn Anthony
Published: January 2023

Immunotherapy with the beta cell antigen glutamic acid decarboxylase (GAD65) or GAD65 peptides delays the onset of type 1 diabetes (T1D) in NOD mice, while alum formulated rGAD65 may be therapeutic in human T1D; nevertheless, the regulatory mechanisms involved in each therapy may be distinct. Here, we sought to discover the factors that contribute to GAD65 peptide 524-538 (p524)-mediated protection from T1D in NOD mice and to determine if IL-13 produced by p524-specific T cells could regulate pathogenic islet-reactive T cells. IL-13 produced by p524-reactive T cells directly modulated IFN-g production by spontaneously arising GAD65 530-543-specific Th1 cells in NOD mice and rIL-13 inhibited proliferative responses of islet-reactive BDC2.5 clonotypic T cells. Contrary to previous observations, both CD4+ and CD8+ T cells in the peripheral lymphoid compartment of NOD and C57BL/6 mice expressed the receptor subunit required for IL-13 signaling, IL-13Rα1. Interestingly, the number of IL13Rα1+ T cells was highest in pancreatic lymph nodes, as compared to inguinal lymph node or splenic T cells, and this expression was uniquely diminished in pancreatic lymph nodes of older prediabetic NOD. Thus, IL-13 may be an important mechanism for regulation of autoreactive T cells in the early stages of insulitis. The loss of IL-13Rα1 expression on T cells in the draining nodes of the pancreas may reflect fading regional immune regulation and progression from insulitis to overt T1D.
The paradoxical role of Th1 effector CD4 T cells during autoimmune chronic inflammation. (143.47)
Authors: Harrington Laurie , Kress Robert , Mollo Sarah , Yeh Wen-I
Published: January 2023

Many autoimmune chronic inflammatory diseases, including multiple sclerosis, have been previously reported as Th1-mediated disorders; however recent data suggests that Th17 cells are responsible for disease. Paradoxically, the principal Th1 cytokine IFNγ does not appear necessary for disease, but the key Th1-associated transcription factor Tbet is required. This conundrum propelled us to investigate the regulation of this transcription factor and effector CD4 T cell functions, as well as the role of Th1 cells during autoimmunity. Following the onset of experimental autoimmune encephalomyelitis (EAE), we observed a preferential upregulation of Tbet by effector CD4 T cells within the CNS, but not the secondary lymphoid organs. These Tbet+ CD4 T cells were capable of producing IFNγ, and a fraction were able to make both IFNγ and IL-17. In contrast, CD4 T cells producing only IL-17 were primarily low for Tbet expression, in keeping with a non-essential role for Tbet during Th17 differentiation. We next examined the requirement of the Th1 promoting cytokines IFNγ and IL-12 for the induction of Tbet+ CD4 T cells during EAE. In the absence of these cytokines, a significant population of CD4 T cells still upregulated Tbet. Consistent with these data, we found that CD4 T cells from IFNγ reporter/IFNγ deficient mice with EAE expressed the Thy1.1 reporter molecule, indicating the presence of Th1-like, Tbet+ CD4 T cells even in the absence of the cardinal Th1 cytokine, IFNγ.
Increased formation of anti-insulin B cells combines with defective central and peripheral tolerance checkpoints to contribute to type 1 diabetes liability in NOD mice (143.5)
Authors: Henry Rachel , Kendall Peggy , Thomas James
Published: January 2023

Insulin-reactive autoantibodies precede disease development in type 1 diabetes (T1D)-prone non-obese diabetic (NOD) mice and humans, consistent with a breakdown in B cell tolerance for this key T1D autoantigen. Accelerated disease occurs in B cell receptor (BCR) transgenic NOD mice which have a small population of anti-insulin B cells. Absence of this specificity in closely related BCR transgene results in disease protection, highlighting the importance of B cell specificity for insulin in promoting disease. The genesis and maturation of anti-insulin B lymphocytes were investigated in NOD mice to identify alterations in tolerance checkpoints which underlie T1D progression. In NOD mice, polymorphic anti-insulin germline Vκ genes promote increased formation of anti-insulin B cells in the bone marrow. In contrast to non-autoimmune mice, failure to cull anti-insulin B cells in NOD mice though receptor editing or deletion in the bone marrow as well as at transitional checkpoints in the periphery permits entry of anti-insulin B cells into T1, follicular, and marginal zone B cell compartments. Entry occurs despite the presence of a large competing repertoire. These findings highlight how multiple points at which B cell tolerance is compromised in NOD mice combine to increase risk of T1D. A broad therapeutic window therefore exists during B cell development, within which successful tolerance restoration could provide effective disease intervention.
MicroRNAs: modulators of T cell phenotype in multiple sclerosis (143.57)
Authors: Smith Kristen , Guerau-de-Arellano Mireia , Williams Jessica , Racke Michael , Lovett-Racke Amy , Whitacre Caroline
Published: January 2023

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system mediated by autoreactive CD4+ T cells of a Th1 and Th17 phenotype. Identifying novel factors that control Th differentiation will lead to a better understanding of disease pathogenesis and the development of new therapeutic strategies. MicroRNAs (miRs) are a class of small non-coding RNAs that negatively regulate post-transcriptional gene expression. MiRs have an established role in immune system homeostasis, and profiling studies have found differential expression of miRs in autoimmune diseases, including MS. Dicer-deficient CD4+ T cells that lack miRs produce more cytokine upon stimulation. Together these data indicate the miR(s) are responsible for controlling cytokine expression and act as negative regulators of inflammation. However, the specific miR(s) responsible for modulating inflammatory cytokine production and Th programming have not been defined. Using target prediction algorithms, we identified miRs predicted to target genes that mediate Th1 specification and function. We have validated these miR-target pairs using gain- and loss-of-function experiments in human Th1-polarized cells. We have also profiled the expression of these miRs in specific CD4+ T cell subsets from MS patients. Our results identify miRs that modulate Th developmental programming and effector function and whose expression is altered in MS. These miRs may be useful immunomodulatory treatments or biomarkers in MS.
Over-expression of the transcription factor Bright alters B cell tolerance (143.9)
Authors: Oldham Athenia , Miner Cathrine , Webb Carol
Published: January 2023

The persistence of circulating auto-antibodies is indicative of a breach in B cell tolerance. Mice that constitutively over-expressed the transcription factor Bright throughout B cell development produced antibodies to nuclear antigens (ANAs) similar to those produced in autoimmune diseases such as lupus. Whereas, normal mice reduce Bright expression in mature B cells and do not produce ANAs. ANAs did not result from global hyperglobulinemia and were IgG demonstrating that they originated from B cells that had transited the germinal center. ANA production was dependent upon Bruton's tyrosine kinase. To determine where tolerance breaches occurred in this autoimmune model, we analyzed transgenic B cells for abnormalities at multiple tolerance checkpoints. We found that early transitional B cells responded to BCR stimulation with exaggerated cell death, suggesting the clonal deletion checkpoint was in tact. Transgenic mice also developed normal numbers of naturally occurring anergic B cells. Interestingly, mature B cell numbers were down by half and those cells were hyper-responsive to proliferative signals. The marginal zone compartment is enriched for self reactive B cell. A bias towards marginal zone versus follicular B cell development was evident both in cell numbers and through crosses with Bcl2 transgenics. Together, these data suggest that appropriate regulation of Bright levels is critical for maintaining normal B cell tolerance.
Automated determination of optimal microarray data processing (144.11)
Authors: Scheuermann Richard , Kong Y. Megan , Wang Siling , Qian Yu , McGee Monnie
Published: January 2023

Gene expression microarrays are widely used to study genome-wide gene expression profiles. Many microarray analysis methods are available, making it challenging to decide which method to use. While the effectiveness of some of these methods has been assessed using artificial spike-in data sets, analytical approaches that work well with spike-in data may not work as well with data from real biological samples. To evaluate these methods we applied Gene Ontology (GO) term co-clustering as a comparative tool to evaluate 300 different data processing pipelines composed of various background correction, normalization and summarization methods using real biological data, based on the premise that an improvement in any step of microarray data analysis should be reflected in improved co-clustering of related genes. Our results suggest that background correction has little affect on GO term co-clustering characteristics, normalization has a big impact, some summarization methods constantly outperform others, some interdependencies exist among these methods, e.g., a good normalization method may work well with one summarization method but not another. An automated method for running and assessing these 300 pipelines has been made available through the Immunology Database and Analysis Portal (ImmPort; www.immport.org) - a public database resource funded by NIAID to support the management and analysis of clinical and mechanistic data generated by their funded investigators.
Use of surface modified microspheres for setting compensation in multicolor flow cytometry (144.15)
Authors: Zhang Yu-Zhong , Langsdorf Chris , Bradford Jolene
Published: January 2023

Yu-Zhong Zhang, Chris L. Langsdorf and Jolene A. Bradford Invitrogne, Eugene, OR 97402, USA Compensation is a process mathematically corrects multiparametric flow cytometry data for spectral overlap. Setting compensation properly requires the use of controls. Recent advances introduced antibody capture beads as substitute for cells in setting compensation. Antibody capture beads can be stained with antibodies, just like cells, and provide a bright uniform signal, regardless of how bright an antibody would stain. We compare two methods of setting compensation for multicolor immunophenotyping, using cells and using antibody capture beads, with both human lymphocytes and mouse splenocytes, and found the antibody capture beads performed comparably. It is essential that a dead cell discriminator be included in each sample, because antibody conjugates bind nonspecifically to dead cells and may lead to erroneous results. We study the need for using a dead cell discriminator in three types of assays: surface immunophenotyping, intracellular testing requiring fixation, and a functional assay. Our data indicated that excluding dead cells from that analysis improved the results. We then used the amine-reactive beads along with the antibody capture beads for setting compensation in a multicolor immunology experiment, and found both types of compensation beads used together provide a consistent, accurate, and easy-to-use technique for the setting of flow cytometry compensation.
Assay development and validation of inflammatory markers in impression cytology specimens (144.19)
Authors: Yafawi Rolla , Sace Frederick , Huang Jing-Feng , John-Baptiste Annette
Published: January 2023

The principle of Impression Cytology (IC) utilizes a non-invasive technique to remove superficial layers of the ocular surface. The application of a filter to the surface of the bulbar conjunctiva allows for repeated collection of superficial cells for the analysis of ocular surface disorders. Evidence suggests expression of the HLA-DR antigen on the surface of conjunctival epithelial cells is associated with allergic conjunctivitis and dry eye syndrome. Performance characteristics of HLA-DR was evaluated through intra, inter and stability assays. Evaluation of the reproducibility and overall performance of the novel methodology would be useful in understanding the tolerance level of the assay. Utilizing established techniques, IC specimens were obtained from 12 donors and assayed over multiple days and timepoints. Conjunctival epithelial cells were extracted and assayed based on differential immunostaining of both conjunctival epithelial cells and leukocytes. The reproducibility across donors over time was consistent, demonstrating conjunctival cells maintained their integrity of cell surface expression of HLA-DR. In conclusion, the studies met all validation criteria, and support the utilization of HLA-DR inflammatory measurements in impression cytology specimens. Utilization of a flow cytometric platform to examine the expression of HLA-DR antigen may help predict clinical outcome, evaluate disease progression, and monitor the effects of treatment.
Long-term functional culture of human tonsil lymphocytes in a three-dimensional bioreactor system (144.32)
Authors: Bottaro Andrea , Kuzin Igor , Sun Hongliang , Moshkani Safiekhatoon , Mantalaris Athanasios , Wu JH David
Published: January 2023

Peripheral lymphoid organs are the primary sites of adaptive immune responses, and harbor characteristic anatomical and histological features reflecting separation of cellular subsets (e.g. T and B lymphocytes) and functional compartments. Disruption of lymphoid organ structure by genetic mutations, pharmacological treatments or infection can lead to defective immunity, highlighting the role of tissue organization in immune responses. The generation of in vitro culture systems recapitulating salient features of peripheral lymphoid organs for experimental, biotechnological and clinical applications would be highly desirable, but has so far been hampered by the complexity of these organ systems and has encountered only limited success. We have previously developed a 3D bioreactor system capable of supporting long-term, functional human bone marrow cultures. Here we show that the same system can be adapted for culture of human primary peripheral lymphoid tissue (tonsil) cells for periods up to several weeks in the absence of specific external growth factors or activators. Cells in this system display extended survival, maintain population diversity and phenotypes largely comparable to primary cells, and organize in cellular aggregates with identifiable separation of T and B cell clusters analogous to primary organs. Strikingly, we also show that these cultures are capable of supporting the generation of antibody-producing cells in response to a panel of diverse antigens.
Magnetic bead-based 28-plex immunoassay for the quantification of rhesus and cynomolgus monkey cytokines (144.34)
Authors: Brodey Mary , Liu Nina , Huuskonen Jarkko , Beernink Hans
Published: January 2023

Non-human primates possess high degrees of homology with humans, and thus are crucial model systems in a number of areas of biomedical research (neuroscience, endocrinology, reproductive physiology, cardiovascular physiology, infectious diseases, vaccine and drug efficacy and safety). Monitoring complex cytokine profiles has proven to be invaluable in these various areas. Using the Luminex® xMAP® platform, Life Technologies has developed a 28-plex magnetic bead-based cytokine immunoassay for the analysis of rhesus monkey (Macaca mulatta) and cynomolgus monkey (Macaca fascicularis) samples. Validated sample types include serum, plasma, and conditioned tissue culture medium. Analytes include: EGF, eotaxin, FGF basic, G-CSF, GM-CSF, HGF, IFN-γ, IL-1β, IL-1RA, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12 p40/p70, IL-15, IL-17, I-TAC, MDC, MIF, MIG, MIP-1α, MIP-1β, MCP-1, RANTES, TNF-α, and VEGF. This magnetic-bead based assay is compatible with robotic washing systems, and thus may be fully automated. Total incubation time is 3.5 hours. The utility of the assay is demonstrated by determining cytokine profiles obtained with LPS-stimulated or PMA plus A23187-stimulated peripheral blood mononuclear cells (PBMCs) from rhesus monkeys and cynomolgus monkeys. Linearity of dilution experiments with tissue culture medium and serum produce R2 values of 0.98 and greater, while sample recovery routinely falls between 80 and 120%. Intra-assay and inter-assay precision is ≤10% CV.
Donor Tr1 cells potently expand in vivo and prevent GVHD after TLI/ATS non-myeloablative conditioning and allogeneic bone marrow transplantation (145.17)
Authors: Pillai Asha , van der Merwe Marie , Ong Taren , Vogel Peter , George Tracy
Published: January 2023

Murine total lymphoid irradiation (TLI) + anti-thymocyte serum (ATS) has been well-translated to human transplantation (Lowsky et al, NEJM 2005). Though host TLI/ATS treatment allows post-transplant donor Foxp3+ Treg expansion, >90% of donor CD4+CD25+ cells are Foxp3neg. We now show that the latter are Tr1-type regulatory T cells. BALB/c (H-2d) hosts receiving TLI/ATS + bone marrow + splenocytes (BMT) from C57BL/6 (H-2b) donors had 90% 60-day survival without GVHD (n=10). BALB/c hosts given 800 cGy or 400 cGy total body irradiation (TBI)/ATS+BMT had 100% 30-day GVHD death (n=10). TLI/ATS+BMT hosts had a 10-fold increase in H-2Kb+CD4+CD25+ cell absolute number in day 6 host spleen versus TBI/ATS controls (p< 0.05). Using Foxp3-GFP transgenic donors, >90% of the H-2Kb+CD4+CD25+ cells after TLI/ATS+BMT are Foxp3(GFP)neg. Supernatant assay on day 6 sorted TLI/ATS+BMT H-2Kb+CD4+CD25+, 72-hour PMA/ionomycin stimulated cells revealed potent IL-10, low IL-2, IL-5, and IFN-γ, and no IL-4 or TNF-α (p< 0.05) secretion. Day 6 TLI/ATS+BMT Tr1 donor cells were potent suppressors of donor-host MLR. Donor CD25 cell depletion pre-TLI/ATS+BMT caused day 6 donor CD8+ T cell-mediated GVHD and loss of donor Tr1 cells, indicating that Tr1 cell expansion protects from GVHD post-TLI/ATS+BMT. We are characterizing a host APC-dependent mechanism for this Tr1 expansion. Our findings delineate a novel mechanism and strategy for in vivo Tr1 expansion for tolerance induction and adoptive immunotherapy.
Stem cell mobilization with G-CSF induces Th-17 differentiation and promotes scleroderma (145.19)
Authors: MacDonald Kelli , Olver Stuart , Kuns Rachel , Varelias Antiopi , Raffelt Neil , Don Alistair , Markey Kate , Wilson Yana , Smyth Mark , Iwakura Yoichiro , Tocker Joel , Clouston Andrew , Hill Geoff
Published: January 2023

The clinical shift toward utilizing G-CSF mobilized stem cell grafts has resulted in a striking increase in chronic GVHD after transplantation although the mechanisms involved are unclear. Comparison of cytokine expression following TCR activation of splenocytes from naïve and G-CSF treated B6 or BALB/c donors (low and high responders respectively) showed little effect of G-CSF on Th1 or Th2 cytokine production. In contrast, IL-17 production was dramatically enhanced in response to G-CSF in both strains. G-CSF induced IL-17 production occurred in both CD4 and CD8 conventional T cells and by using relevant knock-out mice or blocking reagents we demonstrated that this was independent of IL-6, TGF-beta or IL-23 signalling. However, the induction of IL-17 by G-CSF was completely dependent on IL-21 signalling and G-CSF induced IL-21 generation in CD4 T cells independent of IL-17 itself. We utilized multiple models of GVHD using G-CSF mobilized B6 or BALB/c wild-type or IL-17 deficient donors, in both MHC matched and mismatched settings. Surprisingly, IL-17 was critical for the induction of sclerodermatous chronic GVHD occurring after transplant using either donor strain. Importantly, IL-17 controlled the dramatic sequestration of macrophages into skin that coincided with the fibrogenic response. This study provides a logical explanation for the propensity of allogeneic stem cell transplantation to invoke sclerodermatous GVHD and suggests a therapeutic strategy for intervention.
Cytotoxic T Lymphocytes (CTL) specific for CMV, adenovirus, and EBV can be generated from naive T cells for adoptive immunotherapy (145.22)
Authors: Hanley Patrick , Cruz Conrad Russell , Savoldo Barbara , Leen Ann , Decker William , Moldren Jeffrey , Liu Hao , Gee Adrian , Demmler Gail , Heslop Helen , Rooney Cliona , Dotti Gianpietro , Brenner Malcolm , Rodgers John , Shpall Elizabeth , Bollard Catherine
Published: January 2023

Adoptive transfer of virus-specific T cells can effectively restore anti-viral immunity after stem cell transplant. However, standard protocols for eliciting virus-specific T cells from adult seropositive donors have been ineffective when applied to cord blood (CB) or virus-naïve adult donors. We tested alternative techniques for eliciting primary T cell responses in vitro. Culturing APC transduced with an Ad5f35pp65 vector and cytokines IL-2,7,12 and 15, we generated T cells specific for multiple viruses from CB and CMV-seronegative (CMVneg) donors. 9 CB-derived T cell lines contained 87% (range 81-94) CD8+ and 26% (12-40) CD4+ T cells. Specificity was determined in 51Cr release and IFN-γ ELISPOT assays against CMVpp65, adenovirus, and EBV targets. Results showed mean spot forming cells (SFC) following incubation with CMVpp65 (209;range 45-694), Adhexon (74;0-128), and EBV (157;23-291) targets. Further, CMVpp65-specific T cells expanded from 7 CMVneg adult donors showed mean 142(38-410) SFC to CMVpp65. CB and CMVneg-derived T cells derived only from naïve (CD45RA+/CCR7+) T cells and recognized "unconventional" CMVpp65 epitopes as identified by overlapping pp65 peptide pools using IFN-γ ELISPOT, suggesting an inherent difference between naïve and memory T cell responses to CMV. Virus-specific T cells can now be generated from naïve sources and could potentially be used clinically in these highest risk patients after transplant.
Identification of a novel immunosuppressive DC subpopulation in tolerized heart allografts (145.23)
Authors: Diao Jun , Zhao Jun , Mikhailova Anastassia , Cattral Mark
Published: January 2023

The role of dendritic cells (DC) in the maintenance of allograft tolerance is unclear. We analyzed the composition and function of DC infiltrates in tolerized murine heart allografts. Methods. B6 donor splenocytes (Day -7) plus anti-CD4 antibody (Day -1, 2 5 and 7) were injected into BALB/c recipients of B6 heart allografts; mice with heart grafts beating > 80 days were considered tolerant. Syngenic heart grafts (B6 to B6) were used as control. Results: We found that graft-infiltrating Lin-CD11c+MHCII+ cDC contained a novel Gr-1+ cDC subpopulation. B220+CD11c+MHCII+ plasmacytoid DC (pDC) were undetectable. In contrast to allograft Gr-1- cDC, which resemble typical immature tissue cDC, Gr-1+ cDC express lower levels of MHC class II and CD11c, adhere to culture plates, exhibit resistance to maturation stimuli, and stimulate allogeneic lymphocytes poorly. Gr-1+ cDC are distributed uniquely in tolerant allografts but not syngenic grafts or acutely rejecting allografts. Gr-1+cDC are absent in lymphoid tissues. Our previous studies have shown that the cytokine milieu of tumors skews pre-cDC differentiation towards Gr-1+ cDC. We found that pre-cDC exist in heart allografts. It remains to be determined whether tolerized hearts promote pre-cDC to differentiate into Gr-1+ cDC in situ. Conclusion: Tolerized allografts contain an immunosuppressive DC subpopulation that may contribute to the maintenance of long-term allograft survival.
Modulation of influenza-specific humoral recall responses by CD154 blockade and cyclosporine A (145.28)
Authors: Azimzadeh Agnes , Schroeder Carsten , Zhang Tianshu , Ramirez-Medina Karina , Avon Chris , Pasetti Marcella , Pierson N.III Richard
Published: January 2023

Background: Current methods of immunosuppression are broadly inhibitory and thus associated with an increased risk of opportunistic infections. Here we compare the effect of CD154 blockade (αCD154) and Cyclosporine A (CsA) therapy on secondary responses to influenza, when the first immunization was performed before therapy, as might be done in transplant wait-listed patients. Methods. Sixteen cynomologus monkeys were immunized with influenza vaccine in absence of immunosupression and administered a secondary boost immunization with either αCD154 mAb (IDEC-131), CsA, or no treatment. Ab responses against three influenza antigens (H1N1, H3N2, B) were measured by ELISA. An average fold increase >1.4 was considered positive. Results. 14 of 16 animals responded to the primary immunization (I) performed in absence of therapy. In untreated controls, 4/5 animals mounted a strong secondary response (II). With αCD154, 4/6 animals showed mild to strong IgM and IgG, and 2/6 demonstrated either IgM or IgG responses. In contrast, in CsA-treated animals, only 2/5 produced IgM without IgG, and 3/5 mounted low titers of IgG but no IgM. Conclusions. Relative to CsA, αCD154 only moderately suppressed secondary Ab responses to flu antigens. This suggests that αCD154 treatment spares important facets of adaptive immunity against opportunistic infections, potentially conferring a clinically significant advantage over conventional immunosuppression with respect to common pathogens
Detection and ex vivo expansion of dormant anti-viral CTL precursors isolated from recipients of unrelated umbilical cord blood transplant (UCBT). (145.31)
Authors: Szabolcs Paul , Marti Luciana , Leen Ann , Baker Jennifer , Janetzky Sivia
Published: January 2023

Two potentially fatal viruses ,CMV and adenovirus can be detected 2-4 weeks after UCBT. Infection may trigger in vivo priming of the infused na ve T cells despite immunosuppressive (IS)medications. We hypothesized that 1) threshold numbers of CTL precursors can be identified that may influence outcome 2) CTL precursors may reside below the level of detection during IS. Methods: Monocytes infected with an adenovirus vector encoding CMV pp65 (Ad5f35pp65) were used as APC in the presence of IL7. Antiviral CTLp was quantitated by IFNg ELISPOT among 1x10e5 responders after overlapping peptide pool stimulation. Results: 8 infected patients at a median age of 9.9 years have been studied before day 100 . Only 1/3 patients with adenovirus exhibited viremia while all 5 patients with CMV had viremia. In freshly drawn blood, 1/8 patients had any detectable reactivity. However, after 9-12 days of stimulation we could enumerate CTLp in 7/8 patients. For adenovirus, the median hexon-specific response was 52 SFC, and the penton-specific CTLp frequency was 13 SFC. CMV pp65-specific response was 6.7 SFC. No responses were found against CMV EI-1 demonstrating critical role for antigenic restimulation. All patients survive beyond 6 months. Conclusion: To our knowledge these are the first data demonstrating that dormant virus-specific CTLp are present already in the first 3 months in infected UCBT recipients. Although these rare events are below detection ex vivo expansion permits quantitation.
High-mobility group box1 contributes to kidney ischemia reperfusion injury through TLR4 signaling (145.38)
Authors: Wu Huiling , Ma Jin , Wang Peng , Wyburn Kate , Chadban Steven
Published: January 2023

Kidney ischemia reperfusion injury (IRI) is TLR4 dependent. HMGB1 is a TLR4 endogenous ligand expressed within IRI kidney. We aimed to determine 1) whether HMGB1 contributes to kidney IRI; 2) whether this is via a direct HMGB1-TLR4 interaction; 3). therapeutic potential of neutralizing antibody to HMGB1 in kidney IRI. Methods: Wild-type (WT) and TLR4-/- mice received anti-HMGB1 Ab, rHMGB1, or appropriate controls by ip injection 1 hour before 22 min ischemia or immediately after reperfusion. Results: Mice pretreated with anti-HMGB1 Ab were protected against kidney IRI, with lower serum creatinine and less tubular damage versus control mice (p<0.05-0.001). Tubulo-interstitial neutrophil (day 1, p<0.001) and macrophage (day 5, p <0.001) infiltration were less in anti-HMGB1 Ab treated mice versus controls. IL6 & TNF-a mRNA levels were reduced in anti-HMGB1 Ab treated IRI kidneys (p<0.05-0.001). Conversely, administration of rHMGB1 post reperfusion exacerbated kidney IRI in WT mice. TLR4-/- mice were protected against kidney IRI, a finding that was unchanged by administration of either anti-HMGB1 Ab or rHMGB1. As with pretreatment, treatment of neutralizing Ab to HMGB1 after reperfusion also protected against kidney IRI. Conclusion: Endogenous HMGB1 is a mediator of kidney damage following IRI, which may operate through the TLR4 pathway. Administration of a neutralizing antibody to HMGB1, either prior to or soon after IRI, afforded significant protection.
STAT1-deficient bone marrow prevents GVHD by modulating plasmacytoid DCs while preserving vaccine-mediated responses to tumor (145.40)
Authors: Capitini Christian , Larabee Shannon , Qin Haiying , Song Young , Khan Javed , Mackall Crystal , Fry Terry
Published: January 2023

A barrier to using donor lymphocyte infusions (DLI) after allogeneic blood and marrow transplantation (alloBMT) is induction of graft versus host disease (GVHD). We have previously demonstrated that gamma interferon (IFNg) receptor-/- bone marrow abrogates GVHD after T cell depleted (TCD) alloBMT, however the mechanism remains elusive. Since the IFNg receptor signals through STAT1, we hypothesized that STAT1-/- bone marrow could also prevent GVHD. Using TCD minor histocompatibility antigen-mismatched alloBMT with delayed DLI and tumor vaccines, we demonstrate that STAT1-/- whole bone marrow protects against a leukemic and solid tumor challenge as efficiently as wild type marrow, but STAT1-/- marrow prevents GVHD-associated weight loss. Using a Cre-Flox system, we generated mice that had STAT1 selectively deleted in either CD11c+ (Dendritic cells, DCs) or lysozyme+ (macrophage/neutrophils) cells, but as marrow donors neither prevented GVHD, implying other STAT1-/- cell subsets are critical. Prior to DLI administration, an increase in splenic plasmacytoid DCs in recipients of STAT1-/- marrow is observed. Microarray analysis of pDCs from recipients of STAT1-/- marrow shows marked upregulation of S100A8 and S100A9, molecules typically associated with myeloid-derived suppressor cells. Taken together, STAT1-/- marrow replicates the effects of IFNg receptor-/- marrow in terms of preventing GVHD and improved efficacy of DLI and vaccines, but also has immunomodulatory effects on pDCs.
TRAF6, a new member of the proximal signaling complex recruited by BAFFR and TACI in B lymphocytes (34.1)
Authors: Hildebrand Joanne , Bishop Gail
Published: January 2023

The cytokines BAFF (B-cell activating factor of the TNF family, BLyS) and APRIL (a proliferation-inducing ligand) activate several major signaling cascades responsible for B-cell survival and homeostasis. Made primarily by myeloid cells, BAFF binds and activates three cell membrane receptors; BCMA (B-cell maturation antigen), TACI (transmembrane activator and CAML interactor), and BAFF-R (BAFF Receptor, BR3), while APRIL binds TACI and BCMA. Studies of genetically altered mice demonstrate that TACI and BCMA perform niche roles in B-cell Ig isotype switching and plasma cell maintenance. In contrast, BAFF-R deletion in mice results in greater than 90% loss of mature B-cells, revealing it as an essential mediator of B-cell maturation and survival beyond the immature transitional (T1) stage. TNF receptor family proteins utilize characteristic combinations of TNF receptor associated factors (TRAFs) to bridge receptor activation with a multitude of downstream signaling pathways. To date, studies of TRAF recruitment by TACI and BAFFR and their roles in signaling have been limited, and TRAF3 was the only TRAF thought to associate with these receptors. Here we present the novel finding that TRAF6 is directly recruited by TACI and BAFFR in B-cells, and that it forms an essential link to the initiation of the canonical NF-kB pathway and other pro survival signals.
Molecular cloning of human IL-16 gene and its expression in Escherichia Coli (34.17)
Authors: Si Chuanping , Li Shuling , Gao Huijie , Fu Jia
Published: January 2023

Objective: To clone human IL-16 gene, and construct its prokaryotic expression vector, and express it in E. coli DH5. Methods: The total RNA was extracted from peripheral blood mononuclear cells stimulated by histamine. And hIL-16 cDNA was amplified by using RT-PCR and cloned into pUC18 T-vector. After the sequence of hIL-16 cDNA was confirmed, the cDNA was inserted into prokaryotic expression vector pMAL-C2. The recombinant expression plasmid pMAL-IL-16 was identified by endonucleases digestion and PCR, and then transformed into E. coli DH5, human IL-16 expressed in DH5 was identified by SDS-PAGE and Western blotting method. Results: Obtained cDNA of hIL-16 was identical with that published on Genebank, and the prokaryotic expression vector pMAL-IL-16 was constructed correctly. Recombinant protein was expressed in E.coli when induced with IPTG and had a molecular weight of 60kD, wich is consistent with theoretical calculation. Conclusion: The hIL-16 cDNA was cloned and the prokaryotic expression plasmid pMAL-IL-16 was constructed successfully. The E. coli DH5 that stably expressed hIL-16 was obtained, which provide experimental basis for the further studying of functional activities and application of hIL-16. [Key words] Human interleukin-16, Molecular cloning, Prokaryotic expression
DBM1285 suppresses LPS-induced TNF-α production by blocking p38 MAPK/MK2 signaling pathway in macrophages (35.6)
Authors: Park Song-Kyu , Choi In Young , Kang Jong Soon , Han Sang-Bae , Lee Chang Woo , Lee Kiho , Kim Hwan Mook
Published: January 2023

TNF-α is a major inflammatory cytokine that plays an important role in the development of various inflammatory diseases. In this study, we report DBM1285 as a novel inhibitor of TNF-α production and a therapeutic candidate for RA treatment. DBM1285 concentration-dependently inhibited lipopolysaccharide (LPS)-induced TNF-α secretion in various cells of macrophage/monocyte lineage, including bone marrow macrophages, THP-1 cells and RAW 264.7 cells. However, LPS-induced mRNA expression of TNF-α was not affected by DBM1285 in these cells. Further studies demonstrated that the inhibitory effect of DBM1285 on TNF-α production might be mediated by post-transcriptional regulation through the modulation of the p38 mitogen activated protein kinase (MAPK)/MAPKAP kinase 2 (MK2) signaling pathway. We also confirmed that DBM1285 directly inhibits p38 MAPK enzymatic activity. In vivo administration of DBM1285 inhibited LPS-induced increase in serum level of TNF-α in mice. DBM1285 also suppressed progression of RA in an adjuvant-induced arthritis model. These results suggest that DBM1285 might be a promising therapeutic candidate for the treatment of RA.