bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of the innate and adaptive immune system in omental anti-tumor immunity (165.31)
Authors: Sedlacek Abigail , Gerber Scott , Lord Edith
Published: January 2023

Tumor metastasis to the peritoneal cavity is often observed in patients with primary ovarian cancer as well as resected colon cancers. Metastatic cells frequently attach to the omentum, a peritoneal adipose tissue that contains many immune aggregates (IA). Interestingly, it is to these IA that the tumor cells initially bind and proliferate. Our lab has been investigating the capability of harnessing these IA to generate productive anti-tumor immune responses using the tumor cell line Colon38, a colon adenocarcinoma syngeneic to C57BL/6 mice. Immunization of mice with lethally irradiated Colon38 cells grown in serum-free media results in a marked increase in the number of CD8 T cells as well as their expression of activation markers. Challenge of the mice with live Colon38 14 days after immunization results in no observable tumor growth on the omentum and rechallenge experiments also yield no tumor growth. Unexpectedly, when we challenge mice with an alternate tumor cell line, B16.F0, we also observe no tumor growth on the omentum. These results have been repeated in the BALB/c strain of mice using syngeneic tumor cell lines. Our data not only highlight the uniqueness of the peritoneal cavity, but also suggest that this immunization strategy generates an initial non-specific anti-tumor response that likely involves components of the innate immune system as well as a long-term tumor-specific response involving CD8 T cells.
Aminophylline inhibits breast and bladder tumor growth in mice due to CXCR3-dependent adenosine A2B receptor blockade (165.32)
Authors: Cekic Caglar , Sag Duygu , Linden Joel
Published: January 2023

The activation of adenosine A2A or A2B receptors on immune cells inhibits their ability to suppress tumor growth. In this study, we evaluated the anti-tumor effects of aminophyllilne (AMO) a nonselective antagonist of A2A and A2B receptors that is used clinically to treat asthma. Intra-tumor injection of AMO inhibited the growth of syngeneic MB49 bladder cells subcutaneously injected into C57BL/6 mice and the growth and lung metastasis of luminescent syngeneic 4T1 breast tumor cells inoculated into the mammary fat pats of Balb/c mice. Although AMO inhibited the growth of MB49 in A2AR-/- mice it failed to do so in Rag1-/-, A2BR-/- or CXCR3-/- mice. The selective deletion of the A2BR or CXCR3 from bone marrow cells also blocked tumor suppression by AMO. Transcript levels of interferon-γ and CXCL10, and activation of CD4+ and CD8+ T cells, were increased in tumors treated with AMO, while numbers of CD34+/CD45- endothelial cell precursors were decreased. We conclude that: 1) the anti-tumor effects of non-selective adenosine receptor blockade by AMO are primarily mediated by the A2BR; 2) CXCR3 receptors are required for anti-tumor effects of AMO and influence both T cell trafficking and chemokine-mediated angiogenesis; and 3) AMO may be useful for proof-of-principle studies to determine the efficacy of adenosine receptor antagonists as tumor suppressors alone or in conjunction with tumor vaccines.
Molecular regulation of BCL2 to IGH translocation in follicular lymphoma (165.38)
Authors: Bai Jirong , Lange Miles , Xie Wanqin , Hong Song Yang , Huang Lin , Jones Dallas , Yu Zhihong , Ti He , Su Kaihong , Swanson Patrick , McKeithan Timothy , Chan John , Zhang Zhixin , Kai Fu
Published: January 2023

The hallmark of Follicular lymphoma (FL) is the aberrant translocation t(14; 18)(q32; q21) involving the IgH locus on chromosome 14 and BCL2 gene on chromosome 18. Previous analysis showed that approximately 70% of the BCL2/IgH translocations involve the BCL2 major break point region, known as the BCL2-MBR. Accumulating studies suggested that the BCL2-MBR to IgH translocation may be mechanistically similar to IgH DH to JH or VH to DJH recombination. Our analysis of 42 BCL2/IgH break points showed that 60% of them contain clearly identifiable DH genes, suggesting their origin from VH to DJH recombination. Our recent studies showed that the B lineage specific transcriptional factor Pax5 activates IgH VH to DJH recombination through binding to VH gene coding regions and recruiting RAG complexes. Based on this finding, we hypothesize that Pax5 also controls BCL2 to IgH chromosomal translocations. Our initial sequence analyses identified multiple potential Pax5 binding sites within the 150 bp BCL2-MBR. Electrophoresis Mobility Shift Assay (EMSA) demonstrated that Pax5 binds to the individual Pax5 binding sites as well as the full length BCL2-MBR. The BCL2-MBR displays Pax5-dependent enhancer activity when artificially placed in front of a minimal CMV promoter driven luciferase reporter gene. We also found that purified GST-RAG1 and RAG2 core proteins bind to and cleave at the BCL2-MBR. We speculate that Pax5 binding to the BCL2 MBR may enhance BCL2 to IgH recombination.
Epigenetic memory: tolerant T cells cannot escape their fate (165.40)
Authors: Schietinger Andrea , Delrow Jeffrey , Greenberg Philip
Published: January 2023

T-cell tolerance to self antigens is required to prevent autoimmunity. Since most tumor antigens are self antigens a critical challenge is to develop strategies that break T-cell tolerance to such antigens without causing autoimmune injury. A hallmark of tolerant T-cells is the failure to respond to antigen, but the precise regulatory mechanisms remain largely unknown. Previously we showed that strategies which “force” tolerant T-cells to proliferate abrogate tolerance. To understand the mechanisms by which repetitive cell division rescues tolerant T-cells, we studied the effect of homeostasis-driven proliferation (HP) on tolerant T cells. Following adoptive transfer into lymphopenic hosts, tolerant T cells became functional, even in a tolerogenic environment. However, T-cell rescue was only transient and tolerance was re-imposed after the host became lymphoreplete, even if no self antigen was present. The finding that rescued T-cells reacquired tolerance in non-tolerogenic hosts challenges the prevailing paradigm that tolerance maintenance requires continuous exposure of T-cells to self antigen. Instead, mRNA and microRNA profiling revealed that tolerant T-cells harbor a tolerance-specific molecular signature; while the programming can be temporarily overridden, it is eventually reasserted, dictated by an “epigenetic memory”. These studies on the regulation of T-cell tolerance have revealed novel targets for cancer immunotherapy and the treatment of autoimmune disease.
Arginase-I is expressed by ckit+IL-7Ra+ populations in cryptopatches (166.11)
Authors: Bando Jennifer , Liang Hong-Erh , Locksley Richard
Published: January 2023

Arginase-I is expressed by alternatively activated macrophages during type-2 inflammation and is strongly induced by the cytokines IL-4 and IL-13. In order to track arginase-I expression, our lab developed a YFP-arginase-I (Yarg) reporter mouse. In the naive Yarg mouse, we detected arginase-I-expressing cells that were CD45+ckit+IL-7Ra+, but lacked the common myeloid and lymphoid cell surface markers CD11b, CD11c, Gr-1, B220, CD3, and NK1.1. In the small intestine, these cells were found in cryptopatches and expressed RORγt and LTβ RNA, consistent with a lymphoid tissue inducer (LTI) phenotype. Indeed, we found Yarg+RORγt+ cells that were present in adult and one-day-old neonatal mouse small intestine. Interestingly, we also identified Yarg+RORγt- cells that were located in cryptopatches and were still present in the lamina propria of RORγt-deficient mice. These ckit+IL-7Ra+ subsets were the primary Yarg+CD45+ cells in several organs. Thus, ckit+IL-7Ra+ cells are a previously unappreciated arginase-I-expressing population in naive mice.
Differential activation of cytokine signaling pathways in the colon of Crohn’s disease and ulcerative colitis patients. (166.12)
Authors: Christophi George , Rong Rong , Holtzapple Philip , Massa Paul , Landas Steve
Published: January 2023

Cytokine signaling pathways play a central role in the pathogenesis of inflammatory bowel disease (IBD). Numerous clinical studies have shown that several inflammatory cytokines are elevated in active IBD and recently genome-wide association studies further confirmed and advanced our knowledge of intricate immune pathways that confer susceptibility to IBD. This study aims to delineate the cytokine signaling pathways in colonic mucosa of CD and UC patients, explore possible molecular markers for IBD diagnosis, and identify novel immune-mediators of IBD pathogenesis. Based on established clinical diagnosis and strict histopathological criteria, 93 colon biopsy samples were from severe/active UC or CD, quiescent UC or CD, and normal control subjects. Gene expression is analyzed by quantitative real-time RT-PCR. This study analyzes the expression of more than 30 genes, including cytokines, signaling molecules, and downstream inflammatory mediators. Cytokines like IL-17, IL-6 and IL-33 and transcription factors like T-bet, GATA-3, Foxp3are differentially regulated between UC and CD and among disease severity. These data provide insights into the pathogenesis of IBD though identifying novel immune-mediators and elucidating distinct signaling networks in CD and UC. Finally, several biologic agents are novel promising IBD treatments and this study provides an easy approach that can be used to identify molecular markers that can predict the response to those individual treatments.
HSP70-mediated antigen-dependent nonclassical MHC class Ib-restricted anti-tumor responses in the frog Xenopus laevis (170.15)
Authors: Nedelkovska Hristina , Robert Jacques
Published: January 2023

The heat shock proteins (hsps) hsp70 are highly conserved molecular chaperones that elicit potent T cell responses against the antigens (Ags) they chaperone in both mammals and Xenopus. We have shown that frogs immunized with hsp70 generate CD8 T cell responses against the Xenopus thymic tumor 15/0 that expresses several nonclassical MHC class Ib genes, but not classical MHC class Ia. Therefore, we hypothesized that hsp70 can prime class Ib-mediated anti-tumor unconventional CD8 T cells in an Ag-dependent manner. To test this, we produced Xenopus recombinant tagged hsp70 proteins (both the cognate hsc73 and the inducible hsp72) from stable 15/0 tumor transfectants. Antigenic peptides can be easily removed from both hsps by ATP treatment. We used a cross-presentation assay to adoptively transfer hsp-pulsed antigen presenting cells and showed that both hsp72 and hsc73-Ag complexes, but not ATP treated Ag-negative hsp70s, elicit class Ib-mediated CD8 T cells responses resulting in protection from 15/0 tumor challenge. From expression studies, we further postulate that these responses involve one or a few class Ib gene products among which XNC10 is the prime candidate due to its lymphoid tissue distribution. To further elucidate the role of class Ib molecules in anti-tumor immunity, we developed a reverse genetic approach combining transgenesis and siRNA technology and obtained F1 Xenopus clones with silenced expression of class Ib in vivo that will be used for further analysis.
Optimization of immunoassays to monitor canine immune system (170.19)
Authors: Delamere Patricia , Nadwodny Kimberly , DeVinney Brooke , Fuller Claudette
Published: January 2023

Drug related changes to lymphocyte populations and immune functions can indicate immunotoxicity in many species, including the dog. Immunoassays are routinely performed in human, monkey, and rodent samples, but few validated immunoassays specific for the dog exist. Here, we present five immunoassays optimized for functional and phenotypic analysis of canine adoptive and innate immunity. These assays were evaluated in canine peripheral blood and/or tissue to characterize immunomodulation effects and pharmacodynamics of stimuli and/or pharmaceuticals.
Effect of larval thymectomy on T-cell development and immunoglobulin class switch in Xenopus laevis (170.5)
Authors: Mashoof Sara , Du Christina , Criscitiello Michael
Published: January 2023

The gut associate lymphoid tissues (GALT) are the digestive tract’s immune system and play a crucial role in the evolution of the vertebrate adaptive immune system. Mammalian immunity depends on antibody production by B cells with T cell help. Our knowledge about adaptive immunity in cold blooded vertebrates is fragmentary. The frog Xenopus laevis is an ideal model for such studies. Amphibians were the first animals to evolve Ig isotype class switching. It is unclear if T cell help is phylogenetically more fundamental or class switch recombination. To determine whether T cell help is required for gut-specific IgX induction (assumed analogue of mammalian IgA) in X.laevis we need a model with no T -cells. We thymectomize 10-12 day tadpoles which provide us with a useful T -cell deficient model. Thymectomized tadpoles were monitored 15-20 days after surgery to check for any regrowth of thymus. TCR reverse transcriptase PCR was performed from peripheral blood of cleanly thymectomized animals. PCR results show there are no TCR α or γ mRNA transcripts in thymectomized frogs indicating that T-cell development is fully thymus dependent. Thymectomized animals validated by PCR will be orally immunized to test whether it will yield gut specific IgX response, and if IgX is T-dependent. Our preliminary experiments show higher induction of IgX in orally immunized animals with competent thymus.
On the trail of regulatory T cells in blood and tissues of Mycobacterium paratuberculosis infected cattle (170.8)
Authors: Coussens Paul , Murphy Brooke , Sipkovsky Sue , Colvin Chris , Roussey Jon
Published: January 2023

Based on previous observations, it is proposed that induced regulatory T cells (Tregs) form an important aspect of immune responses to Mycobacterium avium subspecies paratuberculosis (MAP) in cattle. Tregs are typically CD4+CD25+ and express the FoxP3 transcription factor. Removal of CD25+ T cells from peripheral blood mononuclear cells (PBMCs) isolated from MAP infected cattle prior to stimulation with MAP antigens enhances production of IFNγ mRNA and reduces production of IL-10 mRNA from remaining cells. To more definitively identify antigen-specific Tregs in MAP infected cattle, we have begun to analyze cell populations using multi-color flow cytometry and intracellular staining. Tregs in tissues have been visualized using multi-color confocal microscopy and immunohistochemical staining. As expected, stimulation of PBMCs from infected cattle with MAP enhances the percent CD3+ cells that are also CD25+ (25%, +/-2.8%), relative to nil stimulated cells (6.4%, +/- 3.8%). Expression of the FoxP3 transcription factor is more variable with some animals showing up regulation following stimulation with MAP (20%-50% of CD25+ cells) relative to nil stimulation (~6%). Thus, in some cattle, CD25+FoxP3+ cells do indeed form a significant proportion of cells responding to MAP antigen stimulation. FoxP3 positive cells are also a significant feature in many lesions associated with MAP infection, although it is not yet known if these cells are antigen specific.
T1D-derived osteoclasts display defective LPS-tolerance (44.1)
Authors: Catalfamo Dana , Neiva Kathleen , Calderon Nadia , Wallet Shannon
Published: January 2023

Bone and joint abnormalities are frequent co-morbidities of type 1 diabetes (T1D). The complexity of T1D itself and added complexity of bone and joint co-morbidities necessitates well-controlled and innovative approaches to assess the totality of potential causes for these co-morbidities. Objective: To elucidate osteoclast-specific mechanisms responsible for increased inflammation induced bone resorption in T1D by evaluating the phenotype and activation of T1D osteoclasts (OCs). Methods: Bone marrow-derived OCs from a T1D mouse model (NOD) and controls (NOR and C57BL/6) were evaluated for their ability for form resorption pits with SEM. Luminex was used to evaluate cyto/chemokine secretion to determine activational status and FACS analysis of calcitonin receptor (CR), integrin beta3, RANK and IL-10R was used to determine phenotype. All experiments were performed in the presence or absence of TLR stimulation. Results: NOD-derived OCs resorb more bone than controls in the presence of LPS while control OCs display LPS-induced tolerance. T1D-derived OCs also secrete higher levels of osteoclastogenic mediators and show increased RANK yet decreased CR expression compared to controls. Conclusions: These data suggest that NOD-derived OCs display defective LPS-tolerance when compared to controls which may be due to defects in osteoclastic regulatory mechanisms.
Secondary light chain gene rearrangements in the generation of anti-DNA autoantibodies in the chronic graft-versus-host model of SLE (44.15)
Authors: Eisenberg Robert , Choudhury Arpita , Luning Prak Eline , Schlissel Mark
Published: January 2023

The loss of B-cell tolerance to nuclear autoantigens is central to the pathogenesis of SLE, but it is not known which tolerance checkpoints fail in the production of anti-DNA autoantibodies. We have utilized the chronic GVH model of SLE to target the loss of tolerance to mature, splenic B cells (JI 179, 5564, 2007). We have now extended these studies to show that the production of these autoantibodies involves the switching of κ-chain alleles in mature B cells. We have distinguished the two κ alleles by utilizing a genetic locus in which the human κ constant region has replaced the mouse κ constant region. Either pure human κ+ CD93- splenic B cells or pure mouse κ+ CD93- splenic B cells, isolated from C57BL/6.huκ/moκ heterozygous mice, transferred to rag1-/- recipients and challenged with CD4+ bm12 T cells, produced both moκ+ and huκ+ anti-DNA and anti-chromatin autoantibodies. Even huκ+ cells that have their moκ locus in the germ line configuration produced moκ+ autoantibodies in this cGVH transfer system. These data suggest that the creation of lupus autoantibodies can occur at least in part by the acquisition of a new immunoglobulin receptor in mature B cells through peripheral secondary light chain genetic recombination (receptor revision). We postulate that abnormal T cell help is required at that stage either to initiate receptor revision and/or to permit the survival and activation of the B cells with their newly acquired autoreactivity.
Contribution of VH replacement products to the generation of autoantibodies in systemic lupus erythematosus (44.19)
Authors: Yu Yangsheng , Fan Run , Wang Zhixin , Yue Yinshi , Huang Lin , Li Song , Su Kaihong , Zhang Zhixin
Published: January 2023

Systemic lupus erythematosus (SLE) is characterized by the over production of high affinity autoantibodies that play important roles in SLE pathogenesis. However, it is not clear how these antibodies are generated and selected during the course of lupus. Our sequence analysis show that VH replacement products are highly enriched in IgH genes derived from different autoimmune diseases, including SLE. To gain insight into the mechanisms responsible for autoantibody generation in SLE, we performed single cell PCR analysis to study the functional antibody repertoires in active SLE patients. Analysis of 102 recombinant antibodies derived from the naive B cells and plasmablasts of the SLE patients show that 4.8% of the antibodies derived from naïve B cells and 26.7% of the antibodies derived from plasmablasts are autoreactive. Detailed sequence analysis reveal that in the plasmablasts of two SLE patients, VH replacement products contribute to more than 30% of IgH genes. These VH replacement products have long CDR3 regions enriched with charged amino acids, and more than 60% of them encode anti-nuclear or poly-reactive autoantibodies. These studies provide the first evidence that VH replacement products directly contribute to the generation of autoantibodies in SLE.
Excess B cell CD40/CD40L signaling promotes CD4 T cell-mediated encephalomyelitis in mice (44.22)
Authors: Shimoda Michiko , Bolduc Anna , Takezaki Mayuko , Tsubata Takeshi , Koni Pandelakis
Published: January 2023

To test the pathogenic role of B cells under excess inflammatory CD40/CD40L signaling, experimental autoimmune encephalomyelitis (EAE) was induced in B cell specific CD40L transgenic (CD40LBTg) and C57BL/6 control mice by immunization with MOG35-55 peptide. Clinical symptoms were monitored using a 0-5 scoring system during days 10-30 after induction. Alternatively, MOG-specific CD4 TCR Tg (2D2) mice were crossed onto a CD40LBTg background and the incidence of spontaneous encephalomyelitis monitored at 4-35 weeks of age. Both CD40LBTg and control mice developed EAE with similar kinetics, with disease onset at around day 15-17 and the peak of disease score at around day 21-25. However, CD40LBTg mice showed significantly more severe disease than wild type mice with average peak clinical scores of 3.0 and 1.5, respectively. Higher frequencies of IL-17+ CD4 T cells were also observed in CD40LBTg mice compared to wild type mice at the EAE disease peak. Also, 60% (6/10) of 2D2 CD40LB double Tg mice showed spontaneous encephalomyelitis-like symptoms (severe hind and/or fore limb paralysis) between 4-6 weeks of age, with IL-17+ and IFNγ+ CD4 T cells in spleen and inguinal lymph nodes. Furthermore, even those without apparent EAE symptoms died before 30 weeks of age. None of the 2D2 or CD40LBTg mice showed such symptoms between 4-35 weeks of age. These results support the notion that excess B cell CD40/CD40L signaling promotes MOG-specific pathogenic CD4 T cell response.
Automated analysis of multi-dimensional B-cell flow cytometry data (44.23)
Authors: Qian Yu , Wei Chungwen , Lee Eun-Hyung , Cambell John , Kaminski Denise , Kong Megan , Liu Yue , Dougall David , Sanz Ignacio , Scheuermann Richard
Published: January 2023

Understanding B cell diversity and its functional correlates requires the combined study of large numbers of surface and intracellular molecules. Advances in multi-parameter flow cytometry (FCM) allow for the independent detection of many fluorochromes on individual cells. However, traditional gating strategies based on single or two-color displays are inadequate for separating cell populations defined in multi-dimensional space. We have developed a novel program, FLOCK, which uses density-based clustering to algorithmically identify cell subsets in multi-dimensional space in an unbiased fashion, reducing operator variability. FLOCK has been used to accurately identify and quantify 17 distinct B cell subsets in human peripheral blood, including novel plasmablast subsets after tetanus vaccination. It is being used to monitor B cell subsets in different autoimmune conditions including systemic lupus erythematosus patients treated with BAFF inhibitors. FLOCK has been assessed by the FlowCAP consortium (Critical Assessment of Population Identification Methods), and achieved encouraging results in a recent FlowCAP competition compared with other automated methods. FLOCK has been implemented in the publically available Immunology Database and Analysis Portal - ImmPort (www.immport.org) for open use by the immunology research community. Core modules of FLOCK will also be integrated into GenePattern of the Broad Institute as a component of the FCM analysis suite.
Interferon alpha causes SLE by expanding CD4- CD8- double negative T cell (44.25)
Authors: Honda Eriko , Akiyama Chieri , Hashiramoto Akira , Shiozawa Shunichi
Published: January 2023

Objective: It has been hypothesized that high interferon alpha (IFNα) levels cause systemic lupus erythematosus (SLE), however, the direct proof for this is lacking. We have shown that sole increase of interferon alpha (IFNα) in doxycycline (Dox)-inducible transgenic (IFNα Tg) mice induces production of anti-dsDNA antibody and renal disease akin to human SLE. In this study, we have investigated how IFNα functions and to what extent IFNα operates in the pathogenesis of SLE. Methods: We made C57BL6/FVBN TetOp-mIFNα/EμSR-tTA transgenic female mice in which IFNα was expressed after the cessation of Dox. We transferred CD4+ (5x106), CD8+ (5x106) and CD4- CD8- double negative (DN) T (3x105) subsets derived from 30 weeks Dox-off IFNα Tg mice into naive recipient mice twice. In additional experiment, CD4+ (8x106), CD8+ (8x106) and DN T (9x105) subsets were transferred. Anti-dsDNA antibody, serum IC, proteinuria and renal lesion of recipient mice were evaluated. Results: Transfer of DN T (3x105) subset of Dox-off IFNα Tg mice induced IC-mediated mesangio-proliferative glomerulonephritis. However, there were no increases in anti-dsDNA antibody, serum IC nor proteinuria after the transfer of DN T (9x105) subset. It was, however, noted that alopecia, was developed by transfer of DN T (9x105) subset. Conclusion: DN T cell is responsible for the induction of lupus glomerulonephritis and alopecia.
DDE and diet-induced obesity alter thymocyte and splenocyte immune cell populations and renal expression of osteopontin and MCP-1 in a murine model (44.31)
Authors: Viselli Susan , McKenna Ryan , Hillhouse David
Published: January 2023

DDE, a metabolic breakdown product of the pesticide DDT, accumulates in adipose tissue and acts as an anti-androgen. Since androgens can protect against autoimmuity, DDE may impact autoimmmune developement. We predicted greater effects of DDE in mice with more adipose tissue. Male C57BL/6J mice were fed either a high-fat (60 kcal% fat) or control low-fat(10 kcal% fat) diet from weaning. At 8 weeks, mice were treated with DDE (200 mg/kg body wt)or vehicle by oral gavage. Mice were sacrificed at 10 weeks or 6 months. Body weights were at least 50% greater in high-fat diet mice. Total spleen and thymus cell numbers were not altered by diet or DDE. Fewer mature thymus T cells (CD3+) were found in the DDE high fat group compared with the DDE low fat group (25.22 +/- 2.76 % vs. 47.55 +/- 3.69%, p<0.05) at 10 weeks. High fat diet, independent of DDE treatment resulted in more CD8+ single-positive thymocytes in high fat controls compared with low fat controls (2.04 +/- 0.9 % vs. 1.13 +/- 0.13%, p<0.05) at 10 weeks and more CD4+ splenocytes in high fat compared with low fat controls (25.63 +/- 2.67% vs. 16.30 +/- 0.79%, p<0.05) at 6 months. DDE caused a 10-15% increase in lymphocytic infiltrates in kidney glomeruli regardless of diet. High fat diet and DDE treatment increased renal expression of MCP-1 while DDE treatment increased OPN expression. In conclusion, diet and obesity influence some effects of DDE on the immune system. Study of additional immune parameters is warranted.
BLyS rescues RF producing B cells from activation induced death (44.34)
Authors: Oropallo Michael , Kiefer Kerstin , Marshak-Rothstein Ann , Cancro Michael
Published: January 2023

Rheumatoid factor (RF) autoantibodies are associated with Systemic Lupus Erythematosus and other autoimmune disorders. Previous research using AM14 mice, which have a transgene encoding a B cell antigen receptor (BCR) typical of pathogenic RF producing B cells, has demonstrated that these cells are induced to divide by chromatin immune complexes (chromatin-ICs). This work revealed a combined requisite for BCR and Toll-like receptor (TLR) signaling in the onset of RF production., We have recently found that although AM14 B cells stimulated with chromatin-ICs indeed divide, they die rapidly within 48-72 hr of the initial stimulus; by contrast, anti-IgM, CpG, or anti-IgM+CpG stimulated cells continue to proliferate within this time frame. Importantly, we have found that the cytokine BLyS (B lymphocyte stimulator) rescues survival of AM14 B cells proliferating in response to chromatin-ICs. We believe our observations may link the immune complex mediated activation of RF producing B cells with the finding that elevated BLyS levels are often seen in SLE. The ability of BLyS to rescue AM14 B cells may involve signaling through either TACI or BR3, as we have found that both of these BLyS binding receptors are upregulated after chromatin-IC stimulation. Finally, we have begun to examine the apoptotic pathway downstream of chromatin-IC stimulation, and have found that it is cell intrinsic and involves caspases 9, 3, and PARP.
Role of bone marrow-derived cells and inflammation in spontaneous cardiac dystrophic calcification (44.36)
Authors: Glass Aaron , Darwish Nedal , Taffet Steven
Published: January 2023

Dystrophic calcification (DC), or extra-osseous deposition of calcium phosphate, is associated with numerous cardiovascular diseases ranging from acute conditions such as myocardial infarction to chronic conditions including atherosclerosis. Development of DC in mouse models is multifactorial, with diet, age and genotype each contributing to the disease phenotype. Although DC has been characterized in a several mouse strains, the relationship between inflammation and DC has not been addressed and no autoimmune component has been identified. We identified a substrain of BALB/c mice (BALB/cByJ) that spontaneously develop epicardial DC lesions containing focal deposits of calcium and infiltrating mononuclear cells. Bone marrow chimera experiments using GFP-expressing donor bone marrow revealed that the lesions are strongly GFP positive, suggesting an inflammatory component of the disease. In particular, there was substantial GFP positive cell infiltration at the site of calcinosis. A characterization of the infiltrating cells by immunohistochemistry confirmed the presence of immune cells. Initiation of experimental autoimmune myocarditis in these animals led to the formation of a more pronounced lesion, where the calcinosis appeared to serve as a focus of the myocarditis. Further study of this model may help elucidate the dynamic interplay between inflammation and calcification in cardiovascular disease.
Antigenic targets in Aire-mediated ovarian autoimmunity (44.41)
Authors: Cheng Mickie , Fan Una , Johannes Kellsey , Anderson Mark
Published: January 2023

Ovarian autoimmunity is a poorly understood cause of premature ovarian failure and infertility among women. Diagnosis of autoimmune ovarian disease (AOD) is challenging due to the lack of specific and sensitive autoantibody tests, and the disease pathogenesis and antigens targeted in AOD remain largely unknown. We have established a novel mouse model of AOD using the Aire-deficient mouse. Like APS1 patients that lack AIRE, Aire-deficient mice develop disease of multiple organs, including the ovary, due to a breakdown in central tolerance. Aire-deficient female mice develop autoimmune oophoritis manifested by 100% incidence of histologic disease and frequent oligoclonal autoantibodies to ovarian tissue, presenting a valuable spontaneous disease model in which to identify ovarian antigens. Initial investigations have identified putative antigenic targets, including a 60 kd ovarian antigen that is targeted early in AOD. Analysis of ovarian infiltrates in Aire-deficient females indicates a central role for Th1 CD4+ T cells in mediating disease. Similar autoantibody profiles and immune infiltrates have also been reported in women with premature ovarian failure, suggesting that common ovarian antigens may be shared. Understanding of antigens and disease pathogenesis in Aire-mediated AOD will define the mechanism by which tolerance is broken in the ovary and will have potential for translation to patients in a significant cause of human ovarian failure and infertility.
Autoreactive Tbet-positive CD4 T cells develop independent of classic Th1 cytokine signaling during EAE (44.45)
Authors: Yeh Wen-I , Harrington Laurie
Published: January 2023

Many autoimmune diseases are associated with the presence of Th1 and Th17 effector CD4 T cells. 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 during autoimmunity. Following the onset of experimental autoimmune encephalomyelitis (EAE), we observed a preferential upregulation of Tbet by CD4 T cells within the CNS, but not the secondary lymphoid organs. These Tbet-positive CD4 T cells were capable of producing the cytokine IFNγ, and a proportion of these cells produced both IFNγ and IL-17A. Interestingly, these Tbet-positive cells were present in high frequencies during disease in IFNγ-deficient mice. Moreover, we found that CD4 T cells from IFNγ-deficient/IFNγ reporter mice upregulated the Thy1.1 reporter, indicating the presence of Th1 or “Th1-like”, Tbet-positive CD4 T cells even in the absence of the cardinal Th1 cytokine, IFNγ. These data support the concept that Th1 cells contribute to EAE in a Tbet-dependent, IFNγ-independent manner. We further examined the requirement of other Th1-associated molecules in controlling Tbet expression during EAE, and noted that STAT1, IL-12, and IFNγ were dispensable for the induction of Tbet in vivo. Hence, this study highlights the complex regulation of Tbet and the potential unrecognized role for Th1 cells during autoimmunity.
Activating transcription factor 3 promotes sublytic C5b-9-induced glomerular mesangial cells apoptosis through up-regulation of Gadd45α and KLF6 (44.5)
Authors: Wang Yingwei , Xu Kuanfeng , Zhou Ying , Liu Xin , Qiu Wen
Published: January 2023

The sublytic C5b-9 complexes can result in glomerular mesangial cells (GMCs) apoptosis, which involved in the initiation and development of rat Thy-1 nephritis, but the mechanism of sublytic C5b-9-mediated GMCs apoptosis remains unclear. Activating transcription factor 3 (ATF3) is an immediate early gene for cells to cope with a variety of stress signals, and our previous study revealed that ATF3 could promote GMCs apoptosis attacked by sublytic C5b-9. However, target genes of ATF3 in GMCs apoptosis triggered by sublytic C5b-9 stimulation have not been identified. In this study, the data showed that the expression of ATF3, growth arrest and DNA damage-45 alpha (Gadd45α), Krüppel-like factor 6 (KLF6) and proliferating cell nuclear antigen (PCNA) in the GMCs induced by sublytic C5b-9 for the indicated time was significantly increased, and ATF3 expression could lead to GMCs apoptosis through up-regulation of Gadd45α and KLF6, but not up-regulation of PCNA. Furthermore, Gadd45α was identified as a downstream target gene regulated by ATF3 directly, and KLF6 might be regulated by ATF3 in an indirect manner.
A critical role of B cells in biliary disease and sialadenitis in the NOD.c3c4 model of autoimmune cholangitis. (44.8)
Authors: Moritoki Yuki , Tsuda Masanobu , Tsuneyama Koichi , Zhang Weici , Yoshida Katsunori , Lian Zhe-Xiong , Yang Guo-Xiang , Ridgway William M. , Wicker Linda S. , Ansari Aftab A. , Gershwin M. Eric
Published: January 2023

The role of B cells in the pathogenesis of PBC in humans has been a controversial problem. On the one hand, the serologic hallmark of PBC are the presence of antimitochondrial antibodies, found in >90% of patients. However, the antibodies do not correlate with disease severity and the titers do not change following therapeutic orthotopic liver transplantation. In addition, related work on a murine model of PBC, the dnTGFβRII mice, suggests that regulatory B cells may be an important contributor to disease pathogenesis. Indeed, the absence of B cells in this model exacerbates biliary pathology. To address this problem, we have taken advantage of the availability of both dnTGFβRII mice and NOD.c3c4 mice, to study the role of B cells in the natural history of disease. In particular, we generated genetically B cell deficient (Igμ-/-) NOD.c3c4 mice and compared the immunopathology of these mice to control B cell sufficient (Igμ+/+) NOD.c3c4 mice. Igμ-/- NOD.c3c4 mice not only had an amelioration of salivary gland inflammation, but also reduced numbers of inflammatory liver infiltrates, ameliorated liver inflammation, and a significantly lower prevalence of biliary cyst formation. B cell deficient mice demonstrated decreased number of non-B cells in the liver accompanied by reduced numbers of activated natural killer cells. In conclusion, B cells play a critical role in promoting liver inflammation and cyst formation as well as salivary gland pathology in autoimmune NOD.c3c4 mice.
FCRLA is an intracellular FcR-related protein that binds IgM, IgG and IgA isotypes in B cells (45.11)
Authors: Burrows Peter , Santiago Teresa , Kulemzin Sergei , Reshetnikova Evdokia , Chikaev Nikolai , Volkova Olga , Mechetina Ludmila , Zhao Meina , Davis Randall , Taranin Alexander , Najakshin Alexander , Hendershot Linda
Published: January 2023

Fc-receptor-like A (FCRLA) is a novel member of the extended Fc receptor (FcR) family. Unlike the classical FcRs and Fc receptor-like (FCRL) molecules, which are typically transmembrane receptors with extracellular ligand binding domains, FCRLA has no predicted transmembrane domain or N-linked glycosylation sites and is positioned intracellularly. We show by confocal microscopy and biochemical assays that FCRLA is a soluble, resident ER protein; however, it lacks a characteristic KDEL retention sequence at its C-terminus. Using a series of deletion mutants, we found that its ER retention is most likely mediated by the amino terminal domain. We have also recently identified ER localized immunoglobulin as an FCRLA ligand. These results indicate that FCRLA is unique among the large FcR family in that can associate with multiple immunoglobulin isotypes, including IgM, IgG, and IgA. To better understand the role of this protein in human disease, B cell chronic lymphocytic leukemia (CLL) samples were assayed for FCRLA expression by flow cytometry. Interestingly, the receptor was more highly expressed by the indolent form of CLL, which is distinguished by somatically hypermutated heavy chain variable region genes, than by the aggressive high-risk form, which features more germline B cell receptor characteristics. These results indicate that FCRLA may thus be a useful diagnostic marker for CLL.
Fast, easy and column-free procedure for the isolation of human memory B Cells (45.14)
Authors: Fairhurst Maureen , Yu Jessie , Thomas Terry
Published: January 2023

Human memory B cells have the unique ability to rapidly proliferate and differentiate when re-exposed to the same antigen. They can be distinguished from naïve B cells by the presence of somatic hypermutations in their Ig-V region sequences. CD27 is widely used as a marker of memory B cells because its expression correlates with the presence of such mutations. Several CD27+ memory subsets collectively make up 20-60% of peripheral B cells. Using a two-step EasySep™ method, memory CD27+ B cells are isolated from fresh or previously frozen peripheral blood nucleated cells. First, non-B cells are targeted for depletion with dextran-coated magnetic particles using a cocktail of tetrameric antibody complexes (TAC). Labeled cells are separated in an EasySep™ magnet without the use of columns and pre-enriched, unlabeled B cells are collected. Next, CD27+ B cells are selected from the pre-enriched fraction using TAC recognizing CD27 and dextran-coated magnetic particles. Labeled cells are separated and remain in the tube in the magnet while unwanted cells are poured off. The unwanted cell fraction may be used to obtain naïve B cells. The selection steps can be fully automated using RoboSep™. Starting with a frequency of 4.1 ± 1.2% CD19+CD27+ B cells, purities of 93.0 ± 3.4% (n=14) can be obtained. Isolation of memory B cells from human samples is increasingly important for the investigation of B cell signaling pathways and regulation mechanisms central to a robust immune response.
Sustained antibody responses are dependent on intrinsic CD28 function in plasma cells (45.15)
Authors: Rozanski Cheryl , Arens Ramon , Carlson Louise , Nair Jayakumar , Boise Lawrence , Chanan-Khan Asher , Schoenberger Stephen , Lee Kelvin
Published: January 2023

Sustained long-term antibody (Ab) levels are the cornerstone of protective immunity, yet it remains unclear how they are maintained. A predominant theory is this occurs via antigen-independent Ab production by a subset of long-lived plasma cells (LLPC) that survive within bone marrow (BM). However, the model's central tenets that BM LLPC are a separate subset defined by intrinsically distinct biology and contribute to long-term Ab titers, have not been demonstrated. We now report that long-term humoral immunity is dependent on plasma cell-intrinsic function of CD28, which selectively supports the survival of BM LLPC but not splenic short-lived PC (SLPC). LLPC and SLPC both express CD28, but CD28 signaling with enhanced survival occurred only in the LLPC. Addition of BMDC improved the survival of WT LLPC 2-fold over that seen with media alone and resulted in a significant increase in IgG production; in contrast to CD28-/- PC when cocultured with BMDC. This suggests a direct role for CD28 in PC-DC interaction. In vivo, even with sufficient T cell help the loss of CD28 or its ligands CD80/CD86 caused significant loss of the LLPC population, reduction of LLPC survival t1/2 from 426 to 63 days, and inability to maintain Ab titers long-term - with no effect on SLPC populations. These findings establish the existence of the distinct BM LLPC subset that is necessary to sustain Ab titers, and uncover a central role for CD28 function in the longevity of PC and humoral immunity.
Host mediated modulation of autoreactive B cell differentiation (45.5)
Authors: Kiefer Kerstin , Marshak-Rothstein Ann
Published: January 2023

Selection of B cells into the mature B cell compartments is based on the affinity of the B cell receptor (BCR) for self-ligands. Strongly self-reactive B cells undergo receptor editing or deletion during this process to avoid autoimmunity. Current dogma suggests that B cells expressing BCRs with low affinity to self become follicular (FO) B cells whereas B cells with intermediate affinity to self develop into marginal zone (MZ) B cells. We have examined this selection process using AM14 BCR gene-targeted mice on either a BALB/c or BALB/c x C57BL/6 F1 background. In BALB/c mice, all mature AM14 B cells move into the FO compartment, are fully functional and respond normally to BCR crosslinking. In contrast, on a BALB/c x C57BL/6 F1 genetic background, AM14 B cells move into the MZ as well as the FO compartment. Importantly, these AM14 B cells appear anergic and do not respond well to BCR crosslinking. In vitro, we found that BALB/c AM14 B cells proliferate in response to RAG-deficient C57BL/6 spleen cells, but not to C57BL/6 cells from the bone marrow or peritoneal cavity. These findings suggest that a ligand expressed by splenic non-T/non-B C57BL/6 cells can modulate the differentiation pathway of AM14 B cells. To further investigate which cell type expresses this self-ligand, we are comparing AM14 B cell engraftment of BALB/c and C57BL/6 F1 mice using mixed bone marrow chimeras. The results of these studies and the implications for MZ B cell selection will be discussed.
Activation induced cytidine deaminase is highly expressed on CD99+Faslow Ramos cells; implication in the early centroblast stage (45.8)
Authors: Lee Hyun Gyu , Park Eunsun , Lee Jae Myun , Park Jeon Han
Published: January 2023

Ramos cell line, derived from Burkitt’s lymphoma with centroblastic phenotype, has been chosen as a good model for studying somatic hypermutation. To delineate maturation stages of B cells in germinal center (GC) with an expression pattern of CD99 that has been reported to be down-regulated in GC, we sorted and subcloned the Ramos cells according to the expression patterns of CD99. CD99+ clones lost CD99 expression to become CD99- cells spontaneously. However, the reverse event has not happened in any cases. We observed that CD99+ clones have earlier centroblastic phenotypes than CD99- ones through experiments such as IgM-induced apoptosis and Fas expression. When the CD99+ clones were subcloned based on Fas expression levels, CD99+Faslow clones generated IgM- population in higher frequency than CD99+Fashi and CD99-Fashi did, then we evaluated the expression level of AID and found that CD99+Faslow clones are the cells at the stage where AID level is the highest. This high expression level of AID in the CD99+Faslow clones is pertinent to the Ramos cells sorted directly and also to different Burkitt’s lymphoma cell lines: Daudi, BJAB, and Raji. These results suggest that AID is differentially expressed between CD99+ and CD99- cells, implying CD99 might be a marker to delineate the maturation stages of B cells in GC. Based on this, we hypothesize that CD99+Faslow cells are immature cells and differentiate into CD99-Fashi with an intermediate step which has CD99+Fashi in GC.
CD99+Faslow Ramos cells, representative of early centroblasts, are sensitive to IgM induced apoptosis (45.9)
Authors: Park Kwang Hwan , Lee Hyun Gyu , Park Eunsun , Lee Jae Myun
Published: January 2023

Ramos cell line, derived from human Burkitt’s lymphoma, expresses a centroblastic phenotype in the early stage of B cell development in germinal center. In previous study, CD99 positive cells were scattered throughout the lymph nodes, but most cells in germinal center had low expression of CD99 in immunohistochemical staining. Here, we hypothesize that CD99+Faslow Ramos cells may be representative of early centroblasts and have sensitivity to IgM induced apoptosis. Using B cells from human tonsils, we have evaluated various markers of centroblastic stage. We found that the expression of Fas was low in the early centroblastic stage and gradually increased during differentiation. Ramos cells that have heterogenous expressions of CD99 were subcloned into CD99+ and CD99-. IgM induced apoptosis in CD99+ clones was higher than that of CD99- clones. CD99+ clones showed heterogeneous patterns of Fas expression from low to intermediate levels, while CD99- clones were homogenous in terms of intermediate expression of Fas. CD99+Faslow and CD99-Fashigh clones were subcloned, and the expression of Fas was gradually increased during the long term culture of CD99+Faslow clones. CD99+Faslow clones were the most sensitive cells for IgM induced apoptosis. All of these results indicate that CD99+Faslow cells may be representative of early centroblasts and sensitive to IgM induced apoptosis. we will investigate the molecular mechanism of this apoptotic signaling pathway in the early centroblast.
Elucidating the mechanisms of CD8 T cell plasticity (46.1)
Authors: Ceccato Christina , Durham Nicholas , Bruno Tullia , Nirschl Christopher , Tam Ada , Drake Charles
Published: January 2023

Christina M Ceccato, Nicholas Durham, Tullia Bruno, Christopher Nirschl, Ada Tam, Charles G Drake. When a naïve CD8 T cell encounters antigen, it acquires a cytokine secreting phenotype in addition to cytotoxic capacities. We and others have shown that CD8 cells cultured in the presence of IL-6 and TGFβ acquire the capacity to secrete IL-17 and lyse specific targets in vivo but not in vitro. We also demonstrated in vivo plasticity of the Tc17 phenotype by showing that adoptively transferred Tc17s convert from an IL-17 to an IFNγ secreting phenotype and are maintained in higher numbers than transferred Tc1. Here, we show that sorted IL-17+IFNγ- (pure Tc17 cells) cultured in vitro can convert to secreting IFNλ in the presence of IL-2 alone. Interestingly even in the presence of Tc17 skewing conditions, IL17-IFNγ+ (pure Tc1 cells) do not convert to IL-17 production. These data confirm our original observations showing the plasticity of the Tc17 phenotype - indeed, IL-2 may be sufficient to drive conversion in vitro. However, the molecular mechanisms behind CD8 T cell plasticity have yet to be elucidated; ongoing studies are examining the role of TCR affinity and various transcription factors in this process.