bluuebunny/crossref_metadata_2025 huggingface dataset:
In vivo therapeutic potential of classes B and R inhibitory oligonucleotides (INH-ODH) for animal lupus (131.18)
Authors: Lenert Petar , Pareigis Elizabeth R. , Seyfer Sarah , Lawry Molly , Ashman Robert F.
Published: January 2023

TLR receptors are implicated in the pathogenesis of systemic lupus erythematosus. Interestingly, MRL mice lacking TLR9 develop more aggressive kidney disease, while those lacking TLR7 show a milder disease. INH-ODNs (linear, Class B) have recently been developed that can block TLR9 activation at low nanomolar concentrations. This holds true particularly for INH-ODNs made with the nuclease-resistant phosphorothioate backbone (PS). Here we show that (PS)-INH-ODNs, in addition to blocking TLR9 stimulation, can also block TLR7 stimulation of mouse macrophages and B cells induced by several (but not all) TLR7 ligands like imiquimod, loxoribine, CL075, CL097 and CL087. Interestingly, certain TLR7/8 ligands escape inhibition by INH-ODNs and may actually enhance B cell G1-M entry, survival and polyclonal IgM secretion. Both inhibition and enhancement are independent of the TLR9 expression. We have recently refined these ODNs to produce INH-ODN sequences capable of forming stable secondary structures. These “innate” INH-ODNs (Class R) showed higher potency for macrophages, dendritic cells and marginal zone B cells over follicular B cells, in vitro. In vivo, both linear and Class R INH-ODNs provided better survival and ameliorated spontaneous lupus disease in MRL-lpr/lpr mice.

This work was supported by NIH grant 1 R56 AI064736-01A2 and by a grant from the Carver Trust fund.
Modulation of CD4+Foxp3− T-cells with a JAK-STAT5 kinase inhibitor (131.2)
Authors: Davoodi-Semiromi Abdoreza , Cheikhi Amin , Xia ChangQing , Litherland Sally , Clare-Salzler Michael
Published: January 2023

In this study we report AG490 treatment reprograms CD4+CD25− Fox3−T-cells into Foxp3 expressing T-cells which possess regulatory function both in-vitro and in-vivo. CD4+CD25− Fox3−T-cells from transgenic BALB/c DO11.10 mice became Foxp3+ when treated in-vitro with AG490. These T-cells possessed regulatory function as they suppressed proliferation of syngenic responding population (CD4+CD25− T-cells) in response to OVA323–329 peptide in-vitro. Purified CD4 T-cells from BALB/c mice treated with AG490 failed to suppress proliferation of transgenic CD4+CD25− T-cells of BALB/c DO11.10 suggesting antigen-specific function of converted population. Our in-vivo preliminary data regarding adoptive co-transfer of bulk CD4+CD25− Foxp3−T-cells treated with AG490 and diabetogenic splenocytes delayed onset of diabetes in 20% of treated mice for 9-weeks. Furthermore we show treatment of diabetic NOD.scid mice (recipient of diabetic splenocytes and CD4+CD25− T-cells) with AG490 increased numbers of CD4+CD25+Foxp3+ T-cells when compared with the same population in B6 and NOD mice and posses regulatory function. Splenocytes of treated mice were anergic to anti-CD3 and CD28 stimulation and purified CD4+CD25+ T-cells suppressed proliferation of responding population in-vitro. In this report, we for the first time uncovered new properties of AG490 in the immune system by demonstrating upregulation of Foxp3 in CD4+CD25− T-cells and propose that NOD islet antigen-specific CD4+Foxp3− T-cells reprograms by AG490 may significantly delay/prevent onset of diabetes in NOD mice.

This study is supported by a JDRF innovative grant awarded to ADS (5-2006-937) and in part by a grant awarded to MCS (JDRF # 1-2004-690).
Fc dependent mechanisms are necessary for ADCC and effective depletion of murine B cells by humanized anti-CD19 mAb. (131.27)
Authors: Groves Christopher J , Wang Y , Sims G P , Kuta E , Rowe D , Ward E , Mittereder N , Carlesso G , Cheng L , Cook K , Tedder T , Damschroeder M , DallAcqua W , Kiener P , Herbst R , Coyle A J
Published: January 2023

B cell depletion is an effective treatment for autoimmune diseases and B cell malignancies. Successful B cell depletion may be limited by the surface expression of the target antigen. CD19 represents an attractive molecular target that is expressed on the surface of early B cell progenitors through the latter stages of B cell differentiation. In this study we developed a fully humanized CD19 monoclonal antibody mAb 19-1 and two variants with either reduced (19-2) or enhanced (19-3) Fc-FcR binding and evaluated their potential to deplete B cells. In vitro antibody-dependent cellular-mediated cytotoxicity (ADCC) activity of 19-3>19-1>19-2 on human peripheral blood B cells as well as B cell lines (Raji, Daudi, and Ramos) by LDH release or Granzyme B assays. We evaluated in vivo depletion in human CD19 transgenic mice using 19-1, 19-2, and 19-3. 19-1 at 50 ug/mouse i.v. depleted most B cells in blood, spleen, and bone marrow after 7 days following treatment. 19-2 showed little depletion of B cells at a high dose, whereas 19-3 efficiently eliminated B cells in the spleen and blood at low dose. These data demonstrate that transgenic mice expressing human antigens provide an excellent model for testing humanized antibodies in depletion studies, and that Fc-FcR interactions represent the most important mechanism. Targeting B cells with anti-CD19 mAb may be a more effective strategy than existing B cell depletion protocols.
The Impact of B Lymphocyte Depletion on the Pathogenesis of Autoimmune Diabetes (131.28)
Authors: Zekavat Ghazal , Migone Thi-Sau , Roses Robert E , Cancro Michael P , Naji Ali , Noorchashm Hooman
Published: January 2023

NOD B cell development is characterized by a defective homeostatic checkpoint in B cell maturation and selection. Having demonstrated that the progression of autoimmune diabetes requires antigen presentation by B-lymphocytes, we hypothesized that B cell depletion may prevent diabetes progression in NOD mice. Here, we employed two distinct approaches for in vivo B lymphocyte depletion:

a cohort of adult hCD20 Tg NOD mice were treated with the B lymphocyte depleting mAb, Rituximab (anti-CD20) anda cohort of adult NOD mice were treated with a neutralizing Hamster anti-mouse mAb specific for the B cell survival factor, BLyS/BAFF.

Treatment with both Rituximab (2mg/injection x3doses) and anti-BLyS mAb (100?g/injection x2 doses) led to B cell depletion within 10 days. BLyS/BAFF neutralization, using anti-BLyS, caused a profound depletion of mature/recirculating B cells in these mice and lasted for >30 days. Diabetes occurred within 2 weeks in all Rituximab treated female hCD20 Tg NOD mice (n=4); despite their early backcross generations (i.e., N4-6F1) onto the NOD genetic background. Conversely, adult female NOD/LtJ mice (n=20), B cell depleted using anti-BLyS/BAFF did not progress to diabetes for >30 days following treatment. Collectively, these results indicate that BLyS/BAFF neutralization may be required for the prevention of autoimmune diabetes following B lymphocyte depletion.
MHC polymorphism translates T cell tolerance into B cell autoimmunity. (131.29)
Authors: Ellis Jason Scott , Bell J. Jeremiah , Khapli Shruti , Divekar Rohit D. , Haymaker Cara L. , Yu Ping , Lee Hyun-Hee , Jain Renu , Tartar Danielle M. , Hoeman Christine M. , Guloglu F. Betul , Zaghouani Habib
Published: January 2023

Experimental allergic encephalomyelitis (EAE) is a mouse model for human multiple sclerosis (MS). While effective treatments of EAE have been developed, adapting these for use in human MS has proven difficult and therefore investigations into this model continue. Here, we examine the effects of MHC polymorphism on the control of tolerance. Our previous studies have shown that treatment of EAE with immunoglobulins (Ig) containing proteolipid protein (PLP) or myelin oligodendrocyte glycoprotein (MOG) epitopes reverses ongoing disease in both SJL/J and C57Bl/6 mice. We used F1(SJL/J x Bl/6) mice to test Ig-PLP and Ig-MOG for reversal of compatible as well as “in trans” EAE wherein disease induced by MOG epitope is treated with Ig-PLP and EAE induced by PLP epitope is treated with Ig-MOG. The findings indicated that EAE induced in F1 mice by PLP is successfully treated with Ig-PLP or Ig-MOG. However, an asymmetrical effect was observed for the treatment of MOG EAE whereby Ig-MOG reverses MOG EAE while Ig-PLP exacerbated MOG induced EAE. The mechanism underlying disease exacerbation correlated with a cytokine-induced Ig class switch among anti-MOG antibodies during treatment with Ig-PLP.

Research supported by grant NS37406 from NIH.
Follistatin Like Protein-1 is a Marker of Inflammation (131.31)
Authors: Clutter Suzanne Davis , Marinov Anthony D , Wilson David C , Vilaiyuk Soamarat , Hirsch Raphael
Published: January 2023

Follistatin-like protein-1 (FSTL-1) is a poorly characterized gene which we have recently shown to be a novel proinflammatory molecule. FSTL-1 is overexpressed in mouse paws during early arthritis, especially at the interface of synovial pannus and eroding bone. In this study we evaluated FSTL-1 expression following inflammation in both mice and humans in order to characterize the role of FSTL-1 in inflammation. Mice inoculated with complete Freunds’ adjuvant in paws, LPS in the peritoneal cavity, or pseudomonas aeruginosa in the lungs displayed an elevation of FSTL-1 protein, similar to that found in arthritic mice. In addition, utilizing quantitative PCR and ELISA specific for FSTL-1, we demonstrated increased FSTL-1 expression in the heart and brain following LPS peritoneal injection. Finally, FSTL-1 is increased in sera of patients with juvenile rheumatoid arthritis and Kawasaki Disease, compared to normal aged-matched control patients. These observations suggest that FSTL-1 is a potential biomarker of inflammation and may be a novel target for therapy.

This work was supported in part by National Institutes of Health grants AI44566 and T32AR052282.
Endogenous Nitric Oxide Carrier S-nitrosoglutathione Attenuates Experimental Autoimmune Encephalomyelitis by Maintaining a Fine Balance between Th1/Th2 Immune Responses (131.32)
Authors: Nath Narender , Giri Shailendra , Morinaga Osamu , Singh Avtar K , Singh Inderjit
Published: January 2023

The carrier of nitric oxide S-nitrosoglutathione (GSNO) is an antioxidant and neutralizes oxidative stress, here we have evaluated its efficacy in EAE and T cell functions. Oral administration of GSNO (0.5 or 1.0 mg/kg) reduced the disease progression in chronic models (SJL or C57BL/6) of EAE induced with PLP(139-151) or MOG(35-55) peptides. The attenuation of EAE was mediated by less infiltration of CD4 T cells in CNS and a balance between Th1/Th2 immune responses. Recall responses and production of Th1 (IFNgamma cytokine was not affected by GSNO treatment in antigen specific T cells however under these condition myelin specific IL4 secreting Th2 cells were present. GSNO treatment neither induces apoptosis nor hampers T cell activation. Consistent with myelin specific T cells, production of IFN from naïve T cells were unaffected whereas IL4 was induced. Further, the phosphorylation of STAT4 or 6 and expression of Tbet or GATA3 were not inhibited suggesting that both Th1/Th2 cell mediated immune responses were intact with GSNO. The 2D2 MOG(35-55) TCR transgenic T cells in naïve host following immunization and GSNO (0.5 or 1.0 mg/kg) treatment differentiated into Th1 and Th2 clones. Further, GSNO (0.5mg/kg) treatment efficiently reduced the clinical severity of EAE in MOG transgenic mice without affecting the production IFNgamma from myelin CD4 cells. These results suggest the efficacy of GSNO in attenuation of EAE by maintaining a balance between Th1/Th2 cell immune responses which can be exploited in the treatment of autoimmune diseases.

This work was supported in parts by grants NS-22576, NS-34741, NS-37766, NS-40810 and NS-40144 from NIH.
Migration inhibitory factor modulates experimental autoimmune encephalomyelitis through a novel combination of suppressive mediators (131.34)
Authors: Kithcart Aaron P , Powell N , Williams J , Gatson N , Papenfuss T , Gienapp I , Shawler T , Satoskar A , Whitacre C C
Published: January 2023

Multiple sclerosis (MS), an autoimmune disease of the central nervous system (CNS), is more prevalent in women than men. Recent studies suggest that the cytokine migration inhibitory factor (MIF) plays a role in the progression of MS and experimental autoimmune encephalomyelitis (EAE). We have shown that MIF−/− mice have decreased EAE severity relative to wt controls. Here, we evaluate the role of MIF in the context of known suppressive hormones. Serum levels of corticosterone (CORT) and testosterone (TEST) were measured prior to and following EAE induction in the presence and absence of MIF. Basal levels of TEST in MIF−/− mice were notably higher than controls; basal levels of CORT were low and similar between groups. Following EAE, there was little change in CORT and TEST in wt mice; however, TEST decreased and CORT increased in MIF−/− mice following immunization. To evaluate the role of TEST, we removed the testes of male MIF−/− mice and induced EAE. We found no difference in disease severity whether MIF−/− mice were gonadectomized or not; control mice had significantly more severe EAE in the absence of TEST. These findings suggest that CORT plays a larger role than TEST in disease progression, although both hormones appear to be influenced by MIF. Further study of the mechanism of MIF could lead to improved therapies for a variety of autoimmune diseases.

(Supported by NIH grant AI 064320 and National MS Society grant RG3272)
Ingestion of lactobacilli delays lupus development by stimulating APC in (NZBxNZW)F1 mice (131.35)
Authors: Manirarora Jean N. , Kosiewicz Michele M. , Parnell Sarah A. , Alard Pascale
Published: January 2023

Various defects in T cells and APC have been associated with lupus in (NZBxNZW)F1 (BWF1) mice. We and others have found that BWF1 mice exhibit lower number of CD4+CD25+ regulatory cells and impaired APC function. Lactobacilli have been shown to prevent colitis and diabetes in autoimmune-prone mice and are used to treat intestinal inflammation and allergic disease in humans. Since viruses and parasites can prevent lupus in BWF1 mice, we examined whether lactobacilli ingestion would have a similar effect. After feeding BWF1 mice with three different strains of lactobacilli, we found that both anti-dsDNA antibody production and lupus severity were delayed. Next, we tested the effect of lactobacilli ingestion on APC in vivo. We found that lactobacilli upregulated B7.1 on both macrophages and dendritic cells (DC) in the LN of BWF1 mice. Finally, we characterized the effect of lactobacilli and lipoteichoic acid (LTA), a bacterial cell wall component, on bone marrow-derived DC in vitro. Interestingly, we found that lactobacilli and LTA restored the expression of all costimulatory molecules and triggered production of high levels of IL-10 vs IL-12. Together, these data suggest that lactobacilli-mediated lupus prevention is associated with induction of IL-10 producing APC which may ultimately restore immune regulation in BWF1 mice.

Supported by the Lupus Research Institute.
Induced Organ-Specific T Regulatory (Treg) Cells Prevent Autoimmunity by Reducing the Ability of Dendritic Cells (DC) to Present Self-Antigen. (131.4)
Authors: DiPaolo Richard J , Davidson Todd S , Andersson John , Brinster Carine , Shevach Ethan M
Published: January 2023

Foxp3− T cells can be converted to Foxp3+ T reg cells by T cell stimulation in the presence of TGFb. TGFb-induced Tregs (iTregs) have been shown to prevent the induction of autoimmunity although little is known about their mechanism of action in vivo. Here we demonstrate that naïve CD4+Foxp3− T cells specific for the gastric parietal cell antigen, H+/K+ ATPase, can be converted to Foxp3+ T regulatory cells following TCR activation in the presence of TGFb. The iTregs were anergic to TCR stimulation, failed to produce effector cytokines, and were suppressive in vitro. When co-transferred with disease inducing CD4+ T cells, the iTreg were long lived in vivo and were able to prevent organ-specific autoimmunity. iTregs prevented the initial activation and expansion of autoreactive T cells in response to H+/K+ ATPase presented by DC in the gastric lymph node. Gastric lymph node DC from animals that were treated with iTreg had a markedly reduced ability to stimulate H+/K+ ATPase-specific T cells in vitro. When stimulated by peptide in vitro, iTregs mediated the downregulation of the costimulatory molecules CD80 and CD86, but not MHC class II or CD40, on the surface of DC. These data demonstrate that the autoantigen presenting DC is the major target for Treg mediated suppression in vivo, and raise the possibility of using TGFb to generate large numbers of organ-specific Tregs to prevent and possibly treat autoimmunity.
How PPARγ Mediates Immunoregulation: An Integral Role for PPARγ in Regulatory T cells (131.6)
Authors: Wohlfert Elizabeth A , Clark Robert B
Published: January 2023

Ligands for the nuclear hormone receptor PPARγ, which are used to treat human type 2 diabetes, also down-regulate APCs and pathogenic T cells. While these effects may underlie the recent demonstration of the efficacy of PPARγ ligands in treating autoimmunity, a role for PPARγ in CD4+CD25+ natural regulatory T cells (TR) has not yet been examined. Strikingly, we found that murine ex-vivo TR express 10-fold higher levels of PPARγ than naive CD4+CD25− T cells (Teff). To examine whether the immunotherapeutic effects of the synthetic PPARγ ligand Ciglitazone (Cgl) involve TR we used the C57BL/6 (WT) Teff → bm12 model of GVHD. Neither ex-vivo TR alone nor Cgl alone was capable of preventing disease. However, Cgl administered with TR significantly ameliorated GVHD. We generated mice with PPARγ-null T cells (T-PPAR) and found that T-PPAR TR were unable to mediate Cgl’s effect. Thus, PPARγ-expressing TR are required for Cgl’s immunotherapeutic effect. We next asked if PPARγ signaling via endogenous ligands, in the absence of synthetic ligands, might also play an important role in TR function. Transfer of in-vitro activated WT TR significantly ameliorated GVHD, with 90% of bm12 mice surviving the disease. In contrast, activated T-PPAR TR were significantly less effective in suppressing disease, with only 40% of bm12 mice surviving. Overall, our results demonstrate for the first time an integral role for PPARγ in TR function.
MHC variant peptide control of experimental autoimmune encephalomyelitis (131.8)
Authors: Rosenthal Kristen M , Evavold Brian D
Published: January 2023

Experimental autoimmune encephalomyelitis is a CD4+ T cell mediated disease of the central nervous system used as a rodent model to study multiple sclerosis. Epitope spread, or reactivity to epitopes distinct from the disease-inducing antigen, in the CNS correlates to relapses in the relapsing-remitting form of both MS and EAE. Treatments specific to the initiating epitope in EAE have been successful in inhibiting the response to that epitope, but not to subsequent epitopes. We have previously shown inhibition of EAE induction, as well as treatment of symptoms, using variants of the initiating peptides with mutations in the MHC contact residues to decrease stability of the peptide:MHC complex. Here, we demonstrate that pre-immunization with an MHC variant of the first epitope results in a significant reduction of disease induced with both the first and second epitope but not the second epitope alone. Using the R-EAE mouse model in SJL mice, an MHC variant of the initiating antigen is able to inhibit subsequent relapses upon treatment during the primary phase of disease symptoms. This data implies that inhibiting one epitope with an MHC variant peptide may also inhibit subsequent epitope-reactivity, suggesting that antigen specific treatment need not include all possible antigens associated with MS to stimulate a response to treatment.

This work is supported by NIAID (R01 AI56017) and NMSS (RG 3683)
Label-free dectection of cytotoxic antigen-specific T cells with semiconductor Nanowires: (132.10)
Authors: Fahmy Tarek M , Stern Eric , Reed Mark
Published: January 2023

Detection of T cell mediated disease and vaccine-induced immunity has been made possible by the discovery of MHC restricted recognition of peptide by T cells. Current approaches for detection of antigen-specific T cells rely on the use fluorescently labeled-soluble forms of multivalent MHC-peptide complexes or functional assays. We have developed a solid-state semiconductor nanowire technology for label-free direct detection of antigen-specific T cell subsets reactive to their specific antigens. The approach is simple, fast, sensitive and integratable into clinical settings. We describe this technology and demonstrate its feasibility in the in vitro detection of 100–1000 murine antigen-specific T cells in a label-free fashion.
Assessment of Immunogenicity of a Peptibody Based Protein Therapeutic Using a Surface Plasmon Resonance based Biosensor Immunoassay using a Biacore 3000 (132.7)
Authors: Hokom Martha M. , Jawa Vibha , Mytych Daniel , Swanson Steve
Published: January 2023

Administration of therapeutic biologic products is often accompanied by an unwanted immune response that requires monitoring. We used a BIAcore based biosensor immunoassay that can detect antibodies against a peptibody protein-based therapeutic and its peptide domain.. The development and validation of the multi-flow cell surface plasmon resonance based technology is described in this presentation. The biosensor immunoassay measures the interaction of serum antibodies to immobilized ligands in real time. This binding interaction is further confirmed to be the result of an antibody by the use of species-specific secondary antibodies (anti-F(ab)’2) to detect the bound antibodies. The binding is further characterized by a competition step using the soluble drug. The fully validated assay has a limit of detection of 50ng/mL and 200 ng/mL for the peptibody and the peptide respectively, and a limit of quantification of 400 and 800 ng/mL respectively.

In summary, we developed and validated a sensitive and robust biosensor immunoassay for the detection of antibodies against a peptibody based drug that meets the requirements necessary to support the clinical development of this therapeutic.
PERIPHERAL BLOOD MONONUCLEAR CELL GENE ARRAY PROFILES IN PATIENTS WITH OVERACTIVE BLADDER (132.8)
Authors: Bluth Martin H , Bluth Mark J , Johns Christopher , Khan Sohail , Lin Yin Yao , Zenilman Michael E , Cheung Wellman
Published: January 2023

Introduction: Overactive bladder (OAB) confirmation of disease often requires invasive approaches. We have previously demonstrated that peripheral blood mononuclear cells (PBMC) can provide a reporter function in solid organ retroperitoneal disease. Here we investigated the utility of using PBMC as a marker for patients with OAB.

Methods: 21 patients were assessed for OAB and structural integrity. Patients with a history of recent surgery or infection were excluded. Whole blood was obtained from patients and appropriately matched controls (n=6) and PBMC were separated by density centrifugation. RNA was isolated from PBMC and converted to aRNA and subjected to microarray analysis (human U133A 2.0).

Results: Microarray analysis revealed that 16 genes were differentially regulated (8 upregulated and 8 downregulated) in all patients with OAB compared with controls. Sex based analysis demonstrated 74 genes differentially regulated in males (25 upregulated and 49 downregulated), and 30 in females (13 upregulated and 17 downregulated). Of these, PDGF, MFAP3L, a microfibrillar-associated protein, and TPM1 (tropomyosin) were downregulated in all sets analyzed.

Conclusions: Microarray analysis revealed many genes which were differentially regulated in PBMC from OAB patients, including regulatory/structural genes; PDGF, MFAP and TPM1 may be important in regulating structural integrity of bladder and supporting tissues. These data suggest that blood derived leukocytes can provide a reporter function for patients with solid organ disease and may serve as a diagnostic marker and/or monitor response to therapy.
A Rapid Automated Method for the Sequential Isolation of CD19, CD3 and CD33 cells from One Tube of Whole Blood. (132.9)
Authors: Fairhurst Maureen A , Nauer Melany , Thomas Terry E.
Published: January 2023

Chimerism analysis is typically performed on small blood samples. Analysis of purified cell subsets requires techniques which can isolate >1 cell type from an entire starting sample rather than a divided cell suspension. High cell recovery is essential. Ficoll steps often result in cell loss of 50% while certain lysis and wash steps can affect granulocyte content. We describe a method of sequential selections, starting with 4–5 mL of human whole blood and using a fully automated pipetting robot (RoboSep®).

CD19 and CD3 and CD33 positive cell fractions were isolated using immunomagnetic, column-free positive and negative selection (EasySep®). Briefly, cells were first labeled with antibody targeting CD19 positive cells. These were then coupled to magnetic nanoparticles and the sample was placed in a magnet. The supernatant with unlabeled cells was removed to a new tube, leaving isolated CD19 positive cells in the magnet. The supernatant was next labeled with anti-CD3 antibody, magnetic nanoparticles, placed in a magnet and the supernatant was removed to a new tube leaving isolated CD3 positive cells. Finally, a cocktail of antibodies was used to label and remove the remaining unwanted cells (CD33 negative) from the supernatant. The resultant CD33 positive cells were collected in the supernatant fraction leaving CD33 negative cells. Assessment by flow cytometry yielded average purities over 90% for all cell types.
T and B Cell Pathways: An Introduction to the Immune System (34.4)
Authors: Paul Mark R. , Budd Ralph C.
Published: January 2023

This presentation will summarize a two-week curriculum designed for an introductory High School Biology Class. This unit meets Vermont Standards for the Human Body (7.14, bbb), The Problem Solving Process (2.2), and Relationships (3.10). This unit also meets Grade Expectations for Human Health and Disease and the Immune system.

Students first learned about different kinds of diseases and the structure and function of viruses and bacteria. Next they read an article and answered questions on Typhoid Mary. They were then given two weeks to solve a case study involving Lyme disease, after which they learned more about Lyme disease and performed an ELISA lab for Lyme.

Students received background information from a textbook and a power point presentation. They also roll played how the immune system responds to a first infection of Borrelia burgdorferi. For a culminating activity students built functional models that simulated either humoral or cell mediated immunity. The basic premise of designing these models was the Mouse Trap Game or Rube Goldberg design using whatever materials they could rustle up. We also used RASMOL so that the student s could view a 3-D model of the Lyme antibody and antigen.
IgH intronic enhancer, Eμ, and associated matrix attachment regions are required for efficient somatic hypermutation (35.1)
Authors: Li Fubin , Eckhardt Laurel A
Published: January 2023

Somatic hypermutation is a critical mechanism for generating antibody with high affinity. Although the enzyme critical to this process, AID, has been identified, the mechanism that largely limits its activity to the Ig loci is not known. Prior studies have shown that IgH gene transcription levels are positively correlated with somatic hypermutation frequency. The intronic enhancer, Eμ, both stimulates IgH transcription and is essential for promoting efficient V region gene assembly in the IgH locus. It consists of both a core region (cEμ) and two flanking matrix attachment regions (MARs). A recent study reported normal levels of somatic hypermutation in the B cells of cEμ-deficient mice. In the present study, we tested whether deletion of both cEμ and MARs had an effect on somatic hypermutation. We found that the mutation frequency for the Eμ-deficient allele was reduced to approximately 1/3 of wild-type levels. This dramatic decrease in mutation frequency suggests that cEμ, together with its associated MARs, plays an important role in somatic hypermutation within the IgH locus.

Supported by NIH grant AI030653 to L.A.E. Research infrastructure and instrumentation at Hunter College is supported by NIH RCMI grant RR-03037.
Ataxia-Telangiectasia, Associated with a Rare 1339C>T ATM mutation, presenting in infancy with Pneumocystis carinii pneumonia, and an elevated serum IgM. (35.11)
Authors: Schaffer Frederick M , Virella-Lowell Isabel , Gatti Richard A , Gossage David L
Published: January 2023

Ataxia-Telangiectasia (A-T) is an autosomal recessive disorder manifested by progressive immunodeficiency, progressive neurologic dysfunction, and an elevated cancer predisposition. Characteristic symptoms are rarely encountered during infancy.

We present the laboratory and clinical studies of a patient with respiratory distress at age 3 months due to pneumocystis pneumonia. Laboratory studies demonstrated an elevated serum IgM (840 mg/dL), recurrent neutropenia, lymphopenia (CD3, CD4, CD8, and CD19), diminished in vitro lymphocyte proliferation, a negative HIV PCR, and normal ADA and PNP levels. Flow cytometry demonstrated normal lymphocyte expression of CD40 and the CD40 ligand. Sequencing, after PCR-amplification of genomic DNA, demonstrated no apparent mutation consistent with type 2 hyper IgM syndrome. Based on a significantly elevated serum alpha-fetoprotein and early neurologic deficits, a diagnosis of A-T was suspected. Subsequently, her cells were found to be radiosensitive, based on a Colony Survival Assay. Western blot analysis of nuclear lysates of patient cells failed to detect ATM protein. Molecular analysis of the ATM gene identified a rare homozygous 1339C>T mutation (previously reported in a Druze A-T family: Hum Mutat 11:69–75, 1998). Notably, our A-T case (AT219LA) demonstrates early and atypical findings of an opportunistic infection and elevated serum IgM. The patient is currently thriving on IVIG and the prophylactic use of Septra.
Amino acid residues in Rag1 crucial for DNA hairpin formation (35.12)
Authors: Lu Catherine Pei-ju , Sandoval Hector , Rice Phoebe A , Roth David B
Published: January 2023

The Rag proteins carry out V(D)J recombination through a process mechanistically similar to cut-and-paste transposition. Specifically, Rag complexes form DNA hairpins through direct transesterification, utilizing a catalytic Asp-Asp-Glu (DDE) triad in Rag1. How is sufficient DNA distortion introduced to allow hairpin formation? We hypothesized that, like certain transposases, the Rag proteins might use aromatic amino acids to stabilize a flipped-out base. Through in vivo and in vitro experiments and structural predictions, we identified residues in Rag1 crucial for hairpin formation. One of these, a conserved tryptophan (Trp893), probably participates in base-stacking interactions near the cleavage site, as do Trp298, Trp265, and Trp319 in the Tn5, Tn10 and Hermes transposases, respectively. Other residues surrounding the catalytic glutamate (Y, K, [E], F, R, K) may share functional similarities with the YREK motif in IS4 family transposases.
Modifications of CD40 regulate defective CD40 signals in B cells from a patient with non-X-linked hyper IgM syndrome (HIGM) (35.13)
Authors: Lu Kristina T. , Covey Lori R.
Published: January 2023

Our previous studies have demonstrated a CD40 signaling defect in B cells from a patient with non-X-linked HIGM (pt1). To elucidate the cause for impaired CD40 signals, we utilized a mini-EBV transformed cell line with inducible latent membrane protein 1 (LMP1) expression (LCLtet). The LMP1 signaling pathway appears to be intact, however CD40 activation was unable to promote cell division and transcriptional synthesis of cMyc in pt1-LCLtet cells. We examined mediators of the CD40 signaling pathway and found impaired localization of CD40 to microdomains, thereby impeding TNF receptor-associated factor recruitment and degradation after 2h of CD40 activation. However, prolonged CD40 ligation was sufficient to induce proper CD40 distribution, suggesting that the pt1 B cells possess a higher CD40 activation threshold that requires a greater magnitude of extensive and sustained CD40 signals to achieve signal transmission. Furthermore, pt1 B cells have a unique CD40 expression profile, indicative of an isoform with post-translational modifications. Specifically, CD40 on pt1 B cells is constitutively hyperphosphorylated and not differentially regulated by LMP1 or CD40 signals. These data collectively suggest that modulation of post-translational modifications, which mediate CD40 function and activity, may provide further insight into the cause of defective CD40 signals in this unique form of HIGM. (AI37081)
Unambiguous HLA Typing for Studies of Human Immunity (35.18)
Authors: Cate Steve , Roselius Kassi , Hildebrand William
Published: January 2023

The Major Histocompatibility Complex (MHC) is recognized as the most polymorphic region of the human genome. Our HLA typing laboratory at the University of Oklahoma Health Sciences Center has performed more than 30,000 HLA typings in the past 10 years. We have systematically overcome various capacity and cost constraints to provide precision, DNA sequence-based, HLA typing data for studies of transplantation, vaccine development, and autoimmunity. A confounding variable in the HLA typing field is data heterogeneity; different HLA laboratories provide different levels of resolution when reporting an HLA type for autoimmune, transplant, and vaccine studies. For example, HLA-A*02, A*0201, or A*0201/09/43N/66/75/83N/89/97 might all be reported for a like sample. Such data heterogeneity hinders data integration and comparison. On the basis of our experience with HLA typing and with functional assays such as the ELISpot and tetramer staining, we propose a system of HLA typing that encompasses known functional regions of the HLA molecule. The DNA sequencing of the appropriate exons at various HLA loci, along with the resolution of known null substitutions, will provide cost efficient HLA typing data that can be integrated among different laboratories. Improved HLA data uniformity will ultimately strengthen studies of human immune responses and disease.
“Allelic” exclusion despite 15 IgH loci in nurse shark (35.2)
Authors: Lee Victor , Malecek Karolina , Hsu Ellen
Published: January 2023

Nurse sharks have an alternative IgH organization consisting of 15 independent miniloci (VH-D1-D2-JH-Cμ). Rearrangement takes place primarily within the locus, and we found there is no strictly regulated order of the three recombination events that occur among the four gene segments. In each B cell only a few IgH rearrange. Aside from the selected functional VDDJ, out-of-frame and intermediate configurations with one or two recombination events are present; these all are transcribed and can become targets of hypermutation. The data are consistent with a stochastic initiation of rearrangement in B cells. In contrast, in thymocytes, multiple IgH each undergo one or two recombination events but are otherwise inert. We speculate there are different levels of activation, one occurring at many IgH in precursor lymphocytes that renders the gene segments accessible to recombinase, and a second B-cell specific factor that, in limiting amounts, leads to efficient V(D)J rearrangement and transcription.
The transcription factor GATA-3 regulates IL-4 responses in B cells (35.23)
Authors: Aud Dee , Pai Sung-Yun , Ho I-Cheng , Peng Stanford L.
Published: January 2023

The GATA-3 transcription factor plays a central role in T helper type 2 development, but its role in B cells has not been well described, even though GATA-3 is present in naïve B cells. To study the role of GATA-3 in B cells, mice with a floxed Gata3 allele (Gata3fl/fl) mice were crossed to CD19-Cre transgenic mice, allowing B cell-specific deletion of GATA-3. B cell development was grossly normal in Gata3fl/fl CD19-Cre, but surprisingly, Gata3fl/fl CD19-Cre B cells showed a decreased ability to proliferate (in an IL-4 dose dependent manner) but an increased ability to produce IgG1 and IgE in response to anti-CD40 and/or IL-4 in vitro. These findings may reflect an interaction between GATA-3 and type 2 Ig-regulating transcription factors such as NF-kB or STAT6, because GATA-3 can modulate the activities of these factors in vitro. These findings demonstrate a critical yet surprising role for GATA-3 in the regulation of IL-4-mediated responses in B cells.
Identification and characterization of a conserved negative regulatory element in the human DAP10 gene (35.27)
Authors: BURGESS STEVEN , MARUSINA ALINA , BORREGO FRANCISCO , COLIGAN JOHN
Published: January 2023

The NKG2D receptor is an activation receptor expressed on all NK cells and the majority of CD8+ T cells that can bind stress-inducible ligands such as MICA/B. Human NKG2D signals through its association with DAP10, which can couple to the PI3-K signaling pathway upon NKG2D ligation leading to cellular activation. Inappropriate activation of NK and/or T cells by NKG2D has been implicated in a variety of human diseases and thus the determination of the factors that regulate NKG2D/DAP10 expression is important. We have embarked on a study in which to understand the molecular mechanisms governing transcription of the DAP10 gene. We have identified a highly conserved intron between exons III and IV within DAP10. By cloning gene fragments of DAP10 into pGLbasic for luciferase reporter assays, we have shown that intron III contains a negative regulatory element capable of completely abolishing the promoter activity of DAP10. Progressive deletions of the conserved intron III allowed us to localize the negative regulatory element to a region about 80 base pairs in size that contains a putative GATA-3 binding site. The putative silencer within intron III appears to be both orientation and location dependent. Using ChIP assays, we have also shown that intron III contains high levels of acetylated chromatin in resting NK cells. Further studies are underway to further characterize the negative regulatory element contained within the conserved intron of the DAP10 gene.
c-maf, NFAT2 and NFkB p65 specifically cooperate to augment HIV-1 transcription in IL-4 producing CD4 T cells (35.28)
Authors: Zhang Mingce , Cron Randy Q.
Published: January 2023

HIV-1, the cause of AIDS, preferentially replicates in IL-4 producing cells, and this coincides with a Th2 switch linked with disease progression. Others suggested differential chemokine receptor expression accounts for increased infectivity of Th2 cells. However, we confirm HIV-1 expression is increased in IL-4 producing CD4 T cells but is irrespective of viral tropism (T-tropic, M-tropic) for different chemokine receptors. Instead, we note increased HIV-1 long terminal repeat (LTR) transcription in IL-4 producing primary CD4 T cells. Using in vitro footprinting, we identified a binding site for the Th2 restricted transcription factor, c-maf (required for IL-4), located adjacent and proximal to the dual NFkB/NFAT sites in the proximal LTR. We show c-maf from activated primary CD4 T cells binds the HIV-1 LTR in vitro (EMSA) and in vivo (ChIP of virally infected cells). Interestingly, c-maf binds in conjunction with NFAT2, but not NFAT1, and with NFkB p65, but not p50. Over-expression of c-maf, NFAT2, and NFkB p65 synergistically augment HIV-1 transcription and p24/gag expression. Conversely, c-maf specific siRNA introduced into IL-4 producing primary CD4 T cells inhibits HIV-1 transcription. Thus, we identified a host factor, c-maf, present in IL-4 producing CD4 T cells that contributes to increased HIV-1 transcription in Th2 cells via binding to the LTR in cooperation with specific NFAT and NFkB family members.
Regulation of epsilon germline transcription and switch region mutations by IgH locus 3′ enhancers in transgenic mice (35.3)
Authors: Severinson Eva , Laurencikiene Jurga
Published: January 2023

Germline (GL) transcription is regulated by specific promoters and Immunoglobulin heavy chain (IgH) 3′ locus enhancers and is necessary for Ig class switch recombination (CSR). We have generated different transgenic lines containing the GL epsilon promoter, switch (S)_psilon region and constant (C)_psilon region with or without the DNase I-sensitive regions (HS)3A-HS1,2 or HS3B-HS4 3′ IgH enhancer pairs. The enhancerless construct was expressed in B cells activated by interleukin (IL)-4 and cluster of differentiation (CD)40, thus resembling regulation of the endogenous gene. Both enhancer-containing transgenes efficiently increased expression in B cells and were strongly up-regulated by stimuli. In addition, S epsilon regions of the transgene containing HS3B-HS4 were mutated in activated, sorted B cells. Such mutations are known to precede CSR and are dependent on activation induced cytidine deaminase (AID). Our findings show that all elements necessary for recruitment of the recombination machinery are present in the transgene containing HS3 and HS4. These enhancers probably provide something more specific than mere increased accessibility of switch regions. We propose that transcription factors binding the enhancers help to target the recombination machinery to the switch regions.

This work was supported by grants from the Swedish Research Council, the Swedish Institute and the Karolinska Institutet
New Sequence of Strategies to Define Gene Transcriptional Territories in Lymphocytes: The Perforin Example (35.32)
Authors: Lichtenheld Mathias Georg , Zúñiga-Pflücker Juan Carlos , Rao Anjana , Pipkin Matthew E.
Published: January 2023

Distal regulatory regions control transcription by promoter interactions that are limited to a territory to avoid non-target genes. Few loci are defined with the scrutiny needed to fully understand how a territory is established, maintained and fenced off. Here we approach the human perforin gene (PRF1). First, we transferred an epigenetically silenced PRF1 within its chromosome into a rodent CTL cell line. Afterward PRF1 expression was turned on equivalently to the endogenous mouse gene without perturbing bordering genes. Ultra long-range DHS assays showed epigenetic remodeling during chromosome transfer and delineated a 150 kb territory with 16 DHS sites for PRF1. When a corresponding BAC was introduced into the CTL, transgene expression proceeded with locus control region activity dependent on 4 distal DHS sites that also protected PRF1 from transcriptional perturbations by bordering genes. Finally, differentiation of transgenic ES cells showed the area contained all regulatory domains required for developmentally appropriate PRF1 expression during in vitro lymphopoiesis. Thus, we defined a new sequence of strategies that reliably characterizes autonomous transcriptional territories in lymphocytes.
Common Transcriptional Control Complexes of Murine CD21 and CD23 Suggests Stage Specific Repression (35.35)
Authors: Debnath Irina , Roundy Kirstin , Weis Janis , Weis John
Published: January 2023

The expression of CD21 and CD23 is coincident in differentiating B cells during the T1 to T2 splenic transition stage. To define the factors regulating the expression of CD21 and CD23, we have used Chromatin Immunoprecipitation to map candidate transcription factors. Total naïve splenocytes showed constitutive binding of Oct1, NFATc3, YY1, NFkB-p52, PU.1, Pax5, E2A and RBP-Jk; to CD21 sequences (promoter and first intron) and NFkB-p52, Pax5, NFATc3, E2A and RBP-Jk to CD23 promoter sequences. Analysis of naïve T and B cell subsets showed constitutive binding of YY1, NFkB-p52, Pax5, Oct1 and NFATc3 proteins to CD21 sequences and NFkB-p52, Pax5, NFATc3 to CD23 promoter in B cells, but no specific binding of NFATc3 or Pax5 in T cell populations. Transcripts and protein for various NFAT species are present in splenic B and T cell (NFATc1, c2, c3, c4, NFAT5), but only a subset of NFAT proteins were defined to form constitutive complexes with the CD21 and CD23 gene sequences of resting T and B cells. The shared transcriptional control elements of CD21 and CD23 did not suggest an obvious means of alternative transcriptional regulation of these genes in marginal zone (MZ) B cells where CD21 cell surface expression and transcription are high and that of CD23 is absent or very low. These data suggest a model where MZ cells are derived directly from T1 splenic precursors.

NIH-AI-24158 to JHW and NIH-AI-32223 to JJW
Identification of a regulatory region which controls inducible expression of the human CD200 gene (35.36)
Authors: Chen Zhiqi , Marsden Philip A , Gorczynski Reginald M
Published: January 2023

CD200 is a type I membrane glycoprotein and plays an important role in prevention of graft rejection, autoimmune diseases and spontaneous abortion by delivering an immunoregulatory signal through interaction with its receptor. The expression of CD200 is not only constitutive but also inducible. We previously documented evidence for C/EBPβ as being important in regulation of constitutive expression of CD200. However, the molecular mechanism(s) controlling inducible expression of CD200 are yet to be defined. Our current studies showed that the expression of CD200 was induced by ConA, IFN-γ and/or TNF-α. To investigate the potential regulatory regions responsible for inducible expression of CD200, a DNase I hypersensitivity assay was performed in Jurkat cells. A prominent hypersensitivity site was detected after stimulation and localized in the region ~ 5.6 kb upstream of the transcriptional start site. Four cis elements were identified in this region: one for NF-κB binding, one IFN-γ activation site (GAS) and two IRF binding sites. To test whether those cis elements play a critical role for inducible expression of CD200, the potential regulatory region was cloned upstream of the CD200 core promoter. Mutation of the binding site for NF-κB, GAS or the first IRF resulted in abolition of the activity of this region, whereas mutation of the second IRF had no effect. A ChIP assay was performed confirming this result.
GATA-6 mediates cell-type specific IFN-γresponse of the HLA-E gene (35.39)
Authors: Grant Quintesia , Barrett David , Zhu Sheng Zu , Ginder Gordon D
Published: January 2023

The human MHC Class Ib gene, HLA-E, has been shown to inhibit both Natural Killer cells and a subset of CD8+ cytotoxic T-cells by engaging the CD94/NKG2A inhibitory receptor. IFN-γ induces the expression of HLA-E as well as Class Ia molecules, which are required for killing of target cells. Since HLA-E has negative effects on immune cell killing of target cells, we have sought to identify locus-specific mechanisms of IFN-γ induction in order to identify molecular targets for selective activation of Class Ia genes, but not HLA-E. We have previously identified a unique upstream IFN-γ response region in the HLA-E promoter and showed that GATA-1 is required for its function in the K562 leukemic cell line. We have now examined the effect of GATA family members on IFN-γ induction of HLA-E in other cell types. HLA-E CAT reporter gene assays demonstrated that only epithelial cells which express GATA factors, as determined by western blot and real-time PCR, mediate a 2-to-4 fold enhanced response to IFN-γ stimulation. Functional analysis of HLA-E CAT reporter gene constructs containing mutations of the core nucleotides in the GATA binding site resulted in a four-fold decreased response to IFN-γ. Tetracycline regulated siRNA knockdown of GATA 6 expression in the SKOV3 ovarian carcinoma cell line revealed a two-to-three fold decrease in IFN-γ response of both HLA-E promoter driven constructs and the endogenous HLA-E gene. We conclude that GATA factors play a tissue specific role in regulation of IFN-γ mediated HLA-E expression.

This work was supported by National Institutes of Health Grant CA87496 and Immunology Training Grant 5T32AI007407-15.