bluuebunny/crossref_metadata_2025 huggingface dataset:
Targeted delivery to tumors in vivo with sigma‐2 ligands
Authors: McDunn Jonathan Edward , Kashiwagi Hiroyuki , Vangveravong Suwanna , Simon Peter O. , Chang Katherine C. , Goedegebuure Peter , Hotchkiss Richard S. , Mach Robert H. , Hawkins William G.
Published: June 2021

Radiolabeled sigma‐2 (σ2) receptor‐specific ligands image pancreas adenocarcinoma allografts in vivo and fluorescently labeled σ2 ligands are rapidly internalized and distributed throughout membrane‐encapsulated organelles. Based on these data, we hypothesized that σ2 ligands could target the delivery of therapeutic compounds to tumors in vivo. To test this hypothesis we synthesized a novel amino acid with a σ2 ligand side chain. This amino acid was incorporated as the N‐terminal residue in a series of pro‐apoptotic peptides without appreciable alteration of the Kd for the σ2 receptor (~10–20 nM). One of these peptides is the BH3‐domain of the pro‐apoptotic Bcl‐2 family member Bim. ~2‐BH3 causes dose‐dependent apoptosis to pancreas adenocarcinoma cells in vitro and is approximately the sum of the apoptotic activity of the σ2 ligand and the TAT‐BH3 peptide (Figure). When administered to tumor bearing mice this chimeric molecule is capable of halting tumor growth without appreciable effects on organ function or blood chemistry. Seven day treatment with this peptide increased median time to death in these mice from 33 days to 45 days (P < 0.0001). These data support the hypothesis that σ2 ligands target the delivery of cancer therapy in vivo and demonstrate a platform approach to delivering peptides selectively to cancer cells in the immunocompetent host.
Microvillar protein trafficking and dynamics imaged by TIRF microscopy in living cells
Authors: Breusegem Sophia Y , Blaine Judith T , Doctor R Brian , Barry Nicholas P , Levi Moshe
Published: June 2021

Polarized epithelial cells have distinct membrane domains. The apical domain consists of microvilli, is separated from the basolateral domain by tight junctions, and contains transporters and ion channels that are regulated by endo‐ and exocytosis from and to the intervillar cleft. Total internal reflection fluorescence (TIRF) microscopy is ideally suited to study endo‐ and exocytosis at the basolateral surface of cells grown on coverslips; however, in this set‐up it can not be used to study apical trafficking as the TIRF excitation field will not reach the apical membrane. We have developed a TIRF method to follow apical endo‐ and exocytosis, growing cells on membrane supports and turning them over onto coverslips. We show that apical TIRF does not lead to de‐differentiation and can be used to follow microvillar trafficking processes and dynamics in living cells with high spatial and temporal resolution. We use this method to study endocytosis of renal sodium/phosphate co‐transporters (NaPi‐2a and NaPi‐2c) in response to parathyroid hormone or high phosphate. We show that microvillar actin turnover occurs on the same time scale as NaPi‐2a internalization. In addition, we show trafficking of Shank2E, a PDZ‐domain containing NaPi‐2a‐binding protein, in the microvilli.

S.Y.B. is supported by a postdoctoral fellowship from the American Heart Association, J.T.B. by a NRSA fellowship, and N.P.B. and M.L. by NIH.
Type‐I interferons inhibit Delta‐like‐1‐dependent T cell development and increase apoptosis of developing thymocytes in vitro
Authors: La Motte‐Mohs Ross , Gauchat‐Feiss Dominique , Baron Marie‐Laurence , Démoulins Thomas , Awong Génève , Michiels Thomas , Perreault Claude , Zúñiga‐Pflücker Juan Carlos , Sékaly Rafick‐Pierre
Published: June 2021

Individuals with HIV/AIDS exhibit a gradual depletion of CD4+ T cells in the periphery and a chronic loss of T cells within the thymus. Strikingly, the thymus from these individuals exhibits increased levels of type‐I interferons (IFNs): α(s) and β, suggesting that developing thymocytes are susceptible to apoptosis through increased production of type‐I IFNs. To determine whether type‐I IFNs directly effect T cell development, we cultured fetal liver‐derived mouse hematopoietic stem cells (HSCs) on OP9‐DL1 cells and examined through flow cytometry the CD4−CD8− double negative (DN) and CD4+CD8+ double positive (DP) T cell populations that emerged. Specifically, addition of type‐1 IFNs reduced cellularity and blocked T cell differentiation at the DN1 to DN2 transition in a dose‐dependent manner. Type‐I IFNs mediated their effects directly to the HSCs independent of the OP9‐DL1 cells, as HSCs obtained from IFN‐receptor deficient (IFNAR−/−) mice, but not IFN‐receptor sufficient (IFNAR+/+) mice, were refractive to IFN treatment, exhibited increased cellularity, and progressed to the DP stage. Increased apoptosis over time was observed in IFN‐susceptible, but not IFN‐insensitive fetal liver cells. These in vitro studies have also been extended to the development of human T cells from cord blood‐derived HSCs.

Supported by grants from the OHTN and CIHR.
Impaired regulation of BAFF production in monocytes of patients with Sjögren's syndrome
Authors: Yoshimoto Keiko , Tanaka Maiko , Setoyama Yumiko , Suzuki Katsuya , Tsuzaka Kensei , Abe Tohru , Takeuchi Tsutomu
Published: June 2021

B cell activating factor belonging to the TNF family (BAFF) is mainly expressed in monocytes and dendritic cells and regulates proliferation, differentiation, and survival of B cells. Accumulating evidence suggests that BAFF plays a pivotal role in the pathogenesis of autoimmune diseases, such as systemic lupus erythematosus (SLE) or Sjögren's syndrome (SS). Therefore, elucidation of the mechanism of BAFF production is important not only to understand the mechanism of pathogenesis of the autoimmune diseases, but also to explore therapeutic possibilities to treat the diseases. In this study, we analyzed the expression of cytokines in peripheral monocytes prepared from SS patients and found that the expression of BAFF was abnormally enhanced upon stimulation with IFN‐gamma, an inducer of IgG, as compared with normal monocytes. IFN‐gamma also induced robust production of BAFF in a human mononuclear cell line, THP‐1, and morphological change of the cells, indicating that a signal transduction pathway triggered by IFN‐gamma is involved in the expression of BAFF in mononuclear cells. Simultaneous induction of the expression of IL‐6, TNF‐alpha and IP‐10 was observed in both SS monocytes and THP‐1 upon stimulation. These results suggest that SS monocytes and THP‐1 share a common mechanism of production of BAFF and hence THP‐1 provides a useful model to investigate the abnormal production of BAFF in SS monocytes.
IFN‐γ Signaling Is Required to Regulate CD8 T Cell Priming and Development in Contact Sensitivity Responses
Authors: Kish Danielle D. , Fairchild Robert L.
Published: June 2021

Contact hypersensitivity (CHS) is a CD8 T cell mediated immune response to skin sensitization and challenge with hapten. In wild‐type (WT) mice, hapten‐primed CD8 T cell populations producing IFN‐γ and IL‐17 are required for elicitation of CHS. The CHS response to 2,4‐dinitrofluorobenzene (DNFB) was tested in mice lacking the IFN‐γ receptor (IFNγR−/−). CHS responses in senstized IFNγR−/−mice were of similar magnitude to WT mice 24 hr after challenge but were markedly extended in duration. Immunohistology and flow cytometry analyses confirmed increased cellular infiltration into the skin challenge site of IFNγR−/− vs. WT mice through 48 hr post‐challenge. These responses were CD8 T cell dependent as depletion of CD8 T cells abrogated CHS responses in both WT and IFNγR−/−mice. In the skin draining lymph nodes of sensitized WT mice, numbers of hapten‐specific CD8 cells producing IFN‐γ peaked at day 5 post‐sensitization and declined thereafter whereas the numbers of these T cells were elevated at day 5 in IFNγR−/− mice and were sustained through day 8. Depletion of CD8 T cells eliminated the CHS in WT, but not IFNγR−/− ,mice. compared to WT, through 48 hrs. post‐challenge. However, KC production in the skin challenge site is similar in WT and IFNγR−/− mice. Moreover, the numbers of hapten‐specific CD8 T cells producing IL‐17 were 5 fold higher in sensitized IFNγR−/− mice. These results indicate that the absence of IFNγ signaling dysregulates CD8 T cell priming for CHS and increases the development of hapten‐specific CD8 T cells producing IL‐17 which are capable of mediating CHS in the absence of IFN‐γ signaling.
Altered Leukocyte Dynamics by PECAM‐deficient mice during Chemical Contact Hypersensitivity
Authors: Schenkel Alan Rowe , Unnithan Ranjana , Early Merideth A , Gilchrist John M. , Schroeder William G.
Published: June 2021

We have shown that Platelet Endothelial Cell Adhesion Molecule (PECAM, CD31) deficient mice in the FVB/n background have altered neutrophil and monocyte trafficking. These studies did not look at other leukocyte populations and relied typically on a primary inflammatory response. In this study, we used 2,4 Dinitrofluorobenzene (DNFB) to first sensitize wild type and PECAM deficient mice. Five days later, DNFB was applied to ears, and leukocytes from the ears were collected two days later. Major leukocyte populations were analyzed using six‐color flow‐cytometry. Some leukocyte populations, like dendritic cells and CD8+ T cells were unaffected by PECAM deficiency. Other populations, CD4+ T cells, gamma‐delta T cells, and DX5+, CD3− Natural Killer cells were markedly reduced in PECAM deficient mice. Another marker of inflammation, ear thickness, was unaffected by PECAM deficiency. These studies show that PECAM is differentially by some various leukocyte populations and may explain why some have little or no detectable PECAM expression in humans. This may have significant effects if anti‐PECAM therapies are used to selectively target certain leukocyte subsets.
Tamalin may cooperate with Cybr in modulating leukocyte migration in vivo
Authors: Coppola Vincenzo , Tessarollo Lino
Published: June 2021

Cybr (aka pscdbp, CASP or CBP) is a scaffold protein that binds cythohesin‐1, a guanine nucleotide exchange factor for the ARF‐GTPases, which affects the actin cytoskeleton remodeling during cell migration. Indeed, we have shown in mouse that Cybr deficiency negatively affects the number of leukocytes circulating in blood and lymphocytes migrating to the lymph nodes especially in response to pro‐inflammatory cytokines in stress conditions. Notably, Cybr‐deficient mice show clear deficits of CTL‐mediated virus‐induced tumor rejection. Cybr is not expressed in the nervous system where its homolog Tamalin (aka Grasp) is expressed the most. Tamalin too is a scaffold protein and shares with Cybr high homology at the PDZ and coiled‐coil domain suggesting common functions. Initially, Tamalin has been described as absent from the immune system. We show that Tamalin, although at low levels compared to Cybr, is expressed in the immune system. Tamalin is co‐expressed with Cybr in specific cell types suggesting a non‐redundant role for these two closely related proteins.

The goal of this study is to investigate in vivo whether Tamalin deficiency affects leukocyte migration and augment defects due to Cybr deficiency. For this purpose, we have generated Tamalin deficient mice and subsequently Cybr/Tamalin double knock‐out mice. We have previously shown that Cybr has a role in cell migration in stress conditions. Here, we show that Tamalin defcient mice do not show any obvious deficit of leukocyte migration. However, preliminary results suggest that Tamalin deficiency may strengthen Cybr migration defect in specific conditions. Further investigation is needed to clarify how these two proteins modulate cell migration in vivo. This research is supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.
DEFECTIVE INNATE IMMUNITY UNDERLIES THE ABSENCE OF CONTACT SENSITIVITY RESPONSES IN gld/perforin−/− MICE
Authors: Kish Danielle D. , Fairchild Robert L.
Published: June 2021

Contact hypersensitivity (CHS) is a CD8 T cell mediated immune response to sensitization and challenge of the skin. Hapten‐primed CD8T cell recruitment into hapten challenge sites requires prior CXCL1/KC‐mediated neutrophil infiltration. Although primed CD8 T cells do not express cytolytic activity, CHS is absent in gld/perforin−/− mice. CD8 T cell priming to sensitization with 2, 4‐dinitrofluorobenzene (DNFB) and T cell and neutrophil infiltration into DNFB challenged skin in gld/perforin−/− mice vs. wild‐type C57BL/6 (WT) mice were tested. ELISPOT analyses indicated equivalent priming of DNFB‐specific CD8 cells in WT and gld/perforin−/−mice. DNFB‐primed CD8 T cells from WT or gld/perforin−/−mice transferred equivalent CHS responses to naïve WT recipients. Production of KC was equivalent in challenged skin of DNFB sensitized WT and gld/perforin−/− mice. However, there was little/no neutrophil infiltration into the challenge site of sensitized gld/perforin−/− mice. CHS responses were restored in gld/perforin−/− mice when WT neutrophils were injected into the challenge site just before hapten challenge. These results indicate that Fas L and perforin are required for the initial neutrophil infiltration into hapten challenge sites that induce subsequent effector CD8 T cell recruitment to mediate CHS responses.
CD38 AND ZAP‐70 REGULATE CXCL12‐MEDIATED CHEMOTAXIS OF CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) CELLS
Authors: Deaglio Silvia , Vaisitti T. , Aydin S. , Bergui L. , D'Arena G. , Bonello L. , Omede' P. , Malavasi F.
Published: June 2021

Absence of mutations in the IgV genes, expression of the receptor CD38 and of the kinase ZAP‐70 are independent markers of unfavorable outcome in CLL patients.

Our group studies the role of CD38 in CLL not only as disease marker but also as pathogenetic agent. We showed that CD38 enhances proliferation of CLL cells through binding CD31, the ligand expressed by stromal and endothelial cells. Further, i) CD38+/ZAP‐70+ cells efficiently migrate towards CXCL12, ii) CD38 engagement leads to tyrosine phosphorylation of ZAP‐70 and iii) CD38 pathway is impaired in CD38+/ZAP‐70− CLL cells.

Collectively, these data point to a role for the CD38/ZAP‐70 pathway in CLL migration towards CXCL12. Indeed, CXCL12‐mediated chemotaxis can be blocked by anti‐CD38 mAbs. Pre‐treatment of CLL with anti‐CD38 mAbs impairs CXCR4 signaling, as indicated by lack of Ca2+ mobilization, ERK1/2 activation and receptor internalization. Further, CD38 and CXCR4 are associated, as shown by confocal microscopy and co‐immunoprecipitation.

In conclusion, these results offer biological evidence explaining why the combination of CD38 and ZAP‐70 results in a more efficient identification of CLL patients with aggressive disease. Further, they provide preliminary rationale for using anti‐CD38 ligands or inhibitors as adjunct therapeutic tools.
The increased expression of the activating Fc receptors in systemic lupus erythematosus
Authors: Su Kaihong , Zhou Huaxin , Rockwell Nicholas A , Chambers James G , Zhang Yan , Zhou Tong , Edberg Jeffrey C , Kimberly Robert P
Published: June 2021

The immunoglobulin G Fc receptors (FcγRs) play an important role in mediating the effector function of IgG. The expression ratio of activating and inhibitory FcγRs determines the threshold for cellular activation. Previous studies have suggested dysregulation of FcγR expression on monocytes and B cells in patients with systemic lupus erythematosus (SLE). However, there is no systematic examination of both activating and inhibitory receptors to determine the activation/inhibition ratio. Using receptor specific monoclonal antibodies, we have examined the cell surface expression levels of the activating FcγRI, FcγRIIa, and FcγRIII and the inhibitory FcγRIIb on circulating monocytes, neutrophils, myeloid dendritic cells and plasmacytoid dendritic cells in SLE patients. Compared to healthy controls, monocytes and myeloid dendritic cells from active SLE patients have increased expression of FcγRI with similar levels of FcγRIIa and FcγRIIb. Plasmacytoid dendritic cells from SLE patients have increased expression of FcγRIIa with no detectable expression of the inhibitory FcγRIIb. We conclude that the upregulation of the activating FcγRs in SLE patients alters the activation/inhibition ratio and may contribute to the immune overactivation in SLE.
IL‐21 Receptor Signaling Is Essential for BXSB‐Yaa SLE Pathogenesis
Authors: Bubier Jason A , Sproule Thomas J , Foreman Oded , Spolski Rosanne , Shaffer Daniel J , Morse Herbert C , Leonard Warren J , Roopenian Derry C
Published: June 2021

Interleukin‐21 (IL‐21) is a cytokine with pleiotropic actions on multiple lympho‐hematopoietic lineages. BXSB‐Yaa mice develop a severe form of a systemic lupus erythematosus (SLE)‐like disease caused by a mutation on the Y‐chromosome. We previously showed that BXSB‐Yaa mice express elevated levels of IL‐21, but the role of IL‐21 in BXSB‐Yaa disease pathogenesis was not determined. To investigate the importance of IL‐21 signaling in this disease, we crossed the BXSB‐Yaa mice onto the Il21r−/− background and monitored the mice for the characteristic BXSB‐Yaa autoimmune syndrome. A deficiency in IL‐21R resulted in the abrogation of all disease phenotypes, including abnormal splenic cell numbers and populations, Ig and anti‐nuclear antibody levels, kidney pathology, and morbidity/mortality. These results demonstrate that IL‐21 signaling is essential for the development of autoimmune disease in BXSB‐Yaa mice and suggest that IL‐21 may be a more broadly critical cytokine in SLE autoimmunity. This work was supported in part by a postdoctoral fellowship from the Arthritis Foundation (to J. Bubier), and NIH DK56597 and R21 DK074463 (to D. Roopenian) and the Intramural Research Program of the NIH, National Institute of Allergy and Infectious Diseases and National Heart, Lung and Blood Institute.
Genetic and clinical parameters determining human systemic lupus erythematosus (SLE) disease flares and complement C4 protein levels
Authors: Yang Yan , Wu Yee Ling , Esack Nazreen , Zhou Bi , Kitzmiller Kathryn , Savelli Stephanie , Nagaraja Haikady N , Birmingham Daniel J. , Rovin Brad H. , Hebert Lee A. , Yu C. Yung
Published: June 2021

Human SLE is characterized by disease flares and remissions. The goal of this study is to determine biomarkers associated with SLE flares. In a longitudinal study that involved 1838 bimonthly visits from 101 patients, clinical parameters associated with SLE disease flares and complement protein levels were studied by Generalized Estimating Equation (GEE) framework. In a cross‐sectional study, complement C4 gene copy‐number (GCN) variation plus plasma protein concentrations of C4 and C3 were determined from 233 European and 73 African American patients with SLE, and 455 healthy first‐degree relatives. The C4 GCN varied from 2 to 6. An increase in one copy of C4 leads to an increase of mean plasma C4 protein conc. by 5 mg/dL in SLE and 6–8 mg/dL in healthy subjects. Mean C4 protein conc. in each GCN was calculated from healthy subjects, and its difference from a SLE patient with the same C4 GCN is termed the “C4‐gap”. GEE analyses revealed that SLE flares are correlated with increased values of C4‐gap (z=2.13; p=0.03) and erythrocyte sedimentation rate (ESR; z=3.71; p=0.0002); and reduced values of age (z= −2.47; p=0.014), C3 levels (z= −3.58; p=0.0003) and interaction between C3 and C4‐gap (z= −2.83; p=0.0046). C4 protein levels were positively associated with C4 GCN, serum levels of C3 and creatinine, ESR and platelet counts; and negatively correlated with antinuclear antibodies and leukocyte counts.
Restoration of CD8 self‐tolerance in NOD mice by protective Idd9 genes
Authors: Hamilton‐Williams Emma , Martinez Javier , Wong Justin , Wicker Linda , Sherman Linda
Published: June 2021

Diabetes development in the NOD mouse is controlled by multiple susceptibility loci. Congenic NOD mice that contain B10.D2 genes within the Idd9 locus are highly protected from diabetes and we have investigated the mechanism of protection in these mice. To test for the presence of islet specific CD8 effector cells mice were infected with vaccinia virus that encodes the CD8 epitope from islet expressed IGRP (Vac‐IGRP). When NOD mice were infected with Vac‐IGRP they elicited strong IGRP specific CD8 responses but Idd9 congenic mice were tolerant. Analysis of radiation chimeric mice revealed that expression of protective Idd9 genes by hematopoietic cells is sufficient to restore IGRP specific CD8 tolerance. Reconstitution of NOD‐scid mice with CD8 cells derived from young Idd9 or NOD mice mixed with total NOD spleen cells showed that high frequencies of IGRP specific CD8 effectors could develop from Idd9 CD8 precursors. Hence (i) thymic tolerance is not responsible for restored CD8 tolerance in Idd9 mice and (ii) no protection is derived from endogenous expression of Idd9 genes within the CD8 cell. Transfer experiments with NOD IGRP specific 8.3 cells showed that early activation in the pancreatic lymph nodes is unaffected in Idd9 mice. When the long‐term survival of transferred 8.3 cells was tested, in NOD hosts IGRP specific 8.3 cells could be expanded 6 weeks post‐transfer but in Idd9 hosts the 8.3 cells were deleted.
The X‐ray Structure of IAd in Complex with a Self Peptide Offers New Insights Into the Basis of Autoimmune Gastritis (AIG)
Authors: Revilleza Maria Jamela R , Levin Ditza , Mage Michael G , Teyton Luc , Robinson Howard , Shevach Ethan M , Natarajan Kannan , Margulies David H
Published: June 2021

We developed a mouse model of autoimmune gastritis in which BALB/c mice expressing a transgenic αβ TCR, designated A51, spontaneously develop the disease. The TCR recognizes a 12‐mer peptide from the alpha subunit of gastric parietal cell H/K ATPase residues 889–900 (PLLCVGLRPQWE (H/K ATPase 889–900)), presented in the context of IAd. The disease, a model of human pernicious anemia, is characterized by slow and incomplete penetrance, anti‐gastric mucosal cell antibodies, inflammatory eosinophilic infiltrates, and parietal cell destruction. To investigate the contribution of MHC/peptide interactions to the initiation and maintenance of AIG, we generated a soluble recombinant version of the IAd/H/K ATPase889‐900 complex for biophysical and crystallographic analyses. We report here the 2.5 Å X‐ray structure of the complex. The structure was solved by molecular replacement and refined to Rfree of 28.9 by standard methods. The identification of peptide anchor residues and of solvent‐exposed side chains as T cell epitopic residues readily explains T cell proliferation data and off‐rate measurements of a panel of truncated, chimeric, and mutant peptides. This first structure of IAd in complex with an autoimmunogenic peptide reveals a heretofore unobserved peptide binding motif for IAd.

This was supported by the Intramural Research Program of the NIAID/NIH.
Inhibition of Activation‐Induced Cytidine Deaminase (AID) Preserved Spontaneous Germinal Centers but Suppressed Autoimmune Disease in BXD2 Mice
Authors: Hsu Hui‐Chen , Yang PingAr , Wu Qi , Job Godwin , Guentert Tanja , Wang John , Mountz John D
Published: June 2021

Increased expression of AID in B cells as a result of extensive spontaneous germinal centers (GCs) has been found to be associated with the development of arthrogenic and nephrogenic autoantibodies in autoimmune BXD2 mice. To determine if inhibition of AID can prevent the generation of pathogenic autoantibodiese, a transgenic (Tg) BXD2 mouse strain expressing a dominant negative (DN) form of AID cloned downstream of the chicken albumin promoter was generated by direct injection of BXD2 single cell embryos. This AID‐DN gene contains the H56R/E58Q mutation in the AID catalytic domain and the S38A mutation at the phosphorylation site that is essential for interaction of AID with replication protein A. At 6–8‐month of age, there was a significant decrease in the IgG isotype of autoantibodies and autoantibody producing B cells against histone, DNA, and BiP in BXD2 AID‐DN Tg+ mice compared to wild‐type (WT) BXD2 mice. At this age, BXD2 AID‐DN Tg+ mice were protected from development of arthritis. There was also decreased IgG2b and IgG2c containing C1q‐circulating immune complexes and decreased IgG deposition in glomeruli in BXD2 AID‐DN Tg+ mice, compared to WT BXD2 mice. The generation of spontaneous GCs in the spleens of BXD2 AID‐DN Tg+ mice, however, remained comparable to that found in the spleens of WT BXD2 mice. Our results suggest that over‐expression and high activity of AID is a down‐stream product of spontaneous autoreactive GCs that plays an essential role in the generation of pathogenic autoantibodies and development of autoimmune disease in BXD2 mice. This work is supported by Arthritis Foundation, VA Merit Review, American College of Rheumatology, and Daiichi‐Sankyo Co., Ltd.
Role of Type I Interferons in Lupus nephritis.
Authors: Bennett Heather Lynn , Sriram Uma , Shivers Debra K , Gallucci Stefania
Published: June 2021

Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by anti‐DNA auto‐antibodies and glomerulonephritis. Type I Interferons (IFNs) are considered pathogenic in this autoimmune disease, by activating the innate and adaptive immune response. Type I IFNs are produced by immune and non‐immune cells upon viral infection or TLR stimulation; they may play an active role in inflammation and destruction of renal tissue. We hypothesize that Type I IFNs role in kidney destruction is fueled by a feedback loop created by the kidney itself. To test our hypothesis, we create chimeric mice in which bone marrow from female Sle1,2,3 lupus prone mice, capable of developing autoAbs and glomerunephritis, are injected into irradiated female Interferon receptor knockout mice (IFNAR‐KO host resistant to Type I IFNs), and C57BL/6 (wild type non autoimmune host) or syngeneic mice (autoimmune host). By measuring the expression of IFN responsive genes and phosphorylation levels of STAT1 and STAT 2 in the immune cells, we can determine the role of the kidney in regulating Type I IFNs. By monitoring auto‐antibody production and kidney pathology, we can assess onset and severity of lupus disease. We predict that chimeric Sle1,2,3 into IFNAR‐KO mice exhibit less severe kidney destruction and milder disease than control chimeras. Our project may suggest kidney‐targeted strategies for lupus treatment.
The IL‐14alpha Transgenic Mouse as a Model for Sjogren Disease
Authors: Shen Long , Suresh Lakshmanan , Kumar Vijay , Ambrus Julian L.
Published: June 2021

Sjogren's disease is a common autoimmune disease with both local (loss of salivary gland function) and systemic manifestations. Current animal models replicate only particular aspects of the disease. We have developed an animal model for Sjogren's disease by expressing IL‐14alpha, a gene expressed at high levels in both human Sjogren's disease and various animal models of Sjogren's syndrome, as a transgene in C57BL/6 mice. The IL‐14alpha transgenic mice (IL‐14a TG) accurately simulate human Sjogren's disease with hypergammaglobulinemia, autoantibodies, infiltration of the submandibular glands followed by the parotid glands with lymphocytes, interstitial lung disease, mild kidney disease and in old age large B cell lymphomas. We further demonstrate the loss of salivary gland function occurs before infiltration of the salivary glands with lymphocytes, but after deposition of immunoglobulins is noted in the salivary glands. We demonstrate that lymphotoxin is present in the salivary gland secretions before salivary gland function is lost. IL‐14aTG mice deficient in lymphotoxin fail to develop loss of salivary gland function or lymphocytic infiltration of their salivary glands. Thus, the IL‐14a TG mice are a valuable new model to study all aspects of Sjogren's disease. Early injury to the salivary glands likely involves cytokines, including lymphotoxin, and various autoantibodies. This work is supported by the Kaleida Health Foundation.
CD8a‐DCs induced upon GM‐CSF treatment can cause expansion of Tregs and suppress experimental autoimmune thyroiditis
Authors: Prabhakar Bellur S , Vasu Chentamarakshan , Ganesh Balaji
Published: June 2021

Experimental Autoimmune Thyroiditis (EAT) is a well characterized mouse model for Hashimoto's thyroiditis. Earlier, we reported that GM‐CSF treatment of mice with EAT can cause an expansion of CD8a‐DCs and CD4+CD25+ T cells with the ability to produce enhanced levels of IL‐10 and suppress the disease. Neither the underlying mechanism of action of GM‐CSF induced CD8a‐DCs (GM‐CD8a‐DCs), nor the regulatory phenotype of CD4+CD25+ T cells was explored. In this study, we show that GM‐CSF treatment can cause significant induction of Foxp3+ and IL‐10+ Tregs. Further, we show that GM‐CD8a‐DCs remain semi‐mature and are more tolerogenic relative to controls. These semi‐mature GM‐CD8a‐DCs showed lower transcript levels for TNF‐á, IL‐12, and IL1â. Antigen presentation by GM‐CD8a‐DCs caused significant expansion of Foxp3+ Tregs that could suppress effector T cell function through enhanced IL‐10. Adoptive transfer of CD8a‐DCs from GM‐CSF treated, but not untreated, SCID mice into CBA/J mice prevented EAT development. Our results show that GM‐CD8a‐DCs are tolerogenic in that antigen presentation by them can cause expansion of IL‐10+ and Foxp3+ Tregs that protect mice from developing EAT.
Immune Dysregulation in Systemic Lupus Erythematosus
Authors: Suggs Jeanann Lovell , Cruse Julius M , Lewis Robert E , Majithia Vikas
Published: June 2021

Forty cases of clinically diagnosed SLE were examined for HLA typing trends as well as composition of immune system cells. The SLE population under consideration was 97% female and 97% African American. Of the 37 cases for which HLA typing was performed, 13 (35%) had at least one 1503 allelic haplotype. All (100%) of these 1503 type cases were African American and 85% were female. Immunologic testing of twelve of these 1503 types showed 8 (67%) with anti‐DNA antibodies, 8 (67%) with antinuclear antibodies, and 4 (33%) with anti‐Sm antibodies. Additionally, 6 cases (16%) had at least one 0804 allelic haplotype. All (100%) of these 0804 type cases were female African Americans. Testing of 5 of these 0804 types showed 4 (80%) with anti‐DNA antibodies, 3 (60%) with anti‐Sm antibodies, and 2 (40%) with antinuclear antibodies. Twelve cases were examined by flow cytometry to determine the mean percentage of lymphocyte population constituents. Across these cases, CD4/CD3 positive Th cells contributed on average to 47% of the lymphocyte population, with an average of 5.1% of these Th cells also being CD25/FoxP3 positive Tregs. Statistical analysis found no significant difference for either mean when comparing 1503 type against all others. Future studies will focus on increasing the number of total SLE cases and incorporating age/gender/race matched controls to determine significance of HLA and regulatory T cell function in SLE.
Dendritic Cell Produced IL‐12 Augments CD8+ T cell Activation through the Production of the Chemokines CCL1 and CCL17
Authors: Henry Curtis James , Grayson Jason M , Ornelles David A , Brzoza Kristina L , Hiltbold Elizabeth M
Published: June 2021

Dendritic cells (DC) shape immune responses by producing cytokines such as Interleukin‐12 (IL‐12). IL‐12 is important in the clearance of the intracellular bacterial pathogen Listeria monocytogenes (Lm) and activates CD8+ T cells (CTL), which are required for sterilizing immunity. Our initial studies on the role of IL‐12 on CTL function determined that IL‐12p40 signals were transduced primarily during the first 48 hours of priming. However, these studies did not address the individual contributions of IL‐12 or IL‐23 nor how they affect the physical interactions of priming.

To address these questions wild‐type, IL‐12p35−/−, or IL‐12p40−/− DC were infected with Lm and then used to prime CTL. IL‐12p40−/− mice immunized with p35−/− DC or p40−/− exhibited a reduced ability to clear Listeria compared to mice immunized with wild‐type DC. We found that CTL primed in vitro by p35−/− or p40−/− DC produced less IFN‐γ with the greatest reduction seen with the p40−/− DC, whereas proliferation was not affected. CTL primed in the absence of IL‐12 engaged in shorter duration interactions with DC which correlated with the absence of the chemokines CCL1 and CCL17. Neutralization of these chemokines resulted in decreased IFN‐γ production by CTL. Thus, CTL priming by DC is influenced more by the activity of IL‐12 than IL‐23 and in part through CCL1 and CCL17 whose production requires IL‐12. 1RO1AI057770‐01A1 and 1F31AI073245‐01A1
Three different subsets of alternatively activated macrophages (M2Mϕ) detected in mice with severe thermal injury
Authors: Shigematsu Kenji , Kobayashi Makiko , Herndon David N , Suzuki Fujio
Published: June 2021

A predominance of M2Mφ in burn mice has been reported in many papers. M2Mφ play a major role on the increased susceptibility of thermally injured hosts to various infections. Since 3 different subsets of M2Mφ (M2a, M2b, M2c) have been recently described, in this study, the properties of M2Mφ appearing in mice with severe thermal injury were examined. Peritoneal Mφ (1 x 106 cells/ml, 92% or more purity) were isolated using magnetic beads coated with anti‐F4/80 mAb from mice 1 to 21 days after burn injury (3rd degree, 25% TBSA burn). Culture fluids of these Mφ, 24 hrs after cultivation without any stimulation, were assayed for CCL5 and IL‐12 (biomarkers of M1Mφ), CCL17 and IL‐10 (biomarkers of M2aMφ), CCL1 and IL‐10 (biomarkers of M2bMφ) and CXCL13 and IL‐10 (biomarkers of M2cMφ) using ELISA. Also, these Mφ preparations were tested for mannose receptor mRNA expression by RT‐PCR. In the results, Mφ from mice 2 to 5 days postburn produced CCL17. Also, Mφ from mice 1 to 14 days postburn produced CXCL13. Mφ from mice 7 to 21 days postburn produced CCL1. Mφ from mice 7 days postburn did not produce CCL17. Mannose receptor mRNA was expressed by Mφ from mice 2 days postburn, while it was not expressed by Mφ from mice 14 days postburn. These results indicate that severe thermal injury induces 3 subsets of M2Mφ with different time courses. M2aMφ are generated in mice early after burn injury (1 to 5 days postburn), M2cMφ are generated in mice 1 to 14 days postburn, and M2bMφ are generated in mice late after burn injury (1 to 3 weeks postburn). This study was supported by SHC NA #8840.
In vitro induction of CCL2‐ and IL‐10‐producing PMN (PMN‐II) using band PMN isolated from exercised volunteers
Authors: Kobayashi Makiko , Shigematsu Kenji , Herndon David N , Suzuki Fujio
Published: June 2021

M2Mφ are known to be inhibitory on the Mφ conversion from resident Mφ to M1Mφ (a major effector cell on host antibacterial innate immunities). As a M2Mφ inducer cell, PMN‐II have been demonstrated in severely burned mice with decreased antibacterial resistances. To analyze the immunosuppressive property of PMN‐II, in this study, we tried to induce PMN‐II in cultures of healthy volunteer PMN. By Ficoll‐Hypaque and dextran sedimentations, PMN were prepared from peripheral blood of healthy volunteers with or without 1 hr‐exercise. Band PMN and segmented PMN were isolated from the PMN preparation using biotin‐conjugated anti‐CD10 mAb and magnetic‐beads coated with streptavidin. Cells obtained (1 x 106 cells/ml) were cultured with SAC, GM‐CSF or VEGF. Culture fluids harvested 18 hrs after cultivation were assayed for CCL2 and IL‐10 (biomarkers for PMN‐II). In the results, segmented and band PMN preparations from volunteers without any exercise did not produce CCL2 and IL‐10 after the cultivation with various stimulants. However, in response to the stimulation with SAC, GM‐CSF or VEGF, band PMN (but not segmented PMN) isolated from volunteers 3 to 24 hrs after 1 hr‐exercise produced CCL2 and IL‐10 into their culture fluids. The highest production of CCL2 was shown when PMN prepared from volunteers 3 hrs after 1 hr‐exercise were stimulated with SAC and GM‐CSF. Also, the numbers of band PMN increased greatly in the peripheral blood of volunteers 3 to 24 hrs after having exercised. These results indicate that, by the stimulation with SAC, GM‐CSF or VEGF, PMN‐II can be inducible from band PMN of exercised volunteers. PMN‐II induced in vitro will be useful in the analytical studies of PMN‐II‐associated immunosuppression. This study was supported by SHC NA #8840.
IFN‐α production by cord blood plasmacytoid dendritic cells is totally deficient.
Authors: Charrier Emily , Dardari R’Kia , Cordeiro Paulo , Michaud Annie , Duval Michel
Published: June 2021

Introduction: The interaction between natural killer cells (NK) and plasmacytoid dendritic cells (pDC) has been suggested to play a role during the early phase of the immune response to viral infections and tumors. NK cells can lyse tumor and virus‐infected cells and their cytolytic activity has long been known to be enhanced by interferon‐alpha (IFN‐α). Human pDC are a major source of IFN‐α following viral stimulation or upon exposure to TLR‐7 or TLR‐9 ligands. Objective: In order to study the cross‐talk between NK and pDC in human newborns, the effect of IFN‐α on NK cells was compared between cord and adult blood, and the response of pDC to CpG or HSV‐1 was studied. Results: We have recently reported that neonatal NK cells are mature and express a reduced cytolytic activity. In this study, we show that IFN‐α‐stimulated‐NK cells display an activated phenotype and a cytolytic activity similar to adult NK cells. Moreover, the production of IFN‐α in response to CpG or HSV‐1 is dramatically impaired in cord blood. Cord blood pDC present a decreased expression of TLR9 and IRF7 which may explain the deficient production of IFN‐α. This lower expression of IRF7 and IFN‐α production can be partially restored by pre‐treatment of IFN‐β. However, although all cord blood show a deficiency in IFN‐α production, most of cord blood samples (2/3) exhibit a normal maturation of pDC in response to CpG, as shown by the up‐regulation of CD80, CD83, CD40, HLA‐DR and CD54. Conclusions: The defect in IFN‐α production by cord blood plasmacytoid dendritic cells can be explained by the low level of IRF7 and TLR9 expression. This defect can be partially restored by increasing IRF7 expression by pre‐treatment of IFN‐β. These findings might explain the increased susceptibility of human newborns to infections.
NKG2A and the functional competence of liver natural killer cells
Authors: Lassen Matthew G. , Hahn Young S.
Published: June 2021

The liver is a unique environment that maintains tolerance to a heavy load of foreign antigens derived from the intestine. The liver is also the site of several chronic viral infections, including hepatitis B & C viruses. Defining the mechanism(s) underlying the tolerogenic environment of the liver will broaden our understanding of normal liver function and contribute to better therapeutic strategies to persistent infection. Natural killer cells (NKs) make up a large percentage of the liver lymphocyte population. NKs play a critical role in innate immunity and generating adaptive immunity via crosstalk with dendritic cells (DCs). To determine the role of liver NK cells in modulating host immunity, we examined cytokine production upon interaction of NKs with DCs. When liver or spleen NKs were co‐cultured with splenic DC in the presence of IL‐12 or IL‐15, IFN‐γ was significantly decreased in the liver NK‐DC coculture compared to the spleen NK‐DC coculture, in the presence of either IL‐12 or IL‐15. We next determined the status of NK cell differentiation and the expression of both inhibitory and activating NK receptors in the liver and spleen on specific NK subsets defined by the expression of CD27 and CD11b. The inhibitory receptor NKG2A was expressed at higher levels on liver NKs compared to spleen NKs. This higher expression was observed in all NK subsets, but most pronounced in the CD27highCD11blow subset. In contrast, expression within all subsets of the activating receptor NKG2D, as well as all Ly49 receptors, was lower on liver NKs compared to spleen NKs. These results suggest that the liver NK cell may be less functionally competent than spleen NK cells, and that the inhibitory receptor NKG2A may play a prominent role in the effector status of NKs within the liver.
TLT2 Functions as an Inhibitory Receptor that Attenuates the Oxidative Burst In Neutrophils Stimulated with FMLP
Authors: Halpert Matthew M , King R. Glenn , Justement Louis
Published: June 2021

Trem Like Transcript 2 (TLT2) is a recently identified member of the Triggering Receptor Expressed on Monocyte (TREM) locus. Analysis of TLT2 expression by cells of the hematopoietic lineage revealed that this receptor is expressed on B lineage cells, making it the only conserved member of the TREM locus to be expressed on lymphocytes. Within the mature B cell compartment, TLT2 expression is highest on the B1 and marginal zone populations, although it is expressed on virtually all follicular B cells as well. Aside from B cells, TLT2 is expressed on macrophages as well as neutrophils in the bone marrow and periphery. Treatment of mice with agents that induce inflammation results in a dramatic increase in the level of TLT2 expressed on activated neutrophils. Because TLT2 is expressed at higher levels on innate‐like B cells, macrophages and neutrophils, and is upregulated in response to inflammatory stimuli, it is likely that this receptor plays a role in innate immunity. Recent studies have demonstrated that ligation of TLT2 inhibits the oxidative burst in neutrophils stimulated with FMLP. These results suggest that TLT2 may function as an inhibitory receptor that attenuates activation of neutrophils, and possibly macrophages, stimulated with agonists that bind to seven transmembrane‐spanning receptors that signal via heterotrimeric G‐proteins.
Hypersensitivity of innate response to TLRs in neonate
Authors: Fu Yang‐Xin , Kim Kwang Dong , Zhao Jie , Auh Sogyong , Yang Xuanming , Tang Hong
Published: June 2021

Neonates have high mortality and morbidity to infection, presumably due to a lack of fully developed adaptive and innate immune system. Injection of low dose of LPS mixed with D‐Galactosamine (D‐GalN) is a traditional “low dose LPS” model to study agonistic effects of this ligand of toll‐like receptor (TLR). Recent studies show that neonates are much more resistant to low dose LPS than adult mice, presumably caused by an impaired innate response in the newborn host. However, we have revealed neonatal mice were much more susceptible to LPS alone than adult mice, and such hypersensitivity extended to other TLR agonists including actual virus infection. We have now revealed that neonates survive better to low dose LPS likely because D‐GalN was more toxic to liver of adult than that of neonatal mice. Our data further showed that an uncontrolled innate immune response to TLR agonists or viral infection would be the direct cause of death in neonates. Therefore, the traditional low dose LPS model might not be an appropriate system to compare the innate responses in different ages or immunogenetic background. Remarkably, through transfer of T cells into neonates or depletion of T cells in young mice, we showed that T cells were necessary and sufficient to temper the early inflammatory response to LPS and protect neonates from death. Therefore, these results suggested that higher morbidity and mortality in neonates were caused by their lower number of T cells to mount sufficient inhibition of the early inflammation response by the innate cells, with important implication for new diagnosis and treatment for neonatal infection.
C1q is a molecular switch dictating the monocyte to dendritic cell (DC) transition and arrests DCs in an immature phenotype
Authors: Hosszu Kinga K , Santiago‐Schwarz Frances , Peerschke Ellinor I , Ghebrehiwet Berhane
Published: June 2021

Complement protein C1q plays a crucial role in regulating peripheral tolerance by facilitating clearance of apoptotic debris and modulating T cell proliferation. Moreover, C1q is emerging as a moderator of dendritic cell (DC) activity. Immature DCs (iDC) are able to secrete C1q. We postulated that such locally secreted C1q regulates the transition from monocytes to DCs. Using flow cytometric analysis we show here that monocyte‐derived DCs cultured in the presence of soluble C1q failed to down regulate CD14 and expressed reduced levels of CD83, DR and CD86. The inhibition of DC maturation was not due to apoptosis, as assessed by lack of annexin V binding and morphological examination. Instead, multiparametric analysis revealed the development of CD14+CD11c+CD16+/− cells representing a subset of iDCs. Monocyte‐DC precursors from the outset expressed gC1qR, the receptor for globular heads of C1q. In contrast, cC1qR, the receptor for the collagen tails of C1q, was lacking on these cells despite the expression of its partner CD91, indicating that inhibition of DC growth by C1q occurs via C1q‐gC1qR interactions. Significantly, pathogens such as hepatitis C virus, which bind gC1qR, may exploit this system by mimicking C1q to escape the acquired immune response. We propose that C1q operates at a critical checkpoint during the monocyte to DC transition and helps maintain immune tolerance by signaling via gC1qR. [Supported by NIH Grant R01 AI‐060866 (to BG) and R03 AR‐05396 (to FSS)]
Identification of C1q receptor associated surface molecules on the monocyte‐like cell line U937
Authors: Menzies Catherine A. , David‐Habiel  , Glassberg Philip F. , Phuong‐Nguyen  , Jolyon‐Jesty  , Peerschke Ellinor I.B. , Berhane‐Ghebrehiwet
Published: June 2021

The C1q receptor (C1qR) comprises of at least two unrelated surface proteins, which bind C1q via either its globular heads (gC1qR) or collagen tail (cC1qR). Although these non‐transmembrane proteins bind C1q and induce biological responses, the mechanism by which they do so is incompletely understood. The present studies were undertaken to identify potential new receptors or associated molecules that may have eluded identification due to limitations of previous techniques. Therefore, we used solubilized membrane proteins from U937 cells (total 2×108 cells) that were first surface biotinylated by a method that targeted either the amino groups or the carbohydrate moieties of proteins. Then, immunocapture ELISA was used to confirm that the known receptors—gC1qR and cC1qR—are surface labeled. Next, we used anti‐gC1qR mAb 74.5.2 to identify membrane proteins that are co‐captured with gC1qR. The results showed the presence of CD44 and β1 integrin. Furthermore, when membrane proteins were incubated with C1q coated wells, both CD44 and β1 integrin were bound to C1q in addition to the expected gC1qR and cC1qR. These results indicate that C1qR probably consists of transmembrane (CD44 and β1 integrin) and non‐transmembrane (gC1qR and cC1qR) surface proteins that together forge a docking/signaling partnership. [Supported by NIH‐NIAID Grant R01 AI‐060866]
Expression of CD21/35 on Follicular Dendritic Cells Expedites Peripheral Prion Accumulation And Neuroinvasion
Authors: Zabel Mark D , Michel Brady , Kranich Jan , von Teichman Adriana , Heikenwalder Mathias , Haas Karen , Tedder Thomas F , Weis John H , Aguzzi Adriano
Published: June 2021

We have studied the role of CD21/35, which bind derivatives of complement factors C3 and C4, in extraneural prion replication and neuroinvasion. Upon administration of small prion inocula, CD21/35−/− mice experienced lower attack rates and delayed disease over both wild‐type (WT) mice and mice with combined C3 and C4 deficiencies. Early after inoculation, CD21/35−/− spleens were devoid of infectivity. Reciprocal adoptive bone‐marrow transfers between WT and CD21/35−/− mice revealed that protection from prion infection resulted from ablation of stromal, but not hematopoietic, CD21/35. Further adoptive transfer experiments between WT mice and mice devoid of both the cellular prion protein PrPC and CD21/35 showed that splenic retention of inoculum depended on stromal CD21/35 expression. Since both PrPC and CD21/35 are highly expressed on follicular dendritic cells (FDCs), CD21/35 appears to be involved in targeting prions to FDCs and expediting neuroinvasion following peripheral exposure to prions.