bluuebunny/crossref_metadata_2025 huggingface dataset:
Golli-Induced Paralysis: A Study in Anergy and Disease
Authors: Clark Lise , Otvos Laszlo , Stein Paul L. , Zhang Xiang-Ming , Skorupa Annette F. , Lesh Georgia E. , McMorris F. Arthur , Heber-Katz Ellen
Published: January 2023

The Golli-MBP transcription unit contains three Golli-specific exons as well as the seven exons of the classical myelin basic protein (MBP) gene and encodes alternatively spliced proteins that share amino acid sequence with MBP. Unlike MBP, which is a late Ag expressed only in the nervous system, Golli exon-containing gene products are expressed both pre- and postnatally at many sites, including lymphoid tissue, as well as in the central nervous system. To investigate whether Golli-MBP peptides unique to Golli would result in neurological disease, we immunized rats and observed a novel neurological disease characterized by mild paralysis and the presence of groups of lymphocytes in the subarachnoid space but not in the parenchyma of the brain. Disease was induced by Th1-type T cells that displayed an unusual activation phenotype. Primary stimulation in vitro induced T cell proliferation with increased surface CD45RC that did not become down-regulated as it did in other Ag-stimulated cultures. Secondary stimulation of this CD45RChigh population with Ag, however, did not induce proliferation or IL-2 production, although an IFN-γ-producing population resulted. Proliferation could be induced by secondary stimulation with IL-2 or PMA-ionomycin, suggesting an anergic T cell population. Cells could adoptively transfer disease after secondary stimulation with IL-2, but not with Ag alone. These responses are suggestive of a chronically stimulated, anergic population that can be transiently activated to cause disease, fall back into an anergic state, and reactivated to cause disease again. Such a scenario may be important in chronic human disease.
The Role of the Human Fc Receptor FcγRIIA in the Immune Clearance of Platelets: A Transgenic Mouse Model
Authors: McKenzie Steven E. , Taylor Scott M. , Malladi Padmini , Yuhan Heena , Cassel Diana L. , Chien Paul , Schwartz Elias , Schreiber Alan D. , Surrey Saul , Reilly Michael P.
Published: January 2023

In humans, the Fc receptor for IgG, FcγRIIA, is expressed on macrophages and platelets and may play an important role in the pathophysiology of immune-mediated thrombocytopenia. Mice lack the genetic equivalent of human FcγRIIA. To better understand the role of FcγRIIA in vivo, FcγRIIA transgenic mice were generated and characterized. One transgenic mouse line expressed FcγRIIA on platelets and macrophages at levels equivalent to human cells, and cross-linking FcγRIIA on these platelets induced platelet aggregation. Immune-mediated thrombocytopenia in this transgenic line was studied using i.v. and i.p. administration of anti-mouse platelet Ab. In comparison with matched wild-type littermates that are negative for the FcγRIIA transgene, Ab-mediated thrombocytopenia was significantly more severe in the FcγRIIA transgenic mice. In contrast, FcR γ-chain knockout mice that lack functional expression of the Fc receptors FcγRI and FcγRIII on splenic macrophages did not demonstrate Ab-mediated thrombocytopenia. We generated FcγRIIA transgenic × FcR γ-chain knockout mice to examine the role of FcγRIIA in immune clearance in the absence of functional FcγRI and FcγRIII. In FcγRIIA transgenic × FcR γ-chain knockout mice, severe immune thrombocytopenia mediated by FcγRIIA was observed. These results demonstrate that FcγRIIA does not require the FcR γ-chain for expression or function in vivo. Furthermore, taken together, the data suggest that the human Fc receptor FcγRIIA plays a significant role in the immune clearance of platelets in vivo.
Cutting Edge: Sustained Expansion of CD8+ T Cells Requires CD154 Expression by Th Cells in Acute Graft Versus Host Disease
Authors: Buhlmann Janet E. , Gonzalez Mercedes , Ginther Brigit , Panoskaltsis-Mortari Angela , Blazar Bruce R. , Greiner Dale L. , Rossini Aldo A. , Flavell Richard , Noelle Randolph J.
Published: January 2023

Brief treatment with αCD154 Ab has been shown to prevent acute graft versus host disease (aGvHD). We extend these data to show that in the absence of CD154 function, donor T cells are unable to expand or generate high level anti-host CTL activity. Using transgenic (Tg) alloreactive CD8+ T cells adoptively transferred into allogeneic recipients, we show that short-term expansion of the CD8+ Tg T cells occurred in the absence of Th cells, and this short-term expansion could be facilitated with an agonistic αCD40. While CD40 agonism could enhance short-term expansion, sustained expansion of CD8+ Tg T cells required bona fide CD154-expressing CD4+ alloreactive Th cells. While CD154 was necessary for CD8+ Tg T cell sustained expansion, IL-2 was also implicated as essential. These observations suggest αCD154 therapy in GvHD is effective because the treatment causes an abortive CD8 alloresponse leading to the exhaustion or deletion of alloreactive CD8+ clones preventing the development of disease.
Cutting Edge: Recruitment of the CD19/CD21 Coreceptor to B Cell Antigen Receptor Is Required for Antigen-Mediated Expression of Bcl-2 by Resting and Cycling Hen Egg Lysozyme Transgenic B Cells
Authors: Roberts Teresa , Snow E. Charles
Published: January 2023

Recruitment of the CD19/CD21 coreceptor is thought to lower the threshold for effective signaling through the B cell Ag receptor. We provide evidence supporting a second role for coreceptor recruitment, and that is to enhance the survival/proliferative potential of the responding B cells. We show that B cell Ag receptor signaling in the absence of coreceptor recruitment induces cellular accumulation of the anti-apoptotic protein Bcl-xL, whereas CD19-mediated signals are required for Bcl-2 accumulation. The expression of both anti-apoptotic proteins correlates with the enhanced responsiveness of both resting and cycling B cells to growth-promoting signals delivered through CD40. These results provide further evidence for the necessity of coreceptor recruitment during Ag-dependent B cell activation and indicate that Ags derived from inflammatory sites function as better thymus-dependent Ags than their counterparts not coated with complement fragments.
MHC Class I-Restricted Presentation of Maleylated Protein Binding to Scavenger Receptors
Authors: Bansal Pratima , Mukherjee Paushali , Basu Sandip K. , George Anna , Bal Vineeta , Rath Satyajit
Published: January 2023

Pathways for loading exogenous protein-derived peptides on MHC class I are thought to be present mainly in monocyte-lineage cells and to involve phagocytosis- or macropinocytosis-mediated antigenic leakage into either cytosol or extracellular milieu to give peptide access to MHC class I. We show that maleylation of OVA enhanced its presentation to an OVA-specific MHC class I-restricted T cell line by both macrophages and B cells. This enhanced presentation involved uptake through receptors of scavenger receptor (SR)-like ligand specificity, was TAP-1-independent, and was inhibited by low levels (2 mM) of ammonium chloride. No peptide loading of bystander APCs by maleylated (maleyl) OVA-pulsed macrophages was detected. Demaleylated maleyl-OVA showed enhanced MHC class I-restricted presentation through receptor-mediated uptake and remained highly sensitive to 2 mM ammonium chloride. However, if receptor binding of maleyl-OVA was inhibited by maleylated BSA, the residual presentation was relatively resistant to 2 mM ammonium chloride. Maleyl-OVA directly introduced into the cytosol via osmotic lysis of pinosomes was poorly presented, confirming that receptor-mediated presentation of exogenous maleyl-OVA was unlikely to involve a cytosolic pathway. Demaleylated maleyl-OVA was well presented as a cytosolic Ag, consistent with the dependence of cytosolic processing on protein ubiquitination. Thus, receptor-specific delivery of exogenous protein Ags to APCs can result in enhanced MHC class I-restricted presentation, suggesting that the exogenous pathway of peptide loading for MHC class I may be a constitutive property dependent mainly on the quantity of Ag taken up by APCs.
Aberrant CD3- and CD28-Mediated Signaling Events in Cord Blood T Cells Are Associated with Dysfunctional Regulation of Fas Ligand-Mediated Cytotoxicity
Authors: Sato Katsuaki , Nagayama Hitomi , Takahashi Tsuneo A.
Published: January 2023

There have been numerous reports of decreased acute and chronic graft-vs-host disease (GVHD) in patients receiving HLA-matched or HLA-disparate umbilical cord transplants. However, little is known about the mechanisms underlying the low incidence of GVHD in umbilical cord blood transplantation (CBT). In this study, we examined CD3- and CD28-mediated functional properties and signaling events in CB T cells (CBTCs). Dual stimulation of peripheral blood TCs (PBTCs) and bone marrow TCs (BMTCs) with mAbs to CD3- and CD28-induced expressions of Fas ligand (FasL), as well as CD25 and CD154 (CD40L), whereas defective induction of these activation-associated cell surface molecules were observed in CBTCs. Engagement of both CD3 and CD28 induced FasL-mediated cytotoxicity in peripheral blood TCs (PBTCs) but not CBTCs; however, both of these tissue sources possess intrinsically similar proliferative responsiveness. Analysis of CD3- and CD28-induced signal transduction revealed a deficiency in signaling events that involved repressed tyrosine phosphorylation and enzymatic activities of a family of mitogen-activated protein kinases, extracellular signal-regulated kinase 2, stress-activated protein kinase/c-jun N-terminal kinase (SAPK/JNK), and p38mapk, as well as p56lck and ZAP-70 in CBTCs compared with those in PBTCs. These results suggest that CD3- and CD28-mediated signaling events blockage in CBTCs may be responsible for dysfunction of FasL-mediated cytotoxicity and lead to the low incidence of severe GVHD in CBT.
Diesel Exhaust Particles Induce NF-κB Activation in Human Bronchial Epithelial Cells In Vitro: Importance in Cytokine Transcription
Authors: Takizawa Hajime , Ohtoshi Takayuki , Kawasaki Shin , Kohyama Tadashi , Desaki Masashi , Kasama Tsuyoshi , Kobayashi Kazuo , Nakahara Kazuhiko , Yamamoto Kazuhiko , Matsushima Kouji , Kudoh Shoji
Published: January 2023

Fine particles derived from diesel engines (diesel exhaust particles, DEP) have attracted attention, since their density in industrial countries seems related to the increased prevalence of pulmonary diseases. Previous studies have suggested that DEP have a potential to directly activate airway epithelial cells to produce and release inflammatory cytokines and mediators, and thus facilitate inflammatory responses in the lung. To elucidate the molecular mechanisms of their action, we studied here IL-8 gene expression, one of the important cytokines in inflammatory responses, by Northern blot analysis and run-on transcription assay. Suspended DEP (1–50 μg/ml) increased the steady state levels of IL-8 mRNA, which was suggested to be largely due to increased transcriptional rates. Electrophoretic mobility shift assay demonstrated that DEP induced increased binding to the specific motif of NF-κB, but not of transcription factor AP-1. The luciferase reporter gene assay using wild-type and mutated NF-κB-binding sequences showed that DEP-induced NF-κB activation was involved in IL-8 transcription. Finally, both N-acetylcysteine and pyrrolidine dithiocarbamate attenuated the action of DEP on IL-8 mRNA expression, suggesting that oxidant-mediated pathway might be involved in its processes. These results suggested that DEP activate NF-κB, which might be an important mechanism of its potential to increase the expression of inflammatory cytokines in vitro.
A Novel β1 Integrin-Dependent Mechanism of Leukocyte Adherence to Apoptotic Cells
Authors: Schwartz Barbara R. , Karsan Aly , Bombeli Thomas , Harlan John M.
Published: January 2023

Adherence of leukocytes to cells undergoing apoptosis has been reported to be dependent on a variety of recognition pathways. These include αVβ3 (CD51/CD61, vitronectin receptor), CD36 (thrombospondin receptor), macrophage class A scavenger receptor, phosphatidylserine translocated to the outer leaflet of apoptotic cell membranes, and CD14 (LPS-binding protein). We investigated the mechanism by which leukocytes adhere to apoptotic endothelial cells (EC). Peripheral blood mononuclear leukocytes and U937 monocytic cells adhered to human or bovine aortic EC induced to undergo apoptosis by withdrawal of growth factors, treatment with the promiscuous protein kinase inhibitor staurosporine, with the protein synthesis inhibitor and protein kinase activator anisomycin, or with the combination of cycloheximide and TNF-α. Expression of endothelial adherence molecules such as CD62E (E-selectin), CD54 (ICAM-1), and CD106 (VCAM-1) was not induced or increased by these treatments. A mAb to αVβ3, exogenous thrombospondin, or blockade of phosphatidylserine by annexin V did not inhibit leukocyte adherence. Further, leukocyte binding to apoptotic EC was completely blocked by treatment of leukocytes but not EC with mAb to β1 integrin. These results define a novel pathway for the recognition of apoptotic cells.
Extracellular Acidification Induces Human Neutrophil Activation
Authors: Trevani Analía S. , Andonegui Graciela , Giordano Mirta , López Daniel H. , Gamberale Romina , Minucci Fernando , Geffner Jorge R.
Published: January 2023

In the current work, we evaluated the effect of extracellular acidification on neutrophil physiology. Neutrophils suspended in bicarbonate-buffered RPMI 1640 medium adjusted to acidic pH values (pH 6.5–7.0) underwent: 1) a rapid transient increase in intracellular free calcium concentration levels; 2) an increase in the forward light scattering properties; and 3) the up-regulation of surface expression of CD18. By contrast, extracellular acidosis was unable to induce neither the production of H2O2 nor the release of myeloperoxidase. Acidic extracellular pH also modulated the functional profile of neutrophils in response to conventional agonists such as FMLP, precipiting immune complexes, and opsonized zymosan. It was found that not only calcium mobilization, shape change response, and up-regulation of CD18 expression but also production of H2O2 and release of myeloperoxidase were markedly enhanced in neutrophils stimulated in acidic pH medium. Moreover, extracellular acidosis significantly delayed neutrophil apoptosis and concomitantly extended neutrophil functional lifespan. Extracellular acidification induced an immediate and abrupt fall in the intracellular pH, which persisted over the 240-s analyzed. A similar abrupt drop in the intracellular pH was detected in cells suspended in bicarbonate-supplemented PBS but not in those suspended in bicarbonate-free PBS. A role for intracellular acidification in neutrophil activation is suggested by the fact that only neutrophils suspended in bicarbonate-buffered media (i.e., RPMI 1640 and bicarbonate-supplemented PBS) underwent significant shape changes in response to extracellular acidification. Together, our results support the notion that extracellular acidosis may intensify acute inflammatory responses by inducing neutrophil activation as well as by delaying spontaneous apoptosis and extending neutrophil functional lifespan.
Soluble Murine IL-1 Receptor Type I Induces Release of Constitutive IL-1α
Authors: Netea Mihai G. , Jan Kullberg Bart , Boerman Otto C. , Verschueren Ineke , Dinarello Charles A. , Van der Meer Jos W. M.
Published: January 2023

IL-1α and IL-1β are proinflammatory cytokines involved in the pathogenesis of many infectious and noninfectious inflammatory diseases. To reduce IL-1 toxicity, extracellular domains of the soluble (s) IL-1R are shed from cell membranes and prevent triggering of cell-bound receptors. We investigated to what extent murine sIL-1RI can neutralize the IL-1 produced by LPS-stimulated macrophages. When mouse peritoneal macrophages were incubated with LPS, addition of sIL-1RI significantly inhibited the bioactivity of IL-1. Stimulation of cells with sIL-1RI alone induced no bioactive IL-1. When immunoreactive cytokine concentrations were measured with specific radioimmunoassays, sIL-1RI alone appeared to induce a significant release of IL-1α in a concentration-dependent manner. This effect was independent of new protein synthesis. The production of IL-1β or TNF-α was not influenced by sIL-1RI. There was no interference of sIL-1RI with the IL-1α radioimmunoassay. In mice, an i.v. injection of sIL-RI alone induced a rapid release of IL-1α, but not of TNF-α or IL-1β. Treatment of mice with sIL-1RI improved the survival during a lethal infection with Candida albicans. In conclusion, sIL-1RI induces a rapid release of IL-1α from cells, as well as into the systemic circulation. Although this IL-1α may be inactivated in circulation by the same sIL-1RI, this phenomenon probably has immunostimulatory effects at local levels where the sIL-1RI-induced IL-1α acts in a paracrine or autocrine manner.
Extracellular-Regulated Kinase 1/2, Jun N-Terminal Kinase, and c-Jun Are Involved in NF-κB-Dependent IL-6 Expression in Human Monocytes
Authors: Tuyt Leonore M. L. , Dokter Wim H. A. , Birkenkamp Kim , Koopmans Sicco B. , Lummen Chantal , Kruijer Wiebe , Vellenga Edo
Published: January 2023

In the present study we investigated the possible involvement of the mitogen-activated protein kinase family members extracellular-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) in mediating IL-6 gene expression in human monocytes, in particular their role in enhancing NF-κB activity. Freshly isolated monocytes treated with the protein phosphatase inhibitor okadaic acid secreted high levels of IL-6 protein, which coincided with enhanced binding activity of NF-κB as well as with phosphorylation and activation of the ERK1/2 and JNK proteins. The ERK pathway-specific inhibitor PD98059 inhibited IL-6 secretion from monocytes. Transient overexpression of inactive mutants of either Raf-1 or JNK1 showed that both pathways were involved in κB-dependent IL-6 promoter activity. By using PD98059, we demonstrated that the Raf1/MEK1/ERK1/2 pathway did not affect the DNA binding of NF-κB but, rather, acted at the level of transcriptional activity of NF-κB. Interestingly, it was shown that NF-κB-mediated gene transcription, both in the context of the IL-6 promoter as well as on its own, was dependent on both serine kinase activity and interaction with c-Jun protein. We conclude that okadaic acid-induced IL-6 gene expression is at least partly mediated through the ERK1/2 and JNK pathway-dependent activation of NF-κB transcriptional capacity. Our results suggest that the JNK pathway may regulate NF-κB-mediated gene transcription through its phosphorylation and activation of c-Jun.
CD26/Dipeptidyl-Peptidase IV Down-Regulates the Eosinophil Chemotactic Potency, But Not the Anti-HIV Activity of Human Eotaxin by Affecting Its Interaction with CC Chemokine Receptor 3
Authors: Struyf Sofie , Proost Paul , Schols Dominique , De Clercq Erik , Opdenakker Ghislain , Lenaerts Jean-Pierre , Detheux Michel , Parmentier Marc , De Meester Ingrid , Scharpé Simon , Van Damme Jo
Published: January 2023

Chemokines attract and activate distinct sets of leukocytes. The CC chemokine eotaxin has been characterized as an important mediator in allergic reactions because it selectively attracts eosinophils, Th2 lymphocytes, and basophils. Human eotaxin has a penultimate proline, indicating that it might be a substrate for dipeptidyl-peptidase IV (CD26/DPP IV). In this study we demonstrate that eotaxin is efficiently cleaved by CD26/DPP IV and that the NH2-terminal truncation affects its biological activity. CD26/DPP IV-truncated eotaxin(3–74) showed reduced chemotactic activity for eosinophils and impaired binding and signaling properties through the CC chemokine receptor 3. Moreover, eotaxin(3–74) desensitized calcium signaling and inhibited chemotaxis toward intact eotaxin. In addition, HIV-2 infection of CC chemokine receptor 3-transfected cells was inhibited to a similar extent by eotaxin and eotaxin(3–74). Thus, CD26/DPP IV differently regulates the chemotactic and antiviral potencies of eotaxin by the removal of two NH2-terminal residues. This physiological processing may be an important down-regulatory mechanism, limiting eotaxin-mediated inflammatory responses.
A Circulating Bovine γδ T Cell Subset, Which Is Found in Large Numbers in the Spleen, Accumulates Inefficiently in an Artificial Site of Inflammation: Correlation with Lack of Expression of E-Selectin Ligands and L-Selectin
Authors: Wilson Eric , Aydintug M. Kemal , Jutila Mark A.
Published: January 2023

Tissue-specific localization of TCR-defined subsets of γδ T cells has been widely reported; however, the mechanisms responsible for this phenomenon are poorly understood. We describe a bovine γδ T cell TCR-associated subset that preferentially localizes in the spleen. This subset was characterized by coexpression of CD8, and was found to lack surface expression of E-selectin ligands, GR Ag ligands, as well as low expression of L-selectin. The CD8-positive γδ T cell subset did not accumulate at sites of inflammation as efficiently as CD8-negative γδ T cells that, in contrast, express E-selectin and GR ligands and high levels of L-selectin. This is the first demonstration of a γδ T cell subset, which exhibits a defined tissue tropism, having a unique adhesion molecule expression profile. These results demonstrate that in some cases tissue-specific accumulation of γδ T cell subsets can be predicted by expression, or lack of expression, of defined homing molecules.
CTLA4 Signals Are Required to Optimally Induce Allograft Tolerance with Combined Donor-Specific Transfusion and Anti-CD154 Monoclonal Antibody Treatment
Authors: Zheng Xin Xiao , Markees Thomas G. , Hancock Wayne W. , Li Yongsheng , Greiner Dale L. , Li Xian Chan , Mordes John P. , Sayegh Mohammed H. , Rossini Aldo A. , Strom Terry B.
Published: January 2023

Sensitization to donor Ags is an enormous problem in clinical transplantation. In an islet allograft model, presensitization of recipients through donor-specific transfusion (DST) 4 wk before transplantation results in accelerated rejection. We demonstrate that combined DST with anti-CD154 (CD40L) therapy not only prevents the deleterious presensitization produced by pretransplant DST in the islet allograft model, it also induces broad alloantigen-specific tolerance and permits subsequent engraftment of donor islet or cardiac grafts without further treatment. In addition, our data strongly indicate that CTLA4-negative T cell signals are required to achieve prolonged engraftment of skin allograft or tolerance to islet allograft in recipients treated with a combination of pretransplant DST and anti-CD154 mAb. We provide direct evidence that a CD28-independent CTLA4 signal delivers a strong negative signal to CD4+ T cells that can block alloimmune MLR responses. In this study immune deviation into a Th2 (IL-4) response was associated with, but did not insure, graft tolerance, as the inopportune timing of B7 blockade with CTLA4/Ig therapy prevented uniform tolerance but did not prevent Th2-type immune deviation. While CTLA4-negative signals are necessary for tolerance induction, Th1 to Th2 immune deviation cannot be sufficient for tolerance induction. Combined pretransplant DST with anti-CD154 mAb treatment may be attractive for clinical deployment, and strategies aimed to selectively block CD28 without interrupting CTLA4/B7 interaction might prove highly effective in the induction of tolerance.
CD28-Mediated Regulation of mRNA Stability Requires Sequences Within the Coding Region of the IL-2 mRNA
Authors: Ragheb Jack A. , Deen Mary , Schwartz Ronald H.
Published: January 2023

Using sequence-tagged genomic reporter constructs, we investigated the contribution of IL-2 sequences to CD28-mediated regulation of mRNA stability. We find that CD28 signaling acts transiently to stabilize the IL-2 mRNA following T cell activation. Such stabilization requires sequences within both exon 2 and the coding region of exon 4. Unexpectedly, CD28 signaling at later times enhances the decay of the IL-2 mRNA. This CD28-dependent decay of IL-2 mRNA requires sequences localized between exon 3 and the stop codon. Our findings demonstrate that the coding region of the IL-2 mRNA contains previously undefined CD28-responsive sequence elements that are critical for the regulation of mRNA stability.
The Guanine-Nucleotide Exchange Factor Vav Is a Crucial Regulator of B Cell Receptor Activation and B Cell Responses to Nonrepetitive Antigens
Authors: Bachmann Martin F. , Nitschke Lars , Krawczyk Connie , Tedford Kerry , Ohashi Pamela S. , Fischer Klaus D. , Penninger Josef M.
Published: January 2023

The proto-oncogene product Vav is required for receptor clustering, proliferation, and differentiation of T cells, and Vav was identified as a substrate in the TCR and B cell receptor signaling pathway. The role of Vav in B cell responses to Ag challenge in vivo is not known. In this study, we show that Vav regulates B cell proliferation following in vitro activation of Ag receptors, but Vav has no apparent role in CD40-, IL-4-, or LPS-induced B cell activation. Increased degrees of Ag receptor cross-linking can partially reverse the proliferative defect in the anti-IgM response of vav−/− B cells. In vivo, vav−/− mice mounted protective antiviral IgM and IgG responses to infections with vesicular stomatitis virus and recombinant vaccinia virus expressing the vesicular stomatitis virus glycoprotein, which harbor repetitive surface epitopes that directly cross-link the Ag receptor and activate B cells in the absence of T cell help. vav−/− B cells also responded normally to the polyvalent, repetitive hapten Ag trinitrophenyl (TNP)-Ficoll that effectively cross-links B cell receptors. However, vav−/− mice failed to mount immune responses to the nonrepetitive, T cell-dependent hapten Ag (4-hydroxy-5-iodo-3-nitrophenyl)acetyl (NIP)-OVA. These results provide the first genetic evidence on the role of the guanine exchange factor Vav in immune responses to viral infections and antigenic challenge in vivo, and suggest that Vav adjusts the threshold for Ag receptor-mediated B cell activation depending on the nature of the Ag.
Bioactive IL-18 Expression Is Up-Regulated in Crohn’s Disease
Authors: Monteleone Giovanni , Trapasso Francesco , Parrello Tiziana , Biancone Livia , Stella Antonella , Iuliano Rodolfo , Luzza Francesco , Fusco Alfredo , Pallone Francesco
Published: January 2023

An imbalance of immunoregulatory factors is believed to contribute to uncontrolled mucosal Th1 cell activation in Crohn’s disease (CD). IL-18, a macrophage-like cell-derived cytokine, is involved in Th1 clone development, and IFN-γ production. Therefore, IL-18 expression was investigated in CD. Whole mucosal intestinal tissue and lamina propria mononuclear cells (LPMC) of 12 CD and 9 ulcerative colitis (UC) patients and 15 non-inflammatory bowel disease (IBD) controls were tested for IL-18 by semiquantitative RT-PCR and Western blot analysis. Transcripts for IL-18 were found in all samples tested. However, increased IL-18 mRNA accumulation was detected in both mucosal and LPMC samples from CD in comparison to UC and controls. In CD, transcripts for IL-18 were more abundant in the mucosal samples taken from involved areas. An 18-kDa band consistent with mature IL-18 was predominantly found in CD mucosal samples. In mucosal samples from non-IBD controls, IL-18 was present as a 24-kDa polypeptide. Consistently, active IL-1β-converting enzyme (ICE) subunit (p20) was expressed in samples from either CD or UC, whereas, in colonic mucosa from non-IBD controls, ICE was synthesized as precursor (p45) only. To confirm that IL-18 produced in CD tissue was functionally active, CD LPMC were treated with a specific IL-18 antisense oligonucleotide. In these cultures, IL-18 down-regulation was accompanied by a decrease in IFN-γ expression. In aggregate, our data indicate that IL-18 up-regulation is a feature of CD and suggest that IL-18 may contribute to the local immunoinflammatory response in CD.
Humoral Response Suppression Observed with CD23 Transgenics
Authors: Payet Margaret E. , Woodward Elaine C. , Conrad Daniel H.
Published: January 2023

CD23, also known as the low affinity IgE receptor (FcεRII), has been hypothesized to have a role in IgE regulation. A new CD23 transgenic mouse was generated using the MHC class I promoter and IgH enhancer to further test the hypothesis that CD23 plays a role in the down-regulation of IgE. Study of three founder lines by FACS showed overexpression to varying extents on both B and T lymphocytes. No alterations in lymphocyte populations was observed. All three founder lines exhibited strong suppression of IgE in response to DNP-keyhole limpet hemocyanin/alum and Nippostrongylus brasiliensis infection compared with that in parental or littermate controls. The founder line exhibiting the highest level of suppression also was less susceptible to Ag-induced systemic anaphylactic shock. Overall, the data support the concept that enhancing CD23 levels can be used to suppress IgE-mediated disease. The mechanism involves decreased IgE synthesis, because the serum half-life of IgE was not altered in transgenics, and enzyme-linked immunospot analysis demonstrated lower IgE-producing cells stimulated by injection of anti-IgD. Transgenics also exhibited significantly decreased IgG1 responses and exhibited lower levels of all Ig isotypes, although this was more variable in different founder lines.
BALB/c Invariant Chain Mutant Mice Display Relatively Efficient Maturation of CD4+ T Cells in the Periphery and Secondary Proliferative Responses Elicited upon Peptide Challenge
Authors: Kenty George , Bikoff Elizabeth K.
Published: January 2023

Allelic differences are known to influence many important aspects of class II biosynthesis, including subunit assembly, Ii chain associations, and DM-mediated peptide loading. Mutant mouse strains lacking Ii chain expression have been previously studied on mixed genetic backgrounds. The present experiments describe cellular and functional characteristics of congenic BALB/c Ii chain mutants. As expected, class II surface expression was markedly decreased, but in contrast to I-Ad-transfected cell lines, serological analysis of BALB/c Ii chain-deficient spleen cells gave no evidence for discordant expression of class II conformational epitopes. Thus, we conclude that properly folded class II molecules are exported via the Ii chain-independent pathway. Functional assays demonstrate consistently superior peptide-loading capabilities, suggesting that these I-Ad molecules are empty or occupied by an easily displaced peptide(s). Defective B cell development was observed for three mutant strains established on diverse genetic backgrounds. Ii chain function is also essential for optimal class II surface expression by mature splenic dendritic cells. Surprisingly, we observe in BALB/c Ii chain mutants, relatively efficient maturation of CD4+ T cells in the periphery and secondary proliferative responses elicited upon peptide challenge. The milder phenotype displayed by BALB/c Ii chain mutants in comparison with class II functional defects previously described for mouse strains lacking Ii chain is likely to have an effect on disease susceptibility.
Enhancer-Blocking Activity Within the DNase I Hypersensitive Site 2 to 6 Region Between the TCR α and Dad1 Genes
Authors: Zhong Xiao-Ping , Krangel Michael S.
Published: January 2023

Although tightly linked, the TCR α and δ genes are expressed specifically in T lymphocytes, whereas the Dad1 gene is ubiquitously expressed. Between TCR α and Dad1 are eight DNase I hypersensitive sites (HS). HS1 colocalizes with the TCR α enhancer (Eα) and is T cell-specific; HS2, -3, -4, -5, and -6 map downstream of HS1 and are tissue-nonspecific. The region spanning HS2–6 was reported to display chromatin-opening activity and to confer copy number-dependent and integration site-independent transgene expression in transgenic mice. Here, we demonstrate that HS2–6 also displays enhancer-blocking activity, as it can block an enhancer from activating a promoter when located between the two in a chromatin-integrated context, and can do so without repressing either the enhancer or the promoter. Multiple enhancer-blocking elements are arrayed across HS2–6. We show that HS2–6 by itself does not activate transcription in chromatin context, but can synergize with an enhancer when located upstream of an enhancer and promoter. We propose that HS2–6 primarily functions as an insulator or boundary element that may be critical for the autonomous regulation of the TCR α and Dad1 genes.
Presentation of the Self Antigen Myelin Basic Protein by Dendritic Cells Leads to Experimental Autoimmune Encephalomyelitis
Authors: Dittel Bonnie N. , Visintin Irene , Merchant Raina M. , Janeway Charles A.
Published: January 2023

Bone marrow (BM)-derived dendritic cells (DC) are potent stimulators of naive CD4+ T cell activation. Because DC are efficient at Ag processing and could potentially present self Ags, we investigated the role of DC in the presentation of an encephalitogenic peptide from myelin basic protein (Ac1–11) in the induction of experimental autoimmune encephalomyelitis (EAE). To determine if DC could prime for EAE, we transferred DC pulsed with Ac1–11 or with medium alone into irradiated mice in combination with CD4+ T cells isolated from a mouse transgenic for a TCR specific for Ac1–11 + I-Au. Mice transferred with Ac1–11-pulsed DC developed EAE 7–10 days later, whereas mice receiving medium-pulsed DC did not. By day 15, all mice given peptide-loaded DC had signs of tail and hind limb paralysis, and by day 20 infiltration of Ac1–11-specific CD4+ T cells was detected in the brain parenchyma. We also demonstrated interactions between Ac1–11-pulsed DC and Ac1–11-specific T cells in the lymph nodes 24 h following adoptive transfer of both cell populations. These data show that DC can efficiently present the self Ag myelin basic protein Ac1–11 to Ag-specific T cells in the periphery of mice to induce EAE.
A Non-AUG-Defined Alternative Open Reading Frame of the Intestinal Carboxyl Esterase mRNA Generates an Epitope Recognized by Renal Cell Carcinoma-Reactive Tumor-Infiltrating Lymphocytes In Situ
Authors: Ronsin Christophe , Chung-Scott Véronique , Poullion Isabelle , Aknouche Nicolas , Gaudin Catherine , Triebel Frédéric
Published: January 2023

A number of Ags recognized by tumor-reactive T cells have been characterized, including nonmutated gene products and a variety of epitopes shown to arise from either mutated or alternatively processed transcripts. Here, we report that the screening of a cDNA library with an HLA-B7-restricted renal cell carcinoma-reactive T cell clone derived from tumor-infiltrating lymphocytes (TILs) that were clonally amplified in vivo (as assessed by TCRBV complementarity determining region-3 length distribution analysis) resulted in the isolation of a nonamer encoded by an alternative open reading frame (ORF) (a +1 frameshift) of the intestinal carboxyl esterase gene. This peptide binds HLA-B*0702-presenting molecules as assessed in an immunofluorescence-based peptide binding assay using transfected T2 cells. Constitutive expression of this alternative ORF protein was observed in all transformed HLA-B7+ renal cell lines that were recognized in cytotoxicity assays by the TILs. The intestinal carboxyl esterase gene is transcribed in renal cell carcinoma tumors as well as in normal liver, intestinal, or renal tissues. Mutation of the natural ATG translation initiation site did not alter recognition, indicating that frameshifting (i.e., slippage of the ribosome forward) and recoding are not involved. In addition, a point mutation of the three AUG codons that may be used as alternative translation initiation sites in the +1 ORF did not abolish recognition, whereas mutation of an upstream ACG codon did, indicating that the latter codon initiates the translation of the alternative ORF. These results further extend the types of Ags that can be recognized by tumor-reactive TILs in situ (i.e., leading to clonal T cell expansion).
Efficient Transfer of a Tumor Antigen-Reactive TCR to Human Peripheral Blood Lymphocytes Confers Anti-Tumor Reactivity
Authors: Clay Timothy M. , Custer Mary C. , Sachs Jessica , Hwu Patrick , Rosenberg Steven A. , Nishimura Michael I.
Published: January 2023

The tumor-associated-Ag MART-1 is expressed by most human melanomas. The genes encoding an αβ TCR from a MART-1-specific, HLA-A2-restricted, human T cell clone have been efficiently transferred and expressed in human PBL. These retrovirally transduced PBL cultures were MART-1 peptide reactive, and most cultures recognized HLA-A2+ melanoma lines. Limiting dilution clones were generated from three bulk transduced PBL cultures to investigate the function of individual clones within the transduced cultures. Twenty-nine of 29 CD8+ clones specifically secreted IFN-γ in response to T2 cells pulsed with MART-1(27–35) peptide, and 23 of 29 specifically secreted IFN-γ in response to HLA-A2+ melanoma lines. Additionally, 23 of 29 CD8+ clones lysed T2 cells pulsed with the MART-1(27–35) peptide and 15 of 29 lysed the HLA-A2+ melanoma line 888. CD4+ clones specifically secreted IFN-γ in response to T2 cells pulsed with the MART-1(27–35) peptide. TCR gene transfer to patient PBL can produce CTL with anti-tumor reactivity in vitro and could potentially offer a treatment for patients with metastatic melanoma. This approach could also be applied to the treatment of other tumors and viral infections. Additionally, TCR gene transfer offers unique opportunities to study the fate of adoptively transferred T cells in vivo.
Cutting Edge: Trimolecular Interaction of TCR with MHC Class II and Bacterial Superantigen Shows a Similar Affinity to MHC:Peptide Ligands
Authors: Redpath Stella , Alam S. Munir , Lin Christina M. , O’Rourke Anne M. , Gascoigne Nicholas R. J.
Published: January 2023

Bacterial superantigens such as Staphylococcus aureus enterotoxin A (SEA) are very potent stimulators of T cells. They bind to the Vβ region of the TCR and to MHC class II, stimulating T cells at nanomolar concentrations. Using surface plasmon resonance measurements, we find that binding between the individual components of the complex (TCR-class II, TCR-SEA, SEA-class II) is very weak, but that the stability of the trimolecular complex is considerably enhanced, reaching an affinity similar to that found for TCR interactions with MHC:peptide ligand. Thus, the potency of SEA in stimulation of T cells is not due to particularly strong affinities between the proteins, but to a cooperative effect of interactions in the TCR-SEA-MHC class II trimolecular complex that brings the kinetics into a similar range to binding of conventional Ags. This range may be the optimum for T cell activation.
Role of Inflammatory Infiltrate in Activation and Effector Function of Cloned Islet Reactive Nonobese Diabetic CD8+ T Cells: Involvement of a Nitric Oxide-Dependent Pathway
Authors: Gurlo Tatyana , Kawamura Kenneth , von Grafenstein Hermann
Published: January 2023

To investigate how CD8+ T cells interact with β cells and local inflammatory cells in islets, we have isolated CD8+ T cell clones from nonobese diabetic (NOD) spleen that recognize and destroy both islets and the NOD insulinoma cell line NIT-1. The clones destroyed NOD islets with pre-existing inflammation better than islets without signs of inflammation. Islets from NOD-scid mice were destroyed only poorly, but that could be improved by adding IL-7 to the assay. Anti-IFN-γ Abs inhibited destruction of infiltrated islets. Single islets were effective stimulators of IFN-γ production by cloned CD8+ T cells, which varied >50-fold depending on the degree of islet infiltration. This effect of the islet mononuclear infiltrate could be mimicked by adding spleen cells to NIT-1 cells, which augmented IFN-γ production above the level stimulated by NIT-1 cells alone. The enhancing effect of spleen cells could be attributed to their macrophage subpopulation and was not MHC restricted, although recognition of islet Ag by cloned CD8+ T cells and subsequent islet destruction was restricted to islets expressing H-2Db molecules. An inhibitor of inducible NO synthase inhibited destruction of inflamed islets by cloned CD8+ T cells. We propose that macrophages in inflamed islets provide a form of bystander costimulation of β cell-specific CD8+ T cells. CD8+ T cells respond to Ag and costimulation by producing IFN-γ that activates macrophages. Activated macrophages facilitate islet destruction by CD8+ T cells through a NO synthesis-dependent pathway.
IL-5 and Rp105 Signaling Defects in B Cells from Commonly Used 129 Mouse Substrains
Authors: Corcoran Lynn M. , Metcalf Donald
Published: January 2023

The use of 129 strain-derived embryonic stem cell lines for targeted gene mutation in mice has led directly to an expanded use of this inbred strain worldwide. It has been noted, however, that the 129 genetic background can make a significant contribution to the severity of a mutant phenotype. In this study, we reveal a specific defect in the IL-5 and Rp105 responses of B lymphocytes from two widely used 129 mouse substrains. The response to stimulation through surface IgM is also diminished, although to a lesser degree, in these mice. The lesion appears to reduce significantly the expression of the α-chain of the IL-5R, but may also influence events downstream of the IL-5R. This phenotype displays a codominant inheritance pattern, and is accompanied by a variable but significant depression of peritoneal B-1 cell numbers in 50% of the mice.
Molecular and Functional Characterization of Mouse Signaling Lymphocytic Activation Molecule (SLAM): Differential Expression and Responsiveness in Th1 and Th2 Cells
Authors: Castro Antonio G. , Hauser Thomas M. , Cocks Benjamin G. , Abrams John , Zurawski Sandra , Churakova Tatyana , Zonin Francesca , Robinson Douglas , Tangye Stuart G. , Aversa Gregorio , Nichols Kim E. , de Vries Jan E. , Lanier Lewis L. , O’Garra Anne
Published: January 2023

Optimal T cell activation and expansion require engagement of the TCR plus costimulatory signals delivered through accessory molecules. SLAM (signaling lymphocytic activation molecule), a 70-kDa costimulatory molecule belonging to the Ig superfamily, was defined as a human cell surface molecule that mediated CD28-independent proliferation of human T cells and IFN-γ production by human Th1 and Th2 clones. In this study, we describe the cloning of mouse SLAM and the production of mAb against it which reveal its expression on primary mouse T and B cells. Mouse SLAM is expressed on highly polarized Th1 and Th2 populations, and is maintained on Th1, but not on Th2 clones. Anti-mouse SLAM mAb augmented IFN-γ production by Th1 cells and Th1 clones stimulated through the TCR, but did not induce IFN-γ production by Th2 cells, nor their production of IL-4 or their proliferation. Mouse SLAM is a 75-kDa glycoprotein that upon tyrosine phosphorylation associates with the src homology 2-domain-containing protein tyrosine phosphatase SHP-2, but not SHP-1. Mouse SLAM also associates with the recently described human SLAM-associated protein. These studies may provide new insights into the regulation of Th1 responses.

1 DNAX is supported by the Schering Plough Research Institute. T.M.H. was initially funded by fellowships from the Swiss National Science Foundation and from the Roche Research Foundation, Switzerland.      Abbreviations used in this paper: SLAM, signaling lymphocytic activation molecule; AP, alkaline phosphatase; h, human; m, mouse; SAP, SLAM-associated protein; SH2, Src homology 2; SHP, SH2 domain-bearing protein tyrosine phosphatase; XLP, X-linked lymphoproliferative disease; PIG, phosphatidylinositol glycan.
IL-2Rβ/IL-7Rα Doubly Deficient Mice Recapitulate the Thymic and Intraepithelial Lymphocyte (IEL) Developmental Defects of γc−/− Mice: Roles for Both IL-2 and IL-15 in CD8αα IEL Development
Authors: Porter Brian O. , Malek Thomas R.
Published: January 2023

IL-7Rα-chain-deficient (IL-7Rα−/−) and common γ chain-deficient (γc−/−) mice both exhibit abnormal thymic and intestinal intraepithelial lymphocyte (IEL) development, but the developmental inhibition is not equivalent. In this report, we assessed whether the defects in T cell development associated with γc−/− mice were due to currently defined γc-dependent cytokines by cross-breeding IL-7Rα−/− mice to mice lacking either IL-2, IL-4, or IL-2Rβ. IL-2/IL-7Rα and IL-4/IL-7Rα double knockout (DKO) mice demonstrated equivalent thymic development to IL-7Rα−/− mice, whereas IL-2Rβ/IL-7Rα DKO mice, which lack IL-2, IL-7, and IL-15 signaling, displayed thymic T cell defects identical to γc−/− mice. Collectively, these data indicate that of the γc-dependent cytokines, only IL-7 and IL-15 contribute to the progression and production of thymic T cells. In the IEL, IL-7Rα−/− mice selectively lack CD8αα TCRγδ cells, whereas IL-2Rβ−/− mice show a significant reduction in all CD8αα cells. IL-2−/− and IL-2/IL-7Rα DKO mice demonstrated a reduction in CD8αα IELs to nearly the same extent as IL-2Rβ−/− mice, indicating that IL-2 functions in CD8αα IEL development. Moreover, IL-2Rβ/IL-7Rα DKO mice lacked nearly all TCR-bearing IEL, again recapitulating the phenotype of γc−/− mice. Thus, these data point to the importance of IL-2, IL-7, and IL-15 as the γc-dependent cytokines essential for IEL development.
The Differentiated State of Intestinal Lamina Propria CD4+ T Cells Results in Altered Cytokine Production, Activation Threshold, and Costimulatory Requirements
Authors: Hurst Stephen D. , Cooper Cristine J. , Sitterding Stephanie M. , Choi Jung-hee , Jump Robin L. , Levine Alan D. , Barrett Terrence A.
Published: January 2023

Intestinal lamina propria (LP) CD4+ T cells are memory-like effector cells that proliferate at relatively low levels and require high levels of TCR signaling and costimulation for full activation in vitro. To study LP CD4+ T cell functional potential we used DO11.10 TCR transgenic (Tg) mice specific for the class II MHC-restricted OVA323–339 peptide and nontransgenic BALB/c mice. Activation of LP Tg+ T cells with Ag using mucosal explants induced high levels of IL-2, IL-4, and IFN-γ. Culturing isolated LP cells with IL-12 enhanced IFN-γ production and down-regulated IL-4 and IL-2, whereas addition of IL-4 maintained IL-4 production without inhibiting IFN-γ production. Systemic administration of relatively high dose (HD; 100 nM) OVA323–339 peptide induced similar levels of bromodeoxyuridine (BrdU) incorporation by LP and splenic Tg+ T cells in vivo, whereas low dose (LD; 4.5 nM) peptide injections induced 4-fold greater levels of BrdU incorporation for LP compared with splenic Tg+ T cells. Coadministration of CTLA-4Ig reduced BrdU incorporation for splenic cells by 70% with HD and LD stimulation, but had little effect on LP responses to HD stimulation. Results of in vivo studies were confirmed in nontransgenic BALB/c mice using HD (200 μg) and LD (10 μg) anti-CD3 mAb+/− CTLA-4Ig. These results suggest that LP T cells are differentiated effector cells that respond at high levels when activated with relatively low levels of Ag- and B7-mediated costimulation in vivo. The reduced activation threshold of LP T cells may facilitate responses to low levels of Ag derived from mucosal pathogens.