bluuebunny/crossref_metadata_2025 huggingface dataset:
Stability of Naive and Memory Phenotypes on Resting CD4 T Cells In Vivo
Authors: Boursalian Tamar E. , Bottomly Kim
Published: December 2022

The reliable identification of naive and memory CD4 T cells is critical to understanding the cellular basis of immunological memory. However, it has long been a controversial issue whether naive and memory phenotypes are stable among resting CD4 T cells in the absence of overt stimulation or whether the proposed memory phenotype is a transient, reversible one that represents recently activated cells. In this study, adoptively transferred, purified populations of naive or memory phenotype CD4 T cells are monitored over time to assess the stability of phenotypes and the functional capabilities of transferred cells. Studying both TCR transgenic and nontransgenic CD4 T cell populations allows one to control for the capacity to respond to environmental Ags in vivo. Several findings are reported. The first is that in the absence of Ag, both naive and memory phenotypes remain unchanged over time. Second, when changes are seen in populations of transferred naive phenotype CD4 T cells, they take place only when there is a potential for antigenic challenge, suggesting that it is an Ag-driven event. Furthermore, when a change from naive to memory phenotype is observed, these transferred donor cells also function as memory cells. Third, the ability of memory CD4 T cells to retain the memory phenotype is independent of specific Ag.
Alleviation of Lipopolysaccharide-Induced Acute Liver Injury inPropionibacterium acnes-Primed IFN-γ-Deficient Mice by a Concomitant Reduction of TNF-α, IL-12, and IL-18 Production
Authors: Tsuji Hirokazu , Mukaida Naofumi , Harada Akihisa , Kaneko Shuichi , Matsushita Eiki , Nakanuma Yasuni , Tsutsui Hiroko , Okamura Haruki , Nakanishi Kenji , Tagawa Yoh-ichi , Iwakura Yoichiro , Kobayashi Ken-ichi , Matsushima Kouji
Published: December 2022

The present study was designed to investigate the role of IFN-γ in LPS-induced liver injury following priming with Propionibacterium acnes. At 1 week after priming BALB/c mice with P. acnes, a large number of macrophages (Mφ) and lymphocytes predominantly infiltrated the portal area, resulting in the intrahepatic formation of granulomas consisting of epithelioid and lymphoid cells. In comparison, in IFN-γ gene-disrupted BALB/c mice (IFN-γ knockout mice), the number of infiltrated Mφ was decreased, with a significant reduction in the number and size of granulomas. Subsequent elicitation with a low dose of LPS induced massive hepatic necrosis in wild-type BALB/c mice, with a marked increase in the serum levels of TNF-α, IL-12, and IL-18 and subsequently of alanine transferase. In contrast, IFN-γ knockout mice developed scattered focal necrosis of the liver with significantly lower levels of serum alanine transferase as well as drastic decreases in TNF-α, IL-12, and IL-18 production. The administration of an anti-IFN-γ neutralizing mAb at the eliciting phase significantly alleviated liver injury and reduced serum IL-12 and IL-18 levels. Thus, endogenously produced IFN-γ is involved in the pathogenesis of this liver injury model by regulating Mφ infiltration and granuloma formation in the priming phase as well as cytokine production in the eliciting phase.
A Role for Perforin in Activation-Induced T Cell Death In Vivo: Increased Expansion of Allogeneic Perforin-Deficient T Cells in SCID mice
Authors: Spaner David , Raju Kaliannan , Rabinovich Brian , Miller Richard G.
Published: December 2022

Despite defective granule exocytosis, T cells from mice whose perforin gene was ablated by homologous recombination (pko mice) caused a similar degree of graft-vs-host disease as normal T cells after injection into sublethally irradiated C.B-17 SCID mice. Moreover host spleens contained significantly greater numbers of T cells from pko mice than from wild-type mice following their i.v. injection. This increase could not be explained by persistence of host APCs that were not cleared by defective donor cytotoxic effector cells. The absence of functional perforin-dependent suppressor cells or an altered cytokine profile of donor T cells could also not account for the behavior of pko cells. Spontaneous and Fas-mediated apoptosis of in vivo activated donor T cells were independent of donor origin. However, pko T blasts exhibited less growth inhibition and cell death after reactivation in vitro. The results are compatible with a model of a defective activation-induced cell death (AICD) pathway, controlled by perforin, accounting for the increased expansion of alloreactive pko T cells.
Rejection of Cardiac Xenografts by CD4+ or CD8+ T Cells
Authors: Lin Yuan , Soares Miguel P. , Sato Koichiro , Takigami Ko , Csizmadia Eva , Anrather Josef , Bach Fritz H.
Published: December 2022

We recently showed that brief complement inhibition induces accommodation of hamster cardiac transplants in nude rats. We have reconstituted nude rats carrying an accommodated xenograft with syngeneic CD4+ or CD8+ T cells to investigate the cellular mechanism of xenograft rejection. We show that CD4+ T cells can initiate xenograft rejection (10 ± 1.7 days) by promoting production of IgG xenoreactive Abs (XAb). These XAb are able to activate complement as well as to mediate Ab-dependent cell-mediated cytotoxicity. Adoptive transfer of these XAb into naive nude rats provoked hyperacute xenograft rejection (38 ± 13 min). The rejection was significantly (p < 0.001) delayed by cobra venom factor (CVF; 11 ± 8 h in four of five cases) but was still more rapid than in control nude rats (3.3 ± 0.5 days). CVF plus NK cell depletion further prolonged survival (>7 days in four of five cases; p < 0.01 vs CVF only). CD8+ T cell-reconstituted nude rats rejected their grafts later (19.4 ± 5.8 days) and required a larger number of cells for transfer as compared with CD4+ T cell-reconstituted nude rats. However, second xenografts were rejected more rapidly than first xenografts in CD8+ T cell-reconstituted nude rats (9 ± 2 days), indicating that the CD8+ T cells had been activated. This study demonstrates that CD4+ and CD8+ T cells can both reject xenografts. The CD4+ cells do so at least in part by generation of helper-dependent XAb that act by both complement-dependent and Ab-dependent cell-mediated cytotoxicity mechanisms; the CD8+ cells do so as helper-independent cytotoxic T cells.
Nuclear Factor-κB p65 Mediates the Assembly and Activation of the TNF-Responsive Element of the Murine Monocyte Chemoattractant-1 Gene
Authors: Ping Dongsheng , Boekhoudt Gunther H. , Rogers Edward M. , Boss Jeremy M.
Published: December 2022

TNF-α transcriptionally regulates murine monocyte chemoattractant protein-1 (MCP-1) expression. Three approaches were used to determine the mechanism by which TNF regulates MCP-1. Mutation analysis showed that two distal κB sites, a novel dimethylsulfate-hypersensitive sequence, and a promoter proximal SP-1 site were required for TNF induction. Although the κB sites and the hypersensitive sequence function as a NF-κB-mediated enhancer, regulating induction by TNF, stereospecific alignment of the κB sites was not critical. Trans-activation studies conducted by cotransfection of p50 and/or p65 expression vectors with MCP-1 constructions showed that TNF regulates MCP-1 through NF-κB. Examination of MCP-1 induction in NF-κB-disrupted embryonic fibroblasts showed that p65 was necessary for both the induction and the TNF-induced protein occupancy of the enhancer in vivo. The action of the antioxidant inhibitor of NF-κB activation, pyrrolidine dithiocarbamate, in wild-type and NF-κB mutant cells was examined. The results suggested that TNF activates NF-κB through both pyrrolidine dithiocarbamate-sensitive and -insensitive mechanisms. This study illustrates the crucial role for NF-κB p65 in the induction of the MCP-1 gene by TNF and in the assembly of a NF-κB dependent enhancer in vivo.
Quantitative Analysis of the Effect of CD16 Ligation on Human NK Cell Proliferation
Authors: Warren Hilary S. , Kinnear Beverley F.
Published: December 2022

CD16 (FcγRIIIA), the low affinity receptor for IgG, is expressed on the majority of human peripheral blood NK cells. Ligation of CD16 with mAb or immune complexes activates NK cell cytotoxicity and cytokine secretion, and stimulates death of activated NK cells by apoptosis. This study uses NK cells labeled with the stable intracytoplasmic fluorescent dye 5- and 6-carboxyfluorescein diacetate succinimidyl ester to provide quantitative data on the effect of CD16 ligation on NK cell division and NK cell survival. When NK cells are cultured with rIL-2 and CD16 is ligated, NK cell division is stimulated, but there also is a substantial loss of NK progenitor cells. When NK cell proliferation is stimulated by coculture with γ-irradiated MM-170 malignant melanoma cells and rIL-2, CD16 ligation enhances entry of NK cells into division. In some cases, CD16 ligation is essential for NK cell proliferation stimulated by MM-170 cells. In these cultures, there is no loss of NK progenitor cells. This study demonstrates that CD16 is an activation receptor for NK cell proliferation, and suggests that cellular costimulation alters the balance between NK cell death and NK cell proliferation stimulated by CD16 ligation.
Novel Splicing of the Human MHC-Encoded Peptide Transporter Confers Unique Properties
Authors: Yan Gang , Shi Lijia , Faustman Denise
Published: December 2022

Presentation of intracellularly derived antigenic peptides to T cells requires their assembly together with MHC class I molecules in the endoplasmic reticulum (ER). Such peptides are delivered to the ER by an MHC-encoded transporter composed of TAP1 and TAP2 protein delivery. Here, the first alternative splicing of Tap2 is described. The human splice variant, termed Tap2iso, lacks exon 11 and original 3′ untranslated region and contains a newly identified exon 12 and 3′ untranslated region. The full-length Tap2iso cDNA (2496 bp) predicts a protein of 653 amino acids. Tap2iso mRNA was normally coexpressed with Tap2 mRNA in all human lymphocyte cell lines examined. Function of TAP2iso was evaluated at multilevel in TAP1/2iso and TAP1/2 cotransfected T2 cells, a mutant cell line deplete of endogenous Tap gene products. The TAP1-TAP2iso transporter facilitated the maturation of MHC class I molecules in the ER and restored surface expression of class I. Importantly, TAP1-TAP2iso transporters expressed in T2 cells exhibited distinct and opposing influences on peptide selectivities, at times exceeding 30-fold differences in competition experiments and attributable to diversity in the 3′-COOH tail. The common coexpression of an alternative splice product of the Tap2 gene may contribute to broaden immune diversity, a mechanism previously described to occur predominantly at the level of the TCR and MHC class I gene products.
Suppression of Host Immune Response by the Core Protein of Hepatitis C Virus: Possible Implications for Hepatitis C Virus Persistence
Authors: Large Mary Kathryn , Kittlesen David J. , Hahn Young S.
Published: December 2022

Hepatitis C virus (HCV) is a major human pathogen causing mild to severe liver disease worldwide. This positive strand RNA virus is remarkably efficient at establishing chronic infections. Although a high rate of genetic variability may facilitate viral escape and persistence in the face of Ag-specific immune responses, HCV may also encode proteins that facilitate evasion of immunological surveillance. To address the latter possibility, we examined the influence of specific HCV gene products on the host immune response to vaccinia virus in a murine model. Various vaccinia/HCV recombinants expressing different regions of the HCV polyprotein were used for i.p. inoculation of BALB/c mice. Surprisingly, a recombinant expressing the N-terminal half of the polyprotein (including the structural proteins, p7, NS2, and a portion of NS3; vHCV-S) led to a dose-dependent increase in mortality. Increased mortality was not observed for a recombinant expressing the majority of the nonstructural region or for a negative control virus expressing the β-galactosidase protein. Examination of T cell responses in these mice revealed a marked suppression of vaccinia-specific CTL responses and a depressed production of IFN-γ and IL-2. By using a series of vaccinia/HCV recombinants, we found that the HCV core protein was sufficient for immunosuppression, prolonged viremia, and increased mortality. These results suggest that the HCV core protein plays an important role in the establishment and maintenance of HCV infection by suppressing host immune responses, in particular the generation of virus-specific CTLs.
Regulation and Functional Involvement of Macrophage Scavenger Receptor MARCO in Clearance of Bacteria In Vivo
Authors: van der Laan Luc J. W. , Döpp Ed A. , Haworth Richard , Pikkarainen Timo , Kangas Maarit , Elomaa Outi , Dijkstra Christine D. , Gordon Siamon , Tryggvason Karl , Kraal Georg
Published: December 2022

The scavenger receptors expressed by macrophages are thought to play an important role in the immune response against bacteria by mediating binding and phagocytosis. A novel member of the class A scavenger receptor family, macrophage receptor with collagenous structure (MARCO), has recently been identified. In this study we have generated a panel of mAbs with specificities for different domains of this receptor. Two of those reacting with the C-terminal cysteine-rich domain block ligand binding of MARCO. The in vivo expression of this murine receptor is normally restricted to distinct populations of macrophages in the spleen and lymph nodes. During bacillus Calmette-Guérin (BCG) infection, during bacterial sepsis, or after the injection of purified LPS, however, the expression of MARCO is rapidly induced on macrophages in other tissues, including Kupffer cells in the liver. Using the mouse macrophage cell line J774.2, it was shown that LPS stimulation up-regulates surface expression of MARCO in a dose- and time-dependent fashion. The proinflammatory cytokines IL-1, IL-6, TNF-α, and IFN-γ had little or no effect. Using inhibitory mAbs, the relevance of MARCO for the clearance of circulating bacteria in vivo was determined. Although the overall elimination of live Escherichia coli and Staphylococcus aureus from the blood did not appear to be affected by treatment with these Abs, the capturing of heat-killed bacteria by macrophages in the marginal zone areas of the spleen was clearly inhibited. This study suggests a role for MARCO in the host antibacterial defense.
Role of IFN-γ-Induced Indoleamine 2,3 Dioxygenase and Inducible Nitric Oxide Synthase in the Replication of Human Cytomegalovirus in Retinal Pigment Epithelial Cells
Authors: Bodaghi Bahram , Goureau Olivier , Zipeto Donato , Laurent Lysiane , Virelizier Jean-Louis , Michelson Susan
Published: December 2022

An in vitro model of human CMV infection of primary retinal pigment epithelial (RPE) cells was used to study the effects of cytokines on CMV replication in these cells, which are targets of CMV infection in vivo. IFN-γ and IFN-β were potent inhibitors of CMV replication in RPE cells, while TNF-α, IL-1β, or TGF-β2 did not affect viral replication. Inhibition by IFN-γ, and to a lesser extent IFN-β, was almost completely reversed by addition of l-tryptophan to the culture medium, strongly implicating the indoleamine 2,3 dioxygenase (IDO) pathway. Polyadenylated IDO mRNA accumulation was detected as early as 2 h after IFN stimulation. Furthermore, CMV blocked the production of nitric oxide by the inducible form of nitric oxide synthase. This inhibition depended on a functional viral genome. However, exogenous nitric oxide significantly inhibited viral protein expression in RPE cells. Thus, CMV infection blocks the inducible nitric oxide synthase pathway activated by IFN-γ and IL-1β, but cannot counteract the IFN-induced IDO pathway, which ultimately controls its replication in primary human RPE cells.
IL-12 Is Not Required for Induction of Type 1 Cytokine Responses in Viral Infections
Authors: Oxenius Annette , Karrer Urs , Zinkernagel Rolf M. , Hengartner Hans
Published: December 2022

To investigate the physiological role of IL-12 in viral infections in terms of T cell cytokine responses involved in virus-specific Ig isotype induction and in antiviral protection, immune responses elicited upon infection of IL-12-deficient mice with lymphocytic choriomeningitis virus (LCMV) or vesicular stomatitis virus (VSV) were studied. Infection of IL-12-deficient mice with LCMV induced a virus-specific type 1 cytokine response as determined by in vitro cytokine secretion patterns as well as by in vivo intracellular cytokine staining of LCMV-specific CD4+ TCR transgenic T cells that had clonally expanded in LCMV-infected IL-12-deficient recipient mice. In addition, LCMV- and VSV-specific IgG responses exhibited normal serum IgG2a/IgG1 ratios, demonstrating again virus-specific CD4+ T cell induction of type 1 phenotype in IL-12-deficient mice upon viral infection. LCMV and VSV immune mice were found to be protected against challenge immunization with recombinant vaccinia viruses expressing either the LCMV- or the VSV-derived glycoprotein, respectively. This protection is known to be mediated by T cell-secreted type 1 cytokines IFN-γ and TNF-α. In contrast, IL-12-deficient mice showed impaired abilities to control infection with the facultative intracellular bacterium Listeria monocytogenes at early time points after infection. However, at later time points of infection, IL-12-deficient mice were able to clear infection. These findings may indicate that viruses are able to induce type 1 T cell responses in the absence of IL-12 as opposed to some bacterial or parasitical infections that are crucially dependent on the presence of IL-12 for the induction of type 1 immune responses.
Timing and Casting for Actors of Thymic Negative Selection
Authors: Dautigny Nicole , Le Campion Armelle , Lucas Bruno
Published: December 2022

We have recently proposed a new model for the differentiation pathway of αβ TCR thymocytes, with the CD4 and CD8 coreceptors undergoing an unexpectedly complex series of expression changes. Taking into account this new insight, we reinvestigated the timing of thymic negative selection. We found that, although endogenous superantigen-driven thymic negative selection could occur at different steps during double-positive/single-positive cell transition, this event was never observed among CD4lowCD8low TCRint CD69+ thymocytes, i.e., within the first subset to be generated upon TCR-mediated activation of immature double-positive cells. We confirm a role for CD40/CD40L interaction, and the absence of involvement of CD28 costimulation, in thymic deletion in vivo. Surprisingly, we found that thymic negative selection was impaired in the absence of Fas, but not FasL, molecule expression. Finally, we show involvement in opposing directions for p59fyn and SHP-1 molecules in signaling for thymic negative selection.
Protein Interactions of Src Homology 2 (SH2) Domain-Containing Inositol Phosphatase (SHIP): Association with Shc Displaces SHIP from FcγRIIb in B Cells
Authors: Tridandapani Susheela , Pradhan Madhura , LaDine James R. , Garber Stacey , Anderson Clark L. , Coggeshall K. Mark
Published: December 2022

Our recent studies revealed that the inositol phosphatase Src homology 2 (SH2) domain-containing inositol phosphatase (SHIP) is phosphorylated and associated with Shc exclusively under negative signaling conditions in B cells, which is due to recruitment of the SHIP SH2 domain to the FcγRIIb. In addition, we reported that SHIP-Shc interaction involves both SHIP SH2 and Shc phosphotyrosine binding domains. These findings reveal a paradox in which the single SH2 domain of SHIP is simultaneously engaged to two different proteins: Shc and FcγRIIb. To resolve this paradox, we examined the protein interactions of SHIP. Our results demonstrated that isolated FcγRIIb contains SHIP but not Shc; likewise, Shc isolates contain SHIP but not FcγRIIb. In contrast, SHIP isolates contain both proteins, revealing two separate pools of SHIP: one bound to FcγRIIb and one bound to Shc. Kinetic studies reveal rapid SHIP association with FcγRIIb but slower and more transient association with Shc. Affinity measurements using a recombinant SHIP SH2 domain and phosphopeptides derived from FcγRIIb (corresponding to Y273) and Shc (corresponding to Y317) revealed an approximately equal rate of binding but a 10-fold faster dissociation rate for FcγRIIb compared with Shc phosphopeptide and yielding in an affinity of 2.1 μM for FcγRIIb and 0.26 μM for Shc. These findings are consistent with a model in which SHIP transiently associates with FcγRIIb to promote SHIP phosphorylation, whereupon SHIP binds to Shc and dissociates from FcγRIIb.
A Critical Tyrosine Residue in the Cytoplasmic Tail Is Important for CD1d Internalization But Not for Its Basolateral Sorting in MDCK Cells
Authors: Rodionov Dmitrii G. , Nordeng Tommy W. , Pedersen Ketil , Balk Steven P. , Bakke Oddmund
Published: December 2022

The CD1 family of polypeptides is divided into two groups, the CD1b and CD1d group. Both groups are involved in stimulation of T cell response. Molecules of the CD1b group can present Ag derived from bacterial cell walls to T cells; the process of Ag acquisition is thought to take place in endosomes. Little is known about Ag presentation by CD1d. We therefore studied the intracellular trafficking of human CD1d in Madin-Darby canine kidney (MDCK) and COS cells. CD1d was found in endosomal compartments after its internalization from the plasma membrane. It is therefore possible that CD1d acquires its yet unidentified exogenous ligand in the same compartments as the MHC class II and CD1b molecules. CD1d contains a tyrosine-based sorting signal in its cytoplasmic tail that is necessary for internalization. Furthermore, the cytoplasmic tail of CD1d also contains a signal for basolateral sorting that is, however, different from the internalization signal.
The Mapping of the Lyn Kinase Binding Site of the Common β Subunit of IL-3/Granulocyte-Macrophage Colony- Stimulating Factor/IL-5 Receptor
Authors: Adachi Tetsuya , Pazdrak Konrad , Stafford Susan , Alam Rafeul
Published: December 2022

It has been shown that a membrane-proximal region within common β (βc) receptor of IL-3/granulocyte-macrophage CSF/IL-5 (amino acids 450–517) is important for Lyn binding. We have shown previously that Lyn kinase is physically associated with the IL-5R βc subunit in unstimulated cells. The result suggests that this association involves binding modules that are not activation or phosphorylation dependent. The objective of this study was to map the exact Lyn binding site on βc. Using overlapping and/or sequential peptides derived from βc 450–517, we narrowed down the Lyn binding site to nine amino acid residues, βc 457–465. The P→A mutation in this region abrogated the binding to Lyn, indicating a critical role of proline residues. We created a cell-permeable Lyn-binding peptide by N-stearation. This cell-permeable peptide blocked the association of Lyn, but not Jak2 with βc in situ. We also investigated the βc binding site of Lyn kinase. Our results suggest that the N-terminal unique domain of Lyn kinase is important for binding to βc receptor. To our knowledge, this is the first molecular identification of the Lyn binding site of βc receptor. This finding may help develop specific inhibitors of Lyn-coupled signaling pathways.
CpG Oligodeoxynucleotides Can Circumvent the Th2 Polarization of Neonatal Responses to Vaccines But May Fail to Fully Redirect Th2 Responses Established by Neonatal Priming
Authors: Kovarik Jiri , Bozzotti Paola , Love-Homan Laurie , Pihlgren Maria , Davis Heather L. , Lambert Paul-Henri , Krieg Arthur M. , Siegrist Claire-Anne
Published: December 2022

Neonatal murine responses to a panel of conventional vaccines differ qualitatively from adult responses by a particular polarization toward a Th2 pattern and a frequent limitation of the Th1 and CTL responses required for protection against intracellular microorganisms. In contrast, DNA vaccines induce adult-like Th1/CTL neonatal responses against the same vaccine Ags. In this report, we show that this can be related to their content in unmethylated CpG motifs. Oligodeoxynucleotides (ODN) containing CpG motifs activate neonatal APCs to produce IL-12 in vitro and induce adult-like Th1 responses to tetanus toxoid and measles Ags in vivo, with production of IgG2a-specific Abs and adult-like secretion of IFN-γ and IL-5 by Ag-specific T cells. However, in spite of their capacity to trigger neonatal B cell proliferation in vitro, CpG-ODN only partially enhanced early life Ab responses. Finally, using Th1-driving CpG-ODN with the boosting dose of a protein vaccine was sufficient to redirect adult but not neonatally primed Th2 responses. These observations could be important for the development of novel vaccines that will have to be effective early in life.
The Inflammatory Response to Nonfatal Sindbis Virus Infection of the Nervous System Is More Severe in SJL Than in BALB/c Mice and Is Associated with Low Levels of IL-4 mRNA and High Levels of IL-10-Producing CD4+ T Cells
Authors: Rowell Jennifer F. , Griffin Diane E.
Published: December 2022

SJL mice are susceptible to inflammatory autoimmune diseases of the central nervous system (CNS), while BALB/c mice are relatively resistant. To understand differences in immune responses that may contribute to autoimmune neurologic disease, we compared the responses of SJL and BALB/c mice to infection with Sindbis virus, a virus that causes acute nonfatal encephalomyelitis in both strains of mice. Clearance of virus was similar, but SJL mice developed a more intense inflammatory response in the brain and spinal cord and inflammation persisted for several weeks. Analysis of lymphocytes isolated from brains early after infection showed an absence of NK cells in SJL mice, while both strains of mice showed CD4+ and CD8+ T cells. During the second week after infection, CD4+ T cells increased in SJL mice and the proportion of CD8+ T cells decreased, while the opposite pattern was seen in BALB/c mice. Expression of IL-10 mRNA was higher and IL-4 mRNA was lower in the brains of infected SJL than in BALB/c mice, while expression of the mRNAs of IL-6, IL-1β, TNFα, and the Th1 cytokines IL-2, IL-12, and IFN-γ was similar. Lymphocytes isolated from the CNS of SJL mice produced large amounts of IL-10. CNS lymphocytes from both strains of mice produced IFN-γ in response to stimulation with Sindbis virus, but not in response to myelin basic protein. These data suggest that IL-10-producing CD4+ T cells are differentially recruited to or regulated within the CNS of SJL mice compared with BALB/c mice infected with Sindbis virus, a characteristic that may be related to low levels of IL-4, and is likely to be involved in susceptibility of SJL mice to CNS inflammatory diseases.
Activated T Cells Acquire Endothelial Cell Surface Determinants During Transendothelial Migration
Authors: Brezinschek Ruth I. , Oppenheimer-Marks Nancy , Lipsky Peter E.
Published: December 2022

Activated T cells acquire endothelial cell (EC) plasma membrane constituents during transendothelial migration. This was assessed using an in vitro model system in which human peripheral blood CD4+ T cells migrated through confluent monolayers of HUVEC. Flow cytometry of migrated CD4+ T cells demonstrated that activated, but not resting, T cells acquired a variety of endothelial surface determinants, including CD31, CD49d, CD54, CD61, and CD62E. The extracellular domains of these molecules were detected on migrated T cells with mAbs, including those directed to the ligand-binding regions. A number of approaches were employed to document that the acquisition of these molecules was uniquely accomplished by activated T cells and clearly involved transfer from both resting and TNF-α-activated EC. Acquisition of endothelial markers by activated T cells occurred as part of the transfer of membrane components, as migrating T cells acquired EC membranes prelabeled with the lipophilic dye, 3,3′-dihexadecyloxacarbocyanine perchlorate (DiOC-16), along with EC surface proteins. Thus, during transendothelial migration, activated T cells acquire endothelial membrane components, and as a result may deliver them to perivascular sites.
Resistance to Fas-Mediated T Cell Apoptosis in Asthma
Authors: Jayaraman Sundararajan , Castro Mario , O’Sullivan Mary , Bragdon M. Janice , Holtzman Michael J.
Published: December 2022

Over activation of CD4+ T cells in the peripheral blood and airway tissues is characteristic of asthma; therefore, we investigated whether activated T cells from asthmatic subjects have altered apoptotic potential through the Fas death receptor. We found that mitogen-stimulated peripheral blood T cells of asthmatic subjects expressed cell surface Fas, but failed to undergo the normal degree of apoptosis after Fas receptor ligation. T cells from asthmatics exhibited normal apoptotic responses to γ-irradiation (dependent on IL-1 converting enzyme family proteases), ceramide, and mitogen challenge, suggesting functional integrity of the apoptotic pathway. Furthermore, the defect in Fas-dependent apoptosis was overcome by prestimulation with allogeneic accessory cells instead of mitogen. Taken together, the findings suggest that selective resistance to Fas-dependent apoptosis reflects altered Ag-driven, accessory cell-dependent signaling and that ineffective activation of Fas signal transduction may contribute to T cell-dependent immunoinflammation in asthma.
Cutting Edge: Alloimmune Responses Against Major and Minor Histocompatibility Antigens: Distinct Division Kinetics and Requirement for CD28 Costimulation
Authors: Song Howard K. , Noorchashm Hooman , Lieu Yen K. , Rostami Susan , Greeley Siri Atma S. , Barker Clyde F. , Naji Ali
Published: December 2022

Comparative study of alloimmune responses against major and minor histocompatibility Ags has been limited by the lack of suitable assays. Here, we use a bioassay that permits tracking of alloreactive CD4+ T cell populations as they proliferate in response to major or minor histocompatibility Ags in vivo. Division of alloreactive CD4+ T cells proceeded more rapidly in response to major histocompatibility Ags than minor Ags, although CD4+ T cells alloreactive to minor Ags had a similar capacity to divide successively up to eight times after stimulation. Allorecognition of minor histocompatibility Ags was highly dependent on CD28 costimulation, with the frequency of CD4+ T cells proliferating in response to minor Ags in the absence of CD28 costimulation reduced up to 20-fold. These findings highlight differences in signaling processes that lead to allorecognition of major and minor histocompatibility Ags and have implications on the design of interventions aimed at abrogating these responses.
Effects of Th2 Cytokines on Chemokine Expression in the Lung: IL-13 Potently Induces Eotaxin Expression by Airway Epithelial Cells
Authors: Li Li , Xia Yiyang , Nguyen Andrea , Lai Yew Hon , Feng Lili , Mosmann Tim R. , Lo David
Published: December 2022

Airway inflammation associated with asthma is characterized by massive infiltration of eosinophils, mediated in part by specific chemoattractant factors produced in the lung. Allergen-specific Th2 cells appear to play a central role in asthma; for example, adoptively transferred Th2 cells induced lung eosinophilia associated with induction of specific chemokines. Interestingly, Th2 supernatant alone administered intranasally to naive mice induced eotaxin, RANTES, monocyte-chemotactic protein-1, and KC expression along with lung eosinophilia. We tested the major cytokines individually and found that IL-4 and IL-5 induced higher levels of macrophage-inflammatory protein-1α and KC; IL-4 also increased the production of monocyte-chemotactic protein-1; IL-13 and IL-4 induced eotaxin. IL-13 was by far the most potent inducer of eotaxin; indeed, a neutralizing anti-IL-13 Ab removed most of the eotaxin-inducing activity from Th2 supernatants, although it did not entirely block the recruitment of eosinophils. While TNF-α did not stimulate eotaxin production by itself, it markedly augmented eotaxin induction by IL-13. IL-13 was able to induce eotaxin in the lung of JAK3-deficient mice, suggesting that JAK3 is not required for IL-13 signaling in airway epithelial cells; however, eosinophilia was not induced in this situation, suggesting that JAK3 transduces other IL-13-mediated mechanisms critical for eosinophil recruitment. Our study suggests that IL-13 is an important mediator in the pathogenesis of asthma and therefore a potential target for asthma therapy.
Tolerance to Antigen-Presenting Cell-Depleted Islet Allografts Is CD4 T Cell Dependent
Authors: Coulombe Marilyne , Yang Huan , Wolf Leslie A. , Gill Ronald G.
Published: December 2022

Pretreatment of pancreatic islets in 95% oxygen culture depletes graft-associated APCs and leads to indefinite allograft acceptance in immunocompetent recipients. As such, the APC-depleted allograft represents a model of peripheral alloantigen presentation in the absence of donor-derived costimulation. Over time, a state of donor-specific tolerance develops in which recipients are resistant to donor APC-induced graft rejection. Thus, persistence of the graft is sufficient to induce tolerance independent of other immune interventions. Donor-specific tolerance could be adoptively transferred to immune-deficient SCID recipient mice transplanted with fresh immunogenic islet allografts, indicating that the original recipient was not simply “ignorant” of donor antigens. Interestingly, despite the fact that the original islet allograft presented only MHC class I alloantigens, CD8+ T cells obtained from tolerant animals readily collaborated with naive CD4+ T cells to reject donor-type islet grafts. Conversely, tolerant CD4+ T cells failed to collaborate effectively with naive CD8+ T cells for the rejection of donor-type grafts. In conclusion, the MHC class I+, II− islet allograft paradoxically leads to a change in the donor-reactive CD4 T cell subset and not in the CD8 subset. We hypothesize that the tolerant state is not due to direct class I alloantigen presentation to CD8 T cells but, rather, occurs via the indirect pathway of donor Ag presentation to CD4 T cells in the context of host MHC class II molecules.
Targeted Deletion of the IgA Constant Region in Mice Leads to IgA Deficiency with Alterations in Expression of Other Ig Isotypes
Authors: Harriman Gregory R. , Bogue Molly , Rogers Pamela , Finegold Milton , Pacheco Susan , Bradley Allan , Zhang Yongxin , Mbawuike Innocent N.
Published: December 2022

A murine model of IgA deficiency has been established by targeted deletion of the IgA switch and constant regions in embryonic stem cells. B cells from IgA-deficient mice were incapable of producing IgA in vitro in response to TGF-β. IgA-deficient mice expressed higher levels of IgM and IgG in serum and gastrointestinal secretions and decreased levels of IgE in serum and pulmonary secretions. Expression of IgG subclasses was complex, with the most consistent finding being an increase in IgG2b and a decrease in IgG3 in serum and secretions. No detectable IgA Abs were observed following mucosal immunization against influenza; however, compared with those in wild-type mice, increased levels of IgM Abs were seen in both serum and secretions. Development of lymphoid tissues as well as T and B lymphocyte function appeared normal otherwise. Peyer’s patches in IgA-deficient mice were well developed with prominent germinal centers despite the absence of IgA in these germinal centers or intestinal lamina propria. Lymphocytes from IgA-deficient mice responded to T and B cell mitogens comparable to those of wild-type mice, while T cells from IgA-deficient mice produced comparable levels of IFN-γ and IL-4 mRNA and protein. In conclusion, mice with targeted deletion of the IgA switch and constant regions are completely deficient in IgA and exhibit altered expression of other Ig isotypes, notably IgM, IgG2b, IgG3, and IgE, but otherwise have normal lymphocyte development, proliferative responses, and cytokine production.
Genome-Wide Linkage Analysis of Chronic Relapsing Experimental Autoimmune Encephalomyelitis in the Rat Identifies a Major Susceptibility Locus on Chromosome 9
Authors: Dahlman Ingrid , Jacobsson Lena , Glaser Anna , Lorentzen Johnny C. , Andersson Magnus , Luthman Holger , Olsson Tomas
Published: December 2022

The immunization of inbred Dark Agouti (DA) rats with an emulsion containing homogenized spinal cord and CFA induces chronic relapsing experimental autoimmune encephalomyelitis (EAE), a disease with many similarities to multiple sclerosis. We report here the first genome-wide search for quantitative trait loci regulating EAE in the rat using this model. We identified one quantitative trait locus on chromosome 9, Eae4, in a [DA(RT1av1) × BN(RT1n)]F2 intercross showing linkage to disease susceptibility and expression of mRNA for the proinflammatory cytokine IFN-γ in the spinal cord. Eae4 had a larger influence on disease incidence among rats that were homozygous for the RT1av1 MHC haplotype (RT1av1 rats) compared with RT1n/av1 rats, suggesting an interaction between Eae4 and the MHC. Homozygosity for the DA allele at markers in Eae4 and in the MHC was sufficient for EAE. Thus, Eae4 is a major genetic factor determining susceptibility to EAE in this cross of DA rats. In addition, there was support for linkage to phenotypes of EAE on chromosomes 1, 2, 5, 7, 8, 12, and 15. The chromosome 12 region has been shown previously to predispose DA rats to arthritis, and the chromosome 2 region is syntenic to Eae3 in mice. We conclude that Eae4 and probably the other identified genome regions harbor genes regulating susceptibility to neuroinflammatory disease. The identification and functional characterization of these genes may disclose critical events in the pathogenesis of multiple sclerosis; understanding these events could be essential for the development of new therapies against the disease.
Surface Expression and Functional Competence of CD3-Independent TCR ζ-Chains in Immature Thymocytes
Authors: Grassi Fabio , Barbier Eliane , Porcellini Simona , von Boehmer Harald , Cazenave Pierre-André
Published: December 2022

In recombinase-deficient (RAG-2−/−) mice, double-negative thymocytes can be stimulated to proliferate and differentiate by anti-CD3 Abs. CD3 molecules are expressed on the surface of these cells in association with calnexin. In this study, we show that ζ-chains can be recovered as phosphorylated proteins in association with phosphorylated ZAP-70 from anti-CD3-stimulated RAG-2−/− thymocytes, even though they are not demonstrably associated with the CD3/calnexin complex. The lack of a physical association of ζ dimers with the CD3 complex in RAG-2−/− thymocytes and also in a pre-TCR-expressing cell line, as well as the efficient association of ζ dimers with ZAP-70 in the RAG-2−/− thymocytes, suggest that these ζ-chain dimers could contribute to pre-TCR signaling. This idea is supported by the finding that in RAG-2−/− ζ-deficient thymocytes, ZAP-70 and p120cbl were only weakly phosphorylated.
Functional Analysis of TRAIL Receptors Using Monoclonal Antibodies
Authors: Griffith Thomas S. , Rauch Charles T. , Smolak Pam J. , Waugh Jennifer Y. , Boiani Norman , Lynch David H. , Smith Craig A. , Goodwin Raymond G. , Kubin Marek Z.
Published: December 2022

mAbs were generated against the extracellular domain of the four known TNF-related apoptosis-inducing ligand (TRAIL) receptors and tested on a panel of human melanoma cell lines. The specificity of the mAb permitted a precise evaluation of the TRAIL receptors that induce apoptosis (TRAIL-R1 and -R2) compared with the TRAIL receptors that potentially regulate TRAIL-mediated apoptosis (TRAIL-R3 and -R4). Immobilized anti-TRAIL-R1 or -R2 mAbs were cytotoxic to TRAIL-sensitive tumor cells, whereas tumor cells resistant to recombinant TRAIL were also resistant to these mAbs and only became sensitive when cultured with actinomycin D. The anti-TRAIL-R1 and -R2 mAb-induced death was characterized by the activation of intracellular caspases, which could be blocked by carbobenzyloxy-Val-Ala-Asp (OMe) fluoromethyl ketone (zVAD-fmk) and carbobenzyloxy-Ile-Glu(OMe)-Thr-Asp (OMe) fluoromethyl ketone (zIETD-fmk). When used in solution, one of the anti-TRAIL-R2 mAbs was capable of blocking leucine zipper-human TRAIL binding to TRAIL-R2-expressing cells and prevented TRAIL-induced death of these cells, whereas two of the anti-TRAIL-R1 mAbs could inhibit leucine zipper-human TRAIL binding to TRAIL-R1:Fc. Furthermore, use of the blocking anti-TRAIL-R2 mAb allowed us to demonstrate that the signals transduced through either TRAIL-R1 or TRAIL-R2 were necessary and sufficient to mediate cell death. In contrast, the expression of TRAIL-R3 or TRAIL-R4 did not appear to be a significant factor in determining the resistance or sensitivity of these tumor target cells to the effects of TRAIL.
Analysis of MHC Class II Genes in the Susceptibility to Lupus in New Zealand Mice
Authors: Rozzo Stephen J. , Vyse Timothy J. , David Chella S. , Palmer Ed , Izui Shozo , Kotzin Brian L.
Published: December 2022

Hybrids of New Zealand Black (NZB) and New Zealand White (NZW) mice spontaneously develop a disease similar to human systemic lupus erythematosus. MHC and non-MHC genes contribute to disease susceptibility in this murine model. Multiple studies have shown that the NZW H2z locus is strongly associated with the development of lupus-like disease in these mice. The susceptibility gene(s) within H2z is not known, but different lines of evidence have pointed to class II MHC genes, either H2-E or H2-A (Ez or Az in NZW). Recent studies from our laboratory showed that Ez does not supplant H2z in the contribution to lupus-like disease. In the present work we generated C57BL/10 (B10) mice transgenic for Aaz and Abz genes (designated B10.Az mice) and used a (B10.Az × NZB)F1 × NZB backcross to assess the contributions of Az genes to disease. A subset of backcross mice produced high levels of IgG autoantibodies and developed severe nephritis. However, no autoimmune phenotype was linked to the Az transgenes. Surprisingly, in the same backcross mice, inheritance of H2b from the nonautoimmune B10 strain was strongly linked with both autoantibody production and nephritis. Taken together with our previous Ez studies, the present work calls into question the importance of class II MHC genes for lupus susceptibility in this model and provides new insight into the role of MHC in lupus-like autoimmunity.
Potent Inhibition of CD4/TCR-Mediated T Cell Apoptosis by a CD4-Binding Glycoprotein Secreted from Breast Tumor and Seminal Vesicle Cells
Authors: Gaubin Muriel , Autiero Monica , Basmaciogullari Stéphane , Métivier Didier , Misëhal Zohar , Culerrier Raphal , Oudin Anne , Guardiola John , Piatier-Tonneau Dominique
Published: December 2022

We previously isolated a CD4 ligand glycoprotein, gp17, from human seminal plasma; this glycoprotein is identical with gross cystic disease fluid protein-15 (GCDFP-15), a factor specifically secreted from primary and secondary breast tumors. The function of gp17/GCDFP-15 in physiological as well as in pathological conditions has remained elusive thus far. As a follow up to our previous findings that gp17 binds to CD4 with high affinity and interferes with both HIV-1 gp120 binding to CD4 and syncytium formation, we investigated whether gp17 could affect the T lymphocyte apoptosis induced by a separate ligation of CD4 and TCR. We show here that gp17/GCDFP-15 is in fact a strong and specific inhibitor of the T lymphocyte programmed cell death induced by CD4 cross-linking and subsequent TCR activation. The antiapoptotic effect observed in the presence of gp17 correlates with a moderate up-regulation of Bcl-2 expression in treated cells. The presence of gp17 also prevents the down-modulation of Bcl-2 expression in Bcl-2bright CD4+ T cells that is caused by the triggering of apoptosis. Our results suggest that gp17 may represent a new immunomodulatory CD4 binding factor playing a role in host defense against infections and tumors.
Involvement of TNF-Related Apoptosis-Inducing Ligand in Human CD4+ T Cell-Mediated Cytotoxicity
Authors: Kayagaki Nobuhiko , Yamaguchi Noriko , Nakayama Masafumi , Kawasaki Akemi , Akiba Hisaya , Okumura Ko , Yagita Hideo
Published: December 2022

TNF-related apoptosis-inducing ligand (TRAIL) has been identified as a member of the TNF family that induces apoptosis in a variety of tumor cells, but its physiological functions are largely unknown. In the present study, we examined the expression and function of TRAIL in human CD4+ T cell clones by utilizing newly established anti-human TRAIL mAbs. Human CD4+ T cell clones, HK12 and 4HM1, exhibited perforin-independent and Fas ligand (FasL)-independent cytotoxicity against certain target cells, including T lymphoma (Jurkat) and keratinocyte (HaCaT) cell lines, which are susceptible to TRAIL-mediated cytotoxicity. In contrast to FasL, the expression of which was inducible upon anti-CD3 stimulation, TRAIL was constitutively expressed on HK12 and 4HM1 cells, and no further increase was observed after anti-CD3 stimulation. Spontaneous cytotoxic activities of resting HK12 and 4HM1 cells against Jurkat and HaCaT cells were blocked by anti-TRAIL mAb but not by anti-FasL mAb, and bystander cytotoxic activities of anti-CD3-stimulated HK12 and 4HM1 cells were abolished by the combination of anti-TRAIL and anti-FasL mAbs. These results indicate a differential regulation of TRAIL and FasL expression on human CD4+ T cell clones and that TRAIL constitutes an additional pathway of T cell-mediated cytotoxicity.
Emergence of T Cell Progenitors Without B Cell or Myeloid Differentiation Potential at the Earliest Stage of Hematopoiesis in the Murine Fetal Liver
Authors: Kawamoto Hiroshi , Ohmura Koichiro , Fujimoto Shinji , Katsura Yoshimoto
Published: December 2022

It has been unclear whether the progenitors colonizing the thymus are multipotent or T cell lineage restricted. We investigated the developmental potential of hematopoietic progenitors in various populations of liver and blood cells from day 12 fetuses using the recently established in vitro experimental system effective in determining the capability of individual progenitors to generate T, B, and myeloid cells. Multipotent progenitors (p-Multi) were exclusively found in the Sca-1 high-positive (Sca-1high) subpopulation of lineage marker (Lin)−c-kit+CD45+ fetal liver cells. Restriction of developmental capacity begins at the Sca-1high stage, and a large majority of progenitors in the Sca-1low or Sca-1− population are restricted to generate T, B, or myeloid cells. Such a lineage commitment or restriction taking place in the fetal liver is independent of the thymus, because no difference in the proportion of different types of progenitors were seen between nu/nu and nu/+ fetuses. T cell lineage-restricted progenitors (p-T) were abundant in the blood of day 12 fetuses, whereas p-Multi were undetectable. It was further shown that the p-Multi generated a large number of B and myeloid cells in the thymic lobe. These results strongly suggest that it is p-T but not p-Multi that migrate into the thymus.