bluuebunny/crossref_metadata_2025 huggingface dataset:
A Genome-Wide Search Identifies Two Susceptibility Loci for Experimental Autoimmune Encephalomyelitis on Rat Chromosomes 4 and 10
Authors: Roth Marie-Paule , Viratelle Carine , Dolbois Laurence , Delverdier Maxence , Borot Nicolas , Pelletier Lucette , Druet Philippe , Clanet Michel , Coppin Hélène
Published: January 2023

Experimental autoimmune encephalomyelitis (EAE) is an autoimmune disease of the central nervous system that exhibits many pathologic similarities with multiple sclerosis. The genetic loci that contribute to mononuclear cell infiltration of the central nervous system and clinical manifestations of EAE in the rat were investigated in the F2 progeny of the highly susceptible Lewis and resistant Brown Norway strains. The data confirmed that the Lewis allele of a MHC-linked gene is necessary, but not sufficient, to confer EAE susceptibility in the F2 progeny. Subsequent analyses were thus restricted to the subset of the F2 animals with EAE-predisposing MHC genotypes. A genome-wide scan approach was performed using 103 microsatellite markers covering 85% of the genome. Two non-MHC regions were identified, one near the centromere of chromosome 4 and the other on the long arm of chromosome 10, that significantly contributed to the disease. In addition, three regions on chromosomes 9, 13, and 17 were suggestive for linkage. Congenic mapping is now needed to reduce the support intervals encoding the loci of interest to sizes amenable to physical mapping and to eventually demonstrate the involvement of some of the candidate genes of immunologic importance localized in these regions.
TCR-Independent Pathways Mediate the Effects of Antigen Dose and Altered Peptide Ligands on Th Cell Polarization
Authors: Grakoui Arash , Donermeyer David L. , Kanagawa Osami , Murphy Kenneth M. , Allen Paul M.
Published: January 2023

We examined the role of the peptide/MHC ligand in CD4+ T cell differentiation into Th1 or Th2 cells using a TCR αβ transgenic mouse specific for hemoglobin (Hb)(64-76)/I-Ek. We identified two altered peptide ligands of Hb(64-76) that retain strong agonist activity but, at a given dose, induce cytokine patterns distinct from the Hb(64-76) peptide. The ability of these peptides to produce distinct cytokine patterns at identical doses is not due to an intrinsic qualitative property. Each peptide can induce Th2 cytokines at low concentrations and Th1 cytokines at high concentrations and has a unique range of concentrations at which these distinct effects occur. The pattern of cytokines produced from limiting dilution of naive T cells demonstrated that the potential to develop an individual Th1 or Th2 cell is stochastic, independent of Ag dose. We propose that the basis for the observed effects on the Th1/Th2 balance shown by the altered peptide ligands and the amount of Ag dose involves the modification of soluble factors in bulk cultures that are the driving force that polarize the population to either a Th1 or Th2 phenotype.
Lymphotactin Acts as an Innate Mucosal Adjuvant
Authors: Lillard James W. , Boyaka Prosper N. , Hedrick Joseph A. , Zlotnik Albert , McGhee Jerry R.
Published: January 2023

Lymphotactin (Lptn) is a C chemokine produced predominantly by NK and CD8-positive (CD8+) T cells including γδ TCR-positive (TCR+) intraepithelial lymphocytes. Lptn is chemotactic for NK and T cells and likely plays an important role in maintaining the integrity of the epithelium and in mucosal immune responses. In this study, we characterized the immune responses to OVA given intranasally with Lptn to mice. This regimen enhanced OVA-specific serum Ab responses and Ab titers in mucosal secretions. Lptn also enhanced OVA-specific Ab-forming cells in mucosal and systemic compartments. CD4-positive (CD4+) T cells isolated from mucosal compartments and spleens of mice intranasally immunized with OVA plus Lptn displayed higher OVA-specific proliferative responses and greater synthesis of IFN-γ, IL-2, IL-4, IL-5, IL-6, and IL-10 than did CD4+ T cells from mice given OVA without Lptn. These studies indicate that Lptn has adjuvant properties and suggest that Lptn present in the mucosa has the potential to enhance mucosal and systemic Ab responses through help provided by Th1- and Th2-type cells to link the initial innate signals of the mucosa with the acquired immune system.
Intranasally Induced Immunological Tolerance Is Determined by Characteristics of the Draining Lymph Nodes: Studies with OVA and Human Cartilage gp-39
Authors: Wolvers Danielle A. W. , Coenen-de Roo Christina J. J. , Mebius Reina E. , van der Cammen Maarten J. F. , Tirion Felicia , Miltenburg André M. M. , Kraal Georg
Published: January 2023

Mucosal tolerance is a naturally occurring immunological phenomenon that prevents harmful inflammatory responses to ingested or inhaled environmental, predominantly nondangerous, Ags. The nasal mucosa is an extremely efficient compartment in the induction of immunological tolerance which can be exploited in Ag-specific treatment of autoimmune disease. With the use of a model Ag (OVA) and an Ag implicated in the autoimmune disease rheumatoid arthritis (human cartilage gp-39), we here show in a mouse model that the superficial cervical and internal jugular lymph nodes that drain the nasal mucosa are instrumental in the induction of tolerance. Removal of these lymph nodes abrogates tolerance induction, which can be restored by transplantation of superficial cervical lymph nodes, but not of peripheral lymph nodes. The results indicate that lymph nodes that directly drain the nasal mucosa constitute a unique microenvironment which favors the induction of immunological tolerance.
CD40 Activation Boosts T Cell Immunity In Vivo by Enhancing T Cell Clonal Expansion and Delaying Peripheral T Cell Deletion
Authors: Maxwell Joseph R. , Campbell Jeff D. , Kim Carol H. , Vella Anthony T.
Published: January 2023

In this report we show that activation of APC with an agonist anti-CD40 mAb profoundly alters the behavior of CD4 T cells in vivo. Stimulation of mice with anti-CD40 2 days before, but not 1 day after, administration of superantigen (SAg) enhanced CD4 and CD8 T cell clonal expansion by approximately threefold. Further, CD40 activation also delayed peripheral T cell deletion after activation. Dying, activated T cells were quantitated by detecting extracellular phosphatidylserine with concomitant staining for SAg-reactive T cells using a TCR Vβ-specific mAb. Upon close examination, it was shown that CD40 activation delayed the death of the activated T cells. Additionally, it was found that enhanced survival of CD4 T cells was equally dependent on APC expression of B7-1 and B7-2. This is in contrast to CD8 T cells, which did not depend as much on B7-1 as B7-2. Thus, CD40 activation indirectly promotes T cell growth and delays the death of SAg-stimulated CD4 T cells in vivo. These data suggest that one way CD40 activation promotes a more robust immune response is by indirectly increasing the production of effector T cells and by keeping them alive for longer periods of time.
IL-2, But Not IL-4 and Other Cytokines, Induces Phosphorylation of a 98-kDa Protein Associated with SHP-2, Phosphatidylinositol 3′-Kinase, and Grb2
Authors: Gadina Massimo , Sudarshan Chitra , O’Shea John J.
Published: January 2023

Binding of IL-2 to its receptor activates several biochemical pathways, including JAK-STAT, Ras-mitogen-activated protein kinase, and phosphatidylinositol 3′-kinase (PI 3′-kinase) pathways. Recently, it has been shown that the SH2-containing phosphatase, SHP-2, becomes phosphorylated in response to IL-2 stimulation, associates with PI3′-kinase and Grb2, and can exert a positive regulatory role in IL-2 signaling. We now report the identification of a prominent 98-kDa protein (p98) found to be phosphorylated in response to IL-2 stimulation and coprecipitated with SHP-2, the p85 subunit of PI 3′-kinase and Grb2. Interestingly, whereas IL-4 is known to activate PI 3′-kinase, we did not observe any p98 phosphorylation in response to IL-4 stimulation. p98 can form a multipartite complex with all these proteins as immunodepleting with anti-p85 antiserum substantially reduced the amount of p98 immunoprecipitated by SHP-2 and Grb2; the converse was also true. Furthermore, phosphorylation of p98 did not occur in cells lacking JAK3, suggesting that it may be a JAK substrate. Finally, deglycosylation of p98 did not alter its migration, suggesting p98 is not a member of the recently described SHP substrate/signal-regulatory proteins family of transmembrane glycoproteins. Thus p98 is a prominent IL-2-dependent substrate that associates with multiple proteins involved in IL-2 signaling and may play an important role in coupling the different signal transduction pathways activated by IL-2.
Immunosuppressive Leflunomide Metabolite (A77 1726) Blocks TNF-Dependent Nuclear Factor-κB Activation and Gene Expression
Authors: Manna Sunil K. , Aggarwal Bharat B.
Published: January 2023

Leflunomide is a novel immunosuppressive and antiinflammatory agent currently being tested for treatment of autoimmune diseases and transplant rejection. NF-κB is a transcription factor activated in response to a wide variety of inflammatory stimuli, including TNF, but whether leflunomide blocks NF-κB activation is not known. In the present report we demonstrate that treatment of a human T cell line (Jurkat) with leflunomide blocks TNF-mediated NF-κB activation in a dose- and time-dependent manner, with maximum inhibition at 5–10 μM. Inhibition was not restricted to TNF-induced activation, because leflunomide also inhibited NF-κB activation induced by other inflammatory agents, including phorbol ester, LPS, H2O2, okadaic acid, and ceramide. Leflunomide blocked the degradation of IκBα and subsequent nuclear translocation of the p65 subunit, steps essential for NF-κB activation. This correlated with inhibition of dual specificity-mitogen-activated protein kinase kinase as well as an Src protein tyrosine kinase, p56lck, by leflunomide. Reducing agents did not reverse the effect of leflunomide. Leflunomide also suppressed the TNF-activated NF-κB-dependent reporter gene expression. Our results thus indicate that leflunomide is a potent inhibitor of NF-κB activation induced by a wide variety of inflammatory stimuli, and this provides the molecular basis for its anti-inflammatory and immunosuppressive effects.
Molecular Mechanisms and Selection Influence the Generation of the Human VλJλ Repertoire
Authors: Farner Nancy L. , Dörner Thomas , Lipsky Peter E.
Published: January 2023

To define the λ light chain repertoire in humans, a single-cell PCR technique using genomic DNA obtained from individual peripheral B cells was employed. Of the 30 known functional Vλ genes, 23 were detected in either the nonproductive or productive repertoires. Specific Vλ genes, including 2A2, 2B2, 1G, and 4B, were overexpressed in the nonproductive repertoire, whereas some Vλ genes, such as 3R, 2A2, 2B2, 1C, 1G, and 1B, were overexpressed in the productive repertoire. Comparison of the nonproductive and productive repertoires indicated that no Vλ genes were positively selected, whereas a number of Vλ genes, including 4C, 1G, 5B, and 4B, were negatively regulated. All four of the functional Jλ segments were found in both repertoires, with Jλ7 observed most often. Evidence of terminal deoxynucleotidyltransferase activity was noted in nearly 80% of nonproductive VλJλ rearrangements, and exonuclease activity was apparent in the majority. Despite this, the mean CDR3 length was 30 base pairs in both productive and nonproductive repertoires, suggesting that it was tightly regulated at the molecular level. These results have provided new insights into the dimensions of the human Vλ repertoire and the influences that shape it.
The Role of Actin Microfilaments in the Down-Regulation of the Degranulation Response in RBL-2H3 Mast Cells
Authors: Frigeri Luciano , Apgar John R.
Published: January 2023

Cross-linking of FcεRI on rat basophilic leukemia (RBL) cells initiates a signaling cascade leading to degranulation of the cells and the release of inflammatory mediators. Inhibitors that disrupt microfilaments, such as latrunculin and cytochalasin D, do not cause any degranulation on their own, but they do enhance FcεRI-mediated degranulation. Dose-response studies show a good correlation between inhibition of actin polymerization and increased degranulation. In RBL cells, latrunculin causes a decrease in basal levels of filamentous actin (F-actin), while cytochalasin D does not. This is particularly evident in the Triton-insoluble pool of F-actin which is highly cross-linked and associated with the plasma membrane. A concentration of 500 nM latrunculin decreases the basal level of Triton-insoluble F-actin by 60–70% and total F-actin levels by 25%. Latrunculin increases both the rate and extent of Ag-induced degranulation while having no effect on pervanadate-induced degranulation. Pervanadate activates the signaling pathways directly and bypasses the cross-linking of the receptor. RBL cells, activated through FcεRI in the presence of latrunculin, show increased phospholipase activity as well as increased tyrosine phosphorylation of Syk and increased tyrosine phosphorylation of the receptor itself by the tyrosine kinase Lyn. This indicates that the very earliest signaling events after receptor cross-linking are enhanced. These results suggest that actin microfilaments may interact, either directly or indirectly, with the receptor itself and that they may regulate the signaling process at the level of receptor phosphorylation. Microfilaments may possibly act by uncoupling Lyn from the cross-linked receptor.
The β-Glucan-Binding Lectin Site of Mouse CR3 (CD11b/CD18) and Its Function in Generating a Primed State of the Receptor That Mediates Cytotoxic Activation in Response to iC3b-Opsonized Target Cells
Authors: Xia Yu , Větvička Václav , Yan Jun , Hanikýřová Margareta , Mayadas Tanya , Ross Gordon D.
Published: January 2023

Mouse leukocyte CR3 (Mac-1, αMβ2 integrin) was shown to function as a receptor for β-glucans in the same way as human CR3. Soluble zymosan polysaccharide (SZP) or pure β-glucans labeled with FITC or 125I bound in a saturable and reversible manner to neutrophils, macrophages, and NK cells. This lectin activity was blocked by anti-CD11b mAb M1/70 or 5C6 and did not occur with leukocytes from CR3−/− (CD11b-deficient) mice. SZP preparations containing primarily mannose or glucose bound to CR3, and the binding of 125I-labeled β-glucan to CR3 was competitively inhibited by β-glucans from barley or seaweed, but not by yeast α-mannan. Also, as with human CR3, the lectin site of mouse CR3 was inhibited by α- or β-methylglucoside (but not d-glucose), α- or β-methylmannoside, and N-acetyl-d-glucosamine. Phagocytosis of zymosan and serum-opsonized zymosan was partially inhibited by anti-CR3 and was reduced to <40% of normal with leukocytes from CR3−/− mice. As with neutrophils from patients with CD18 deficiency, neutrophils from CR3−/− mice exhibited no phagocytosis of particulate β-glucan. SZP or β-glucans primed CR3 of neutrophils, macrophages, and NK cells for cytotoxicity of iC3b-opsonized tumor cells that otherwise did not trigger killing. β-Glucan priming for cytotoxicity was inhibited by anti-CR3 and did not occur with leukocytes from CR3−/− mice. The primed state of macrophage and NK cell CR3 remained detectable for 18 to 24 h after pulsing with β-glucans. The similarity of mouse and human CR3 in response to β-glucans highlights the utility of mouse tumor models for development of therapeutic β-glucans.
Expression and Function of the Chemokine Receptors CXCR1 and CXCR2 in Sepsis
Authors: Cummings C. James , Martin Thomas R. , Frevert Charles W. , Quan Joanne M. , Wong Venus A. , Mongovin Steven M. , Hagen Tonja R. , Steinberg Kenneth P. , Goodman Richard B.
Published: January 2023

Neutrophils (polymorphonuclear neutrophils; PMN) and a redundant system of chemotactic cytokines (chemokines) have been implicated in the pathogenesis of the acute respiratory distress syndrome in patients with sepsis. PMN express two cell surface receptors for the CXC chemokines, CXCR1 and CXCR2. We investigated the expression and function of these receptors in patients with severe sepsis. Compared with normal donors, CXCR2 surface expression was down-regulated by 50% on PMN from septic patients (p < 0.005), while CXCR1 expression persisted. In vitro migratory responses to the CXCR1 ligand, IL-8, were similar in PMN from septic patients and normal donors. By contrast, the migratory response to the CXCR2 ligands, epithelial cell-derived neutrophil activator (ENA-78) and the growth-related oncogene proteins, was markedly suppressed in PMN from septic patients (p < 0.05). Ab specific for CXCR1 blocked in vitro migration of PMN from septic patients to IL-8 (p < 0.05), but not to FMLP. Thus, functionally significant down-regulation of CXCR2 occurs on PMN in septic patients. We conclude that in a complex milieu of multiple CXC chemokines, CXCR1 functions as the single dominant CXC chemokine receptor in patients with sepsis. These observations offer a potential strategy for attenuating adverse inflammation in sepsis while preserving host defenses mediated by bacteria-derived peptides such as FMLP.
Anti-ICAM-1 Monoclonal Antibody R6.5 (Enlimomab) Promotes Activation of Neutrophils in Whole Blood
Authors: Vuorte Juha , Lindsberg Perttu J. , Kaste Markku , Meri Seppo , Jansson Sten-Erik , Rothlein Robert , Repo Heikki
Published: January 2023

R6.5 (BIRR-1, Enlimomab), a murine IgG2a mAb to the human ICAM-1, inhibits leukocyte adhesion to the vascular endothelium, thereby decreasing leukocyte extravasation and inflammatory tissue injury. In initial clinical trials, R6.5 proved to be beneficial in reducing both disease activity in refractory rheumatoid arthritis and the incidence of acute rejection after kidney and liver allograft transplantations. However, adverse effects such as fever, leukopenia, or cutaneous reactions were not infrequent. We studied the effects of R6.5 on neutrophil function in whole blood samples ex vivo. Surprisingly, at the concentrations achieved in clinical trials, R6.5 activated neutrophilic granulocytes, as indicated by a significant increase in expression of the adhesion molecule β2-integrin CD11b, a concurrent decrease in L-selectin expression, and an enhancement of the oxidative burst activity. Neutrophil activation was not exerted by an anti-ICAM-1 mAb of the IgG1 isotype, by isotype-matched, irrelevant anti-2-phenyloxazolone mAb, or by F(ab′)2 fragments of R6.5. Neutrophil activation was completely inhibited by soluble complement receptor type 1. We conclude that in whole blood, R6.5 activates resting neutrophils in a complement-dependent manner. This finding can explain, at least in part, the side effects associated with R6.5 therapy.
IFN-α 2B Enhances Th1 Cytokine Responses in Bladder Cancer Patients Receiving Mycobacterium bovis Bacillus Calmette-Guérin Immunotherapy
Authors: Luo Yi , Chen Xiaohong , Downs Tracy M. , DeWolf William C. , O’Donnell Michael A.
Published: January 2023

Combination therapy with intravesical bacillus Calmette-Guérin (BCG) plus IFN-α for superficial bladder cancer has been demonstrated to be more effective than either single agent alone in animal studies and of suggested greater efficacy in clinical studies. However, the mechanism by which IFN-α enhances BCG-mediated antitumor activity is poorly understood. Using PBMCs from bladder cancer patients, IFN-α was found to substantially enhance the efficacy of BCG to induce IFN-γ production. Among 34 patients tested, 80% showed >4-fold increase. This effect of IFN-α was observed in both initial and memory responses to BCG. In addition, IFN-α up-regulated BCG-induced IL-12 and TNF-α and down-regulated BCG-induced IL-10. Neutralizing endogenous IL-10 or adding exogenous IL-12 provided further synergy for IFN-γ production. In clinical practice, intravesical IFN-α 2B (50 million units (MU)/dose) was observed to accelerate urinary IFN-γ production to low-dose BCG (one-tenth or one-third of a full dose) in patients treated with combination therapy compared with BCG alone. These results suggest that IFN-α is a potent BCG enhancer that polarizes the BCG-induced immune response toward the cellular immune pathway by promoting Th1 cytokine expression and reducing Th2 cytokine expression. This study provides an immunological basis for future rational use of IFN-α in conjunction with intravesical BCG for bladder cancer immunotherapy.
Genetic Dissection of SLE Pathogenesis: Adoptive Transfer ofSle1Mediates the Loss of Tolerance by Bone Marrow-Derived B Cells
Authors: Sobel Eric S. , Mohan Chandra , Morel Laurence , Schiffenbauer Joel , Wakeland Edward K.
Published: January 2023

Sle1 is a potent autoimmune susceptibility locus on chromosome 1 originally identified in a genome scan of testcross progeny between the systemic lupus erythematosus-prone NZM2410 strain and C57BL/6. We subsequently produced B6.NZMc1, a congenic strain carrying the NZM2410-derived Sle1 genomic interval on the B6 background and demonstrated that Sle1 mediated the loss of tolerance to chromatin in both the B and T cell compartments. In this communication, we show by adoptive transfer experiments that the autoimmune phenotypes of Sle1 are completely reconstituted in B6 radiation chimeras receiving B6.NZMc1 bone marrow but not by the reciprocal reconstitution, demonstrating that Sle1 is functionally expressed in B cells. In additional experiments, cotransfer of mixtures of bone marrow derived from B6.NZMc1 and nonautoimmune congenic B6 mice carrying allelic T and B cell markers showed that only B cells derived from B6.NZMc1 bone marrow produced anti-chromatin autoantibodies. In contrast, increased expression of CD69 was equivalent in CD4+ T cells derived from either B6.NZMc1 or congenic B6 bone marrow, suggesting that either T cell population could be activated subsequent to loss of tolerance in the B cell compartment. These findings indicate that the expression of Sle1 in B cells is essential for the development of autoimmunity.
Extracellular HIV-1 Tat Protein Up-Regulates the Expression of Surface CXC-Chemokine Receptor 4 in Resting CD4+ T Cells
Authors: Secchiero Paola , Zella Davide , Capitani Silvano , Gallo Robert C. , Zauli Giorgio
Published: January 2023

Here we report that synthetic HIV-1 Tat protein, immobilized on a solid substrate, up-regulates the surface expression of the CXC-chemokine receptor 4 (CXCR4), but not of the CC-chemokine receptor 5 in purified populations of primary resting CD4+ T cells. The Tat-mediated increase of CXCR4 occurred in a well-defined range of concentrations (1–10 nM of immobilized Tat) and time period (4–8 h postincubation). Moreover, the increase of CXCR4 was accompanied by an increased entry of the HXB2 T cell line-tropic (X4-tropic), but not of the BaL macrophage-tropic strain of HIV-1. The ability of Tat to up-regulate CXCR4 expression was abrogated by the protein synthesis inhibitor cycloheximide, clearly indicating the requirement of de novo synthesis. As Tat protein is actively released by HIV-1 infected cells, our data indicate a potentially important role for extracellular Tat in rendering bystander CD4+ T cells more susceptible to infection with X4-tropic HIV-1 isolates.
In Vivo and In Vitro Activation of T Cells After Administration of Ag-Negative Heat Shock Proteins
Authors: Breloer Minka , Fleischer Bernhard , Bonin Arne von
Published: January 2023

Heat shock proteins (HSP) Hsp70 and gp96 prime class I-restricted cytotoxic T cells against Ags present in the cells from which they were isolated. The immunization capacity of HSPs is believed to rely on their ability to bind antigenic peptides. In this study, we employed the well-established OVA and β-galactosidase (β-gal) antigenic model systems. We show that in vitro long-term established OVA and β-gal-specific CTL clones release TNF-α and IFN-γ when incubated with Ag-negative Hsp70 and gp96. In the absence of antigenic peptides, HSP-mediated secretion of TNF-α and IFN-γ requires cell contact of the APC with the T cell but is not MHC-I restricted. Moreover, Hsp70 molecules purified from Ag-negative tissue, e.g., negative for antigenic peptide, are able to activate T cells in vivo, leading to significant higher frequencies in OVA-specific CD8+ T cells. In unprimed animals, these T cells lyse OVA-transfected cell lines and produce TNF-α and IFN-γ after Ag stimulus. Taken together our data show that, besides the well-established HSP/peptide-specific CTL induction and activation, a second mechanism exists by which Hsp70 and gp96 molecules activate T cells in vivo and in vitro.
Reciprocal Expression in CD4 or CD8 Subsets of Different Members of the Vα11 Gene Family Correlates with Sequence Polymorphism
Authors: Sim Bee-Cheng , Gascoigne Nicholas R. J.
Published: January 2023

Previous staining studies with TCR Vα11-specific mAbs showed that Vα11.1/11.2 (AV11S1 and S2) expression was selectively favored in the CD4+ peripheral T cell population. As this phenomenon was essentially independent of the MHC haplotype, it was suggested that AV11S1 and S2 TCRs exert a preference for recognition of class II MHC molecules. The Vα segment of the TCR α-chain is suggested to have a primary role in shaping the T cell repertoire due to selection for class I or II molecules acting through the complementarity determining regions (CDR) 1α and CDR2α residues. We have analyzed the repertoire of Vα11 family members expressed in C57BL/6 mice and have identified a new member of this family; AV11S8. We show that, whereas AV11S1 and S2 are more frequent in CD4+ cells, AV11S3 and S8 are more frequent in CD8+ cells. The sequences in the CDR1α and CDR2α correlate with differential expression in CD4+ or CD8+ cells, a phenomenon that is also observed in BALB/c mice. With no apparent restriction in TCR Jα usage or CDR3α length in C57BL/6, these findings support the idea of Vα-dependent T cell repertoire selection through preferential recognition of MHC class I or class II molecules.
Development of CD8+ Effector T Cells Is Differentially Regulated by IL-18 and IL-12
Authors: Okamoto Iwao , Kohno Keizo , Tanimoto Tadao , Ikegami Hakuo , Kurimoto Masashi
Published: January 2023

We investigated the effects of IL-18 on the development of CD8+ effector T cells in DBA/2 anti-BDF1 whole spleen cell MLC and compared the results with those of IL-12. Addition of IL-18 to the MLC resulted in a twofold increase in CD8/CD4 ratios compared with the control cultures when cells were expanded in IL-2-containing medium following MLC. Purified CD8+ T cells recovered from the IL-18-stimulated MLC produced 20- to 30-fold more IFN-γ after secondary stimulation with C57BL/6 spleen cells or anti-CD3 mAb, and exhibited strong allospecific CTL activity. Neither IL-18 nor IL-18-supplemented culture supernatants from DBA/2 anti-BDF1 MLC induced type I CD8+ effector T cells when purified CD8+ T cells were used as responder cells in primary MLC. Furthermore, CD4+ T cell depletion from the responder cells abrogated the IL-18-induced increase in secondary IFN-γ production by CD8+ T cells, suggesting that IL-18-induced type I effector CD8+ T cell development was CD4+ T cell dependent. In marked contrast, adding IL-12 to primary MLC decreased CD8/CD4 ratios by 50% and suppressed secondary IFN-γ production and CTL activity by CD8+ T cells regardless of concentration, whereas Th1 development was promoted by IL-12. Moreover, both IL-12 and IL-18 efficiently induced type I CD8+ effector T cells in C57BL/6 anti-BDF1 MLC. These findings show that IL-18 plays an important role in the generation of type I CD8+ effector T cells, and further suggest that functional maturation of CD8+ T cells is differentially regulated by IL-18 and IL-12.
Evidence for Distinct Intracellular Signaling Pathways in CD34+ Progenitor to Dendritic Cell Differentiation from a Human Cell Line Model
Authors: St. Louis Daniel C. , Woodcock Juliana B. , Fransozo Guido , Blair Patrick J. , Carlson Louise M. , Murillo Maria , Wells Mark R. , Williams Amanda J. , Smoot Douglas S. , Kaushal Sumesh , Grimes Janelle L. , Harlan David M. , Chute John P. , June Carl H. , Siebenlist Ulrich , Lee Kelvin P.
Published: January 2023

Intracellular signals that mediate differentiation of pluripotent hemopoietic progenitors to dendritic cells (DC) are largely undefined. We have previously shown that protein kinase C (PKC) activation (with phorbol ester (PMA) alone) specifically induces differentiation of primary human CD34+ hemopoietic progenitor cells (HPC) to mature DC. We now find that cytokine-driven (granulocyte-macrophage CSF and TNF-α) CD34+ HPC→DC differentiation is preferentially blocked by inhibitors of PKC activation. To further identify intracellular signals and downstream events important in CD34+ HPC→DC differentiation we have characterized a human leukemic cell line model of this process. The CD34+ myelomonocytic cell line KG1 differentiates into dendritic-like cells in response to granulocyte-macrophage CSF plus TNF-α, or PMA (with or without the calcium ionophore ionomycin, or TNF-α), with different stimuli mediating different aspects of the process. Phenotypic DC characteristics of KG1 dendritic-like cells include morphology (loosely adherent cells with long neurite processes), MHC I+/MHC IIbright/CD83+/CD86+/CD14− surface Ag expression, and RelB and DC-CK1 gene expression. Functional DC characteristics include fluid phase macromolecule uptake (FITC-dextran) and activation of resting T cells. Comparison of KG1 to the PMA-unresponsive subline KG1a reveals differences in expression of TNF receptors 1 and 2; PKC isoforms α, βI, βII, and μ; and RelB, suggesting that these components/pathways are important for DC differentiation. Together, these findings demonstrate that cytokine or phorbol ester stimulation of KG1 is a model of human CD34+ HPC to DC differentiation and suggest that specific intracellular signaling pathways mediate specific events in DC lineage commitment.
Fas-Mediated Suicide of Tumor-Reactive T Cells Following Activation by Specific Tumor: Selective Rescue by Caspase Inhibition
Authors: Zaks Tal Z. , Chappell Dale B. , Rosenberg Steven A. , Restifo Nicholas P.
Published: January 2023

CD8+ T lymphocytes that specifically recognize tumor cells can be isolated and expanded ex vivo. While the lytic properties of these cells have been well described, their fate upon encounter with cognate tumor is not known. We performed reverse 51Cr release assays in which the lymphocyte effectors rather than the tumor cell targets were radioactively labeled. We found that melanoma tumor cells caused the apoptotic death of tumor-specific T cells only upon specific MHC class I-restricted recognition. This death was entirely blockable by the addition of an Ab directed against the Fas death receptor (APO-1, CD95). Contrary to the prevailing view that tumor cells cause the death of anti-tumor T cells by expressing Fas ligand (FasL), our data suggested that FasL was instead expressed by T lymphocytes upon activation. While the tumor cells did not express FasL by any measure (including RT-PCR), functional FasL (as well as FasL mRNA) was consistently found on activated anti-tumor T cells. We could successfully block the activation-induced cell death with z-VAD-fmk, a tripeptide inhibitor of IL-1β-converting enzyme homologues, or with anti-Fas mAbs. Most importantly, these interventions did not inhibit T cell recognition as measured by IFN-γ release, nor did they adversely affect the specific lysis of tumor cell targets. These results imply that Fas-mediated activation-induced cell death could be a limiting factor in the in vivo efficacy of adoptive transfer of class I-restricted CD8+ T cells and provide a means of potentially enhancing their growth in vitro as well as their function in vivo.
Dissociation Between IFN-α-Induced Anti-Viral and Growth Signaling Pathways
Authors: Arora Taruna , Floyd-Smith Georgia , Espy Mark J. , Jelinek Diane F.
Published: January 2023

The ability of IFN-α to induce an anti-viral state in a wide variety of cell types as well as to inhibit cellular growth has long been appreciated. It is less clear, however, whether both these effects lie downstream of a common signaling pathway. In this study we have taken advantage of an atypical human myeloma cell line (KAS-6/1) displaying a dramatic proliferative response to IFN-α in an effort to resolve the signaling requirements for IFN-α-induced anti-viral and growth regulatory effects. Thus, we have analyzed the ability of IFN-α to induce a number of known receptor-initiated events in this cell line and have compared these responses with those exhibited by a cell lineage- and maturation stage-matched myeloma cell line (ANBL-6) that displays typical IFN-α responsiveness. Despite the widely contrasting effects of IFN-α on cellular proliferation, IFN-α was shown to be comparable in its ability to induce the expression of early response genes as well as induce resistance to viral infection in both cell lines. By contrast, the effects of IFN-α on the activation of mitogen-activated protein kinase (MAPK) were strikingly distinct. Finally, although inhibition of MEK and MAPK activation had no effect on the induction of the anti-viral response, it completely blocked IFN-α-stimulated proliferation of the KAS-6/1 cells. In summary, our analysis of the role of the MAPK and anti-viral signaling pathways using these two cell lines suggests that the anti-viral and growth regulatory effects of IFN-α display a differential requirement for activation of the MAPK pathway.
IL-2-Mediated Cell Cycle Progression and Inhibition of Apoptosis Does Not Require NF-κB or Activating Protein-1 Activation in Primary Human T Cells
Authors: Iacobelli Milena , Rohwer Forest , Shanahan Paul , Quiroz Jose A. , McGuire Kathleen L.
Published: January 2023

The IL-2 growth hormone is the major growth factor of activated T lymphocytes during a developing immune response. IL-2 is required not only for cell cycle progression but also to protect Ag-activated T cells from programmed cell death. In several cell types, activation of NF-κB and/or activating protein-1 (AP-1) has been demonstrated to be extremely important in blocking apoptosis. To determine whether either or both of these transcription factors are involved in cell survival or cell cycle progression in response to IL-2, primary human T cells responsive to the growth factor were analyzed for NF-κB and AP-1 activation. The current study clearly demonstrates that IL-2 does not induce IκBα degradation or NF-κB activation in primary human T cells that respond to IL-2 by entering the cell cycle and avoiding apoptosis. Similarly, IL-2 neither activates JNK nor increases AP-1 binding activity to a consensus o-tetradecanoylphorbol 13-acetate (TPA) response element. On the other hand, the growth factor does induce the activation of STAT3 and STAT5 in these cells, as has been previously demonstrated. These data show that neither NF-κB nor AP-1 activation is required for IL-2-mediated survival or cell cycle progression in activated primary human T cells.
Suppressive Immunization with DNA Encoding a Self-Peptide Prevents Autoimmune Disease: Modulation of T Cell Costimulation
Authors: Ruiz Pedro J. , Garren Hideki , Ruiz Irene U. , Hirschberg David L. , Nguyen Louis-Vu T. , Karpuj Marcela V. , Cooper Minton T. , Mitchell Dennis J. , Fathman C. Garrison , Steinman Lawrence
Published: January 2023

Usually we rely on vaccination to promote an immune response to a pathogenic microbe. In this study, we demonstrate a suppressive form of vaccination, with DNA encoding a minigene for residues 139–151 of myelin proteolipid protein (PLP139–151), a pathogenic self-Ag. This suppressive vaccination attenuates a prototypic autoimmune disease, experimental autoimmune encephalomyelitis, which presents clinically with paralysis. Proliferative responses and production of the Th1 cytokines, IL-2 and IFN-γ, were reduced in T cells responsive to PLP139–151. In the brains of mice that were successfully vaccinated, mRNA for IL-2, IL-15, and IFN-γ were reduced. A mechanism underlying the reduction in severity and incidence of paralytic autoimmune disease and the reduction in Th1 cytokines involves altered costimulation of T cells; loading of APCs with DNA encoding PLP139–151 reduced the capacity of a T cell line reactive to PLP139–151 to proliferate even in the presence of exogenous CD28 costimulation. DNA immunization with the myelin minigene for PLP-altered expression of B7.1 (CD80), and B7.2 (CD86) on APCs in the spleen. Suppressive immunization against self-Ags encoded by DNA may be exploited to treat autoimmune diseases.
B Cell Maintenance in Aged Mice Reflects Both Increased B Cell Longevity and Decreased B Cell Generation
Authors: Kline Gregory H. , Hayden Tracy A. , Klinman Norman R.
Published: January 2023

In aged mice the population of mature peripheral B cells is maintained despite a severalfold decrease in the population of bone marrow B cell progenitors. The analysis of the rate of accumulation of 5′-bromo-2-deoxyuridine (BrdU)-labeled splenic B cells in mice fed BrdU for 8 days to 8 wk demonstrated a severalfold increase in the half-life of mature B cells in aged mice. Consistent with a role for decreased B cell turnover in maintaining the mature B cell population of aged mice, several findings indicate that fewer newly generated B cells enter the spleen from the bone marrow in aged vs young adult mice. These include 1) a fourfold decrease in the population of relatively immature splenic B cells, defined as cells that express high levels of heat-stable Ag and accumulate BrdU within 8 wk of labeling; and 2) an equivalent decrease in the population of bone marrow cells representative of later stages of B cell maturation (sIgD−sIgMint-high). Surprisingly, despite a four- to sixfold decrease in pre-B cells, the population of least mature bone marrow B cells (IgD−sIgMvery low) remains intact. Because this population accumulates BrdU-labeled cells more slowly in aged mice than in younger mice, and bone marrow B cells at more mature developmental stages are diminished, it appears that in aged mice B cell development beyond the sIgMvery low stage may be retarded and that cells, therefore, accumulate within this population.
Effect of Fever-Like Whole-Body Hyperthermia on Lymphocyte Spectrin Distribution, Protein Kinase C Activity, and Uropod Formation
Authors: Wang Xiang-Yang , Ostberg Julie R. , Repasky Elizabeth A.
Published: January 2023

Regional inflammation and systemic fever are hallmarks of host immune responses to pathogenic stimuli. Although the thermal element of fever is thought to enhance the activity of immune effector cells, it is unclear what the precise role of increased body temperatures is on the activation state and effector functions of lymphocytes. We report here that mild, fever-like whole body hyperthermia (WBH) treatment of mice results in a distinct increase in the numbers of tissue lymphocytes with polarized spectrin cytoskeletons and uropods, as visualized in situ. WBH also induces a coincident reorganization of protein kinase C (PKC) isozymes and increased PKC activity within T cells. These hyperthermia-induced cellular alterations are nearly identical with the previously described effects of Ag- and mitogen-induced activation on lymphocyte spectrin and PKC. Immunoprecipitation studies combined with dual staining and protein overlay assays confirmed the association of PKCβ and PKCθ with spectrin following its reorganization. The receptor for activated C kinase-1 was also found to associate with the spectrin-based cytoskeleton. Furthermore, all these molecules (spectrin, PKCβ, PKCθ, and receptor for activated C kinase-1) cotranslocate to the uropod. Enhanced intracellular spectrin phosphorylation upon WBH treatment of lymphocytes was also found and could be blocked by the PKC inhibitor bisindolylmaleimide I (GF109203X). These data suggest that the thermal element of fever, as mimicked by these studies, can modulate critical steps in the signal transduction pathways necessary for effective lymphocyte activation and function. Further work is needed to determine the cellular target(s) that transduces the signaling pathway(s) induced by hyperthermia.
Normal Development in Porcine Thymus Grafts and Specific Tolerance of Human T Cells to Porcine Donor MHC
Authors: Nikolic Boris , Gardner Jason P. , Scadden David T. , Arn J. Scott , Sachs David H. , Sykes Megan
Published: January 2023

The induction of T cell tolerance is likely to play an essential role in successful xenotransplantation in humans. In this study, we show that porcine thymus grafts in immunodeficient mice support normal development of polyclonal, functional human T cells. These T cells were specifically tolerant to MHC Ags of the porcine thymus donor and responded to nondonor porcine xenoantigens and alloantigens. Exogenous IL-2 did not abolish tolerance, suggesting central clonal deletion rather than anergy as the likely tolerance mechanism. Our study suggests that the thymic transplantation approach to achieving tolerance with restoration of immunocompetence may be applicable to xenotransplantation of pig tissues to humans.
Differential Sensitivity of Distinct Chlamydia trachomatis Isolates to IFN-γ-Mediated Inhibition
Authors: Perry Linda L. , Su Hua , Feilzer Karen , Messer Ron , Hughes Scott , Whitmire William , Caldwell Harlan D.
Published: January 2023

Resistance to the mouse pneumonitis (MoPn) strain of Chlamydia trachomatis has been mapped to MHC class II-restricted, IL-12-dependent CD4+ T cells that secrete a type 1 profile of proinflammatory cytokines, which includes IFN-γ and TNF-α. The relative contribution of IFN-γ is controversial, however, due to variation in results presented by different laboratories. To determine whether C. trachomatis strain differences contributed to this apparent conflict, the relative resistance of IFN-γ-deficient mice to murine and human strains of C. trachomatis was compared. All human serovars were much more sensitive to the direct inhibitory actions of IFN-γ than the MoPn strain. Furthermore, genital clearance of human serovar D in the C57BL/6 mouse was mediated by class II-independent mechanisms that probably involved local production of IFN-γ by cells of the innate immune system. TNF-α also contributed indirectly to host resistance against all strains tested. The differential susceptibility of distinct C. trachomatis strains to effector cytokines such as IFN-γ could not have been predicted by interstrain biologic variation or by the profile of cytokines stimulated during infection. These findings indicate that strain variation should be considered in situations where related isolates of a given parasite produce conflicting data in models of infection and immunity. They also suggest that stimulation of mucosal IFN-γ activity is a relevant goal for a human chlamydial vaccine.
IFN-γ Up-Regulates the A2B Adenosine Receptor Expression in Macrophages: A Mechanism of Macrophage Deactivation
Authors: Xaus Jordi , Mirabet Maribel , Lloberas Jorge , Soler Concepció , Lluis Carme , Franco Rafael , Celada Antonio
Published: January 2023

Adenosine is a potent endogenous anti-inflammatory agent released by cells in metabolically unfavorable conditions, such as hypoxia or ischemia. Adenosine modulates different functional activities in macrophages. Some of these activities are believed to be induced through the uptake of adenosine into the macrophages, while others are due to the interaction with specific cell surface receptors. In murine bone marrow-derived macrophages, the use of different radioligands for adenosine receptors suggests the presence of A2B and A3 adenosine receptor subtypes. The presence of A2B receptors was confirmed by flow cytometry using specific Abs. The A2B receptor is functional in murine macrophages, as indicated by the fact that agonists of A2B receptors, but not agonists for A1, A2A, or A3, lead to an increase in cAMP levels. IFN-γ up-regulates the surface protein and gene expression of the A2B adenosine receptor by induction of de novo synthesis. The up-regulation of A2B receptors correlates with an increase in cAMP production in macrophages treated with adenosine receptor agonist. The stimulation of A2B receptors by adenosine or its analogues inhibits the IFN-γ-induced expression of MHC class II genes and also the IFN-γ-induced expression of nitric oxide synthase and of proinflammatory cytokines. Therefore, the up-regulation of the A2B adenosine receptor expression induced by IFN-γ could be a feedback mechanism for macrophage deactivation.
Dynamic Association of L-Selectin with the Lymphocyte Cytoskeletal Matrix
Authors: Evans Sharon S. , Schleider David M. , Bowman Lori A. , Francis Michelle L. , Kansas Geoffrey S. , Black Jennifer D.
Published: January 2023

L-selectin mediates lymphocyte extravasation into lymphoid tissues through binding to sialomucin-like receptors on the surface of high endothelial venules (HEV). This study examines the biochemical basis and regulation of interactions between L-selectin, an integral transmembrane protein, and the lymphocyte cytoskeleton. Using a detergent-based extraction procedure, constitutive associations between L-selectin and the insoluble cytoskeletal matrix could not be detected. However, engagement of the L-selectin lectin domain by Abs or by glycosylation-dependent cell adhesion molecule-1, an HEV-derived ligand for L-selectin, rapidly triggered redistribution of L-selectin to the detergent-insoluble cytoskeleton. L-selectin attachment to the cytoskeleton was not prevented by inhibitors of actin/microtubule polymerization (cytochalasin B, colchicine, or nocodozole) or serine/threonine and tyrosine kinase activity (staurosporine, calphostin C, or genistein), although L-selectin-mediated adhesion of human PBL was markedly suppressed by these agents. Exposure of human PBL or murine pre-B transfectants expressing full-length human L-selectin to fever-range hyperthermia also markedly increased L-selectin association with the cytoskeleton, directly correlating with enhanced L-selectin-mediated adhesion. In contrast, a deletion mutant of L-selectin lacking the COOH-terminal 11 amino acids failed to associate with the cytoskeletal matrix in response to Ab cross-linking or hyperthermia stimulation and did not support adhesion to HEV. These studies, when taken together with the previously demonstrated interaction between the L-selectin cytoplasmic domain and the cytoskeletal linker protein α-actinin, strongly implicate the actin-based cytoskeleton in dynamically controlling L-selectin adhesion.
Elimination of the Immunogenicity of Therapeutic Antibodies
Authors: Gilliland Lisa K. , Walsh Louise A. , Frewin Mark R. , Wise Matt P. , Tone Masahide , Hale Geoff , Kioussis Dimitris , Waldmann Herman
Published: January 2023

The immunogenicity of therapeutic Abs limits their long-term use. The processes of complementarity-determining region grafting, resurfacing, and hyperchimerization diminish mAb immunogenicity by reducing the number of foreign residues. However, this does not prevent anti-idiotypic and anti-allotypic responses following repeated administration of cell-binding Abs. Classical studies have demonstrated that monomeric human IgG is profoundly tolerogenic in a number of species. If cell-binding Abs could be converted into monomeric non-cell-binding tolerogens, then it should be possible to pretolerize patients to the therapeutic cell-binding form. We demonstrate that non-cell-binding minimal mutants of the anti-CD52 Ab CAMPATH-1H lose immunogenicity and can tolerize to the “wild-type” Ab in CD52-expressing transgenic mice. This finding could have utility in the long-term administration of therapeutic proteins to humans.