bluuebunny/crossref_metadata_2025 huggingface dataset:
Alloantigen-Reactive Th1 Development in IL-12-Deficient Mice
Authors: Piccotti Joseph R. , Li Kewang , Chan Sherri Y. , Ferrante Jessica , Magram Jeanne , Eichwald Ernst J. , Bishop D. Keith
Published: December 2022

IL-12p70, a 70- to 75-kDa heterodimer consisting of disulfide-bonded 35-kDa (p35) and 40-kDa (p40) subunits, enhances Th1 development primarily by its ability to induce IFN-γ production by NK and Th1 cells. Although homodimers of the p40 subunit of IL-12 are potent IL-12 receptor antagonists in some systems, we have reported that p40 homodimer may accentuate alloreactive CD8+ Th1 function. To test the role of endogenously produced p40 in alloimmunity, Th1 development was assessed in either IL-12 p35 knockout (p35−/−) mice, the cells of which are capable of secreting p40, or p40 knockout (p40−/−) mice. Compared with IL-12 wild-type controls, splenocytes obtained from both p35−/− and p40−/− mice produced markedly less IFN-γ after in vitro stimulation with Con A or alloantigens. Interestingly, in vivo-sensitized Th1 were detected in both p35−/− and p40−/− cardiac allograft recipients. However, in vivo Th1 development was enhanced in p35−/− recipients compared with p40−/− animals, suggesting that endogenous p40 produced in p35−/− mice may stimulate alloreactive Th1. Indeed, neutralizing endogenous p40 with anti-IL-12 p40 mAb reduced Th1 development in p35−/− allograft recipients to that seen in p40−/− mice. To determine whether Th1 development that occurred in the absence of IL-12p70 and p40 required IFN-γ, p40−/− allograft recipients were treated with anti-IFN-γ mAb. Neutralizing IFN-γ did not inhibit in vivo Th1 development in p40−/− recipients and resulted in a unique pathology of rejection characterized by vascular thromboses. Collectively, these data suggest that 1) endogenous p40 may substitute for IL-12p70 in alloantigen-specific Th1 sensitization in vivo and 2) in vivo alloreactive Th1 development may occur independent of IL-12 and IFN-γ, suggesting an alternate Th1-sensitizing pathway.
Migration of Lymphoid Cells from Vaginal Epithelium to Iliac Lymph Nodes in Relation to Vaginal Infection by Herpes Simplex Virus Type 2
Authors: King Nicholas J. C. , Parr Earl L. , Parr Margaret B.
Published: December 2022

To determine whether lymphocytes and Langerhans cells in vaginal epithelium are migratory, we stained mouse vaginal epithelium, including its lymphoid cells, by intraluminal administration of H33342, a fluorescent, vital dye. Stromal staining was superficial, and no free dye reached the iliac lymph nodes. The numbers and phenotypes of H33342-stained cells that migrated from the vagina to the iliac lymph nodes during the next 48 h were determined in four groups: normal mice, mice infected intravaginally with wild-type herpes simplex virus type 2 (HSV-2), mice that were immune to vaginal HSV-2 infection, and immune mice that received vaginal challenge with HSV-2. H33342-stained cells migrated from the vaginal epithelium to the iliac lymph nodes in all groups and were mainly Thy-1.2+ cells and B220+ cells. The number of migrating Thy-1.2+ cells was similar to the sum of CD4+ and CD8+ cells in all groups and was not significantly different from the number of CD44+ cells, suggesting that most of the migrating T cells were memory cells. B lymphocytes comprised 31, 32, 43, and 68% of the migrating cells in the four groups, respectively. We found no evidence that Langerhans cells or macrophages were migrating. Thus, most MHC class II+ cells in all groups were accounted for by B cells, and migrating cells did not express B7.1 or F4/80 or exhibit indented nuclei or dendritic processes. We suggest that the migrating T cells and B cells probably belonged to a pool of lymphocytes that recirculates from blood to tissues and back to the lymph nodes via their afferent lymphatics.
Cholera Toxin and Cholera Toxin B Subunit Induce IgA Switching Through the Action of TGF-β1
Authors: Kim Pyeung-Hyeun , Eckmann Lars , Lee Wha Jung , Han Wonkyo , Kagnoff Martin F.
Published: December 2022

Cholera toxin (CT) and its B subunit (CTB) are potent immunogens and adjuvants that, either alone or linked to protein Ags, can stimulate mucosal immune responses, modulate the induction of oral tolerance, and stimulate IgA isotype switching. The present studies addressed the mechanisms by which CT and CTB promote IgA switching. CT and rCTB, in the presence of IL-2, significantly increased IgA isotype switching at the clonal level in populations of purified and LPS-activated murine surface IgA− spleen B cells, as determined by ELISA, enzyme linked immunospot assays, and limiting dilution analysis. The IgA stimulatory effects of CT and CTB were independent of the A subunit of CT. CTB and CT did not increase the secretory rate of IgA-producing cells or the clonal burst size of IgA clones, and did inhibit B cell growth. Because TGF-β1 also inhibits B cell growth and promotes IgA switching, further studies tested whether the activity of CTB and CT on IgA isotype switching was mediated through TGF-β1. Anti-TGF-β Ab and soluble TGF-β1 type IIR inhibited CTB- and CT-stimulated IgA isotype switching. Furthermore, increased TGF-β1 mRNA levels and bioactive TGF-β1, within a range shown to induce IgA isotype switching, were detected in cultures of surface IgA− B cells stimulated with CT or CTB and IL-2. These data indicate that CTB- and CT-stimulated IgA isotype switching are mediated through TGF-β1. The finding that CTB up-regulates TGF-β1 activity has important implications for understanding the mechanisms by which CTB promotes both IgA mucosal immunity and oral tolerance.
Vascular Endothelial Growth Factor Affects Dendritic Cell Maturation Through the Inhibition of Nuclear Factor-κB Activation in Hemopoietic Progenitor Cells
Authors: Oyama Tsunehiro , Ran Sophia , Ishida Tadao , Nadaf Sorena , Kerr Lawrence , Carbone David P. , Gabrilovich Dmitry I.
Published: December 2022

Vascular endothelial growth factor (VEGF), produced by almost all tumor cells, affects the ability of hemopoietic progenitor cells (HPC) to differentiate into functional dendritic cells (DC) during the early stages of their maturation. In this study we demonstrate specific binding of VEGF to HPC. This binding was efficiently competed by placenta growth factor (PlGF), a ligand reportedly specific for the Flt-1 receptor. The number of binding sites for VEGF decreased during DC maturation in vitro associated with decreased levels of mRNA for Flt-1. VEGF significantly inhibited nuclear factor-κB (NF-κB)-dependent activation of reporter gene transcription during the first 24 h in culture. The presence of VEGF significantly decreased the specific DNA binding of NF-κB as early as 30 min after induction with TNF-α. This was followed on days 7 to 10 by decreases in the mRNA for RelB and c-Rel, two subunits of NF-κB. Blockade of NF-κB activity in HPC at early stages of differentiation with an adenovirus expressing a dominant IκB inhibitor of NF-κB reproduced the pattern of effects observed with VEGF. Thus, NF-κB plays an important role in maturation of HPCs to DC, and VEGF activation of the Flt-1 receptor is able to block the activation of NF-κB in this system. Blockade of NF-κB activation in HPCs by tumor-derived factors may therefore be a mechanism by which tumor cells can directly down-modulate the ability of the immune system to generate effective antitumor immune responses.
Functional Analysis by Site-Directed Mutagenesis of the Complex Polymorphism in Rat Transporter Associated with Antigen Processing
Authors: Deverson Edward V. , Leong Louise , Seelig Angela , Coadwell W. John , Tredgett Evelyn M. , Butcher Geoffrey W. , Howard Jonathan C.
Published: December 2022

The transporter associated with Ag processing, TAP, is an endoplasmic reticulum resident heterodimeric member of the ATP-binding cassette transporter family. TAP transports short peptides from cytosol to the endoplasmic reticulum lumen for loading into recently synthesized class I MHC molecules. In the rat, two alleles of the TAP2 chain differ in their permissiveness to the transport of peptides with small hydrophobic, polar, or charged amino acids at the C terminus, and this correlates with differences between the peptide sets loaded into certain class I molecules in vivo. We have used segmental exchanges and site-directed mutagenesis to identify the residues in rat TAP2 responsible for differential transport between the two alleles of peptides terminating above all in the positively charged residue, arginine. Of the 25 residues by which the two functional TAP2 alleles differ, we have localized differential transport of peptides with a C-terminal arginine to two adjacent clusters of exchanges in the membrane domain involving a total of five amino acids. Each cluster, transferred by site-directed mutagenesis from the permissive to the restrictive sequence, can independently confer on TAP a partial ability to transport peptides with arginine at the C terminus. The results suggest that the permissive TAP2-A allele evolved in at least two steps, each partially permissive for peptides with charged C termini.
Variable Region Domain Exchange Influences the Functional Properties of IgG
Authors: Morrison Sherie L. , Porter Stephen B. , Trinh K. Ryan , Wims Letitia A. , Denham Jerrod , Oi Vernon T.
Published: December 2022

In the present study we have characterized a family of anti-dansyl Abs with the variable region of the heavy chain on human Cκ and the variable region of the light chain on different human γ constant regions (creating inside-out molecules). Although fully assembled molecules were secreted, this variable region exchange slowed the kinetics of Ab assembly. Although the variable region exchange does not lead to a detectable change in the microenvironment of the combining site, it did alter the kinetic parameters of binding to immobilized Ag, slowing both the on and off rates. When effector functions were evaluated, inside-out IgG1 and IgG3 were more effective in complement-mediated cytolysis than their wild-type counterparts. Variable region domain exchange may be one approach to obtaining Abs of identical specificity with altered binding characteristics.
Peptide Selection by an MHC H-2Kb Class I Molecule Devoid of the Central Anchor (“C”) Pocket
Authors: Molano Alberto , Erdjument-Bromage Hediye , Fremont Daved H. , Messaoudi Ilhem , Tempst Paul , Nikolić-Žugić Janko
Published: December 2022

The peptide-binding site of the murine MHC class I molecule H-2Kb contains a deep C pocket, that is critical for peptide binding, as it accepts the anchor phenylalanine or tyrosine residue located in the middle (position 5, P5F/Y) of H-2Kb binding peptides. H-2Kb predominantly binds octameric peptides. By both criteria, H-2Kb is unique among the known murine and human class I molecules, none of which have a deep C pocket or preferentially select octamers. We investigated the relative importance of the C pocket in peptide selection and binding by the MHC. An MHC class I H-2Kb variant, KbW9, predicted to contain no C pocket, was engineered by replacing valine at MHC9 with tryptophan. This mutation drastically altered the selection of peptides bound to KbW9. The KbW9 molecule predominantly, if not exclusively, bound nonamers. New peptide anchor residues substituted for the loss of the P5F/Y:C pocket interaction. P3P/Y, which plays an auxiliary role in binding to Kb, assumed the role of a primary anchor, and P5R was selected as a new primary anchor, most likely contacting the E pocket. These experiments demonstrate that the presence of a deep C pocket is responsible for the selection of octameric peptides as the preferred ligands for Kb and provide insight into the adaptation of peptides to a rearranged MHC groove.
Gene Structure, Promoter Characterization, and Basis for Alternative mRNA Splicing of the Human CD58 Gene
Authors: Wallich Reinhard , Brenner Christiane , Brand Yvonne , Roux Matthias , Reister Manuel , Meuer Stefan
Published: December 2022

The 60-kDa lymphocyte function-associated Ag-3 (LFA-3/CD58), a highly glycosylated adhesion molecule that serves as ligand for the T cell-restricted glycoprotein CD2, is encoded by a gene at the human chromosome locus 1p13. We have elucidated the exon-intron organization of the entire human CD58 gene, including ∼2.5 kilobases (kb) of 5′-flanking DNA. Four overlapping genomic clones, spanning ∼65 kb, contained the entire ∼1-kb coding sequence of CD58 and consisted of six separate exons, which varied from 72 to 294 bp in size. At least two different CD58 mRNA precursors can be generated from the human gene as a result of alternative choice of one of the two acceptor splice sites located within exon 5. DNA sequence analysis of about 2.5 kb of 5′-flanking sequence of the CD58 gene indicated the absence of a CAAT box. However, potential binding sites for the transcriptional activators AP-2, GATA, PU.1, and Sp-1 are present. Two consensus TATAA elements, located ∼2.4 kb upstream of the transcriptional start site, have been identified. The 2.5-kb CD58 promoter sequence displayed functional activity in transient transfection assays in the hepatocellular carcinoma cell line HepG2. Comparing the response of CD58 promoter-driven luciferase plasmids to several cytokines and other agents suggests that the CD58 promoter is regulated by up-regulatory, enhancer-like and down-regulatory, silencer-like elements. Further analysis of this region should allow researchers to gain insight into the molecular mechanisms by which this gene is regulated, e.g., during inflammatory responses.
Oligoclonality of Vδ1 and Vδ2 Cells in Human Peripheral Blood Mononuclear Cells: TCR Selection Is Not Altered by Stimulation with Gram-Negative Bacteria
Authors: Shen Jian , Andrews David M. , Pandolfi Franco , Boyle Lenora A. , Kersten Catalina M. , Blatman Robert N. , Kurnick James T.
Published: December 2022

Despite the enormous potential repertoire of γδ T cells, there are several observations which suggest that the expressed γδ repertoire in the periphery of normal individuals is often quite restricted. To assess selective expansions among γδ T cells from both adult and newborn blood samples, PBMC from 12 normal adults and cord blood from 15 normal newborns were analyzed for TCRDV1 and TCRDV2 junctional diversity by CDR3 size spectratyping and single-strand conformational polymorphism. Although TCRBV usage showed extensive heterogeneity in adults and newborns, both populations often showed CDR3 region restriction for TCRDV1 and TCRDV2. Analysis of the CDR3 spectratype patterns of newborn twins suggested that clonal selection for TCRDV is independent of genetic background. The possible role of Gram-negative bacteria in driving selective responsiveness of γδ T cells in PBMCs from adults was examined by in vitro stimulation with Escherichia coli and Pseudomonas aeruginosa. Donors whose TCRDV repertoire was highly clonal in the unstimulated blood cells showed the same predominant clones among the bacteria-stimulated cultures. In individuals whose γδ T cells were less restricted, in vitro stimulation did not select for clonality; rather, the TCRDV repertoires were similar before and after bacterial stimulation. Together, these data indicate that γδ T cells are often clonally restricted in adults as well as in newborns and suggest that the prominent stimulatory activity of Gram-negative bacteria does not by itself account for the restriction or diversity of the γδ T cell repertoire.
Androstenediol Antagonizes Herpes Simplex Virus Type 1-Induced Encephalitis Through the Augmentation of Type I IFN Production
Authors: Daigle Jennifer , Carr Daniel J. J.
Published: December 2022

Dehydroepiandrosterone and androstenediol (AED) have previously been found to protect mice from viral-induced encephalitis resulting in an increased survival rate of the animals. These hormones have been shown to antagonize corticosteroids, which have immunosuppressive effects in vivo and in vitro, suggesting the antiviral effect of DHEA and AED may be linked to the anticorticosteroid action. The present study was undertaken to address the immune response to herpes simplex virus type 1 (HSV-1) during the acute ocular infection with and without AED treatment focusing on the early immune events in the eye and trigeminal ganglion. AED treatment was found to significantly improve the survival of HSV-1-infected mice in a dose-dependent fashion. While AED did not antagonize the elevated serum corticosterone levels following acute infection, AED enhanced the expression of IFN-α mRNA and decreased the expression of HSV-1-infected cell polypeptide 27 mRNA in the trigeminal ganglion during the acute (day 6 postinfection) infection of mice, as determined by reverse transcription-PCR. However, there was no change in the viral load from the eye or trigeminal ganglion when comparing the AED-treated with the vehicle-treated mice. Neutralization Abs to IFN-α, -β, or -α/β, but not control Ab, blocked the protective effect following AED exposure, confirming the involvement of type I IFN in the enhancement of survival in AED-treated mice. Collectively, these results identify innate immunity as a key component in augmenting the survival of HSV-1-infected mice following AED treatment.
Cutting Edge: Dichotomous Effects of β-Chemokines on HIV Replication in Monocytes and Monocyte-Derived Macrophages
Authors: Kelly Mark D. , Naif Hassan M. , Adams Susan L. , Cunningham Anthony L. , Lloyd Andrew R.
Published: December 2022

The role of β-chemokines in the pathogenesis of HIV disease remains undefined. Given the potent capacities of these proteins to attract mononuclear cells to inflammatory sites, such as lymph nodes of patients with HIV disease, the effects of exposure of monocytes and monocyte-derived macrophages to β-chemokines before HIV infection were compared with their effects when added either simultaneously with or after HIV infection. In this system, HIV replication was substantially increased in cells that had been exposed to β-chemokines before HIV infection. These effects were pertussis toxin sensitive. By contrast, HIV replication was inhibited in cells that had been exposed to β-chemokines either simultaneously with or after HIV infection. These effects were not pertussis toxin sensitive. In view of this potent capacity of β-chemokines to stimulate HIV replication, treatment approaches for HIV disease based on the apparent inhibitory activity of these proteins on viral replication should be undertaken with caution.
Discoordinate Surface Expression of IFN-γ-Induced HLA Class II Proteins in Nonprofessional Antigen-Presenting Cells with Absence of DM and Class II Colocalization
Authors: Muczynski Kimberly A. , Anderson Susan K. , Pious Donald
Published: December 2022

We compared HLA class II expression in a human melanoma line (a nonprofessional APC), induced by IFN-γ or by stable transfection with CIITA, with constitutive class II expression in an EBV-transformed B lymphoblastoid cell line (a professional APC) from the same donor. IFN-γ-induced and CIITA-transfected melanoma cells expressed DR, DP, and DQ at levels similar to those expressed by the professional APC; however, DP and DQ proteins and DM-dependent DR epitopes were delayed in appearing on the cell surface when induced by IFN-γ. The delay in cell surface expression of some IFN-γ-induced class II epitopes was observed even though Northern blots demonstrated class II and DM genes to be coordinately transcribed and their mRNA levels to be equivalent to that in B lymphoblastoid cells. Confocal microscopy suggests that discoordinate cell surface expression of class II results from different intracellular trafficking for IFN-γ-induced class II proteins in the melanoma line compared with that in professional APCs. Specifically, although DR and DM proteins were present 2 days after IFN-γ induction, colocalization of DR and DM proteins intracellularly was not apparent in cells at any time after induction. Failure of DR and DM proteins to colocalize suggests that IFN-γ-induced cells lack an intracellular MIIC-like compartment. The absence of a compartment containing DR and DM to facilitate interaction between the two proteins may account for the delayed surface expression of class II epitopes whose formation requires both class II and DM.
In Vivo IL-4 Responses to Anti-IgD Antibody Are MHC Class II Dependent and β2-Microglobulin Independent and Develop Normally in the Absence of IL-4 Priming of T Cells
Authors: Morris Suzanne C. , Coffman Robert L. , Finkelman Fred D.
Published: December 2022

A crucial role for CD1-responsive, MHC class II-unrestricted T cells in the generation of T cell IL-4 responses is suggested by the: 1) requirement for IL-4 to prime in vitro IL-4 responses by naive CD4+ T cells; 2) ability of TCR cross-linking to induce CD1-responsive T cells, but not conventional naive T cells, to produce IL-4; 3) failure of anti-IgD Ab to induce an IL-4-dependent IgE response in β2-microglobulin-deficient mice, which lack CD1; and 4) reported ability of MHC class II-deficient mice to make IgE responses to anti-IgD Ab. In contrast, the Ag specificity of cytokine and Ab responses in anti-IgD-injected mice and the normal IgE responses made by anti-IgD-treated CD1-deficient mice are difficult to reconcile with this view. We now find that the failure of β2-microglobulin-deficient mice to make an IgE response to anti-IgD Ab is caused by their rapid degradation of anti-IgD; sustained anti-IgD treatment induces them to make relatively normal IL-4 and IgE responses. Furthermore, in our study, MHC class II-deficient mice make little or no IL-4 or IgE responses to anti-IgD Ab and β2-microglobulin-deficient mice make large in vivo IL-4 responses to anti-CD3 mAb. Finally, although IL-4 priming of T cells for IL-4 production is Stat6 dependent, Stat6-deficient mice make normal IL-4 responses to anti-IgD. Thus, CD1-responsive T cells and other β2-microglobulin-dependent T cells are not required to prime conventional CD4+ T cells to make IL-4 responses to anti-IgD in vivo; in fact, the large IL-4 response made in this system does not require IL-4 priming.
A B7.1-Antibody Fusion Protein Retains Antibody Specificity and Ability to Activate Via the T Cell Costimulatory Pathway
Authors: Challita-Eid Pia M. , Penichet Manuel L. , Shin Seung-Uon , Poles Tina , Mosammaparast Nima , Mahmood Kutubuddin , Slamon Dennis J. , Morrison Sherie L. , Rosenblatt Joseph D.
Published: December 2022

We describe the construction and characterization of an Ab fusion protein specific for the tumor-associated Ag HER2/neu linked to sequences encoding the extracellular domain of the B7.1 T cell costimulatory ligand. The Ab domain of the fusion molecule will specifically target HER2/neu-expressing tumor cells, while the B7.1 domain is designed to activate a specific immune response. We show that the B7.1 fusion Ab retained ability to selectively bind to the HER2/neu Ag and to the CTLA4/CD28 counter-receptors for B7.1. Specific T cell activation was observed when the B7.1 Ab fusion protein was bound to HER2/neu-expressing cells. The use of the B7.1 Ab fusion protein may overcome limitations of gene transfer and/or standard Ab therapy and represents a novel approach to the eradication of minimal residual disease.
Dominant-Negative Effect of the Lymphocyte Function-Associated Antigen-1 β (CD18) Cytoplasmic Domain on Leukocyte Adhesion to ICAM-1 and Fibronectin
Authors: Rey-Ladino Jose A. , Pyszniak Andrew M. , Takei Fumio
Published: December 2022

The cytoplasmic domains of LFA-1 (CD11a/CD18) are thought to play an important role in the regulation of LFA-1 function. To further elucidate the role of the LFA-1 cytoplasmic domains, we transfected chimeric proteins consisting of the extracellular domain of CD4 fused with the transmembrane and cytoplasmic domains of LFA-1 into T and B cell lines, EL-4 and A20, respectively, and examined their effects on LFA-1-mediated cell adhesion. The CD4/18, but not CD4/11a, chimera profoundly inhibited LFA-1-mediated cell adhesion to ICAM-1, as well as cell spreading following cell adhesion. Unexpectedly, cell adhesion to fibronectin was also inhibited by the CD4/18 chimera. The CD4/18 chimera did not affect the expression of endogenous LFA-1 or the association of CD11a and CD18. Truncation of the carboxyl-terminal 13 amino acid residues of the CD18 cytoplasmic domain of the chimera completely abrogated the inhibitory effect on LFA-1. Among these amino acid residues, the carboxyl-terminal six residues were dispensable for the inhibitory effect in EL-4 cells, whereas it significantly reduced the inhibitory activity of CD4/18 in A20 cells. A larger truncation of the CD18 cytoplasmic domain was needed to fully abrogate the inhibitory effects of CD4/18 on the adhesion to fibronectin. These results show that 1) the CD4/18 chimera has dominant-negative effects on cell adhesion mediated by LFA-1 as well as fibronectin receptors, and 2) amino acid residues of the CD18 cytoplasmic domain involved in the inhibition of LFA-1 seem to be different from those for fibronectin receptors.
The Role of the CD40 Pathway in Alloantigen-Induced Hyporesponsiveness In Vivo
Authors: Niimi Masanori , Pearson Thomas C. , Larsen Christian P. , Alexander Diane Z. , Hollenbaugh Diane , Aruffo Alejandro , Linsley Peter S. , Thomas Elaine , Campbell Kim , Fanslow William C. , Geha Raif S. , Morris Peter J. , Wood Kathryn J.
Published: December 2022

Resting B (rB) cells are known to be incompetent APCs in vitro, which alone can induce specific unresponsiveness to single minor histocompatibility (miH) Ags and, when combined with CD40 pathway blockade, can induce hyporesponsiveness to MHC molecules in vivo. Here we show that anti-CD40 ligand (CD40L) mAb does not prevent the expression of B7-2 on allogeneic rB cells in vivo but did prolong donor-specific cardiac allograft survival. Moreover, pretreatment with professional APCs combined with anti-CD40L mAb induced hyporesponsiveness to alloantigens in vivo. rB cells from CD40 knockout mice were unable to induce unresponsiveness, while graft prolongation was achieved in CD40L knockout recipients pretreated with wild-type rB cells. These data suggest that CD40-CD40L interactions in the recipient play a critical role in the induction of hyporesponsiveness to alloantigens in vivo and that the effect of the CD40 pathway may be independent of its effect on the B7 costimulatory pathway.
Alterations in Cytokine Production Following Intraocular Injection of Soluble Protein Antigen: Impairment in IFN-γ and Induction of TGF-β and IL-4 Production
Authors: Kosiewicz Michele M. , Alard Pascale , Streilein J. Wayne
Published: December 2022

Immune deviation induced by intraocular injection of soluble protein Ag, referred to as anterior chamber-associated immune deviation (ACAID), is characterized by impairment of delayed hypersensitivity (DH). Two populations of splenic regulatory cells that impair the induction and expression phases of DH are involved in the ACAID response and may mediate their effects through cytokines. The purpose of the present study was to evaluate the role that cytokines play in ACAID. IFN-γ production in draining lymph nodes induced by conventional immunization with protein Ag and adjuvant was suppressed after intraocular injection of protein Ag administered either before or after sensitization; IL-12 production in these mice was not decreased, suggesting that suppression of IL-12 may not be the mechanism involved in the impairment in IFN-γ production. Surprisingly, although significant amounts of IL-4 (but not IL-10) were produced by spleen and lymph node cells from several different strains of mice, experiments in IL-4 knockout mice showed that impairment of neither DH nor IFN-γ production required IL-4. Interestingly, significant levels of TGF-β were detected in cultures of spleen cells from mice with ACAID. As determined by quantitative RT-PCR, TGF-β was produced primarily by the splenic CD4 and non-T cells and was of the TGF-β1 type. These results suggest that the Th1 response is impaired in ACAID by a mechanism(s) that does not require Th2-type cytokines, but may involve TGF-β at several different (including the effector) phases during the response.
IL-2Rα on One Cell Can Present IL-2 to IL-2Rβ/γc on Another Cell to Augment IL-2 Signaling
Authors: Eicher Donald M. , Waldmann Thomas A.
Published: December 2022

IL-2Rα augments IL-2 signaling. Although this is generally believed to occur only when the three known components of IL-2R are associated within a single cell membrane, we demonstrate here an intercellular interaction. Cocultivation of cells individually expressing chimerae incorporating the extracellular domain of IL-2Rα alone with cells expressing chimerae of IL-2Rβ alone permitted IL-2 dose-dependent oligomerization of the chimerae. Likewise, native IL-2Rα-bearing cells augmented the IL-2 proliferative response of ex vivo large granular lymphocytic leukemia cells expressing IL-2Rβ/γc but lacking IL-2Rα. In both cases, the response was inhibitable by an Ab to IL-2Rα. Intercellular augmentation of cytokine effects, acting in trans, has important implications for biology and medicine.
Early IL-4 Induction in Bone Marrow Lymphoid Precursor Cells by Mycobacterial Lipoarabinomannan
Authors: Collins Helen L. , Schaible Ulrich E. , Kaufmann Stefan H. E.
Published: December 2022

IL-4 is produced promptly in response to certain infections and plays a key role in the Th1/Th2 T cell dichotomy; however, the cellular source remains a matter of debate. Here we describe the induction of IL-4 in bone marrow cells of normal and RAG−/− mice by both Mycobacterium tuberculosis and its major cell wall glycolipid, lipoarabinomannan. Characterization of the cell type responsible indicated that it was distinct from the NK1+ or CD4+ T cell previously ascribed the function of rapid IL-4 secretion. Cell-sorting experiments identified CD19+/B220+ precursor cells, presumably pre-B cells that produced IL-4 constitutively and whose frequency was rapidly and markedly up-regulated by lipoarabinomannan. Thus, pathogenic mycobacteria and their glycolipids may influence hemopoiesis by rapidly inducing IL-4 secretion in the bone marrow.
Optimization of Codon Usage of Plasmid DNA Vaccine Is Required for the Effective MHC Class I-Restricted T Cell Responses Against an Intracellular Bacterium
Authors: Uchijima Masato , Yoshida Atsushi , Nagata Toshi , Koide Yukio
Published: December 2022

In an attempt to study codon usage effects of DNA vaccines on the induction of MHC class I-restricted T cell responses against an intracellular bacterium, Listeria monocytogenes, we designed two plasmid DNA vaccines encoding an H-2Kd-restricted epitope of listeriolysin O (LLO) of L. monocytogenes, LLO 91–99. One DNA vaccine, p91wt, carries the wild-type DNA sequence encoding LLO 91–99, and the other one, p91mam, possesses the altered DNA sequence in which the codon usage was optimized for murine system. Our read-through analyses with LLO 91–99/luciferase fusion genes confirmed that the optimized 91mam DNA sequence showed extremely higher translation efficiency than the wild-type sequence in murine cells. Consistent with this, i.m. injections of p91mam, but not of p91wt, into BALB/c mice were capable of inducing specific CTL- and IFN-γ-producing CD8+ T cells able to confer partial protection against listerial challenge. Taken together, these observations suggest that optimization of codon should be taken into consideration in the construction of DNA vaccines against nonviral pathogens.
The Efficiency of Antigen Recognition by CD8+ CTL Clones Is Determined by the Frequency of Serial TCR Engagement
Authors: Hudrisier Denis , Kessler Benedikt , Valitutti Salvatore , Horvath Clotilde , Cerottini Jean-Charles , Luescher Immanuel F.
Published: December 2022

Using H-2Kd-restricted CTL clones, which are specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS252–260 (SYIPSAEKI) and permit assessment of TCR-ligand interactions by TCR photoaffinity labeling, we have previously identified several peptide derivative variants for which TCR-ligand binding and the efficiency of Ag recognition deviated by fivefold or more. Here we report that the functional CTL response (cytotoxicity and IFN-γ production) correlated with the rate of TCR-ligand complex dissociation, but not the avidity of TCR-ligand binding. While peptide antagonists exhibited very rapid TCR-ligand complex dissociation, slightly slower dissociation was observed for strong agonists. Conversely and surprisingly, weak agonists typically displayed slower dissociation than the wild-type agonists. Acceleration of TCR-ligand complex dissociation by blocking CD8 participation in TCR-ligand binding increased the efficiency of Ag recognition in cases where dissociation was slow. In addition, permanent TCR engagement by TCR-ligand photocross-linking completely abolished sustained intracellular calcium mobilization, which is required for T cell activation. These results indicate that the functional CTL response depends on the frequency of serial TCR engagement, which, in turn, is determined by the rate of TCR-ligand complex dissociation.
A Novel Type II Complement C2 Deficiency Allele in an African-American Family
Authors: Zhu Zeng-Bian , Atkinson T. Prescott , Volanakis John E.
Published: December 2022

A 9-yr-old African-American male presenting with severe recurrent pyogenic infections was found to have C2 deficiency (C2D). Analysis of his genomic DNA demonstrated that he carried one type I C2D allele associated with the HLA-A25, B18, DR15 haplotype. Screening all 18 exons of the C2 gene by exon-specific PCR/single-strand conformation polymorphism indicated abnormal bands in exons 3, 7, and 6, the latter apparently caused by the 28-bp deletion of the typical type I C2D allele. Nucleotide (nt) sequencing of the PCR-amplified exons 3 and 7 revealed a heterozygous G to A transition at nt 392, causing a C111Y mutation, and a heterozygous G to C transversion at nt 954, causing a E298D mutation and a polymorphic MaeII site. Cys111 is the invariable third half-cystine of the second complement control protein module of C2. Pulse-chase biosynthetic labeling experiments indicated that the C111Y mutant C2 was retained by transfected COS cells and secreted only in minimal amounts. Therefore, this mutation causes a type II C2D. In contrast, the E298D mutation affected neither the secretion of C2 from transfected cells nor its specific hemolytic activity. Analysis of genomic DNA from members of the patient’s family indicated that 1) the proband as well as one of his sisters inherited the type I C2D allele from their father and the novel type II C2D allele from their mother; 2) the polymorphic MaeII site caused by the G954C transversion is associated with the type I C2D allele; and 3) the novel C111Y mutation is associated in this family with the haplotype HLA-A28, B58, DR12.
Alterations in Cell Surface Carbohydrates on T Cells from Virally Infected Mice Can Distinguish Effector/Memory CD8+ T Cells from Naive Cells
Authors: Galvan Marisa , Murali-Krishna Kaja , Ming Lisa Lau , Baum Linda , Ahmed Rafi
Published: December 2022

Glycosylation changes on surface molecules of T cells affect cell trafficking and function and may be useful in discriminating between naive, effector, and memory T cells. To analyze oligosaccharide structures on T cells activated in vivo, we examined alterations in sialic acid residues on T cells following infection of mice with lymphocytic choriomeningitis (LCMV), vaccinia virus, and vesicular stomatitis virus. We found that the majority of CD8 T cells from mice acutely infected with these viruses showed increased binding to peanut agglutinin (PNA). All of the PNAhighCD8 T cells from infected mice were CD44high, indicating that glycosylation changes were occurring on activated T cells. There was also an increase in the PNAhighCD4 T cell population in virally infected mice. Increased PNA binding to activated CD8 T cells correlated with higher endogenous neuraminidase levels in these cells. This higher neuraminidase activity most likely contributed to the PNAhigh phenotype by cleaving sialic acid residues off the core-1 O-glycans or glycoproteins destined for the cell surface. A PNAhighCD8 T cell population persisted in immune mice that had cleared the LCMV infection. When spleen cells from immune mice were sorted into PNAhigh and PNAlow populations, >95% of the LCMV-specific memory CD8 T cells segregated with the PNAhigh population. This shows that virus-specific memory CD8 T cells remain hyposialylated and can be distinguished from naive CD8 T cells based on PNA binding. Thus, PNA can be used as a marker for Ag-experienced T cells.
HIV-Tat Protein Activates c-Jun N-Terminal Kinase and Activator Protein-1
Authors: Kumar Ashok , Manna Sunil K. , Dhawan Subhash , Aggarwal Bharat B.
Published: December 2022

Human immunodeficiency virus-1 tat (HIV-tat) protein, like other proinflammatory cytokines (such as TNF), activates a wide variety of cellular responses, some of which play a critical role in progression of HIV infection. Whether HIV-tat, like TNF, also activates c-Jun N-terminal kinase (JNK) and the transcription factor activator protein (AP)-1 is not known. We show that treatment of human histiocytic lymphoma U937 cells with the HIV-tat protein causes activation of JNK and AP-1 in a time- and dose-dependent manner. Transfection of a T cell line, H9 cells with the HIV-tat gene also resulted in an activation of JNK that was not further increased by treatment of cells with exogenous HIV-tat protein. Neutralizing Ab against HIV-tat inhibited the HIV-tat-mediated JNK activation. The activation of JNK by HIV-tat appears to be mediated through generation of free radical species, since pretreatment of cells with N-acetylcysteine (NAC) abolished the effect. Overall our results demonstrate that HIV-tat activates JNK and AP-1, which may contribute to the pathogenesis of AIDS.
Regulatory Elements in the Promoter of a MurineTCRD VGene Segment
Authors: Kienker Laura J. , Ghosh Maya R. , Tucker Philip W.
Published: December 2022

TCRD V segments rearrange in an ordered fashion during human and murine thymic development. Recombination requires the accessibility of substrate gene segments, and transcriptional enhancers and promoters have been shown to regulate the accessible chromatin configuration. We therefore investigated the regulation of TCRD V rearrangements by characterizing the promoter of the first TCRD V segment to be rearranged, DV101S1, under the influence of its own enhancer. Sequences required for full promoter activity were identified by transient transfections of normal and mutated promoters into a human γδ lymphoma, and necessary elements fall between −86 and +66 nt, relative to the major transcription start site. They include a cAMP responsive element (CRE) at −62, an Ets site at −39, a TATA box at −26, the major transcriptional start site sequence (−8 to −5 and −2 to +11), and a downstream sequence (+12 to +33). Gel shift analyses and in vitro DNase I footprinting showed that nuclear proteins bind to the functionally relevant CRE, Ets, +1 to +10 sequence, and the +17 to +21 sequence. Nuclear proteins also bind to an E box at −52, and GATA-3 binds to a GATA motif at −5, as shown by Ab ablation-supershift experiments, but mutations that abrogated protein binding to these sites failed to affect DV101S1 promoter activity. We conclude that not all protein-binding sites within the DV101S1 minimal promoter are important for enhancer driven TCRD gene transcription. Further, the possibility remains that the GATA and E box sites function in enhancer independent DV101S1 germline transcription.
Use of Complete Eluted Peptide Sequence Data from HLA-DR and -DQ Molecules to Predict T Cell Epitopes, and the Influence of the Nonbinding Terminal Regions of Ligands in Epitope Selection
Authors: Godkin Andrew J. , Davenport Miles P. , Willis Anthony , Jewell Derek P. , Hill Adrian V. S.
Published: December 2022

In diseases with a strong association with an HLA haplotype, identification of relevant T cell epitopes may allow alteration of the pathologic process. In this report we use a reverse immunogenetic approach to predict possible HLA class II-restricted T cell epitopes by using complete pool sequencing data. Data from HLA-DR2(B1*1501), -DR3(B1*0301), -DQ2(A1*0501, B1*0201), and -DQ8(A1*0301, B1*0302) alleles were used by a computer program that searches a candidate protein to predict ligands with a relatively high probability of being processed and presented. This approach successfully identified both known T cell epitopes and eluted single peptides from the parent protein. Furthermore, the program identified ligands from proteins in which the binding motif of the HLA molecule was unable to do so. When the information from the nonbinding N- and C-terminal regions in the pool sequence was removed, the ability to predict several ligands was markedly reduced, particularly for the HLA-DQ alleles. This suggests a possible role for these regions in determining ligands for HLA class II molecules. Thus, the use of complete eluted peptide sequence data offers a powerful approach to the prediction of HLA-DQ and -DR peptide ligands and T cell epitopes.
The CXC Chemokines IP-10 and Mig Are Necessary for IL-12-Mediated Regression of the Mouse RENCA Tumor
Authors: Tannenbaum Charles S. , Tubbs Raymond , Armstrong David , Finke James H. , Bukowski Ronald M. , Hamilton Thomas A.
Published: December 2022

The role of the non-ELR-containing CXC chemokines IP-10 and Mig in antitumor activity induced by systemic treatment with IL-12 was examined in mice bearing the murine renal adenocarcinoma RENCA. IL-12 treatment produces a potent antitumor effect that is associated with tumor infiltration by CD8+ T lymphocytes. The regression of tumor is associated with the elevated expression of the IFN-γ-inducible chemokines IP-10 and Mig within the tumor tissue. IP-10 and Mig have been shown to function as chemoattractants for activated T lymphocytes. In animals treated with rabbit polyclonal Abs specific for IP-10 and for Mig, the IL-12-induced regression of RENCA tumors was partially abrogated. This effect was associated with a dramatic inhibition of T cell infiltration. Thus, it appears that IL-12-dependent, T cell-mediated antitumor activity requires the intermediate expression of IP-10 and Mig to recruit antitumor effector T cells to the tumor site.
Cutting Edge: A Conditionally Active Form of STAT6 Can Mimic Certain Effects of IL-4
Authors: Kamogawa Yumiko , Lee Hyun Jun , Johnston James A. , McMahon Martin , O’Garra Anne , Arai Naoko
Published: December 2022

Binding of IL-4 to its cognate receptor leads to the activation of a number of signaling pathways within the cell. Activation of the transcription factor STAT6 by JAK family protein tyrosine kinases has been shown to be essential for the full response of cells to IL-4. To elucidate the role of STAT6 in IL-4 signaling, we have constructed and expressed in cells a conditionally active form of the protein (STAT6:ER*) by fusing STAT6 to a modified form of the hormone-binding domain of the estrogen receptor. Activation of STAT6:ER* by 4-hydroxytamoxifen leads to specific activation of STAT6-regulated gene expression including the activation of a STAT6 reporter construct and induction of CD23 in B cell lines. Interestingly, in contrast to native STAT6, activation of STAT6:ER* occurs in the absence of detectable tyrosine phosphorylation of the fusion protein. This type of conditional system will be helpful in dissecting the mechanisms and specificity of transcriptional regulation by the STAT family of transcription factors.
Kinetics of Expression of Costimulatory Molecules and Their Ligands in Murine Relapsing Experimental Autoimmune Encephalomyelitis In Vivo
Authors: Issazadeh Shohreh , Navikas Vaidrius , Schaub Meike , Sayegh Mohamed , Khoury Samia
Published: December 2022

We studied the kinetics of expression of costimulatory molecules and cytokines in the central nervous system (CNS) in murine relapsing experimental autoimmune encephalomyelitis (EAE). During the natural course of EAE, B7-2 expression in the CNS correlated with clinical signs, while B7-1 was exclusively expressed during remissions. Interestingly, B7-1 was expressed on infiltrating mononuclear cells as well as neuronal cells in the CNS. In the periphery, B7-1 expression on APCs peaked with clinical disease but decreased on T cells. CD28 and CTLA4 molecules, the two known ligands for B7-1 and B7-2, had distinct expression patterns in the CNS; CD28 was highly expressed and correlated with B7-2 expression on APCs (macrophages/microglia as well as astrocytes) and with the clinical signs of EAE. CTLA4, on the other hand, was expressed by substantially fewer cells during the effector phase of disease and peaked during remission, which is consistent with the emerging role of this molecule in the termination of immune responses. The expression of CD40 and CD40L in the CNS was increased during clinical attacks. The expression of IL-12, IFN-γ, and TNF-α correlated with disease activity and severity, while TGF-β was the only factor that was up-regulated during the recovery phase. Interestingly, TGF-β was also expressed by neurons during remission. This is the first study demonstrating the kinetics of the in vivo expression of costimulatory molecules, their ligands, and cytokines in an autoimmune disease model characterized by remissions and relapses. Our data suggest that the targeting of costimulatory molecules to block an immune response must take into account the expression patterns in the target organ.
CD4 Regulation of TCR Signaling and T Cell Differentiation Following Stimulation with Peptides of Different Affinities for the TCR
Authors: Leitenberg David , Boutin Yvan , Constant Stephanie , Bottomly Kim
Published: December 2022

To define the role of CD4 in modulating T cell signaling pathways and regulating Th1 and Th2 differentiation, we have examined the activation and differentiation characteristics of naive T cells from CD4 mutant mice. Using peptides with differing affinities for the moth cytochrome c-specific TCR, we test the hypothesis that differences in coreceptor recruitment and signaling explain the qualitatively distinct signaling pathways seen in CD4 T cells following high affinity agonist and low affinity altered peptide ligand (APL) ligation. We find that the absence of CD4 signaling during stimulation with a strong agonist peptide does not qualitatively change the pattern of early TCR-mediated biochemical signaling events into a pattern resembling the response of CD4+ T cells to APLs. In contrast, the response to APL stimulation, by T cells bearing the same TCR, does require a component of CD4 signaling. The proliferative response and calcium signals normally seen following APL stimulation are markedly diminished in the absence of CD4. In addition, we find that naive T cell differentiation into Th2 effector cells is impaired in the absence of CD4. These data suggest that the altered pattern of biochemical signals generated by APLs require CD4 coreceptor function and that some of these signals may be required to initiate Th2 differentiation.