bluuebunny/crossref_metadata_2025 huggingface dataset:
Loss of Original Antigenic Specificity in T Cell Hybridomas Transduced with a Chimeric Receptor Containing Single-Chain Fv of an Anti-Collagen Antibody and FcεRI-Signaling γ Subunit
Authors: Annenkov Alexander E. , Moyes Sasha P. , Eshhar Zelig , Mageed Rizgar A. , Chernajovsky Yuti
Published: December 2022

T cell hybridomas HCQ6 and MD.45 acquired Ab-type specificity to collagen type II, when engrafted with a chimeric cell surface receptor, scC2Fv/γ, which includes the single-chain Fv domain (scFv) of the anti-collagen type II mAb C2 and the signaling γ subunit of the FcεRI. When transduced into MD.45 cells, scC2Fv/γ or its mutated form lacking immunoreceptor tyrosine-based activation motif (ITAM), scC2Fv/γIC−, formed mainly homodimers. A small proportion of these molecules formed heterodimers with endogenous CD3ζ in these hybridoma cells. By contrast, in HCQ6 cells, the majority of scC2Fv/γ and scC2Fv/γIC− molecules formed heterodimers with CD3ζ, and only a small proportion of them was expressed as homodimers. Stimulation with plastic-immobilized collagen induced IL-2 production in scC2Fv/γ-transduced MD.45 cells, but not in MD.45 cells transduced with the ITAM-less chimera scC2Fv/γIC−. HCQ6 cells transduced with scC2Fv/γ responded to plastic-bound collagen. Due to the high content of CD3ζ-associated chimeras, HCQ6 cells transduced with the ITAM-less scC2Fv/γIC− chimera were also responsive to plastic-bound collagen. When cells were stimulated with collagen in solution, MD.45 cells transduced with scC2Fv/γ produced IL-2, whereas transduced HCQ6 cells were unresponsive, hence suggesting that the ability of cells transduced with scC2Fv chimeras to respond to soluble collagen correlated with predominant expression of divalent scC2Fv/γ homodimers, but not monovalent scC2Fv/γ-CD3ζ or scC2Fv/γIC−-CD3ζ heterodimers. Of interest, expression of CD3 subunits in hybridomas transduced with scC2Fv chimeras was reduced, resulting in decreased response to cognate Ags.
T Cell Proliferation-Augmenting Activities of the Gene 3 Protein Derived from a Phage Library Clone with CD80-Binding Activity
Authors: Fukumoto Takahiro , Torigoe Naohiko , Ito Yuji , Kajiwara Yasuhiro , Sugimura Kazuhisa
Published: December 2022

We have isolated a phage clone, F2, by panning a phage library with a CTLA4-conformation recognizing mAb (anti-CTLA4 mAb). The unique sequence of 15 amino acids with an internal disulfide bond was inserted in the gene 3 proteins of F2 phage clone (F2-g3p). We show here that 1) F2-g3p was recognized with anti-CTLA4 mAb but not with anti-CD28 mAb, and 2) F2-g3p bound to CD80 but not to CD86. The surface plasmon resonance analysis showed that F2-g3p strongly bound CD80. F2-g3p inhibited the binding of CTLA4 to CD80 but not to CD86. In contrast, F2-g3p weakly inhibited the binding of CD28 with CD80. When hen egg lysozyme (HEL)-primed lymph node cells were stimulated with HEL in the presence of F2-g3p in vitro, cell proliferation was highly potentiated. In the absence of antigenic stimulation, F2-g3p induced no T cell proliferation, indicating the costimulatory nature of F2-g3p. The T cell-augmenting activity of the F2 clone was eliminated when the F2 clone was preincubated with CD80-Ig before the addition to the cultures, indicating the involvement of CD80-binding in the F2-g3p-mediated immunopotentiation. Thus, the F2 motif conferred CD80-binding activity and an immunoregulatory function to the g3p.
Efficient Lymphocyte Migration Across High Endothelial Venules of Mouse Peyer’s Patches Requires Overlapping Expression of L-Selectin and β7 Integrin
Authors: Steeber Douglas A. , Tang Mimi L. K. , Zhang Xiu-Qin , Müller Werner , Wagner Norbert , Tedder Thomas F.
Published: December 2022

Lymphocyte migration into lymphoid organs is regulated by adhesion molecules including L-selectin and the β7 integrins. L-selectin and α4β7 are predominantly hypothesized to direct the selective migration of lymphocytes to peripheral lymph nodes and the gut-associated lymphoid tissues, respectively. To further characterize interactions between L-selectin and β7 integrins during lymphocyte recirculation, mice deficient in both receptors (L-selectin/β7 integrin−/−) were generated. The simultaneous loss of L-selectin and β7 integrin expression prevented the majority of lymphocytes (>95% inhibition) from attaching to high endothelial venules (HEV) of Peyer’s patches and other lymphoid tissues during in vitro binding assays. Moreover, the inability to bind HEV eliminated the vast majority of L-selectin/β7 integrin−/− lymphocyte migration into Peyer’s patches during short-term and long-term in vivo migration assays (>99% inhibition, p < 0.01). The lack of lymphocyte migration into Peyer’s patches correlated directly with the dramatically reduced size and cellularity (99% reduced) of this tissue in L-selectin/β7 integrin−/− mice. High numbers of injected L-selectin/β7 integrin−/− lymphocytes remaining in the blood of wild-type mice correlated with markedly increased numbers of circulating lymphocytes in L-selectin/β7 integrin−/− mice. Loss of either L-selectin or the β7 integrins alone resulted in significant but incomplete inhibition of Peyer’s patch migration. Collectively, the phenotype of L-selectin/β7 integrin−/− mice demonstrates that these two receptors primarily interact along the same adhesion pathway that is required for the vast majority of lymphocyte migration into Peyer’s patches.
Transcriptional Suppression of Matrix Metalloproteinase-2 Gene Expression in Human Astroglioma Cells by TNF-α and IFN-γ
Authors: Qin Hongwei , Moellinger Jason D. , Wells Alan , Windsor L. Jack , Sun Yi , Benveniste Etty N.
Published: December 2022

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that function in the turnover of extracellular matrix components during development. In addition, MMPs also contribute to pathological conditions associated with inflammation, angiogenesis, and tumor invasion. A 72-kDa type IV collagenase, also referred to as gelatinase A or MMP-2, has been proposed to potentiate the invasion and metastasis of malignant tumors. In particular, MMP-2 activity has been shown to constitute an important component of human astroglioma invasion. We investigated the influence of various cytokines, both proinflammatory and immunosuppressive, on MMP-2 gene expression in two human astroglioma cell lines (U251-MG and CRT). Our results indicate that the cell lines constitutively express high levels of MMP-2 mRNA, protein, and bioactivity as assessed by ribonuclease protection assay, immunoblotting, and zymography assays, respectively. The proinflammatory cytokines TNF-α and IFN-γ individually can inhibit constitutive MMP-2 expression, and function in an additive manner for near-complete inhibition of MMP-2 expression. Inhibition of MMP-2 mRNA levels by TNF-α and IFN-γ is not due to destabilization of the MMP-2 message; rather, inhibition is mediated at the transcriptional level. Furthermore, TNF-α/IFN-γ inhibition of MMP-2 expression results in decreased invasiveness of the human astroglioma cells through an extracellular matrix. These results raise the possibility that TNF-α and IFN-γ may have beneficial effects in attenuating astroglioma invasive properties.
Structural Basis of the gp120 Superantigen-Binding Site on Human Immunoglobulins
Authors: Karray Saoussen , Juompan Laure , Maroun Rachid C. , Isenberg David , Silverman Gregg J. , Zouali Moncef
Published: December 2022

B cell superantigens (SAg) interact with normal human nonimmune Igs (Igs), independently of the light chain isotype, and activate a large proportion of the B cell repertoire. Recently, the major envelope protein of HIV-1, gp120, was found to exhibit SAg-like properties for B cells with potential pathologic consequences for the infected host. This unconventional mode of interaction contrasts with its binding to immunization-induced Abs, which requires the tertiary structure of the heavy and light chain variable regions. In this report, we have examined the structural basis of the interaction between human Igs and gp120. We found that gp120 binding is restricted to Igs from the VH3 gene family and that the two VH genes 3-23 and 3-30, known to be overutilized during all stages of B cell development, frequently impart gp120 binding. We also provide evidence that the viral gp120 SAg can interact with only a subset of the human VH3+ Igs that can convey binding to the prototypic bacterial B cell SAg protein A from Staphylococcus aureus. Finally, we have identified amino acid positions present primarily in the first and third framework regions of the Ig heavy chain variable region, outside the conventional hypervariable loops, which correlate with gp120 binding. In a three-dimensional sequence-homology model, these residues partially overlap with the predicted SAg protein A binding site for VH3+ Igs.
Leishmania mexicanaCysteine Proteinase-Deficient Mutants Have Attenuated Virulence for Mice and Potentiate a Th1 Response
Authors: Alexander James , Coombs Graham H. , Mottram Jeremy C.
Published: December 2022

Leishmania mexicana mutants lacking cysteine proteinase genes cpa (Δcpa), cpb (Δcpb), or both cpa and cpb (Δcpa/cpb) have been generated by targeted gene disruption. Δcpa mutants produce a disease phenotype in BALB/c mice close to that of wild-type L. mexicana, but Δcpb mutants are much less infective, producing very slowly growing small lesions, and Δcpa/cpb double mutants do not induce lesion growth. Immunologic analysis of Ab isotype during infection and splenocyte IFN-γ, IL-2, and IL-4 production following stimulation with Leishmania Ag or Con A indicates that there was a significant shift from a predominantly Th2-associated immune response in mice infected with wild-type L. mexicana to a Th1-associated response in mice inoculated with Δcpb or Δcpa/cpb. Significantly, Δcpa altered the balance of the immunologic response to a lesser extent than did the other mutants. Similar disease outcomes and switches in the Th1/Th2 balance were also observed when other L. mexicana-susceptible mouse strains were infected with the mutants. BALB/c and C57BL/6 mice vaccinated with Δcpa/cpb and CBA/Ca mice vaccinated with Δcpb or Δcpa/cpb were subsequently more resistant, to varying degrees, than were untreated mice to infection with wild-type parasites, as measured by development of lesions and parasite burden. These data implicate leishmanial cysteine proteinases not only as parasite virulence factors but also in modulation of the immune response and provide strong encouragement that cysteine proteinase-deficient L. mexicana mutants are candidate attenuated live vaccines.
Matrix Metalloproteinases Generate Angiostatin: Effects on Neovascularization
Authors: Cornelius Lynn A. , Nehring Leslie C. , Harding Elizabeth , Bolanowski Mark , Welgus Howard G. , Kobayashi Dale K. , Pierce Richard A. , Shapiro Steven D.
Published: December 2022

Angiostatin, a cleavage product of plasminogen, has been shown to inhibit endothelial cell proliferation and metastatic tumor cell growth. Recently, the production of angiostatin has been correlated with tumor-associated macrophage production of elastolytic metalloproteinases in a murine model of Lewis lung cell carcinoma. In this report we demonstrate that purified murine and human matrix metalloproteinases generate biologically functional angiostatin from plasminogen. Macrophage elastase (MMP-12 or MME) proved to be the most efficient angiostatin-producing MMP. MME was followed by gelatinases and then the stomelysins in catalytic efficiency; interstitial collagenases had little capacity to generate angiostatin. Both recombinant angiostatin and angiostatin generated from recombinant MME-treated plasminogen inhibited human microvascular endothelial cell proliferation and differentiation in vitro. Finally, employing macrophages isolated from MME-deficient mice and their wild-type littermates, we demonstrate that MME is required for the generation of angiostatin that inhibits the proliferation of human microvascular endothelial cells.
The First Subcomponent of Complement, C1q, Triggers the Production of IL-8, IL-6, and Monocyte Chemoattractant Peptide-1 by Human Umbilical Vein Endothelial Cells
Authors: van den Berg Rocco H. , Faber-Krol Maria C. , Sim Robert B. , Daha Mohamed R.
Published: December 2022

We and others have demonstrated previously the occurrence of cC1qR/CaR, a receptor for the collagen-like stalks of complement component C1q, on endothelial cells. In the present study we investigated whether binding of C1q to endothelial cells resulted in enhancement of cytokine or chemokine production. HUVEC produced 82 ± 91 pg/ml of IL-8, 79 ± 113 pg/ml of IL-6, and 503 ± 221 pg/ml of monocyte chemoattractant peptide-1 (MCP-1) under basal conditions. Incubation with C1q resulted in a time- and dose-dependent up-regulation of IL-8 (1012 ± 43 pg/ml), IL-6 (392 ± 20 pg/ml), and MCP-1 (2450 ± 101 pg/ml). This production is dependent on de novo protein synthesis, as demonstrated by the detection of specific mRNA after C1q stimulation, and inhibition of peptide production in the presence of cycloheximide. The production of all factors was inhibited (69 ± 7%) by the collagenous fragments of C1q, while the C1q globular heads only induced 13 ± 11% inhibition. When HUVEC were incubated with C1q in the presence of aggregated IgM, enhanced production of IL-8 (2500 ± 422 pg/ml), IL-6 (997 ± 21 pg/ml), and MCP-1 (5343 ± 302 pg/ml) was found. Furthermore, F(ab′)2 anti-calreticulin partially inhibited the production of IL-8, confirming at least the involvement of cC1qR/CaR. These experiments suggest that in an inflammatory response C1q not only is able to activate the complement pathway, but when presented in a proper fashion also might induce the production of factors that contribute to acute phase responses and recruitment of inflammatory cells.
Early Development and Spreading of Autoantibodies to Epitopes of IA-2 and Their Association with Progression to Type 1 Diabetes
Authors: Naserke Heike E. , Ziegler Anette-G. , Lampasona Vito , Bonifacio Ezio
Published: December 2022

Autoimmunity precedes clinical type 1 diabetes, and indicators of maturing autoimmune responses may be useful markers for disease prediction. To study this, epitope maturation of autoantibodies to the related protein tyrosine phosphatase (PTP)-like autoantigens IA-2 and IA-2β was examined in sequential samples from birth in a cohort of 21 offspring developing multiple islet autoantibodies and a similar cohort of 48 relatives of patients with type 1 diabetes recruited at an older age. Initial reactivity in offspring was heterogeneous against the IA-2 juxtamembrane region (10/21) and PTP domains (13/21), and both specificity and extent of initial IA-2/IA-2β autoantibodies were associated with HLA class II genotype. Intra-IA-2 epitope spreading and/or intermolecular spreading to IA-2β epitopes were observed in seven offspring. In contrast, in older relatives, IA-2/IA-2β Ab reactivity was stable and spreading rare. Development of diabetes in children was associated with the presence of Abs to the IA-2 juxtamembrane region (risk by age 5 yr, 52% vs 0% in those with PTP domain Abs only; p < 0.02), and 5 of 26 relatives who developed diabetes had IA-2 Abs only against the juxtamembrane region. The findings show that autoantibody reactivity to IA-2/IA-2β is dynamic in the young, show that the juxtamembrane region of IA-2 is an early IA-2 autoantibody target, and suggest that these Abs are a risk factor for development of type 1 diabetes in infancy.
Cutting Edge: Detection of a High Frequency of Virus-Specific CD4+ T Cells During Acute Infection with Lymphocytic Choriomeningitis Virus
Authors: Varga Steven M. , Welsh Raymond M.
Published: December 2022

Lymphocytic choriomeningitis virus (LCMV), like many viruses, induces a profound activation and expansion of CD8+ T cells. In contrast, CD4+ T cells do not increase in total number during the acute infection. We show here that mice infected with LCMV have a low but detectable frequency (<1/300) of CD4+ T cells, as detected by IL-2 production in limiting dilution assays, to each of two class II peptides during the peak of the acute LCMV response and into long-term memory. However, during the peak of the acute CD4+ T cell response, >20% of the CD4+ T cells secreted IFN-γ after stimulation with PMA and ionomycin, and >10% of the CD4+ T cells secreted IFN-γ after stimulation with the LCMV peptides. Thus, these new sensitive assays reveal a heretofore unappreciated, yet profound Ag-specific CD4+ T cell response during viral infections.
Urocanic Acid Enhances IL-10 Production in Activated CD4+ T Cells
Authors: Holáň Vladimír , Kuffová Lucia , Zajícová Alena , Krulová Magdaléna , Filipec Martin , Holler Petr , Jančárek Alexander
Published: December 2022

The immunosuppressive effects of UV radiation have been well documented. This suppression has been attributed to the action of the cis form of urocanic acid (UCA), a photoproduct of trans-UCA, a natural constituent of the skin. Here, we show that mouse spleen cells preincubated with cis-UCA have a diminished proliferative response to allogeneic cells in MLC and to stimulation with anti-CD3 mAb. Cells preincubated with cis-UCA also had a decreased ability to serve as APC and to stimulate the proliferation of allogeneic lymphocytes in MLC. Simultaneously, the production of IL-2 and IFN-γ by cells preincubated with cis-UCA was decreased. However, IL-10 gene expression and IL-10 protein secretion by spleen cells stimulated in the presence of cis-UCA were significantly enhanced. The principal cell population displaying the cis-UCA-induced elevated production of IL-10 was CD4+ T cells, which were shown to be a direct target of cis-UCA action. This was also supported by the observation that production of IL-10 by stimulated splenic non-T cells or by macrophages was not altered by cis-UCA. The enhanced production of IL-10 by activated CD4+ T cells may represent a novel pathway of UVB radiation-induced, cis-UCA-mediated immunosuppression. We suggest that the elevated production of IL-10 by activated CD4+ T cells may account for the suppressor T cell phenomena described in UV-irradiated recipients.
Chemical Chaperones Enhance Superantigen and Conventional Antigen Presentation by HLA-DM-Deficient as well as HLA-DM-Sufficient Antigen-Presenting Cells and Enhance IgG2a Production In Vivo
Authors: Ghumman Birinder , Bertram Edward M. , Watts Tania H.
Published: December 2022

Chemical chaperones, first defined in studies of mutant cystic fibrosis transmembrane conductance regulator proteins, are small molecules that act as stabilizers of proteins in their native state and have the ability in some cases to rescue protein-folding mutants within cells. HLA-DM is an MHC II-specific molecular chaperone that facilitates peptide loading onto MHC II proteins and also stabilizes empty MHC II molecules prior to their acquisition of antigenic peptides. APC that lack HLA-DM exhibit quantitative defects in protein Ag as well as superantigen presentation. Here we show that both the superantigen and protein presentation defect in MHC II-transfected, HLA-DM-deficient T2 cells can be partially overcome by treating the APC with the chemical chaperones glycerol, DMSO, or trimethylamine oxide. These chemical chaperones also enhance superantigen and conventional Ag presentation by wild-type APC. In vivo, glycerol was found to act as an adjuvant and resulted in enhanced IgG2a production to trinitrophenyl-keyhole limpet hemocyanin (TNP-KLH). In vitro, the enhancement of Ag presentation by chemical chaperones was found to take place at the level of the APC and took several hours to develop. Subcellular fractionation experiments show that HLA-DM enhances presentation of peptides by dense endosome fractions whereas chemical chaperones enhance presentation by light membrane fractions (early endosome or plasma membrane). The mechanism by which these chemical chaperones augment Ag presentation is not defined, but flow cytometric analysis suggests that the enhancement may be due to a subtle effect on the stability of several different proteins at the cell surface.
Selective Ability of Mouse CD1 to Present Glycolipids: α-Galactosylceramide Specifically Stimulates Vα14+ NK T Lymphocytes
Authors: Burdin Nicolas , Brossay Laurent , Koezuka Yasuhiko , Smiley Stephen T. , Grusby Michael J. , Gui Ming , Taniguchi Masaru , Hayakawa Kyoko , Kronenberg Mitchell
Published: December 2022

Mouse CD1 (mCD1) glycoproteins are known to present peptides, while human CD1 molecules present glycolipids. In mice, mCD1-autoreactive NK T cells play critical roles in various immune responses, through the secretion of high amounts of cytokines. This study was initiated to determine whether glycolipids are involved in the autorecognition of mCD1 by NK T cells. α-Galactosylceramide (α-GalCer) was the only glycolipid tested capable of eliciting an mCD1-restricted response by splenic T cells. Moreover, splenic T cells derived from mCD1-deficient mice were not stimulated by α-GalCer, suggesting that the responsive T cells are selected by mCD1. Using cytoflow techniques, we confirmed that, in response to α-GalCer, IFN-γ-secreting cells displayed an NK T cell phenotype. The predominance of IFN-γ vs IL-4, however, is determined by the type of mCD1+ APC, suggesting the potential for APC regulation of cytokine production by NK T cells. Among a panel of 10 mCD1-autoreactive T cell hybridomas, only the ones that express the typical Vα14Jα281 TCR rearrangement of NK T cells responded to α-GalCer. Fixation or treatment of mCD1+ APCs with an inhibitor of endosomal acidification and the use of mCD1 mutants unable to traffic through endosome still allowed α-GalCer to stimulate NK T cells. Thus, endosomal trafficking and Ag processing are not required for glycolipid recognition. In summary, α-GalCer might be the autologous ligand, or a mimic of a glycolipid ligand, involved in the mCD1-mediated stimulation of NK T cells.
Fine Specificity of the Myelin-Reactive T Cell Repertoire: Implications for TCR Antagonism in Autoimmunity
Authors: Anderton Stephen M. , Manickasingham Shivanthi P. , Burkhart Christoph , Luckcuck Tracy A. , Holland Sam J. , Lamont Alan G. , Wraith David C.
Published: December 2022

The use of altered peptide ligands (APL) to modulate T cell responses has been suggested as a means of treating T cell-mediated autoimmune disorders. We have assessed the therapeutic potential of TCR antagonist peptides in autoimmunity using murine experimental autoimmune encephalomyelitis (EAE) as a model. The Tg4 transgenic mouse expresses an MHC class II-restricted TCR specific for the immunodominant encephalitogenic epitope of myelin basic protein, Ac1–9 (acetylated N-terminal nonamer). We have used T cell lines derived from Tg4 mice to define the TCR contact residues within Ac1–9. APL with appropriate substitutions at the primary TCR contact residue were effective antagonists of Tg4 T cells. These antagonist APL, however, were found to induce EAE in susceptible, nontransgenic strains of mice. Underlying this, the Ac1–9-specific T cell repertoire of normal mice, rather than reflecting the Tg4 phenotype, showed considerable diversity in fine specificity and was able to respond to the Tg4 antagonist APL. Defining antagonist APL in vitro using T cell clones, therefore, was not a reliable approach for the identification of APL with EAE-suppressing potential in vivo. Our findings highlight the complexities of the autoreactive T cell repertoire and have major implications for the use of APL in autoimmune diseases.
Mechanisms of Hyaluronan-Induced Up-Regulation of ICAM-1 and VCAM-1 Expression by Murine Kidney Tubular Epithelial Cells: Hyaluronan Triggers Cell Adhesion Molecule Expression Through a Mechanism Involving Activation of Nuclear Factor-κB and Activating Protein-1
Authors: Oertli Beat , Beck-Schimmer Beatrice , Fan Xiaohong , Wüthrich Rudolf P.
Published: December 2022

The matrix constituent hyaluronan (HA) markedly accumulates in inflammatory lesions. To gain insight into the biologic significance of this phenomenon we tested the hypothesis that HA could regulate cell adhesion molecule expression in epithelial cells. Using a clonal line of mouse cortical tubular (MCT) cells we found that fragmented intermediate m.w., but not high m.w., HA markedly increased ICAM-1 and VCAM-1 steady state mRNA and cell surface expression. Up-regulation of ICAM-1 and VCAM-1 mRNA by HA was preceded by a marked increase in NF-κB and activating protein-1 DNA binding activity in MCT cells. Transcript levels for the NF-κB inhibitor IκBα and for the activating protein-1 constituents c-jun and c-fos also increased in response to HA stimulation of tubular cells. Inhibition of NF-κB with the serine protease inhibitor N-tosyl-l-phenylalanine chloromethyl ketone blocked the HA-mediated expression of ICAM-1 and VCAM-1 in MCT cells. In conclusion, HA displays proinflammatory effects by directly stimulating the expression of the cell adhesion molecules ICAM-1 and VCAM-1 in mouse kidney epithelial cells. HA could thereby play an important role in leukocyte adhesion in inflammatory renal diseases.
Differential Usage of VH Gene Segments Is Mediated bycisElements
Authors: Yu Calvin C. K. , Larijani Mani , Miljanic Ivana N. , Wu Gillian E.
Published: December 2022

Ig diversity is generated in large part by the combinatorial joining of the Ig gene segments, VH, D, and JH, that together encode the variable domain of Ig. The final Ig repertoire, however, not only reflects the diversity generated through V(D)J recombinatorial joining, but it is also the product of a number of developmental restraints and selections. To avoid such restrictions and assess the recombination potential of individual Ig gene segments, we constructed Ig heavy (H) chain microlocus plasmids, each of which contain germline coding, recombination signal, and flanking sequences of a VH, D, and JH gene segment. These plasmids allow us to assess the recombination potential of the segments in the context of their natural flanking DNA sequences, but in the absence of any higher order chromatin structure or cellular selection. We found that the frequency and extent of deletions and additions at the recombination breakpoints are similar to those observed at rearranged Ig H chain loci in intact animals. The relative frequencies of the types of rearrangements—VD-J, V-DJ, VinvD-J (invD = inverted D), and VDJ—however, differ strongly. Moreover, V81x, the most used VH gene segment in intact mice, also is overused in this plasmid assay, 15 to 30 times that of another VH segment. This result indicates that the overuse of V81x in the early B cell repertoire can be a consequence of its DNA sequence and not of cellular activities.
Molecular Mapping with Functional Antibodies Localizes Critical Sites on the Human IL Receptor Common γ (γc) Chain
Authors: Raskin Natalya , Jakubowski Aniela , Sizing IreneDougas , Olson Dian L. , Kalled Susan L. , Hession Catherine A. , Benjamin Christopher D. , Baker Darren P. , Burkly Linda C.
Published: December 2022

The IL receptor common γ (γc) chain is required for the formation of high affinity cytokine receptor complexes for IL-2, IL-4, IL-7, IL-9, and IL-15, and for signals regulating cell survival, growth, and differentiation. Our current understanding of how γc chain associates with multiple ligands and receptor subunits is drawn largely from its structural homology to the human growth hormone (hGH) receptor and known structure of the hGH/hGH receptor complex. These receptors share distinct features in their extracellular portions and are believed to function by a mechanism of ligand-induced association of receptor subunits. Here, we report the first directed mutational analysis of the human γc chain by alanine scanning conducted across seven regions likely to contain residues required for intermolecular contact. Functionally distinct, neutralizing anti-γc mAbs were employed to define critical residues. One particular mAb, CP.B8, unique in its ability to inhibit IL-2-, IL-4-, IL-7-, and IL-15-induced proliferation and high affinity cytokine binding of normal T cells as an intact mAb and as a Fab fragment, localized critical residues to four noncontinuous stretches, namely residues in loops AB and EF of domain 1, in the interdomain segment, and in loop FG of domain 2. Notably, these residues form a contiguous patch on the γc chain surface in a three-dimensional structural model. These results provide functional evidence for the location of contact points on γc chain required for its association with multiple ligands.
B Cell Responses to a Peptide Epitope. V. Kinetic Regulation of Repertoire Discrimination and Antibody Optimization for Epitope
Authors: Nayak Bishnu P. , Tuteja Renu , Manivel Venkatasamy , Roy Rajendra P. , Vishwakarma Ram A. , Rao Kanury V. S.
Published: December 2022

The influence of imposing various conformational constraints on immune responses to a model epitope within a synthetic peptide immunogen was examined in mice. Although overall immunogenicity was affected, the model epitope (sequence DPAF) remained the predominant recognition site regardless of the conformation in which it was presented. A comparison of anti-DPAF mAbs obtained in response to two analogue peptides, PS1CT3 and CysCT3, in which the DPAF segment was either unconstrained or held within a cyclic loop, respectively, revealed a significant homology in the paratope composition. At one level a subset of anti-PS1CT3 and anti-CysCT3 mAbs was found to share a common heavy chain variable region. In addition, nucleotide sequence homology comparisons of both heavy and light chain variable regions identified the presence of anti-PS1CT3 and anti-CysCT3 mAbs that collectively appeared to derive from a common progenitor, but with nonidentical somatic mutations. Interestingly, however, no bias toward homologous Ag could be discerned on measurement of relative affinities of the mAbs for the two peptides. In contrast, mAb binding on-rates clearly discriminated between peptides representing the homologous vs the heterologous confomer of the DPAF epitope. Thus, it would appear that the kinetics of Ag recognition dominate over equilibrium binding criteria both in epitope-driven repertoire selection and Ab maturation in a humoral response.
Characterization of Human γ4 Switch Region Polymorphisms Suggests a Meiotic Recombinational Hot Spot Within the Ig Locus: Influence of S Region Length on IgG4 Production
Authors: Pan Qiang , Rabbani Hodjattallah , Hammarström Lennart
Published: December 2022

Human γ4 gene RFLPs, revealed after BamHI digestion, show IGHG4 alleles of 9.0 (9.2), 9.4, and 9.6 kb at various frequencies in different ethnic populations. Studies in immunodeficient individuals have previously suggested that the 9.4 BamHI allele is associated with a higher serum level of IgG4 than the 9.0 (9.2) BamHI allele, but it is not clear whether this is associated with the S region itself or other control elements. In addition, a duplication of the 9.4-kb γ4 allele has recently been observed in a high proportion of normal donors. We therefore undertook a study of the structural basis for the difference in Ab levels in the various γ4 alleles. We demonstrate that the Sγ4 alleles differ in length due to deletions and insertions of a varying number of 79-bp Sγ4 repeat units. Two novel RFLPs, 8.8 and 9.1 kb, were also observed. The alleles are likely to be generated by unequal crossing over, and the breakpoints cluster in Sγ4 repeat units that contain chi-like motifs, implicating chi-like sequences in the meiotic recombination. Our data support the idea that the 9.4-kb BamHI allele is more productive than the 9.0 (9.2)-kb allele in normal healthy donors, possibly due to the extended switch regions, whereas duplication of the γ4 gene has no effect on switching and IgG4 serum levels.
Structural Basis of Specificity and Degeneracy of T Cell Recognition: Pluriallelic Restriction of T Cell Responses to a Peptide Antigen Involves Both Specific and Promiscuous Interactions Between the T Cell Receptor, Peptide, and HLA-DR
Authors: Doherty Derek G. , Penzotti Julie E. , Koelle David M. , Kwok William W. , Lybrand Terry P. , Masewicz Susan , Nepom Gerald T.
Published: December 2022

TCR engagement of peptide-MHC class II ligands involves specific contacts between the TCR and residues on both the MHC and peptide molecules. We have used molecular modeling and assays of peptide binding and T cell function to characterize these interactions for a CD4+ Th1 cell clone, ESL4.34, which recognizes a peptide epitope of the herpes simplex type 2 virus virion protein, VP16 393–405, in the context of several HLA-DR alleles. This clone responded to VP16 393–405 in proliferation and cytotoxicity assays when presented by DRB1*0402, DRB1*1102, and DRB1*1301, which share a common amino acid sequence, ILEDE, at residues 67–71 in the α-helical portion of the DRβ polypeptide, but not when presented by other DR4, DR11, and DR13 alleles that are negative for this sequence. Using a panel of APCs expressing DR4 molecules that were mutagenized in vitro at individual residues within this shared epitope and using peptide analogues with single amino acid substitutions of predicted MHC and TCR contact residues, a unit of recognition was identified dependent on DRβ residues 67–71 and relative position 4 (P4) of the VP16 393–405 peptide. The interactions of this portion of the peptide-DR ligand with the ESL4.34 TCR support a structural model for MHC-biased recognition in some Ag-specific and alloreactive T cell responses and suggest a possible mechanism for autoreactive T cell selection in rheumatoid arthritis.
Up-Regulation by Ammonium Trichloro(Dioxoethylene-0,0′) Tellurate (AS101) of Fas/Apo-1 Expression on B16 Melanoma Cells: Implications for the Antitumor Effects of AS101
Authors: Kalechman Yona , Strassmann Gideon , Albeck Michael , Sredni Benjamin
Published: December 2022

It was recently reported that human and mouse melanoma cells express Fas ligand (FasL) but almost no Fas, which may contribute to their immune privilege. AS101 (ammonium trichloro(dioxoethylene-0,0′)tellurate), a synthetic immunomodulator with minimal toxicity, was found to have antitumor effects in various tumor models. Our present study shows that AS101 has direct and indirect effects on tumor cells; AS101 inhibits the clonogenicity of B16 melanoma cells in vitro. Moreover, wild-type P53 expression, which is required for induction of Apo-1 expression, increased significantly in AS101-treated cells. We therefore investigated Fas expression in AS101-treated B16 cells and found that Fas, but not FasL, expression was significantly increased; moreover, Fas receptors were functional. Longer incubation with AS101 resulted in spontaneous apoptosis triggered by the Fas-FasL system. To explore the relationship of these results to the antitumor effects of AS101, we injected B16-F10 mouse melanoma cells into syngeneic C57BL/6 mice carrying the lpr mutation in the Fas gene and to gld mutant mice that lack functional FasL. Tumor development in control groups was lowest in the lpr mice, while no difference was observed between gld and wild-type mice. Among the AS101-treated groups, the most pronounced effect appeared in the lpr mice, while the lowest was seen in the gld mutant mice. Our study suggests that AS101 may render melanoma tumor cells more sensitive to Fas/FasL-induced apoptosis and may therefore have clinical potential.
Controlled Lipidation and Encapsulation of Peptides as a Useful Approach to Mucosal Immunizations
Authors: Mora Ana L. , Tam James P.
Published: December 2022

To generate a useful strategy for mucosal immunization, we have developed an approach of lipidating a multiple Ag peptide (MAP) containing part of the V3 loop from HIV-1 gp120IIIB. In this work, we compare two delivery systems, lipidated MAP in PBS and encapsulation in poly(dl-lactide-co-glycolide) microparticles. Subcutaneous immunization, followed by intragastric administration of MAP peptide entrapped or not entrapped in microparticles, induced mucosal and systemic immune responses at local and distant sites, including mucosal IgA in saliva, vaginal secretions and feces, and IgG in blood. However, lipidated Ag delivered in microparticles induced higher levels of mucosal Abs, particularly of intestinal IgA, and generated CTL responses. In contrast, lipidated MAP delivered by nasal route microparticles was less effective in inducing CTL responses. These results demonstrate the feasibility of using a lipidated multimeric peptide for mucosal immunization to stimulate both systemic and mucosal immune systems, including the genital tract, irrespective of the route or method of delivery and without requiring the use of a carrier or an extraneous adjuvant.
Stem Cell Factor Augments FcεRI-Mediated TNF-α Production and Stimulates MAP Kinases via a Different Pathway in MC/9 Mast Cells
Authors: Ishizuka Tamotsu , Kawasome Hideki , Terada Naohiro , Takeda Katsuyuki , Gerwins Pär , Keller Gordon M. , Johnson Gary L. , Gelfand Erwin W.
Published: December 2022

Mast cells express the receptor tyrosine kinase kit/stem cell factor receptor (SCFR) which is encoded by the proto-oncogene c-kit. Ligation of SCFR induces its dimerization and activation of its intrinsic tyrosine kinase activity leading to activation of Raf-1, phospholipases, phosphatidylinositol 3-kinase, and extracellular signal-regulated kinases. However, little is known about the downstream signals initiated by SCFR ligation except for activation of extracellular signal-regulated kinases. The murine mast cell line, MC/9, synthesizes and secretes TNF-α following the aggregation of high affinity Fc receptors for IgE (FcεRI). Ligation of SCFR or FcεRI on MC/9 cells resulted in the activation of all three MAP kinase family members, extracellular signal-regulated kinases, c-Jun amino-terminal kinase (JNK), and p38. Stem cell factor (SCF)-induced activation of JNK and p38 was insensitive to wortmannin, cyclosporin A, and FK506 whereas activation of these kinases through FcεRI was sensitive to these drugs. Coligation of SCFR augmented FcεRI-mediated activation of MAP kinases, especially JNK activation, and SCF augmented FcεRI-mediated TNF-α production in MC/9 cells, although SCF alone did not induce TNF-α production. This augmentation by SCF was regulated at the level of transcription, at least in part, since the promoter activity of TNF-α was enhanced following addition of SCF. These results demonstrate that SCF can augment FcεRI-mediated JNK activation and cytokine gene transcription but via pathways that are regulated differently than the ones activated through FcεRI.
Dust Mite Proteolytic Allergens Induce Cytokine Release from Cultured Airway Epithelium
Authors: King Cecile , Brennan Siobhan , Thompson Phillip J. , Stewart Geoffrey A.
Published: December 2022

Endogenous proteolytic enzymes have been shown to be potential sources of airway inflammation inducing proinflammatory cytokine release from respiratory epithelial cells; however, whether any of the exogenous proteases from important allergen sources such as the house dust mite present in our environment behave in a similar fashion is unclear. To this end, we have investigated whether the mite cysteine and serine proteolytic allergens, Der p 1 and Der p 9, respectively, induced cytokine production from primary human bronchial epithelial cells and from the epithelial cell line BEAS-2B. Cells were exposed to mite proteases, and cells or supernatants were assayed for cytokine release, cytokine mRNA expression, and modulation of intracellular calcium ion concentration. Both proteases induced concentration- and time-dependent increases in the release of granulocyte-macrophage (GM)-CSF, IL-6, and IL-8 as well as an increase in the expression of IL-6 mRNA. Cytokine release and mRNA expression were first observed at 8 h and 2 h after protease exposure, respectively. The minimum concentration of each protease that was required to stimulate GM-CSF, IL-6, and IL-8 release was ∼10 ng/ml. Cytokine release was initiated by 1 to 2 h of protease exposure, although maximum concentrations were detected only after a 24-h incubation. IL-6, but not IL-8 and GM-CSF, was shown to be degraded by both proteases at concentrations of >2 μg/ml. The proteases also stimulated changes in the intracellular calcium ion concentration. All mite protease-induced phenomena were inhibited using appropriate protease inhibitors. These results suggest that the proteolytic activity of an allergen may stimulate the release of proinflammatory cytokines from human bronchial epithelium.
CXCR4 and CCR5 Expression Delineates Targets for HIV-1 Disruption of T Cell Differentiation
Authors: Berkowitz Robert D. , Beckerman Karen P. , Schall Thomas J. , McCune Joseph M.
Published: December 2022

HIV-1 disease is often associated with CD4+ T lymphopenia as well as quantitative reductions in naive CD8+ T cells and cytopenias involving nonlymphoid hemopoietic lineages. Studies in HIV-1-infected humans as well as in animal models of lentivirus disease indicate that these effects may be secondary to infection and destruction of multilineage and lineage-restricted hemopoietic progenitor cells. To define the stages of T cell differentiation that might be susceptible to HIV-1, we performed flow cytometric analysis of the surface expression of CXCR4 and CCR5 on T cells and their progenitors from fetal tissue, cord blood, SCID-hu Thy/Liv mice, and adult peripheral blood. We found that CXCR4 is expressed at low levels on hemopoietic progenitors in the bone marrow, is highly expressed on immature (CD3−CD4+CD8−) T cell progenitors in the thymus, and then is down-regulated during thymocyte differentiation. As thymocytes leave the thymus and enter the peripheral circulation, the expression of CXCR4 is again up-regulated. In contrast, CCR5 is undetectable on most hemopoietic progenitors in the bone marrow and on intrathymic T progenitor cells. It is up-regulated when thymocytes coexpress CD4 and CD8, then down-regulated either in the thymus (CD4+ cells) or during exit from the thymus (CD8+ cells). These results indicate that discrete, lineage-related populations of T cell progenitors may vary widely in their potential to respond to chemokines and to be infected by HIV-1, and that T lymphoid differentiation is particularly vulnerable to CXCR4-using viruses.
Cutting Edge: Cells That Carry A Null Allele for Toll-Like Receptor 2 Are Capable of Responding to Endotoxin
Authors: Heine Holger , Kirschning Carsten J. , Lien Egil , Monks Brian G. , Rothe Mike , Golenbock Douglas T.
Published: December 2022

Toll-like receptor (TLR) 2 and TLR4 have been implicated in the responses of cells to LPS (endotoxin). CD14-transfected Chinese hamster ovary (CHO)-K1 fibroblasts (CHO/CD14) are exquisitely sensitive to endotoxin. Sequence analysis of CHO-TLR2, compared with human and mouse TLR2, revealed a single base pair deletion. This frameshift mutation resulted in an alternative stop codon, encoding a protein devoid of transmembrane and intracellular domains. CHO-TLR2 cDNA failed to enable LPS signaling upon transient transfection into human epithelial kidney 293 cells. Site-directed mutagenesis of CHO-TLR2 enabled expression of a presumed full-length hamster TLR2 that conferred LPS responsiveness in human epithelial kidney 293 cells. Genomic TLR2 DNA from primary hamster macrophages also contained the frameshift mutation found in CHO fibroblasts. Nevertheless, hamster peritoneal macrophages were found to respond normally to LPS, as evidenced by the induction of cytokines. These results imply that expression of TLR2 is sufficient but not essential for mammalian responses to endotoxin.
Human Anti-Porcine T Cell Response: Blocking with Anti-Class I Antibody Leads to Hyporesponsiveness and a Switch in Cytokine Production
Authors: DerSimonian Harout , Pan Luying , Yatko Chris , Rodrigue-Way Amelie , Johnson Eric , Edge Albert S. B.
Published: December 2022

Intervention in the molecular interactions that lead to an immune response is possible at various stages of Ag recognition and T cell activation. Perturbation of the interaction of the TCR with the MHC/peptide ligand complex is one approach that has shown promise for autoimmunity and graft rejection in blocking T cell-activated responses. In this study, we investigated the effect of altering the target MHC class I molecule by blocking with Abs. We established a system that analyzed the human T cell response against MHC class I+/class II− porcine stimulatory cell targets. The primary human response against porcine smooth muscle cells was CD8+ T cell dependent. In the presence of F(ab′)2 fragments of the MHC class I-reactive Ab, PT-85, the proliferative response was inhibited and production of IL-2 and IFN-γ was blocked. Moreover, in a secondary response, proliferation was reduced and type 1 cytokine levels were inhibited. In contrast, levels of IL-10 and IL-4 were sustained or slightly increased. These findings indicate that Ab against MHC class I blocked the recognition of porcine cells by the human CD8+ T cells and altered the cytokine secretion profile. Thus, a single treatment with PT-85 F(ab′)2 directed against the MHC class I molecule provides an attractive approach to the induction of T cell tolerance that may provide long-term graft survival in porcine-to-human cell transplantation.
CD28-Independent Costimulation of T Cells by OX40 Ligand and CD70 on Activated B Cells
Authors: Akiba Hisaya , Oshima Hideo , Takeda Kazuyoshi , Atsuta Machiko , Nakano Hiroyasu , Nakajima Atsuo , Nohara Chiyoko , Yagita Hideo , Okumura Ko
Published: December 2022

OX40 and its ligand (OX40L) have been implicated in T cell-dependent humoral immune responses. To further characterize the role of OX40/OX40L in T-B cell interaction, we newly generated an anti-mouse OX40L mAb (RM134L) that can inhibit the costimulatory activity of OX40L transfectants for anti-CD3-stimulated T cell proliferation. Flow cytometric analyses using RM134L and an anti-mouse OX40 mAb indicated that OX40 was inducible on splenic T cells by stimulation with immobilized anti-CD3 mAb in a CD28-independent manner, while OX40L was not expressed on resting or activated T cells. OX40L was inducible on splenic B cells by stimulation with anti-IgM Ab plus anti-CD40 mAb, but not by either alone. These activated B cells exhibited a potent costimulatory activity for anti-CD3-stimulated T cell proliferation and IL-2 production. Anti-CD80 and anti-CD86 mAbs partially inhibited the costimulatory activity, and further inhibition was obtained by their combination with RM134L and/or anti-CD70 mAb. We also found the anti-IgM Ab- plus anti-CD40 mAb-stimulated B cells exhibited a potent costimulatory activity for proliferation of and IL-2 production by anti-CD3-stimulated CD28− T cells from CD28-deficient mice, which was substantially inhibited by RM134L and/or anti-CD70 mAb. These results indicated that OX40L and CD70 expressed on surface Ig- and CD40-stimulated B cells can provide CD28-independent costimulatory signals to T cells.