bluuebunny/crossref_metadata_2025 huggingface dataset:
Selective tolerance to the E1 protein of rubella virus in congenital rubella syndrome.
Authors: Mauracher C A , Mitchell L A , Tingle A J
Published: December 2022

Rubella virus (RV) infection of the fetus in the first trimester of pregnancy usually results in severe birth defects collectively termed Congenital Rubella Syndrome (CRS) and is frequently associated with prolonged RV persistence in the infant. Immunological tolerance to RV is believed to contribute to viral persistence, but the mechanism for this is unknown. In this study, RV-specific antibody responses in CRS patients and healthy controls who had experienced Rubella infection postnatally were compared to determine if there were differences that might account for RV persistence in the former group. Levels and functional affinities of IgG specific for individual RV proteins (E1, E2, and C) were measured by enzyme immunoassay (EIA). Relative amounts of RV protein-specific IgG directed to linear and topographic epitopes were compared by immunoblots run under reducing or nonreducing conditions, respectively, and biological activity was determined by hemagglutination inhibition (HAI) assay. Results showed that both CRS patients and control subjects had comparably high levels of IgG directed to whole RV and to RV E2 and C proteins as measured by EIA. However, in contrast to the controls, CRS patients were found to have significantly reduced levels of antibodies directed to RV E1 protein and its linear (but not topographic) epitopes. Also, functional affinities of specific IgG directed to whole RV and E1 protein, as well as hemagglutination inhibition titers, were found to be significantly lower in CRS patients than in controls. The data suggest that intrauterine exposure to RV may result in selective immunological tolerance to the RV E1 protein. A model is presented that accommodates the serological findings of this investigation within a proposed mechanism of RV persistence resulting from selective immunological tolerance to RV E1 protein.
T cell epitope expression of mycobacterial and homologous human 65-kilodalton heat shock protein peptides in short term cell lines from patients with Behçet's disease.
Authors: Pervin K , Childerstone A , Shinnick T , Mizushima Y , van der Zee R , Hasan A , Vaughan R , Lehner T
Published: December 2022

T cell epitopes of the 65-kDa heat shock protein (HSP) were mapped in patients with Behçet's disease (BD), by stimulating T cells with the overlapping synthetic peptides derived from the sequences of the Mycobacterium tuberculosis 65-kDa HSP. Significant lymphoproliferative responses were stimulated with four HSP peptides in BD, as compared with the related disease (recurrent oral ulcers), unrelated disease, and healthy controls (p < 0.05 to 0.005). In order to assess the relative frequency of sensitized lymphocytes by these peptides, 7353 short term cell lines were generated from the lymphocytes of patients and controls. Peptides 111-125, 154-172, and 311-325 (p < 0.001) and peptide 219-233 (p < 0.02) yielded significantly greater frequency of STCL in BD than in healthy and disease controls. All but peptide 154-172 stimulated only the CD4+ subset of T cells, although there was no evidence that reactivity to the selected peptides is restricted by DR2 to DR7 Ag. HLA-B51 is significantly associated with BD, but there was no evidence that B51 was a restricting element, when B51+ patients were compared with B51- patients with BD, and with B51+ healthy control subjects. A comparative investigation was then carried out between the corresponding mycobacterial and human HSP peptides. Similar or higher lympho-proliferative responses were stimulated by the human peptides compared with the mycobacterial peptides. These results suggest that the four peptide determinants within the 65-kDa HSP might be involved in the pathogenesis of BD. Whereas the high microbial load and associated stress proteins found in oral ulceration of BD may initiate an immune response to these conserved epitopes, expression of autoreactive T cell clones might be stimulated by immunodominant T cell epitopes of endogenous HSP which may induce immunopathologic changes.
Costimulation with integrin ligands intercellular adhesion molecule-1 or vascular cell adhesion molecule-1 augments activation-induced death of antigen-specific CD4+ T lymphocytes.
Authors: Damle N K , Klussman K , Leytze G , Aruffo A , Linsley P S , Ledbetter J A
Published: December 2022

Integrin ligands intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) can efficiently costimulate proliferation of resting T cells but not that of Ag-specific T cells. In contrast, CD28 ligand B7 and CD2 ligand leukocyte function-associated Ag (LFA-3) can support IL-2 synthesis and proliferation of Ag-specific T cells more efficiently than that of resting T cells. The molecular basis for this differential costimulation of T cells is poorly understood. In this study, using mAb and soluble IgC gamma 1 chimeras of these adhesion molecules, we demonstrate that coligation of the TCR and CD11a/CD18 (LFA-1/beta 2 integrin) or CD29/CD49d (very late activation Ag-4/beta 1 integrin) using anti-TCR mAb and either ICAM-1 or VCAM-1 induces activation-dependent death of DRw6-specific CD4+ T cells. Similar coligation of the TCR with CD2 or CD28 using either mAb or ligands LFA-3 or B7 not only lacked the ability to induce death but also failed to reverse or inhibit integrin-facilitated death of DRw6-specific T cells. Each of these ligands augmented anti-TCR mAb-induced transcription of IL-2 and IL-4 genes. Exogenous addition of IL-2 and IL-4 did not reverse the integrin-supported T cell death. The death-promoting costimulatory effects of ICAM-1 and VCAM-1 were observed with Ag-specific chronically stimulated T cells but not with either resting T cells or those activated in short-term cultures. Treatment of T cells with cyclosporin A or a protein tyrosine kinase inhibitor herbimycin A inhibited ICAM-1 or VCAM-1-promoted activation-induced T cell death. The Ag-specific T cells that survived death-promoting effects of ICAM-1 or VCAM-1 proliferated efficiently upon restimulation with these ligands. Exposure of DRw6-specific T cells to DRw6+ B7+ ICAM-1+ LFA-3+ VCAM-1+ APC but not DR3+ B7+ ICAM-1+ LFA-3+ VCAM-1+ APC induced death of these T cells. This effect was blocked by pretreatment of T cells with mAb directed at CD18 or CD29 but not with those against CD2 or CD28. Taken together, these results suggest that TCR-directed engagement of integrins by their ligands ICAM-1 or VCAM-1 induces activation-dependent death of some perhaps more differentiated Ag-specific T cells and this may be an important homeostatic mechanism by which functional expression of Ag-specific T cells is regulated during an ongoing immune response.
CD30 is a signal-transducing molecule that defines a subset of human activated CD45RO+ T cells.
Authors: Ellis T M , Simms P E , Slivnick D J , Jäck H M , Fisher R I
Published: December 2022

CD30 has been extensively studied as a cell surface marker expressed by Reed-Sternberg cells of Hodgkin's disease and other hematologic malignancies, although little is known about its expression by normal lymphoid cells. We therefore characterized the requirements for the induction of CD30 expression and identified the subsets of T cells that express CD30. CD30 is inducible on approximately 15% of normal PBMC stimulated with any of a variety of nonspecific T cell activators, including PHA, Con A, anti-T11(2) + T11(3), and anti-CD3; ionomycin alone induced lower percentages of CD30+ T cells (3 +/- 2%) compared to other stimuli. Maximal numbers of CD30+ cells were observed at 48 to 72 h of activation and the addition of rIL-2 did not affect these kinetics. However, CD30 expression was enhanced by the addition of exogenous rIL-2 to any of the stimuli tested, although rIL-2 alone did not lead to CD30 expression. The induction of CD30 during anti-CD3 mitogenesis was completely inhibitable by anti-IL-2 antibodies and partially inhibitable by rIL-4, indicating a requirement for both TCR triggering and IL-2 for its expression. Dual immunofluorescence analysis revealed that CD30+ cells were confined to CD3+ T cells that coexpressed higher levels of the p55 IL-2 receptor (CD25) than the CD30- population. Furthermore, CD30 expression was restricted to a subset of cells derived from CD45RO+ T cell precursors. Cell cycle analysis showed that CD30+ expression was not cell cycle dependent. Cross-linking of membrane CD30 induced Ca2+ in TCR+, but not TCR- Jurkat T cells. These results demonstrate that CD30 can serve as a T cell signal-transducing molecule and expressed by a unique subset of activated CD45RO+ T cells.
Glycopeptides bind MHC molecules and elicit specific T cell responses.
Authors: Harding C V , Kihlberg J , Elofsson M , Magnusson G , Unanue E R
Published: December 2022

Carbohydrates are T cell independent antigens because they do not bind to MHC molecules. However, glycopeptides might potentially bind to MHC molecules via their peptide component for presentation to T cells. We have conjugated the disaccharide galabiose [Gal alpha (1-4)Gal beta] to the amino terminus of a T cell peptide determinant from hen egg-white lysozyme [HEL(52-61)]. The resulting glycopeptide (Gal2-52-61) and a nonglycosylated analogue containing tyrosine and glutamic acid at the amino-terminus (YE-52-61) bound equally well to purified I-Ak. T cell hybridomas were produced after immunization with Gal2-52-61. Many of the T cell hybridomas were glycopeptide-specific and responded to Gal2-52-61 but not to nonglycosylated synthetic peptides or to HEL presented by APC, indicating that the carbohydrate moiety influenced T cell recognition. Recognition was lost with the amino terminal attachment of the disaccharide to a peptide six amino acids longer at the amino terminus than HEL(52-61). Recognition also was lost with peptides containing only a single galactosyl residue or with galabiose bound to a different I-Ak binding peptide. T cells directed to Gal2-52-61 recognized glycopeptides having significant variation in the disaccharide structure, such as HEL(52-61) glycopeptides carrying lactose, cellobiose, or hepta-o-acetylated galabiose. Peptide residues were important features of the T cell epitope; Ala substitutions of two critical T cell contact residues of HEL(52-61) (Tyr53 and Leu56) abrogated T cell reactivity to the glycopeptides without affecting binding to I-Ak. In conclusion, we propose that these T cells recognize a peptide conformation specific to glycopeptide-I-Ak complexes and that this recognition does not involve specific interaction between the carbohydrate moiety and the T cell receptor.
Regulation of CD27 expression on subsets of mature T-lymphocytes.
Authors: Hintzen R Q , de Jong R , Lens S M , Brouwer M , Baars P , van Lier R A
Published: December 2022

CD27, a lymphocyte-specific member of the TNF/NGF-R family, is expressed on the majority of peripheral blood T cells. Activation of T cells via TCR/CD3 induces high CD27 surface expression and the release of a soluble extracellular part of the molecule. After prolonged activation in vitro, CD27 becomes gradually switched off. There is evidence that also in vivo, CD4+ cells that have persistently been stimulated by Ag, accumulate within the CD45RA-CD27- subset. In addition, an increase of CD27- T cells has been observed under certain immunopathologic conditions and during aging. This study was undertaken to analyze the regulation of CD27 on different T-cell subsets and to determine whether the loss of CD27 expression is an irreversible event and may thereby mark T-cell differentiation. In agreement with earlier findings, all CD4+CD45RA+CD45RO- T cells were found to express CD27, whereas a small fraction of the CD4+CD45RA-CD45RO+ subset lacks the molecule. In contrast, within the CD8+ compartment CD27- subsets were found both in the CD45RA+ and CD45RA- subpopulations. After stimulation with CD3 mAb, both CD27 membrane expression and release was equally up-regulated in CD4+ and CD8+ subpopulations. This stimulus, however, provoked a strikingly predominant up-regulation of membrane CD27 on CD45RO- cells as compared with CD45RA- cells. On CD4+CD45RA-CD27- T cells and long-term grown CD45RA-CD27- TLC, CD27 expression could not be reinduced after stimulation of the TCR/CD3 complex, neither at the protein nor at the mRNA level. Comparison of CD27 expression with its structural homologue FAS/APO-1 showed that down-regulation after prolonged activation is not a general feature of TNF/NGF-R family members. The CD27 ligand was recently identified and was shown to give a co-stimulatory signal to PHA-activated T cells. The restricted up-regulation of CD27 on CD45RA+ cells after T-cell stimulation may point at a discrete role of CD27-CD27 ligand interaction during transition of CD45RO- to CD45RA- T cells. In addition, the CD27 negative phenotype seems a stable reflection of differentiation rather than of activation.
Medullary thymic epithelium expresses a ligand for CTLA4 in situ and in vitro.
Authors: Nelson A J , Hosier S , Brady W , Linsley P S , Farr A G
Published: December 2022

A fusion protein consisting of the extracellular domain of CTLA4 and an Ig C gamma 1 chain (CTLA4-Ig) was used to examine the distribution of the ligands for CTLA4 within the murine thymus and to characterize the nature of these ligands. Two-color immunofluorescence of thymus tissue revealed binding of the fusion protein to medullary thymic epithelial cells and dendritic cells within the corticomedullary and medullary areas of the thymus. Medullary cells binding the fusion protein also expressed MHC class II products and ICAM-1. Thymus tissue sections treated with cross-linking fixatives, such as glutaraldehyde, paraformaldehyde, or 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide no longer bound the CTLA4 fusion protein, indicating that binding was very sensitive to the tertiary structure of the tissue ligand. The ability of thymic tissue to bind the fusion protein was developmentally regulated. At day 14 of gestation, only scattered single cells were labeled. Clusters of labeled cells, which were detected by day 16 of gestation, increased in frequency with advancing gestational age. Consistent with the in situ labeling studies, CTLA4-Ig also labeled several thymic epithelial cell lines previously shown to have a medullary phenotype. Polymerase chain reaction analysis of mRNA extracted from these cells indicated they contained mRNA for B7, a known counter receptor for CTLA4 and CD28. Immunoprecipitation of 125I-labeled thymic epithelial cells with the CTLA4-Ig detected a M(r) 65,000 to 70,000 species under reducing conditions, consistent with previous studies of B7. These data suggest that the ligand for CTLA4 expressed by thymic epithelial cells in vitro is B7 and that the expression of this ligand in situ is largely restricted to the medullary compartment and is associated with epithelial cells and dendritic cells.
Properdin, a positive regulator of complement activation, is expressed in human T cell lines and peripheral blood T cells.
Authors: Schwaeble W , Dippold W G , Schäfer M K , Pohla H , Jonas D , Luttig B , Weihe E , Huemer H P , Dierich M P , Reid K B
Published: December 2022

Properdin plays an important regulatory role in the activation of the complement system. Here we report the biosynthesis of properdin in four different human T cell lines and in T cells purified from peripheral blood. Cell sorting experiments, in conjunction with Northern blotting, showed that both CD4- and CD8-bearing populations of T cells have the potential to synthesize properdin. The functional activity of properdin secreted from these T cell lines was determined in a hemolytic assay. In view of the numerous ways in which complement activation may influence cellular immune response, the present results indicate, for the first time, a possible interaction between the complement and the T cell system.
Low concentrations of Gm allotypic subsets G3 mg and G1 mf in homozygotes and heterozygotes.
Authors: Seppälä I J , Sarvas H , Mäkelä O
Published: December 2022

Serum concentrations of IgG3, IgG1, and of the Gm allotypic subsets of these two isotypes were measured in adult homozygotes and heterozygotes. Alleles G3 mb and G3 mst of the IgG3 locus, and alleles G1 ma and G1 max of the IgG1 locus were found to associate with a high concentration of the allotypic product. Alleles G3 mg (IgG3) and G1 mf (IgG1) were associated with a low concentration of the product. This was true regardless of the haplotype; for example, allele G3 mb was associated with a high concentration of the product in all haplotypes f;n+;b f;n-;b and fa;n+;b. One dose of allele G3 mg was associated with a characteristic mean concentration of the product (g-type IgG3). This rule was valid regardless of the other allele of the subject, thus, heterozygotes and G3 mg/g homozygotes had mean concentrations of 0.10 and 0.20 g/liter, respectively, of g-type IgG3. Products of the IgG1 alleles were also simply additive: one dose of allele G1 ma(x) or G1 mf was associated with mean concentrations of 3.63 and 2.84 g/liter, respectively, and two doses with twice these amounts. Only allele G3 mb did not completely follow this rule. We also studied the serum concentrations and the allotype distribution of 41 IgG1 and 31 IgG3 myeloma proteins. The results suggested that the allotype-associated differences in serum concentrations are caused by different numbers of B cells producing allotypic subsets of IgG1 or IgG3, not by different rates of synthesis per B cell.
Genomic organization and transcriptional regulation of the RANTES chemokine gene.
Authors: Nelson P J , Kim H T , Manning W C , Goralski T J , Krensky A M
Published: December 2022

RANTES is a member of a large supergene family of pro-inflammatory cytokines called CC chemokines that appear to play a fundamental role in inflammatory processes. The RANTES protein causes release of histamine from basophils and is a chemoattractant for CD45RO/CD4+ "memory" T lymphocytes, monocytes, and eosinophils. Although expression of RANTES was first thought to be limited to activated T cells, recent data have shown that it is produced by a variety of tissue types in response to specific stimuli. RANTES mRNA is expressed late (3 to 5 days) after activation of resting T cells whereas in fibroblasts, renal epithelial and mesangial cells, RANTES mRNA is quickly up-regulated by TNF-alpha stimulation. In order to gain a better understanding of the molecular mechanisms that regulate expression of the RANTES locus, we have characterized the RANTES gene and determined a putative promoter region. The RANTES gene spans approximately 7.1 kb and is composed of three exons of 133, 112 and 1075 bases and two introns of approximately 1.4 and 4.4 kb with the position of intron/exon boundaries conserved relative to the other CC chemokine family members. Approximately 1 kb of DNA from the immediate 5' upstream region of RANTES was sequenced and found to contain a large number of potential consensus elements for specific T cell/hemopoietic, myeloid, muscle, and ubiquitously expressed DNA-binding factors. RANTES-promoter-luciferase gene fusion assays demonstrate high levels of reporter gene activity in a "mature" T cell line Hut78, the erythroleukemic cell line HEL, and the rhabdomyosarcoma cell line RD, with little or no activity in the "early" T cell line Jurkat, the gamma delta T cell line PEER, the thymic tumor Molt4, or the pre-erythroid cell line K562. Deletion analysis of the promoter region indicates that different transcriptional mechanisms control expression of RANTES in the various tissues studied.
The soluble pool of beta 2-microglobulin free HLA class I alpha-chains. Qualitative and quantitative characterization.
Authors: Pickl W F , Majdic O , Faé I , Reuschel R , Holter W , Knapp W
Published: December 2022

Previous studies have demonstrated that HLA class I heterodimers are present in plasma and cell culture supernatants. They can be precipitated by mAb the binding of which is dependent on the proper association of the polymorphic alpha-chain with beta 2-microglobulin (beta 2-m). The molecular mass of the alpha-chain ranges from 45 to 35 kDa with a number of intermediate products. We report on the identification of 35-kDa soluble beta 2-m free HLA class I H chains immunoprecipitated by mAb LA45 from cell culture media of activated B and T cells. Furthermore, a peptide-based competitive immunosorbent assay was established to determine the amounts of soluble HLA class I alpha-chains. By means of this assay, we formally proved the specificity of mAb LA45 for a linear epitope on HLA class I H chains centered on residues arginine-asparagine at positions 62 and 63 of the alpha 1-domain. PHA or rIL-2 were identified as efficient stimuli for PBMC leading to the generation of soluble beta 2-m free HLA class I H chains. Testing of cell lines representing distinct stages of hematopoietic differentiation demonstrated a significant correlation between cell surface expression of beta 2-m free HLA class I H chains and amounts of soluble LA45 reactive molecules. However, three of six human T lymphotropic virus type I transfected cell lines, although expressing beta 2-m free H chains, do not generate soluble molecules. Finally, human sera were found to contain considerable amounts of beta 2-m free HLA class I H chains. The average amount of these molecules in sera of individuals with one positive LA45 allele was determined to be 46.9 +/- 38.6 nM/liter.
Humanization of an antibody directed against IgE.
Authors: Presta L G , Lahr S J , Shields R L , Porter J P , Gorman C M , Fendly B M , Jardieu P M
Published: December 2022

IgE antibodies bind to specific high-affinity receptors on mast cells, leading to mast cell degranulation and release of mediators, such as histamine, which produce symptoms associated with allergy. Hence, anti-IgE antibodies that block binding of IgE to its high-affinity receptor are of potential therapeutic value in the treatment of allergy. These antibodies must also not bind to IgE once it is bound to the receptor because this would trigger histamine release. This study describes the humanization of a murine antibody, MaE11, with these characteristics. Variants of the humanized antibody were evaluated to probe the importance of framework residues on antibody binding and to determine which charged residues in the CDR interacted with IgE. We found that only five changes in human framework residues were required to provide for binding comparable to that of the original murine antibody.
Extraembryonic expression of the human MHC class I gene HLA-G in transgenic mice. Evidence for a positive regulatory region located 1 kilobase 5' to the start site of transcription.
Authors: Schmidt C M , Ehlenfeldt R G , Athanasiou M C , Duvick L A , Heinrichs H , David C S , Orr H T
Published: December 2022

Trophoblast, the only fetal tissue in direct contact with maternal cells, fails to express the polymorphic HLA class I molecules HLA-A and -B, but does express the nonpolymorphic class I molecule HLA-G. It is thought that HLA-G may provide some of the functions of a class I molecule without stimulating maternal immune rejection of the fetal semiallograft. As a first step in identifying the cis-acting DNA regulatory elements involved in the control of class I expression by extraembryonic tissue, several types of transgenic mice were produced. Two HLA-G genomic fragments were used, 5.7 and 6.0 kb in length. These included the entire HLA-G coding region, 1 kb of 3' flanking sequence, and 1.2 or 1.4 kb of 5' flanking sequence, respectively. A hybrid transgene, HLA-A2/G, was produced by replacing the 5' flanking sequence, first exon, and early first intron of HLA-G with the corresponding elements of HLA-A. Comparison of transgene mRNA expression patterns seen in HLA-A2/G and HLA-G transgenic mice suggests that 5' flanking sequences are largely responsible for the differing patterns of expression typical of the classical class I and HLA-G genes. Studies comparing the extraembryonic HLA-G expression levels of founder embryos transgenic for either the 5.7- or 6.0-kb HLA-G transgene showed that the 6.0-kb transgene directed HLA-G expression far more efficiently than did the 5.7-kb HLA-G transgene, producing extraembryonic HLA-G mRNA levels similar to those seen in human extraembryonic tissues. The results of these studies suggest that the 250-bp fragment present at the extreme 5' end of the 6.0-kb HLA-G transgene and absent from the 5.7-kb HLA-G transgene contains an important positive regulatory element. This 250-bp fragment lies further upstream than any of the previously documented class I regulatory regions and may function as a locus control region.
Effects of subunit mutation on the localization to coated pits and internalization of cross-linked IgE-receptor complexes.
Authors: Mao S Y , Pfeiffer J R , Oliver J M , Metzger H
Published: December 2022

IgE receptors of mast cells, Fc epsilon RI, localize to coated pits and internalize after cross-linking. We investigated whether any one of the receptor's four distinctive cytoplasmic domains regulates these phenomena. COS cells, which lack Fc epsilon RI entirely, and P815 mouse mastocytoma cells that lack the alpha and beta subunits of the tetrameric Fc epsilon RI (alpha beta gamma 2), were transfected with wild-type, incomplete, or variant Fc epsilon RI. IgE-receptor complexes were observed by electron microscopy. Before cross-linking with anti-IgE gold particles, receptors were not preferentially localized to coated pits, which occupy approximately 1% of the cell surface. After cross-linking, up to 10 to 20% of the wild-type and most other receptor variants were in coated pits in transfected P815 cells at any one time. beta-less variants localized normally but, surprisingly, receptors containing a variant beta subunit showed reduced localization. "Receptors" consisting simply of the lipid-anchored ectodomains of the human alpha subunit failed to localize to coated pits. In general, cross-linked receptors that localized to coated pits were progressively internalized, whereas receptors that failed to accumulate in coated pits were not. We conclude that no single cytoplasmic domain of the Fc epsilon RI uniquely controls its ligand-induced localization to coated pits and internalization.
Synergism between human monocyte chemotactic and activating factor and bacterial products for activation of tumoricidal properties in murine macrophages.
Authors: Singh R K , Berry K , Matsushima K , Yasumoto K , Fidler I J
Published: December 2022

Chemotactic factors regulate the recruitment of monocytes-macrophages to inflammatory sites and neoplastic tissues. The purpose of this study was to determine whether MCAF also influences the activation of C3H/HeN macrophages to become tumoricidal. Several metastatic and nonmetastatic clones of the K-1735 murine melanoma cells syngeneic to C3H/HeN mice were transfected with expression vectors containing the human MCAF gene or control DNA. Tumor cells producing high levels of MCAF were significantly lysed by macrophages treated with LPS, whereas parental or control transfected cells were not. Control-activated macrophages incubated with both IFN-gamma and LPS lysed all the melanoma cells regardless of MCAF production. Pretreatment of macrophages with MCAF significantly enhanced their response to low concentrations of LPS, muramyl tripeptide, and a synthetic bacterial LPP, as measured by lysis of murine melanoma cells. These data suggest that in addition to being a chemotactic factor, MCAF can prime macrophages to respond to endotoxins and other bacterial products and therefore may regulate several levels of macrophage-tumor interactions in situ.
Allogenic MHC class II determinant(s) in MRL/Ipr autoimmune disease-prone mice. Unusual expression of an L1 transposable element creates molecular mimicry.
Authors: Bobé P , Benihoud K , Kiger N
Published: December 2022

In some aged MRL/Ipr autoimmune disease-prone mice, unexpected reactivity has been observed between lymphoid cells and mAb or polyclonal antibodies directed against the A beta d class II chain of the MHC. However, Southern blot analysis of their genomic DNA, using different class I and class II MHC-specific probes, confirmed their inbred character and their H-2k genotype. In this study, immunoprecipitation experiments with an anti-A beta d mAb indicated that the 45 (and 12)-kD molecule(s) recognized by an anti-A beta d mAb differed from a class II chain. After specific antibody screening of a lambda gt11 expression library constructed with MRL/Ipr "A beta d-positive" lymphoid cells, we cloned cDNA that share sequences with high homology (> 80%) to the 3' end of a 7-kb L1Md (L1) element propagated in the mouse genome via retrotransposition. Northern blot analysis showed an overtranscription of these L1 sequences in the MRL/Ipr spleen cells used for the construction of the cDNA library, in comparison to the MRL ancestor strains. Therefore, the autoimmune MRL/Ipr strain could express a translation product of L1 open reading frame 2, which mimics a "highly antigenic" epitope of an allogenic MHC class II Ag.
Parameters of tolerance induction by antigen targeted to B lymphocytes.
Authors: Eynon E E , Parker D C
Published: December 2022

We have shown that a foreign protein Ag (the F(ab) fragment of rabbit IgG) becomes a particularly effective tolerogen when it is targeted to B lymphocytes in vivo. This is done by injecting the Ag intravenously into mice in the form of the F(ab) fragment of rabbit anti-mouse IgD antibody (F(ab)) rabbit anti-delta). Our hypothesis is that resting B cells are tolerogenic APC for CD4+ T cells in unprimed animals, and induce Ag-specific nonresponsiveness in the Th cell compartment by presenting Ag without appropriate costimulatory signals. In this report, we find that Ag-activated T cells appear to be resistant to tolerance induction, because F(ab) rabbit anti-delta given 5 days after challenge with rabbit F(ab) in alum adjuvant has little or no effect of the subsequent antibody response. Tolerance also fails in this model when B cells are activated independently of Ag by simultaneous injection of activating concentrations of divalent, IgG mouse anti-delta at the same time as F(ab) rabbit anti-delta. Finally, nonresponsiveness in the T cell compartment is not limited to a lesion in T cell help for the antibody response, because T cells from mice treated with F(ab) rabbit anti-delta and then primed with rabbit F(ab) fragments in CFA show reduced T cell proliferation and IL-2 production when restimulated with Ag in vitro.
A B lymphocyte surface molecule mediating activation and protection from apoptosis via calcium channels.
Authors: Santos-Argumedo L , Teixeira C , Preece G , Kirkham P A , Parkhouse R M
Published: December 2022

A rat mAb (NIM-R5) was prepared against a 42-kD B cell activation Ag (p42). The expression of p42 is increased upon activation. NIM-R5 induces an increase of intracellular Ca2+, due to influx from the exterior milieu via calcium channels. This stimulation does not prejudice further stimulation with anti-Ig, and thus p42 constitutes an activation signal independent of membrane Ig. The antibody induces increased expression of class II molecules on resting B lymphocytes and prepares the cells for "spreading" when interacted with immobilized anti-class II antibody. The antibody alone is weakly mitogenic and comitogenic with IL-4 on resting B cells. Of particular interest, NIM-R5 induces proliferation and rescue from apoptosis in B cells activated in vitro. In conclusion, NIM-R5 induces an Ig-independent activation and proliferation of resting and activated B cells. This antibody does not recognize other known B cell activation Ag such as CD23, CD40, or CD72. We therefore propose that the p42 Ag is a glycoprotein with an important role in the regulation of B lymphocyte activation and survival.
Granulocyte-macrophage colony-stimulating factor, but not macrophage colony-stimulating factor, suppresses basal and lipopolysaccharide-stimulated complement factor production in human monocytes.
Authors: Høgåsen A K , Hestdal K , Abrahamsen T G
Published: December 2022

Monocyte/macrophage contribution of C biosynthesis is important, particularly during inflammation. Since granulocyte-macrophage CSF (GM-CSF) and macrophage-CSF (M-CSF) exert a variety of stimulatory effects on monocyte/macrophage functions in vitro, we studied their impact on the biosynthesis of the C components C3 and factor B by human monocytes in culture. GM-CSF at doses of 10 ng/ml and higher inhibited the basal C3 synthesis. This effect was most pronounced when the cytokine was added to freshly isolated monocytes. No effect was found on the basal production of factor B. Furthermore, GM-CSF abrogated the LPS-stimulated production of both C3 and factor B. These suppressive effects were neutralized by a polyclonal anti-GM-CSF antibody. Moreover, when anti-GM-CSF was added to unstimulated or LPS-stimulated cells, their C3 production increased. This indicates that both spontaneous and LPS-triggered release of monocyte-produced GM-CSF has an autocrine function in regulating monocyte C3 biosynthesis. GM-CSF also down-modulated the expression of CD14 at an early stage of cell culture. This might be the mechanism through which the LPS-effects are suppressed because CD14 has been shown to be a LPS receptor. Contrary to this, M-CSF at doses of 100 U/ml and higher stimulated the synthesis of C3, whereas the basal production of factor B and the LPS-stimulated production of C3 and factor B were unaffected. Granulocyte-CSF (G-CSF) did not influence monocyte C biosynthesis, and neither anti-M-CSF nor anti-G-CSF influenced the LPS-induced C3 production. The effects of GM-CSF and M-CSF on C biosynthesis may be important in regulating the availability of C components during an inflammatory response, and these observations may also have implications for the clinical use of CSF.
Human mast cells produce IL-8.
Authors: Möller A , Lippert U , Lessmann D , Kolde G , Hamann K , Welker P , Schadendorf D , Rosenbach T , Luger T , Czarnetzki B M
Published: December 2022

Recruitment of neutrophils is a common feature in diseases that are associated with mast cell activation. The mechanisms that mediate neutrophil activation are not well understood. IL-8 is a recently described potent chemotactic factor that might be pathogenetically involved in this process. We therefore studied the human mast cell line HMCI and human skin mast cells for their ability to produce IL-8 using various stimuli. IL-8-mRNA was expressed in a stimulus- and time-dependent fashion as detected by Northern blot analysis with an IL-8-specific cDNA probe. The molecular mass of HMCI-derived IL-8 was determined to be about 8 kDa by immunoblot analysis. Immunoreactive and biologically active IL-8 protein was measured in the cell culture supernatants of HMCI cells by an ELISA and a chemotaxis assay, respectively. On immunoelectron microscopy of stimulated skin mast cells, IL-8 was found along cytoplasmatic membranes and in intracellular granules. Our data indicate that mast cells may contribute to neutrophil recruitment by secretion of IL-8.
Retina and retinal pigment epithelial cell autoantibodies are produced during murine coronavirus retinopathy.
Authors: Hooks J J , Percopo C , Wang Y , Detrick B
Published: December 2022

The murine coronavirus, mouse hepatitis virus (MHV), JHM strain, induces a biphasic retinal disease in adult BALB/c mice. In the early phase of the disease, day 1 to 7, a retinal vasculitis is noted and is associated with the presence of virus particles. In the late phase of the disease, day 10 to 140, a retinal degeneration is observed and is associated with the absence of both virus particles and inflammatory cells. We show that the retinal degenerative process is also associated with the presence of antiretinal autoantibodies. In total, 22 of 23 sera collected from 10 to 70 days after JHM virus inoculation contained antiretinal autoantibodies. These autoantibodies are not found in sera from normal or mock-injected mice. Antibodies to retinal tissue were identified as two distinct patterns of immunoperoxidase staining on frozen sections of normal rat eyes, retinal autoantibodies and retinal pigment epithelium (RPE) autoantibodies. The antiretinal autoantibodies first appeared as IgM class antibodies that shifted to IgG class autoantibodies. The anti-RPE cell autoantibodies were predominantly of the IgG class. Sera that were positive for these autoantibodies did not stain with liver or kidney sections but 2 of 3 did react with rat brain sections. A second mouse strain, CD-1, was also evaluated because these animals respond to JHM virus inoculation by developing only the early phase of this disease, i.e. vasculitis. On day 10 postinoculation, the retina architecture has a normal appearance. In these mice, which are free of a retinal degeneration, antiretinal autoantibodies are not produced. However, just as is noted in the BALB/c mice, antivirus neutralizing antibodies are produced in the infected CD-1 mice. These findings suggest a role for autoimmunity in the pathogenesis of murine coronavirus induced retinal degeneration. This study establishes an animal model for the study of humoral autoimmune responses in human retinal degenerations.
Intracellular transport of class I MHC molecules in antigen processing mutant cell lines.
Authors: Anderson K S , Alexander J , Wei M , Cresswell P
Published: December 2022

Intracellular transport and stability of class I MHC glycoproteins depends on the assembly of H chain, beta 2-microglobulin, and peptide. The Ag processing mutant cell lines T2 and RMA-S have defects in peptide loading of class I, resulting in reduced cell surface expression of class I molecules. Expression of class I molecules in the murine cell line RMA-S can be induced at 26 degrees C, suggesting that they are transported to the cell surface, but are unstable. However, most human class I molecules in T2 are poorly expressed at the cell surface, even at 26 degrees C. To directly compare the transport of human and mouse alleles in RMA-S and T2, the human alleles HLA-A2, A3, and B27 were transfected into RMA-S along with human beta 2-microglobulin, and the mouse alleles H-2Kb and Db were transfected into T2. Surface expression of HLA-A3 and B27 in RMA-S remained less than 10% of wild-type levels at 26 degrees C. H-2Kb and Db in both cell lines, however, were expressed at 20 to 30% wild-type levels at 37 degrees C and could be induced to wild-type levels at 26 degrees C or with peptides. The selective expression of murine class I glycoproteins at the cell surface of T2 is not because of their greater stability when associated with human beta 2m, since H-2Kb and Db H chain/human beta 2m complexes dissociate more rapidly in vitro than HLA-A3 and B27 complexes. These results suggest that the difference in transport between human and mouse class I in T2 reflects a fundamental structural property of the class I glycoproteins.
Selection of the T cell receptor repertoire in Lpr mice.
Authors: Herron L R , Eisenberg R A , Roper E , Kakkanaiah V N , Cohen P L , Kotzin B L
Published: December 2022

The development of double-negative (CD4-, CD8-) T cells and other T cells subsets in lymphoproliferation (lpr) mice continues to be poorly defined. Recent studies indicate that lpr is a mutation of a receptor mediating apoptosis. It has thus been hypothesized that T cell development in the thymus should be abnormally affected. In this study, we analyzed the TCR V beta repertoire of double-negative T cells as well as CD4+ and CD8+ single-positive subsets in various lpr and matched non-lpr strains. Particular comparisons were made to determine the influence of different class I and class II molecules on repertoire formation. The data demonstrate that positive and negative selection of the CD4+ and CD8+ subsets are normal in lpr mice when compared with non-lpr congenic mice. Surprisingly, the result also suggest that double-negative T cells are mostly selected on class I MHC molecules in a pattern similar to the CD8+ population, and that T cells positively selected on class II MHC antigens may be absent from the double-negative population. In all lpr strains, we also found an increased percentage of double-negative V beta 8.3+ cells out of proportion to levels in the CD4+ or CD8+ subsets. Longitudinal studies and studies in thymectomized animals showed that this increase reflects a peripheral process selectively affecting V beta 8.3+ double-negative T cells. Together, these repertoire data provide new insight into the effect of the lpr genetic defect on T cell development and the derivation of double-negative T cells. Despite the role of Fas in apoptosis and the abnormal expression of this gene in lpr mice, the present results support the hypothesis that thymic events are relatively normal in lpr mice, and that the double-negative T cells are mostly class I MHC selected and expanded by abnormal peripheral processes.
Processing of endogenously synthesized hen egg-white lysozyme retained in the endoplasmic reticulum or in secretory form gives rise to a similar but not identical set of epitopes recognized by class II-restricted T cells.
Authors: Adorini L , Guéry J C , Fuchs S , Ortiz-Navarrete V , Hämmerling G J , Momburg F
Published: December 2022

To study the processing and presentation of endogenously synthesized Ag to class II MHC-restricted T cells, hen egg lysozyme (HEL), either tagged with a peptide that confers retention in the endoplasmic reticulum (HEL.KDEL), or in the secretory form (HELs), was stably expressed in LK-35.2 B hybridoma cells. Presentation of HEL peptides bound to class II molecules was assessed by activation of specific T cell hybridomas recognizing seven different epitopes derived from exogenous HEL. The presentation of endogenously synthesized HEL was not caused by reuptake of secreted of shed Ag. All the HEL epitopes examined were efficiently presented after processing of endogenous HEL by HELs-transfected LK-35.2 cells. Processing of HEL tagged with KDEL, however, gave rise to presentation of only six of the seven HEL epitopes. The epitope included in the HEL sequence 112-124 was not presented by HEL.KDEL-transfected B cells. In addition, two of the four T cell hybridomas recognizing HEL 116-129 together with I-Ak molecules were not activated by HEL.KDEL, and three other epitopes were presented with lower efficiency as compared with HELs. Thus, endogenously synthesized HEL in secretory form gives rise to a set of class II-binding epitopes indistinguishable from exogenous HEL, whereas endoplasmic reticulum-retained HEL generates a similar but not identical set of epitopes. The endosomal protease inhibitor leupeptin prevented presentation of the epitope 108-116, but not 46-61, both by HELs and HEL.KDEL transfected cells, indicating a requirement for endosomal processing in both cases. In addition, the presentation of peptides derived from endogenously synthesized, either secretory or endoplasmic reticulum-retained HEL, could be inhibited by lysosomotropic amines, further indicating that the intracellular route of class II molecules presenting peptides derived from endogenous Ag intersects the acidic endosomal compartment.
Endogenous IL-1 receptor antagonist protein (IRAP) regulates schistosome egg granuloma formation and the regional lymphoid response.
Authors: Chensue S W , Bienkowski M , Eessalu T E , Warmington K S , Hershey S D , Lukacs N W , Kunkel S L
Published: December 2022

This study examined the role of IL-1 receptor antagonist protein (IRAP) in the regulation of the immune/inflammatory response to schistosome eggs. Initial screening for IRAP-specific mRNA transcripts by reverse transcriptase and primer-directed polymerase chain reactions suggested significant endogenous IRAP synthesis in lungs with Schistosoma mansoni egg-induced hypersensitivity granulomas but not in normal lungs or lungs with non-immune bead granulomas. Direct detection using mIRAP-specific antibodies corroborated the RNA studies. Both ELISA and immunohistochemical studies revealed significant spontaneous IRAP production in cultures of isolated egg granulomas and regional reactive lymphoid tissue that could be localized largely but not exclusively to macrophages. Synchronously developing secondary schistosome egg granulomas showed accelerated and augmented IRAP production compared with primary lesions, paralleling granuloma cellularity and growth kinetics. Nonimmune T cell-independent bead lesions produced the least amounts of IRAP. In draining lymphoid tissue the onset of IRAP production corresponded with local cell proliferation in both primary and secondary egg responses. Next, the in vivo role of IRAP was tested by administration of anti-IRAP antisera which caused 40-50% increases in egg granuloma area. Moreover, treatment increased (50-100%) IL-2, IL-4, IL-10, and IFN-gamma production in the primary response and all but IFN in secondary response lymph node cultures. Our results suggest that IRAP is an endogenous regulatory protein and may limit the activity of IL-1 during the Ag-specific granulomatous response to schistosome eggs. Furthermore, our findings provide in vivo support for the notion that Ag-elicited lymphocyte-derived products probably augment IRAP production and block the action of IL-1.
Identification of human melanoma peptides recognized by class I restricted tumor infiltrating T lymphocytes.
Authors: Storkus W J , Zeh H J , Maeurer M J , Salter R D , Lotze M T
Published: December 2022

Using a newly described pH 3.3 acid elution technique, peptides were extracted by denaturation of class I molecules on the surface of human melanomas. HPLC fractionation of this material revealed six T cell epitopes (termed P1-P6) recognized by HLA-A2-restricted, melanoma-specific tumor infiltrating lymphocyte (TIL) lines. Three of these fractions (P1, P2, and P4) appeared to represent shared/immunodominant melanoma Ag recognized in the context of HLA-A2 because they were expressed by 4/4 HLA-A2+ melanoma cell lines and were each recognized by all four oligoclonal HLA-A2-restricted TIL lines examined. Interestingly, P1 and P2 (but not P3-P6) could also be recognized by these same TIL when presented by the HLA-Aw69 class I molecule, which is closely related to HLA-A2. P3, P5, and P6 displayed more restricted expression and were differentially recognized by the four oligoclonal TIL lines. These results suggest that synthetic peptide derived from P1, P2, and P4 sequences (when deduced) may form the basis of effective prophylactic or therapeutic melanoma vaccines by stimulating CD8+ CTL in HLA-A2+ individuals. This approach of identifying T cell epitopes presented by class I molecules should prove generally applicable to the study of other tumors recognized by class I-restricted CTL.