bluuebunny/crossref_metadata_2025 huggingface dataset:
Oral tolerance in experimental autoimmune uveoretinitis. Distinct mechanisms of resistance are induced by low dose vs high dose feeding protocols.
Authors: Gregerson D S , Obritsch W F , Donoso L A
Published: December 2022

Studies of oral tolerance in LEW rat models of autoimmune diseases including S-antigen (S-Ag)-mediated experimental autoimmune uveoretinitis (EAU), and myelin basic protein-induced experimental autoimmune encephalomyelitis have produced conflicting evidence for the roles of clonal anergy and suppression. Using subpeptides from a region of S-Ag known to induce oral tolerance a protective site was localized to a nonamer of residues 347-355. This site was also uveitogenic, providing the basis for testable hypotheses for tolerance to be due to clonal anergy in pathogenic T cells specific for that site, or to suppression. Evidence for suppression was strongly supported by several observations. 1) Induction of oral tolerance with low dose feedings (250 micrograms/feeding) of peptide 343-362 conferred resistance to EAU induction by intact S-Ag, which should not be possible if only T cells specific for epitopes in 343-362 were rendered unresponsive, since there are several other pathogenic sites in S-Ag. 2) Low dose feeding induced resistance to EAU induction by a distinct, spatially separate peptide, residues 270-289, of S-Ag. 3) The requirement for linked recognition was shown by the inability of tolerance induced by feeding 343-362 to protect from EAU induction by a peptide, residues 521-540, derived from interphotoreceptor retinoid binding protein, a different uveitogenic retinal protein. 4) Resistance could be adoptively transferred. Conversely, induction of tolerance with high doses of peptide (5 mg/feeding) resulted in loss of resistance to EAU induced by S-Ag, although disease induction by the fed peptide was inhibited; observations that are consistent with clonal anergy. The apparent lack of suppression after high dose feeding could mean that suppressor T cells can also be rendered unresponsive or that induction of T suppressor cells is dependent on CD4+ Th cells, which were rendered anergic, leading to lack of T suppressor development. We suggest that oral tolerance operates by at least two distinct mechanisms that depend on the feeding dose; low doses induce suppression, whereas high doses induce unresponsiveness.
Identification of human pre-T/NK cell-associated genes.
Authors: Ranes-Goldberg M G , Hori T , Mohan-Peterson S , Spits H
Published: December 2022

We have used a combination of subtractive cloning and differential screening techniques to identify genes preferentially expressed in early stages of human T/NK cell development compared with mature T and NK cells. A fetal liver-derived cytoplasmic (c) CD3+ membrane (m) CD3- clone, FL508, which expresses markers characteristic of pre-T and pre-NK cells served as a cell source for our cloning experiments. A cDNA library enriched for genes expressed in FL508 was constructed by removal of cDNA that hybridized to mRNA from a B cell line, JY. One-tenth of the resulting library of 5000 clones was screened by differential hybridization with cDNA probes from JY and a mature CD4+ T cell clone, B21. The relative expression levels of six selected clones were analyzed in 17 different cell/tissue types by semiquantitative polymerase chain reaction. Four of these clones are expressed at higher levels in thymocytes than in mature T or NK cells, and three clones are expressed at higher levels in fetal liver cells than in either mature T or NK cells. Partial and complete DNA sequence information suggests that these six cDNA correspond to previously unidentified genes. Genes identified in this study may be useful not only as markers for early stages of T/NK cell ontogeny, but also as tools for understanding novel developmental events.
Characterization of type I complement C2 deficiency MHC haplotypes. Strong conservation of the complotype/HLA-B-region and absence of disease association due to linked class II genes.
Authors: Truedsson L , Alper C A , Awdeh Z L , Johansen P , Sjöholm A G , Sturfelt G
Published: December 2022

Fourteen individuals with complete C2 deficiency from 11 families and 3 heterozygous C2-deficient individuals from two families were investigated. In all the 24 independent C2-deficient haplotypes, the complotype S042 was present and the majority (21/24) was [HLA-B18,S042,DR2]. All carried the type I C2 deficiency C2 pseudogene with its characteristic 28 bp deletion. All but two haplotypes had 10 AC/GT repeats in the TNF alpha microsatellite polymorphism and all but one of the haplotypes were identical at or near HLA-B as assessed by RFLP using BstEII digestion and two genomic probes, R5A and M20A, located 100 and 38 kb centromeric to HLA-B, respectively. The exceptional haplotype was HLA-B40 with four AC/GT repeats at TNF-alpha. Three of the haplotypes were not DR2 based on generic and sequence-specific oligonucleotide typing. Another four haplotypes showed different DO-variants detected by RFLP analysis using BglIIand Mspl digestion. Thus, the [HLA-B18,S042,DR2] haplotype appears to be more fixed in the region between the complement genes and the HLA-B locus (96%) than in the region between the complement genes and DR (88%) and DO loci (71%). Of the 14 individuals studied, six had SLE or SLE-like syndromes and six had a history of severe infections although two were apparently healthy. Three of the six SLE patients and two individuals with repeated infections were homozygous for [HLA-B18,S042,DR2] and also homozygous for DQB1*0602 and the common DO variant. Thus, MHC class II genes linked to the C2 pseudogene do not appear to determine different clinical consequences of C2 deficiency.
Specific IgG autoantibodies are potent inhibitors of autoreactive T cell response to phytohemagglutinin-activated T cells.
Authors: Wolf-Levin R , Azuma T , Aoki K , Yagami Y , Okada H
Published: December 2022

PHA-activated human T cells express MHC class II molecules and have been shown to stimulate autoreactive T cells in autologous mixed lymphocyte reaction (T-T AMLR). We now demonstrate that normal human serum can dramatically suppress the proliferative response in T-T AMLR. The inhibitory factor was detected in the IgG fraction, and the activity was found to be mediated through the F(ab')2 portion of the IgG molecule, implying that the inhibitor could be specific. All homologous as well as autologous sera tested contained the inhibitory activity. Human serum was also found to suppress allogenic MLR stimulated by PHA-activated T lymphocytes but not allogenic MLR stimulated by nonactivated PBMC, indicating that the mechanism of inhibition is related to PHA activation of the stimulator cells. Moreover, pretreatment of the PHA-activated T lymphocytes with human serum resulted in a significant inhibition of T-T AMLR, as opposed to pretreatment of nonactivated PBMC, indicating that PHA-activated stimulators are functionally involved in the inhibition. Cytofluorometric analysis revealed that autologous IgG specifically binds to PHA-activated T lymphocytes and not to nonactivated CD3+ T cells. Furthermore, serum absorbed by PHA-activated T lymphocytes substantially lost its inhibitory activity whereas serum absorbed by nonactivated PBMC did not, suggesting that a surface molecule(s) expressed during activation of the stimulator cells is involved in the inhibition. The addition of human serum later in the culture period (> 3 days) resulted in a marked decrease in inhibition, implying that the presence of IgG in the early recognition phase of T-T AMLR is essential for maximum inhibitory effect. These results raise the possibility that natural autoantibodies present in normal human IgG may play an important role in regulating immune response mediated by autoreactive T cells.
Unexpected expansions of CD8-bearing cells in old mice.
Authors: Callahan J E , Kappler J W , Marrack P
Published: December 2022

As mice age, spontaneous changes occur in the receptor repertoire of their T cells. The receptor repertoire of CD4+ T cells does not change with age. By contrast, however, the percentage of alpha beta+, CD8+ T cells bearing particular V elements varies considerably between individual aged mice, although it is remarkably consistent among individual young animals within a given strain. Changes of receptor V element use among CD8+ T cells in individual mice are unpredictable. However, when a large number of mice of the same strain is analyzed, strain-specific trends in V element skewing are found. Old C3H.SW and B10.BR mice have mono- or oligoclonal expansions of CD8+ T cells. These expansions of peripheral CD8+ T cells with age are probably due to deregulation of proliferation of individual CD8+ T cells after recognition of viral or environmental Ag, accompanied, perhaps, by partial transformation of particular T cell clones. Another phenomenon documented herein is the fact that the CD4/CD8 ratio drops steadily as a function of age. Shifts in CD4/CD8 ratio were not due to increased numbers of CD8+ T cells in spleen and lymph nodes, rather the CD4+ T cells disappeared from aging mice faster than CD8+ T cells.
Down-regulation of macrophage transforming growth factor-beta messenger RNA expression by IL-7.
Authors: Dubinett S M , Huang M , Dhanani S , Wang J , Beroiza T
Published: December 2022

Transforming growth factor-beta (TGF-beta) is a potent inhibitor of immune responses. Macrophage-derived products, including TGF-beta, have been suggested as inhibitors of the antitumor immune response. We hypothesized that IL-7, a cytokine with antitumor properties, may exert its immunoregulatory effects in part through the down-regulation of TGF-beta. To test this hypothesis we analyzed IL-2-stimulated murine macrophage TGF-beta mRNA expression following exposure to IL-7 both in vitro and in vivo. IL-7 down-regulated IL-2-induced TGF-beta expression by macrophages in vitro, as well as after i.p. injections of IL-2 and IL-7 in vivo. The IL-7-mediated inhibition of TGF-beta mRNA expression did not require new protein synthesis and therefore appears to be a direct effect of IL-7. IL-7 had no significant effect on the stability of TGF-beta mRNA. Nuclear run-on assays revealed that the suppression of IL-2-induced TGF-beta gene expression by IL-7 is mediated at the level of transcription. Also, IL-7 decreased TGF-beta secretion as measured by bioassay. We conclude that IL-7 down-regulates TGF-beta and suggest that some of the proliferative and cytolytic activities mediated by cells exposed to IL-7 may be caused by a decrement in macrophage-derived TGF-beta.
Immunoglobulin secretion and phosphorylation of common proteins are induced by IL-2, IL-4, and IL-6 in the factor responsive human B cell line, SKW6.4.
Authors: Goldstein H , Kim A
Published: December 2022

After the binding of IL-2, IL-4, or IL-6 to their respective receptors on activated human B cells, a multistep cascade of intracellular events is initiated that results in the secretion of Ig. However, it is not known whether these different cytokine receptors use common or divergent signal transduction pathways to stimulate Ig secretion. Therefore, we examined the signaling mechanisms used by a human lymphoblastoid cell line arrested at a late stage of differentiation, SKW6.4, that secretes IgM following stimulation with IL-2, IL-4, or IL-6 alone. Our study demonstrated that IL-2, IL-4, and IL-6-stimulation of IgM secretion by SKW6.4 cells was inhibited by either the serine/threonine kinase inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperizine dihydrochloride (H7) or the tyrosine kinase inhibitor, genistein. To investigate the early phosphorylation events initiated by these cytokines, a membrane-enriched preparation from SKW6.4 cells was isolated in a manner that minimized the disruption of membrane protein complexes and then incubated with IL-2, IL-4, or IL-6 in the presence of [gamma-32P]ATP. IL-2, IL-4, and IL-6 stimulated the rapid serine/threonine phosphorylation of 47-, 49-, and 91-kDa proteins. However, in contrast to the 47- and 49-kDa proteins that remained phosphorylated for up to 30 min poststimulation, the 91-kDa protein was rapidly dephosphorylated within 15 min of stimulation. The observation that serine/threonine phosphorylation of the same proteins was stimulated by IL-2, IL-4, and IL-6 suggested that the cytokines activated either different protein kinases with the same substrate specificity or the same protein kinase. In addition, stimulation of intact SKW6.4 cells with either IL-2, IL-4, or IL-6 induced the phosphorylation of two proteins with molecular masses of 45- to 50-kDa and 85 to 90-kDa. Taken together, our data demonstrate that activation of both a serine/threonine kinase and a tyrosine kinase is involved in the IL-2, IL-4, and IL-6-stimulation of IgM secretion by SKW6.4 cells and activation of the same or a similar serine/threonine protein kinase is an early step in the signal transduction pathway used by these cytokines.
CD44 expression on activated B cells. Differential capacity for CD44-dependent binding to hyaluronic acid.
Authors: Hathcock K S , Hirano H , Murakami S , Hodes R J
Published: December 2022

CD44 expression and the functional capacity for CD44-dependent binding of hyaluronic acid (HA) were analyzed on unstimulated B cells and on B cells stimulated with a variety of polyclonal B cell activators. Whereas essentially all LPS-activated and anti-IgD-dextran-activated B cells and a subpopulation of IL-5-activated B cells expressed increased levels of cell surface CD44 relative to unstimulated B cells, only IL-5-activated CD44hi B cells constitutively bound to FITC-conjugated hyaluronic acid (FITC-HA). Preincubation of LPS or anti-IgD-dextran-activated B cells with the CD44-specific mAb IRAWB14.4 (IRA) induced a high degree of FITC-HA binding in these populations; preincubation of unstimulated B cells with this CD44-specific mAb induced minimal FITC-HA binding. In contrast, preincubation with mAb IRA failed to induce FITC-HA binding by the IL-5-activated CD44lo B cell subset. Neither the amount of constitutive FITC-HA binding nor the level of IRA-inducible FITC-HA binding correlated simply with the overall level of CD44 expressed by the different B cell populations. Biochemical analysis of immunoprecipitated CD44 molecules revealed that relative to CD44 isolated from all other populations examined, CD44 isolated from IL-5-activated B cells was of a lower molecular weight. Treatment with N-Glycanase eliminated this observed difference in molecular weight, indicating that it reflected differences in N-glycosylation of CD44 on activated B cells. Polymerase chain reaction analysis of amplified cDNA showed that each B cell population expressed a common dominant CD44 mRNA. These findings suggest that post-translational modification of CD44 and/or differential association of CD44 with other cellular components plays a critical role in activation-specific ligand binding by CD44.
T cell receptor-beta mRNA splicing during thymic maturation in vivo and in an inducible T cell clone in vitro.
Authors: Qian L , Vu M N , Carter M S , Doskow J , Wilkinson M F
Published: December 2022

The expression of TCR-beta mRNAs competent to encode functional V(D)JC beta proteins requires the activation of programmed DNA rearrangement events. It is not known whether other regulatory mechanisms control the steady-state levels of mature TCR-beta transcripts during thymic ontogeny. In this report, we demonstrate that TCR-beta pre-mRNAs accumulate in T cells, thus implicating RNA splicing as another potential level of regulation. Three methods were used to characterize the intron content of these pre-mRNA: Northern blot analysis, ribonuclease H mapping, and reverse transcription polymerase chain reaction analysis. Using these methods, we demonstrate that intron-containing TCR-beta transcripts derived from both the JC beta 1 and JC beta 2 loci accumulate in murine fetal and adult thymus. (VD)JC beta 1 pre-mRNAs that accumulate in the thymus possess unusually long poly(A) tails (> or = 300 nucleotides) and contain different combinations of four introns: the large intron between the J beta 1 and C beta 1 elements and the three introns within the C beta 1 element. The presence of an unusual transcript possessing IVS2C beta 1 at the 5' terminus suggests that cleavage of its splice acceptor is inefficient or negatively regulated. The profile of incompletely spliced TCR-beta transcripts present in the thymus in vivo is identical in intron content to those that we previously showed accumulate in the nucleus of the immature SL12.4 T lymphoma cell clone. An unstable negative regulatory protein may control TCR-beta expression in this cell clone because fully spliced TCR-beta transcripts are dramatically induced in the cytoplasm after treatment with any of five different protein synthesis inhibitors (cycloheximide, anisomyosin, emetine, puromycin, and pactamycin), all of which act by distinct mechanisms to inhibit protein synthesis.
A human monoclonal antibody specific for the N terminus of the hepatitis C virus nucleocapsid protein.
Authors: Cerino A , Boender P , La Monica N , Rosa C , Habets W , Mondelli M U
Published: December 2022

PBMC from a patient with chronic hepatitis C virus (HCV) infection were immortalized with EBV and plated by limiting dilution. Cultures secreting antibodies reactive in a commercial HCV II generation ELISA, which incorporates Ag derived from the nucleocapsid, NS3, and NS4 regions, were repeatedly cloned in the presence of feeder cells and growth factors. Of 23 initially immunoreactive cultures, only one cloned line, designated B12.F8, secreted HCV nucleoprotein-specific IgG1(kappa), whereas no reaction with recombinant polypeptides derived from NS3, NS4, and NS5 regions were documented. Human mAb (hmAb) B12.F8 was shown to recognize the native HCV nucleoprotein expressed in eukaryotic cells transfected with a core cDNA construct by immunofluorescence. The fine specificity of this hmAb was evaluated using synthetic oligopeptides covering the entire HCV nucleocapsid region. A weak but consistent reactivity was observed by PEPSCAN using a 12-mer encompassing residues 34-45 of the HCV-deduced amino acid sequence. Such weak reactivity is indicative for conformational epitopes and, in concurrence with this assumption, we found that longer peptides from the region containing residues 27-59 were more efficiently recognized and effectively inhibited binding of hmAb B12.F8 to recombinant nucleocapsid protein. Several overlapping immunoreactive fragments from the nucleocapsid region were selected from a random cDNA library consisting of DNase I fragments of recombinant core Ag. Best reactive recombinants were identified within residues 1-78 of the HCV sequence, in agreement with the results obtained using synthetic peptides. Comparative experiments on the fine specificity of sera from HCV-infected patients with anticore antibodies invariably showed recognition of peptides 8-40 and 27-59, as well as recombinant fragments spanning from residues 1 to 73, suggesting that hmAb B12.F8 identifies a major B cell epitope within the immunodominant nucleoprotein amino terminal subregion.
T cells are responsive to the simian virus 40 large tumor antigen transgenically expressed in pancreatic islets.
Authors: Geiger T , Soldevila G , Flavell R A
Published: December 2022

The qualities of a peripheral Ag that determine whether T cells will be tolerant of or responsive to it are poorly understood. To approach this problem, we studied the T cell response in a line of transgenic mice selectively expressing an oncoprotein in the islets of Langerhans. The SV40 large tumor Ag (SV40-T) is directed to islet beta-cells in Rip1-Tag3 (RT3) mice by a hybrid insulin promoter-SV40-T construct. Ag is first detected on these cells between 10 and 12 wk after birth. RT3 mice were bred with mice expressing a transgenic rearranged TCR recognizing SV40-T in the context of the class I MHC molecule, H-2Kk. T cell response in the resultant RT3/TCR-double transgenic mice was then analyzed. T cells are fully responsive to SV40-T in RT3/TCR-transgenic mice, and T cells infiltrate the islets of both RT3 and RT3/TCR-transgenic mice. This work demonstrates that T cells may remain responsive to self-Ag expressed outside the thymus, and that this responsiveness may result in autoimmunity. The developmentally delayed expression or the oncogenic nature of SV40-T in the RT3-transgenic mice may be important in determining this T cell response.
Evidence for a specific IgA receptor in rat and human mesangial cells.
Authors: Gómez-Guerrero C , González E , Egido J
Published: December 2022

Previous works have demonstrated that mesangial cells (MC) possess Fc receptors for IgG. We have investigated whether serum IgA could bind to MC in culture through a specific receptor. The binding of 125I-IgA (4 degrees C, 60 min) to MC was dose dependent and saturable, and the Scatchard analysis revealed a population of 1 x 10(5) binding sites per cell with a Ka of 7.8 x 10(8) M-1. This receptor was specific for IgA because unlabeled IgA and its Fc fragment were able to inhibit the binding, whereas IgG, IgM, and the IgA F(ab) fragment were not. Parallel experiments showed that human IgA bound in a similar manner both in a homologous system (human MC) and in heterologous systems (rat MC and peritoneal macrophages). In saturation studies using neuraminidase-desialylated IgA (desIgA), which has more carbohydrate residues exposed to binding, similar results to those of untreated IgA were obtained. Incubation with several carbohydrates decreased the IgA and desIgA binding to MC, obtaining maximal inhibition with simultaneous addition of galactose and N-acetyl-galactosamine. The possible IgA receptor (IgA-R) was purified from MC by affinity chromatography on an IgA-Sepharose column and appeared on SDS-PAGE gels as a 60-kDa band. The expression of IgA-R molecules on MC was enhanced by the presence of IgA in the culture medium. Finally, a band comparable with that of U937 cells was shown by Northern blot assay hybridizing mRNA from rat and human MC with a cDNA probe for the Fc alpha R. Taken together, these results strongly suggest the existence of a 60-kDa protein on the surface of MC that is able to bind IgA with high affinity.
Recruitment of inflammatory cells to the pleural space. Chemotactic cytokines, IL-8, and monocyte chemotactic peptide-1 in human pleural fluids.
Authors: Antony V B , Godbey S W , Kunkel S L , Hott J W , Hartman D L , Burdick M D , Strieter R M
Published: December 2022

Pleural effusions secondary to various diseases are associated with the presence of different inflammatory cells. The role of selective chemotactic cytokines in the recruitment of phagocytes to the pleural space is unclear. IL-8 and monocyte chemotactic peptide-1 (MCP-1) are recently described cytokines that are chemotactic for neutrophils and monocytes, respectively. We prospectively studied 63 patients, using strictly defined criteria for their selection. IL-8 concentrations were elevated in both empyema fluid (9.15 +/- 0.89 ng/ml) and parapneumonic effusions (4.7 +/- 0.697 ng/ml) when compared with pleural effusions secondary to other diseases. IL-8 levels were higher in empyema fluid than in parapneumonic effusions (p = 0.01). There was a significant correlation between IL-8 levels and the total numbers of neutrophils in empyema fluids (r = 0.80). Chemotactic activity for neutrophils was elevated in empyema fluid and the addition of IL-8 neutralizing serum decreased bioactivity by 32.22%. Malignant pleural effusions had the highest levels of MCP-1 (12.0 +/- 3.7 ng/ml) when compared with others. Cytology-positive pleural fluids (n = 10) had a higher level of MCP-1 than cytology-negative effusions (p = < 0.05). Malignant pleural fluid MCP-1 levels correlated (r = 0.70) with the absolute number of monocytes in the pleural fluid. Neutralization of monocyte chemotactic activity of malignant pleural fluid by specific neutralizing serum caused a 70.3% inhibition of bioactivity. Immunohistochemical staining of malignant pleural fluid localized antigenic MCP-1 to malignant cells. We conclude that both IL-8 and MCP-1 play major but not exclusive roles in the recruitment of neutrophils and monocytes from the vascular compartment to the pleural space.
Epitopes of myelin basic protein that trigger TGF-beta release after oral tolerization are distinct from encephalitogenic epitopes and mediate epitope-driven bystander suppression.
Authors: Miller A , al-Sabbagh A , Santos L M , Das M P , Weiner H L
Published: December 2022

We have been studying the suppression of experimental autoimmune encephalomyelitis in the Lewis rat after oral administration of myelin basic protein (MBP). Suppression is mediated by CD8+ T cells that adoptively transfer protection and suppress immune responses in vitro. This suppression is mediated by secretion of TGF-beta following triggering by the fed antigen. In the present study, we tested the ability of overlapping 20 amino acid peptides from MBP to trigger suppression mediated by spleen cells from Lewis rats orally tolerized to MBP. Using a transwell system, we found that spleen cells from MBP orally tolerized animals stimulated by residues 21-40, 51-70 and 101-120 of MBP suppress proliferative responses of an ovalbumin specific cell line. This suppression correlated with secretion of TGF-beta by cells stimulated with the peptide. In addition, T cells from animals fed the tolerogenic peptide 21-40 alone secreted TGF-beta whereas no TGF-beta release or in vitro suppression was observed in animals fed the MBP encephalitogenic determinant 71-90. The 71-90 peptide triggered proliferation of MBP primed cells from animals immunized with MBP/CFA whereas the suppressor epitopes identified above did not. Furthermore, oral administration of peptide 21-40 suppressed disease induced by peptide 71-90. DTH responses to 71-90 were not affected by oral administration of peptide 21-40 whereas DTH responses to whole MBP were suppressed. These results demonstrate that distinct suppressor determinants exist on MBP which are separate from encephalitogenic determinants, and that epitope-driven bystander suppression plays an important role in down-regulation of tissue specific autoimmune processes following oral tolerization. These findings have important implications for the design of tissue specific targeted immunotherapy by oral tolerization in humans.
Expression of 5HT1a receptors on activated human T cells. Regulation of cyclic AMP levels and T cell proliferation by 5-hydroxytryptamine.
Authors: Aune T M , McGrath K M , Sarr T , Bombara M P , Kelley K A
Published: December 2022

The neurotransmitter, serotonin (5-hydroxytryptamine, 5HT), has been shown to affect function of cells of the immune system. More recently, specific 5HT receptors have been identified and partially characterized on Jurkat cells. Results presented here characterize the receptor on Jurkat cells as the 5HT1a receptor subtype and show that mitogen-activated but not resting human T cells also express the 5HT1a receptor subtype. Analysis of mRNA in Jurkat cells and activated and resting T cells by PCR or by Northern analysis revealed the presence of 5HT1a receptor. Pharmacologic analysis of this receptor demonstrated that the receptors on Jurkat cells and activated T cells are similar to each other and that they resemble the 5HT1a receptor found in the brain. Analysis of the second messenger pathways activated by the 5HT1a receptor show that ligand-binding to the Jurkat cell 5HT1a receptor results in elevation of intracellular inositol phosphates and Ca2+ and that ligand binding to the 5HT1a receptor on activated T cells modulated intracellular levels of cAMP.
Characterization of the production of monocyte chemoattractant protein-1 and IL-8 in an allogeneic immune response.
Authors: Christensen P J , Rolfe M W , Standiford T J , Burdick M D , Toews G B , Strieter R M
Published: December 2022

Delayed-type hypersensitivity and allograft rejection are dependent upon the generation of Ag-specific T cell immune responses. The mixed lymphocyte reaction is a model of alloantigen-driven immunity and has provided significant insight into the mechanisms of T cell proliferation. Recently, soluble mediators, including TNF-alpha have been shown to be involved in the full expression of this response. In order to elucidate the potential mechanisms of cellular recruitment in the context of either delayed-type hypersensitivity or allograft rejection, we employed a mixed lymphocyte reaction to study the production of chemokines, monocyte chemoattractant protein-1 (MCP-1), and IL-8. Time-course experiments demonstrated that significant levels of MCP-1 and IL-8 were produced in allogeneic cultures as early as day 1, and that this relationship persisted over 6 days in culture. Northern blot analysis of chemokine mRNA confirmed the early induction of these genes in the allogeneic response. The levels of MCP-1 and IL-8 protein from mixed lymphocyte reaction supernatants as measured by specific ELISA were positively correlated with the proliferative response as measured by [3H]TdR uptake. Although significant MCP-1 and IL-8 were produced during a mixed lymphocyte reaction, these molecules did not participate in the proliferative response, as neutralizing antibodies to either MCP-1 or IL-8 had no effect on proliferation. In order to ascertain the mechanism for the induction of MCP-1 and IL-8 during the mixed lymphocyte reaction, experiments were performed in the presence of neutralizing anti-human TNF antibodies. Neutralization of TNF resulted in significant abrogation of MCP-1 and IL-8 production (75 +/- 5 and 87 +/- 2 percent, respectively). Cells isolated from the mixed lymphocyte reaction at day 6, demonstrated that MCP-1 and IL-8 Ag were localized to mononuclear phagocytes. These results demonstrate that potent chemokines, MCP-1 and IL-8, are produced during the evolution of a mixed lymphocyte reaction, and their induction is TNF-dependent.
IL-10 inhibits macrophage costimulatory activity by selectively inhibiting the up-regulation of B7 expression.
Authors: Ding L , Linsley P S , Huang L Y , Germain R N , Shevach E M
Published: December 2022

We have previously demonstrated that the inhibitory effects of IL-10 on ConA-induced T cell proliferation or IL-2 production by resting murine T cells were only observed when macrophages, but not when activated B cells, dendritic cells, or L cells, were used as accessory cells. To further elucidate the mechanism of action of IL-10 on the inhibition of macrophage costimulatory activity, we have used a system in which macrophages can develop into effective costimulator cells and the effect of IL-10 on this process can be studied in the absence of T cells. After fixation, resting macrophages have no costimulatory activity for soluble anti-CD3-induced T cell proliferation nor do they express the activation Ag B7/BB1. In contrast, macrophages activated by culture alone, or by culture with IFN gamma or LPS for 24 h, and then fixed, were effective accessory cells, expressed B7, and their costimulatory activity correlated with their level of cell surface B7 expression. Addition of IL-10 during the process of macrophage activation resulted in both a marked reduction in costimulatory activity and in B7 expression. IL-4 and transforming growth factor-beta that suppress many macrophage functions did not inhibit the induction of B7 expression. The inhibitory effect of IL-10 on the up-regulation of B7 was selective because the up-regulation of intercellular adhesion molecule-1 and MHC class II Ag was not affected. Direct evidence that the lack of B7 is the relevant limiting defect for IL-10-treated macrophage accessory cell function was obtained from studies in which the costimulatory capacity of IL-10-treated macrophages could be completely restored by the addition of B7 transfected, but not nontransfected, L cells to the assays.
IFN-gamma inhibits the replication of Listeria monocytogenes in hepatocytes.
Authors: Gregory S H , Wing E J
Published: December 2022

We previously reported that the bulk of Listeria monocytogenes injected intravenously into mice is taken up in the liver and replicates within the parenchymal cells (hepatocytes). Although IFN-gamma is known to play an important role in host defenses to listerial infections of the liver, the mechanism(s) that underlies this role remains to be fully delineated. In the initial experiments presented here, we demonstrated the elevated expression of IFN-gamma message in the livers of mice during primary listerial infections. Subsequent experiments showed that the listerial burden of the hepatocyte population was increased significantly in mice administered monoclonal anti-IFN-gamma. Conversely, the administration of murine rIFN-gamma resulted in a marked (2 log10) decrease in the number of hepatocyte-associated Listeria. In vitro, IFN-gamma stimulated the listericidal activity of purified hepatocytes. Infected hepatocytes incubated in the presence of > 0.1 U/ml murine rIFN-gamma exhibited a significant reduction in intracellular Listeria. The elevated antilisterial activity of IFN-gamma-treated hepatocytes in culture was abrogated by the presence of compounds that scavenged or inhibited the production of reactive oxygen intermediates. Taken together, these findings suggest that activation of the oxygen-dependent, antimicrobial activity of hepatocytes may constitute a principal effector function of IFN-gamma in host defenses to listerial infections of the liver.
Detection of shared MHC-restricted human melanoma antigens after vaccinia virus-mediated transduction of genes coding for HLA.
Authors: O'Neil B H , Kawakami Y , Restifo N P , Bennink J R , Yewdell J W , Rosenberg S A
Published: December 2022

To detect shared human melanoma Ag that are recognized by HLA-A2 restricted, melanoma-specific CTL derived from tumor infiltrating lymphocytes, we have developed a convenient method to insert and express foreign HLA genes capable of presenting Ag on target cell lines. Seventeen melanoma cell lines and 11 nonmelanoma cell lines were infected with recombinant vaccinia virus containing the HLA-A2.1 gene. Infection by the vaccinia virus resulted in expression of functional HLA-A2 molecules on the cell surface of virtually 100% of infected cells within a 3.5-h period. The results showed that 11 of 17 (65%) naturally HLA-A2- melanoma cell lines were specifically lysed by the HLA-A2-restricted, melanoma-specific TIL after infection with the vaccinia-HLA-A2.1 virus. None of the nine human nonmelanoma cell lines tested (three colon cancer, four breast cancer, or two immortalized non-tumor cell lines) or two murine melanoma cell lines were lysed by the HLA-A2-restricted TIL after vaccinia-HLA-A2.1 infection. Coinfection of the vaccinia virus containing the beta 2-microglobulin gene with the vaccinia-HLA-A2.1 virus increased the surface expression of HLA-A2 and subsequent lysis by melanoma-specific tumor infiltrating lymphocytes. With this new method we could extend previous findings demonstrating that shared melanoma Ag recognized by HLA-A2-restricted tumor infiltrating lymphocytes exist among melanoma cells from different patients regardless of HLA type. These Ag represent excellent candidates for the development of vaccines to induce T cell responses for the immunotherapy of patients with melanoma.
Recombinant soluble CD14 mediates the activation of endothelial cells by lipopolysaccharide.
Authors: Haziot A , Rong G W , Silver J , Goyert S M
Published: December 2022

Recent studies have suggested that soluble CD14 found in serum is involved in the LPS-induced activation of endothelial cells (EC). To more fully investigate the relevance of sCD14 to LPS-induced activation of EC, we have used recombinant soluble CD14 (rsCD14) and have examined, under serum-free conditions, its role in the LPS-induced EC response in the presence of LPS alone as well as in the presence of LPS-binding protein. Our studies show that EC can be activated by high concentrations of LPS in the presence of rsCD14 alone. However, at low concentrations of LPS (5 and 10 ng/ml), the rsCD14-stimulated activation is strongly enhanced by LPS-binding protein. In addition, we show that LPS binds to rsCD14 directly; in the presence of low concentrations of LPS this binding is enhanced by the presence of LPS-binding protein. These results show that while the membrane form of CD14 can function as a receptor, its soluble form can function as a co-ligand with LPS in the EC-LPS response.
Transendothelial migration activity of lymphokine-activated killer (LAK) cells.
Authors: Kitayama J , Juji T , Atomi Y , Kuroda A , Muto T , Kobayashi M , Mitsui Y , Minami M
Published: December 2022

With an in vitro static system using HUVEC (human umbilical vein-derived endothelial cells) cultured on type I collagen gel, we investigated the transendothelial migration activities of I1-2 activated killer (LAK) cells. Our results indicate that in comparison with unstimulated T cells, LAK cells exhibit strong transendothelial migration activity, as well as increased adhesiveness to HUVEC. Pretreatment of HUVEC for 24 h with rINF-gamma, rTNF-alpha and rIL-1 beta enhanced the LAK cell migration. The increase in the percentage of migration of LAK cells was greater than that of the percentage of adhesion but significantly less than the increase in the percentage of migration of resting T cells. The results of blocking studies using mAb strongly suggest that the enhanced migration of LAK cell was probably attributed to nonspecifically increased binding to HUVEC and markedly enhanced chemokinetic activity that was dependent primarily on the LFA-1 molecule. Among LAK cells, there were considerable differences in the migration activities of the various phenotypes. CD8+T-LAK migrated preferentially to CD4+T-LAK. CD16+ NK-LAK showed increased adhesion but somewhat decreased migration activities. However, rINF-gamma treatment of HUVEC for 24 h promoted vigorous migration of CD16+ NK-LAK, which suggests that endothelium regulate the migration of LAK cells. Based on these observations, we proposed that LAK cells, if transferred into tumor feeding vessels, can migrate into tumor tissue in considerable numbers and efficiently make contact with individual tumor cells to produce preferable clinical effects.
T-cell receptor V region beta-chain gene expression in the autoimmune thyroiditis of non-obese diabetic mice.
Authors: Matsuoka N , Bernard N , Concepcion E S , Graves P N , Ben-Nun A , Davies T F
Published: December 2022

The non-obese diabetic (NOD) mouse develops both a spontaneous T-cell-mediated autoimmune insulitis and, in addition, a well characterized thyroiditis. We have examined the repertoire of murine T-cell receptor (TCR) variable (V) beta-chain genes used by intrathyroidal T cells with specific oligonucleotides that amplified 17 murine V beta gene families in cDNA samples prepared from intact NOD thyroid tissues. Normal NOD thyroid tissue contained only low levels of TCR V gene mRNA. In contrast, NOD mice with histologic thyroiditis showed the marked expression of up to 3 TCR V beta genes consistent with a restricted T-cell invasion. Sequencing of amplified TCR V beta cDNA showed that within each NOD thyroid sample at least one of the overexpressed V beta gene families was clonally expanded. However, the clonally expanded T-cell V gene family was not consistent in all animals. Even within the same TCR V beta gene families, various D and J segments had been rearranged with open reading frames and together with insertions and deletions gave no significant homology at the nucleotide or amino acid level. In summary, these data showed that the intrathyroidal T-cell infiltrate in NOD mice was markedly biased towards the use of a single, but variable, TCR V gene family within each animal. It also appeared that the choice of the TCR V beta chain determined the intrathyroidal infiltrative process rather than the choice of D and/or J regions. However, there was no consistent use of a single TCR V beta chain. As thyroiditis does not occur uniformly in apparently genetically homogeneous animals, reared under similar environmental conditions, it may not be surprising that different TCR V genes are involved in different animals.
Circulating soluble CR1 (CD35). Serum levels in diseases and evidence for its release by human leukocytes.
Authors: Pascual M , Duchosal M A , Steiger G , Giostra E , Pechère A , Paccaud J P , Danielsson C , Schifferli J A
Published: December 2022

C receptor type 1 (CR1, CD35) is present in a soluble form in plasma (sCR1). Soluble CR1 was measured with a specific ELISA assay in normal individuals and in patients with different diseases. The mean serum concentration of sCR1 in 31 normal donors was 31.4 +/- 7.8 ng/ml, and was identical in plasma. An increase in sCR1 was observed in 36 patients with end-stage renal failure on dialysis (54.8 +/- 11.7 ng/ml, p < 0.0001), and in 22 patients with liver cirrhosis (158.3 +/- 49.9 ng/ml, p < 0.0001). The mean sCR1 levels dropped from 181 +/- 62.7 to 52.1 +/- 24.0 ng/ml (p < 0.001) in nine patients who underwent liver transplantation, and was 33.5 +/- 7.3 in 10 patients with functioning renal grafts, indicating that the increase in sCR1 was reversible. Soluble CR1 was elevated in some hematologic malignancies (> 47 ng/ml), which included B cell lymphoma (12/19 patients), Hodgkin's lymphoma (4/4), and chronic myeloproliferative syndromes (4/5). By contrast, no increase was observed in acute myeloid or lymphoblastic leukemia (10) or myeloma (5). In two patients with chronic myeloproliferative syndromes, sCR1 decreased rapidly after chemotherapy. The mean concentration of sCR1 was not significantly modified in 181 HIV-infected patients at various stages of the disease (34.8 +/- 14.4 ng/ml), and in 13 patients with active SLE (38.3 +/- 19.6 ng/ml), although in both groups the number of CR1 was diminished on E. There was a weak but significant correlation between sCR1 and CR1 per E in HIV infection and SLE (r = 0.39, p < 0.0001, and r = 0.60, p < 0.03 respectively). In vitro, monocytes, lymphocytes, and neutrophils were found to release sCR1 into culture supernatants. In vivo, sCR1 was detected in the serum of SCID mice populated with human peripheral blood leukocytes. The sCR1 levels correlated with those of human IgG (r = 0.97, p < 0.0001), suggesting synthesis of sCR1 by the transferred lymphocytes. The mechanisms underlining the increased levels of sCR1 and its biologic consequences remain to be defined.
Rapid Il-2-induced adherence of human natural killer cells. Expression of mRNA for cytokines and IL-2 receptors in adherent NK cells.
Authors: Vitolo D , Vujanovic N L , Rabinowich H , Schlesinger M , Herberman R B , Whiteside T L
Published: December 2022

Natural killer (NK) cells selected by IL-2-induced rapid adherence to plastic and called A-NK cells represent a phenotypically and functionally distinct subset of mature peripheral blood NK cells. To further characterize this subset of NK cells functionally, their potential to express mRNA for the IL-2R and various cytokines after IL-2 activation was examined. Highly purified normal human peripheral blood resting NK (R-NK) cells were obtained by negative immunoselection using OKT3 mAb and magnetic beads coated with goat anti-mouse Ig. By two-color flow cytometry, > 90% of these R-NK cells were either CD3-CD56+CD16+ or - or CD3-CD56-CD16+. R-NK cells were activated in the presence of 6000 IU/ml (22 nM) of IL-2 for different periods of time. After 1, 3, 5, or 24 h, plastic-adherent (A) and nonadherent (NA) NK cells were separated and compared for the expression of the IL-2R or cytokine mRNA by in situ hybridization, using 35[S]-cDNA probes. Only low proportions of R-NK cells expressed genes for IL-2Rp55 (16%) or cytokines IL-2 (20%), IFN-gamma (18%), TNF-alpha (16%), and TGF-beta (7%). Thus, the genes for the IL-2Rp55 and these cytokines were not constitutively expressed by most human R-NK cells, and there was no indication that the NK cells used in these experiments were activated in vivo or during the purification procedure. However, larger proportions of R-NK cells showed expression of mRNA for IL-1-beta (35%) and IL-6 (40%), which indicates that genes for these cytokines may be constitutively expressed in a substantial proportion of normal human circulating NK cells. When R-NK cells were incubated in the presence of 22 nM of IL-2 for 1 to 24 h and separated into A-NK cells and NA-NK cells, a large proportion of A-NK cells became positive for IL-2R and cytokine gene expression. In contrast, the proportion of mRNA-positive NA-NK cells was similar or lower than that observed for R-NK cells, with the exception of an increase in TGF-beta.(ABSTRACT TRUNCATED AT 400 WORDS)
Role of the Q10 class I regulatory element region 1 in controlling tissue-specific expression in vivo.
Authors: Marine J B , Shirakata Y , Wadsworth S A , Hooley J J , Handy D E , Coligan J E
Published: December 2022

The MHC class I regulatory element (CRE) region 1 has been previously described as a positive cis-acting regulatory element essential for class I gene expression. We have generated transgenic mice (CBA x C57BL/6) with the MHC class I gene H-2Dd driven by two different 400-bp promoter regions of Q10, a nonpolymorphic MHC class I gene expressed in the liver, kidney, and fetal yolk sac. One transgene contained the wild-type Q10 promoter (Q10WT/Dd). The second construct (Q10M3/Dd) had 2 bp substitutions introduced in region 1 of the CRE that reconstituted the CRE inverted repeat present in classical class I genes. Mice containing the wild-type Q10/Dd gene expressed membrane-bound H-2Dd molecules in a tissue-restricted expression pattern similar to that observed for endogenous Q10. In mice containing the mutant construct (Q10M3/Dd), H-2Dd was also expressed in the thymus, a tissue not normally associated with Q10 expression but, surprisingly, the Dd was not expressed in other lymphoid tissues. Furthermore, thymic expression was greatest on double positive (CD4+ CD8+) thymocytes. Thymic Dd expression was correlated with the presence of what appears to be a previously unidentified transcription factor in thymocytes that is capable of interacting with the CRE-inverted repeat. These results show that the mutations in region 1 altered the tissue-specific regulation of the Q10 promoter in vivo, although an intact inverted repeat did not restore the ubiquitous pattern of expression characteristic of classical class I genes. Thus, these results indicate that elements in addition to CRE region 1 in the Q10 promoter region serve to limit ubiquitous tissue expression.