bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure of the VH and VL segments of monoreactive and polyreactive IgA autoantibodies to DNA in patients with systemic lupus erythematosus.
Authors: Kasaian M T , Ikematsu H , Balow J E , Casali P
Published: December 2022

Anti-DNA IgA autoantibodies play an important immunopathologic role in SLE patients. To analyze the cellular origin and the VH and VL structure of anti-DNA IgA autoantibodies, we generated five IgA1 mAbs to DNA using B lymphocytes from three SLE patients. Two mAbs bound to ssDNA only and one to both ssDNA and dsDNA (monoreactive antibodies). The remaining two mAbs bound to DNA (one to ssDNA and the other to both ssDNA and dsDNA) and to other self and foreign Ag (polyreactive antibodies). The IgA mAb relative avidity for DNA ranged from 7.5 x 10(-8) to 8.0 x 10(-10) g/microliters. The anti-DNA IgA mAb used VH segments of the VHI(VI-3b), VHII (VH2-MC2), VHIII (WHG16G and VH26c), and VHIV (V71-2) families in conjunction with V kappa I, V kappa IIIb, or V lambda I segments. All IgA mAb VH segments were juxtaposed with JH4b segments. The heavy chain CDR3 sequences were divergent in composition and length. When compared with those of the closest reported germ line genes, the IgA mAb VH and VL gene sequences displayed a number of differences. That these differences represented somatic point mutations was formally proved in both the monoreactive IgA mAb 412.67.F1.3 and the polyreactive IgA mAb 412.66.F1 VH segments by differential PCR amplification and cloning and sequencing of genomic DNA from the mAb-producing cell lines and autologous polymorphonuclear cells. The sequences of the germ line genes that putatively gave rise to the mAb 412.67.F1.3 and mAb 412.66.F1 VH segments were identical with those of the WHG16G and VH26c genes, respectively. In not only the monoreactive mAb 412.67.F1.3 but also the polyreactive mAb 412.66.F1 and mAb 448.9G.F1 VH segments, the higher concentration of replacement (R) mutations and the higher R:S (silent) mutation ratios in the complementarity-determining region (infinity; 19:0) than in the framework region (1.0) (p = 0.00001, chi 2 test) were highly consistent with selection by Ag. In the five IgA mAb VH and VL segments, the putative and verified somatic point mutations yielded 68 amino acid replacements, of which 38 were nonconserved. Twenty of these yielded positively charged or polar residues that play a major role in DNA binding, including seven Arg, five Lys, three Tyr, two Gln, two His, and a Thr. The conserved amino acid changes included seven Asn. These findings suggest that anti-DNA IgA autoantibodies use a broad selection of VH and VL genes and enhance their fit for Ag by undergoing somatic hypermutation and Ag selection.(ABSTRACT TRUNCATED AT 400 WORDS)
Induction of circulating antagonists to IL-1 and TNF by IL-2 administration and their effects on IL-2 induced cytokine production in vitro.
Authors: Tilg H , Shapiro L , Vannier E , Poutsiaka D D , Trehu E , Atkins M B , Dinarello C A , Mier J W
Published: December 2022

The objectives of this study were to determine whether intensive immunotherapy with IL-2 results in detectable levels of circulating IL-1 and TNF antagonists and whether the levels achieved in vivo are sufficient to affect the generation of secondary proinflammatory cytokines such as IL-1 beta and TNF-alpha. We also sought to determine the extent to which endogenous TNF mediates the generation of an IL-1 antagonist by IL-2-activated PBMCs. In patients undergoing high dose IL-2 immunotherapy, plasma IL-1 receptor antagonist (IL-1ra) levels rose dramatically after the first IL-2 injection, reaching a plateau of 11.03 +/- 0.92 ng/ml within 4 h. TNF-soluble receptor p55 (TNFsRp55) was also detected in the plasma shortly after initiating treatment, and the levels progressively increased throughout the treatment course. PBMCs exposed to IL-2 expressed IL-1ra mRNA and secreted the IL-1ra protein, but neither PBMCs nor neutrophils shed TNFsRp55 in response to IL-2 or supernatants from IL-2-activated PBMCs. IL-1ra at concentrations achieved in the plasma during IL-2 immunotherapy (approximately 10 ng/ml) inhibited the in vitro production of IL-1 beta and TNF-alpha by IL-2-activated PBMCs by 65% and 30%, respectively. Although the monomeric receptor TNFsRp55 at concentrations achieved in the plasma had no effect on the in vitro production of IL-1ra, TNF-alpha, or IL-1 beta, the bivalent TNFsRp75-Fc chimera suppressed the generation of TNF and IL-1. IL-1ra synthesis was unaffected. These results suggest that the amount of IL-1ra generated in response to IL-2 is most likely sufficient to down-modulate the production of proinflammatory cytokines.
Clustering of class I HLA molecules on the surfaces of activated and transformed human cells.
Authors: Matko J , Bushkin Y , Wei T , Edidin M
Published: December 2022

We surveyed cells for the clusters of class I HLA molecules, HLA-I, which we have previously found on JY lymphoblasts. Two fluorescence techniques, fluorescence resonance energy transfer and electron exchange quenching, detected clustered HLA-I molecules on activated normal B and T cells, on cells of B and T lymphoblast lines, and on transformed fibroblasts. No HLA-I clusters were detectable in the surfaces of resting B or T cells or normal fibroblasts. HLA clustering correlates perfectly with the presence of the HC-10 epitope of beta 2-microglobulin (beta 2m)-free heavy chains at the cell surface although not with the amount of this epitope expressed. Clustering was reversed by exogenous beta 2m, but this did not change the amount of HC-10 bound. This suggests that a form of beta 2m-free heavy chain in equilibrium with both native HLA molecules and fully denatured HC-10-positive heavy chains is involved in HLA-I cluster formation.
Cyclic AMP inhibits phosphatidylinositol-coupled and -uncoupled mitogenic signals in T lymphocytes. Evidence that cAMP alters PKC-induced transcription regulation of members of the jun and fos family of genes.
Authors: Tamir A , Isakov N
Published: December 2022

T lymphocyte stimulation via the Ag receptor results in activation of phospholipase C gamma 1 that catalyses the hydrolysis of phosphatidylinositol (PI). The hydrolysis generates inositol phosphate and diacylglycerol, which in turn, increase intracellular Ca2+ concentration and activates protein kinase C, respectively. Agonists operating via the adenylate cyclase pathway or cell permeable cAMP analogues inhibit T cell activation by interfering with the PI-turnover. We have shown that dbcAMP inhibits PI-independent mitogenic signals in T cells after stimulation with TPA plus ionomycin. dbcAMP inhibited the TPA plus ionomycin-induced transcription of IL-2 and IL-2R genes in EL4 cells, suggesting interference with biochemic events downstream to PI hydrolysis and upstream to transcription of early activation genes. Because many of the early genes operating in T cell mitogenesis possess a TPA-response element (TRE) in their promoter region, we tested the effect of cAMP on the TRE-binding protein, TPA-response element (TRE) in their promoter region, we tested the effect of cAMP on the TRE-binding protein, AP-1. dbcAMP increased the binding activity of nuclear proteins consisting of Fos:Jun heterodimers to a TRE-containing oligonucleotide, but altered the composition of Jun proteins in the AP-1. Furthermore, the TPA plus ionomycin-induced transcription program of members of the jun and fos family of genes was altered by dbcAMP, suggesting that inhibition of T cell proliferation by dbcAMP is a consequence of intervention in transcriptional regulation by TRE-binding proteins.
Recognition of a single amino acid change on the surface of a major transplantation antigen is in the context of self peptide.
Authors: Pullen J K , Tallquist M D , Melvold R W , Pease L R
Published: December 2022

The transcripts encoding two strongly alloantigenic class I mutant molecules, Kdm4 and Kdm5, were characterized and found to encode products that differ from the parental Kd glycoprotein by single amino acid substitutions. The Kdm4 molecule has an amino acid change at position 114, an integral component of a beta-sheet associated with pockets D and E of the peptide binding site. The basis for strong alloantigenicity of the variant molecule can be attributed to differences in peptide binding that were visualized by HPLC analysis of eluted peptides. In contrast, the Kdm5 molecule differs from the parent at position 158, a component of the alpha-helix that is not associated with any of the pockets of the peptide binding site. No differences in peptide binding by Kdm5 in comparison with the parent Kd molecule were seen by HPLC, suggesting that the variant and parent molecules bind the same set of peptides. The ability of (dm4 x dm5) F1 hybrid mice to recognize and lyse BALB/c stimulator cells indicates that the alloantigenic properties determined by the 158 substitution result from the interactions of the alpha-helix regions (changed in dm5) with the pockets of the binding site (changed in dm4). We conclude that self peptides shared by the F1 hybrid and the BALB/c stimulator cells are recognized in the context of structural features of the helices of the Ag-presenting molecule as alloantigenic determinants.
Induction of IL-8 gene expression in human polymorphonuclear neutrophils by recombinant IL-2.
Authors: Wei S , Liu J H , Blanchard D K , Djeu J Y
Published: December 2022

Induction of IL-8 gene expression was investigated in IL-2-stimulated circulating peripheral blood polymorphonuclear neutrophils (PMN). Brief exposure of normal PMN to human rIL-2 enhanced both transcriptional and translational expression of IL-8. The IL-8 mRNA was first detectable by 3 h, followed by a continuous maintenance of high mRNA levels up to 18 h. Maximal transcription was obtained with 1000 U/ml of IL-2, which achieved the level observed with known neutrophil-activating factors such as granulocyte macrophage-CSF and Candida albicans. The protein synthesis inhibitor, cycloheximide, had no detectable effect on levels of IL-8 mRNA expression in PMN incubated in medium alone; however, cycloheximide could selectively modulate IL-8 mRNA transcription in PMN, depending on the cytokine used. Cycloheximide did not affect or alter IL-8 mRNA induction in IL-2-treated PMN but abrogated it in granulocyte macrophage-CSF-treated PMN and super-induced the level of IL-8 mRNA in C. albicans-treated PMN. Of significance was the observation that IL-2 has no direct chemotactic effect on PMN, whereas the cell-free supernatants from IL-2-stimulated PMN show potent chemotaxis for freshly isolated PMN, which can be specifically blocked by anti-IL-8 Abs. These findings suggested that the induction of IL-8 gene expression in PMN by IL-2 may be involved in the recruitment of PMN into tissues during local IL-2 therapy in human cancer and in part contribute to tumor rejection.
Regulatory mechanisms for the expression of type IV collagenases/gelatinases in murine macrophages.
Authors: Xie B , Dong Z , Fidler I J
Published: December 2022

The purpose of this study was to determine the regulation of type IV collagenase expression in murine peritoneal macrophages (PEM) after they are incubated with LPS. LPS stimulated the production of the latent forms of 92-kDa (MMP-9) and 72-kDa (MMP-2) type IV gelatinases in a dose-dependent (> 10 ng/ml) and serum-dependent manner. Time course analyses revealed that LPS regulated the expression of MMP-9 and MMP-2 via discordant kinetics. Prolonged treatment of PEM with LPS decreased MMP-9 but not MMP-2 activities. IFN-gamma decreased the production of both gelatinases by PEM responding to LPS. TGF-beta stimulated production of both matrix metalloproteinases but blocked the LPS-mediated secretion of MMP-9. LPS-stimulated MMP-9 production was suppressed by genistein and tyrphostin, two specific tyrosine kinase inhibitors, as well as H-7, a serine/threonine protein kinase inhibitor, but not by HA1004, a relatively selective inhibitor for PKA and PKG. Our data demonstrate that the secretion of MMP-2 and MMP-9 by murine PEM is differentially regulated, suggesting a distinct in vivo role for these two otherwise analogous type IV gelatinases in macrophage-mediated connective tissue destruction at sites of immunologic challenges.
Recombinant and native zymogen forms of human complement factor D.
Authors: Yamauchi Y , Stevens J W , Macon K J , Volanakis J E
Published: December 2022

We have expressed a full-length cDNA clone encoding human factor D by using a baculovirus expression system. The purified recombinant protein reacted with Ab against native factor D, but was hemolytically inactive and slightly larger than factor D. These results suggested that the recombinant protein was the elusive zymogen of factor D. Amino acid sequencing demonstrated that the recombinant factor D consisted of two proenzyme forms with respective activation peptides, AAPPRGR and APPRGR. Catalytic amounts of trypsin converted recombinant profactor D to its enzymatically active form, exhibiting SDS-PAGE mobility and specific hemolytic activity similar to those of native factor D. About 90% of trypsin-activated recombinant profactor D had the same NH2-terminus as factor D. Human thrombin, kallikrein, and plasmin could also activate recombinant profactor D, but relatively high concentrations of these enzymes were required and the specific hemolytic activity of the "activated" profactor D was about one-third that of native factor D. Trypsin-activatable profactor D was also purified from the urine of a patient with Fanconi's syndrome. This native profactor D represented less than 1.0% of the total antigenic factor D in the patient's urine and had a Gly-Arg dipeptide as the activation peptide. Apparently, urine profactor D was produced by cleavage of pre-profactor D at Arg-(-3) by a serine protease with trypsin-like specificity, which probably is different from the putative leader peptidase that produces the recombinant profactor D. Urine profactor D was inhibited by diisopropyl fluorophosphate although the recombinant proenzyme was resistant to this inhibitor.
Specific down-regulation of proliferative T cell alloresponsiveness by interference with CD2/LFA-3 and LFA-1/ICAM-1 in vitro.
Authors: Böhmig G A , Kovarik J , Holter W , Pohanka E , Zlabinger G J
Published: December 2022

T cell activation requires Ag contact with the TCR in the presence of costimulatory signals provided by APCs. When Ag is presented without costimulation, T cells are functionally inactivated. Here we demonstrate that interference with distinct adhesion molecules during Ag contact using mAbs leads to Ag-specific functional inactivation of alloreactive T cells. We found that the presence of a mixture of mAbs specific for CD2, LFA-3, LFA-1 alpha- and beta-chain, and ICAM-1 during primary MLC leads to down-regulation of secondary proliferative responses to Ag from the donor used for priming. Cells from pretreated cultures proliferated well, however, when stimulated with Ag from third party donors, mitogens, or mitogenic CD3 mAb. Because specific reactivity could be restored by addition of IL-2 to restimulation cultures, altered secondary responsiveness appeared to be caused by anergy and not by elimination of specific clones. Furthermore, specific down-regulation of alloresponsiveness was prevented by addition of IL-2 to primary cultures in the presence of mAb. Interference by mAb with either CD2/LFA-3, LFA-1/ICAM-1, CD2/LFA-1, or LFA-3/ICAM-1 had a substantial, though less pronounced effect on secondary responsiveness. After pretreatment with the Ab mixture, CTL generation was substantially but incompletely down-regulated against the original and third party donors. Because a decrease in the reactivity of unstimulated responders by culture was also observed, these findings might be explained by the loss of cytotoxic precursors after culturing under nonstimulating conditions. In conclusion, our data demonstrate that T cells enter a state of anergy when T cell activation is modulated by simultaneous interference with distinct adhesion molecules during Ag contact, which thus might reflect at least partly overlapping functions of particular receptor-ligand pairs in T cell costimulation.
Anti-CD2 mAbs suppress cytotoxic lymphocyte activity by the generation of Th2 suppressor cells and receptor blockade.
Authors: Chavin K D , Qin L , Yon R , Lin J , Yagita H , Bromberg J S
Published: December 2022

The mechanism by which anti-CD2 mAbs inhibit hapten-specific and alloantigen specific CTL was explored. In vivo administration of alpha-CD2 mAbs resulted in 80 to 100% inhibition of alloantigen specific CTLs. Mixing cells from control animals with cells from alpha-CD2-treated groups demonstrated transferable suppression of CTL (40-67% suppression). These suppressor cells were CD4+CD8- and associated with increased IL-4 and TGF-beta in culture as compared with controls. Anti-CD2 mAbs added at the initiation of culture resulted in 60 to 72% inhibition of trinitrophenyl-CTL, whereas mAbs added at the time of assay resulted in less than 50% inhibition of trinitrophenyl-CTLs. F(ab')2 and Fab alpha-CD2 produced inhibition similar to intact mAbs when added at the time of the lytic assay, whereas both produced only modest inhibition in vivo or when added at the initiation of culture. Alloantigen-specific CTLs were not affected by Ab addition to either culture or assay. The immunosuppressive effects were generalizable because a panel of alpha-CD2 mAbs were all comparably effective in suppressing hapten-specific CTLs when administered in vivo. The results demonstrate that the inhibitory effects are the result of blockade of receptor adhesion function during Ag priming or target recognition, Fc-related effects, and the generation of a negative regulatory, CD4+CD8-, IL-4- and TGF-beta-producing TH2 suppressor T cell.
TCR cross-linking induces CTL death via internal action of TNF.
Authors: Wang J , Stohlman S A , Dennert G
Published: December 2022

TCR-mediated stimulation can result in either activation or apoptosis, raising the question of what the mechanisms of these opposing effects might be. Here we demonstrate that microinjection of Ab specific for TNF inhibits activation-induced cell death, implicating TNF in anti-CD3-induced apoptosis. Induction of CTL in the presence of antisense TNF oligodeoxynucleotides inhibits TNF secretion and cell death after stimulation with anti-CD3. Incubation of cells with IL-2 for 48 h before stimulation protects from anti-CD3-induced death, whereas the presence of IL-2 during anti-CD3 treatment is without effect.
The regulatory complex of HLA class I promoters exhibits locus-specific conservation with limited allelic variation.
Authors: Cereb N , Yang S Y
Published: December 2022

The extensive genetic polymorphism of the classical MHC class I molecules provides an important distinguishing feature of the host's immune system. They influence the selection and function of effector cells against tumor and virally infected cells. In these cells, class I molecules are often selectively suppressed. This suppression is locus specific and, in certain cases, allele specific. We analyzed the HLA class I promoter sequences that include class I regulatory complex (CRC) in a total of 41 well-characterized HLA homozygous B lymphoblastoid cell lines, using locus-specific oligonucleotide probes complementary to the overlapping CRC elements. These include kappa B1, kappa B2, IFN response sequence, the putative negative regulatory element, and the HLA counterpart of the H-2RII region that contains the retinoid x receptor beta binding motif. The CRC of HLA promoters displayed locus-specific conservation; however, limited allelic variation was observed in each of the cis elements. In some, variations were apparently generated by gene conversion. The palindromic kappa B1 site, which has an active role in enhancing promoter activity, was found to be conserved in almost all HLA-A and -B alleles, but not in HLA-C. The core DNA binding motif for retinoid x receptor beta was absent within the HLA CRC region. Sequence analysis of promoters from HLA-A31, -B13, and -Cw1 genomic clones, as well as pairwise interallelic and intra-allelic comparison with those of published alleles, showed that locus-specific conservation extended throughout the promoter sequences. Locus specificity and the allelic variation seen in the CRC regions may provide a structural basis for the selective modulation of HLA class I genes.
CD3 delta enhancer. CREB interferes with the function of a murine CD3-delta A binding factor (M delta AF).
Authors: Gupta A , Terhorst C
Published: December 2022

Tissue-specific expression of the murine TCR/CD3-delta gene is regulated by multiple factors. Earlier, we reported that this gene has a tissue-specific enhancer at its 3' end that consists of two cis-acting elements, M delta A and M delta B. This study demonstrates that at least two independent factors bind to the 22 nucleotide long M delta A element. A T cell-specific factor termed M delta AF recognizes a 10-bp sequence TTCCATGACA, whereas the cAMP response element binding protein (CREB) recognizes an overlapping sequence TGACATCA. In vitro studies indicated that the 135-kDa M delta AF and the 43-kDa CREB competed for binding to the M delta A element. To test this competition in vivo, murine T cells (EL4) were cotransfected with a CREB expression plasmid and a reporter plasmid containing the M delta A element. The presence of an excess of CREB protein in the cell inhibited the CAT activity completely. This inhibitory effect of CREB could be overcome by cotransfection of an additional vector containing the somatostatin CRE site that prevented the overexpressed CREB protein from binding to the M delta A element. It is possible that the murine CD3-delta gene in T cells is regulated by the relative levels of the T cell-specific DNA binding protein M delta AF and the ubiquitous CREB in conjunction with other factors.
CD8 cells play a critical role in delayed type hypersensitivity to intact Cryptococcus neoformans.
Authors: Mody C H , Paine R , Jackson C , Chen G H , Toews G B
Published: December 2022

Although cell-mediated immunity is critical for optimal host defense to C. neoformans, the role of T lymphocyte subsets is complex and poorly understood. CD8 cells are important both for optimal host defense against C. neoformans, and for expression of delayed type hypersensitivity (DTH). Because host defense correlates with the ability to mount a DTH response to C. neoformans, the current studies were performed to determine the mechanism by which CD8 cells participate in DTH. Mice were immunized by the intratracheal route with live C. neoformans, or by the subcutaneous route with heat-killed C. neoformans. Mice were depleted of CD8 cells in vivo by administration of mAb. After challenge with soluble cryptococcal Ag, the DTH response was quantified as footpad swelling. We found that mice depleted of CD8 cells before immunization were unable to express DTH. Mice depleted of CD8 cells after immunization but before challenge also were unable to express DTH. Splenocytes of mice depleted of CD8 cells in vivo, before immunization, failed to transfer DTH to naive, undepleted mice. Immune splenocytes depleted of CD8 cells in vitro also failed to transfer DTH to naive, undepleted mice. These data indicate that CD8 cells were necessary during the challenge and immunizing phases of DTH, and were necessary for expression of DTH. However, CD8 cell depletion did not abrogate DTH in mice immunized with either soluble cryptococcal Ag in complete Freund's adjuvant, or sheep red blood cells, which are mediated by CD4 cells. These data suggest that CD8 cells play a critical role in the cell-mediated immune response to C. neoformans. Based on this information, it may be possible to protect hosts with deficiencies of CD4 cells, such as in AIDS, by designing immunizing strategies for stimulating CD8 cells.
Complement-inhibiting activities of human CD59 and analogues from rat, sheep, and pig are not homologously restricted.
Authors: van den Berg C W , Morgan B P
Published: December 2022

Human erythrocyte CD59 and analogues isolated from erythrocytes of rat, sheep, and pig were examined for their ability to protect erythrocytes from various species against lysis by C from homologous and heterologous sources. In all cases, incorporation of human CD59 or analogues from rat, sheep, and pig efficiently protected guinea pig erythrocytes against lysis by C homologous with the CD59. However, each of the CD59 analogues also conferred on guinea pig erythrocytes protection against C from most heterologous species. These results demonstrate that none of the CD59 analogues tested were species specific in their C-inhibiting activity. Erythrocytes from species other than guinea pig could not be protected by incorporation of any of the available CD59 analogues despite similar incorporation in all erythrocytes tested. We suggest that the presence of endogenous inhibitors on these other erythrocytes masks the activity of incorporated CD59. Evidence that is supportive of this hypothesis was provided by demonstrating that blocking the endogenous CD59 with mAbs rendered erythrocytes susceptible to inhibition by high dosages of incorporated CD59.
Nitric oxide regulates endotoxin-induced TNF-alpha production by human neutrophils.
Authors: Van Dervort A L , Yan L , Madara P J , Cobb J P , Wesley R A , Corriveau C C , Tropea M M , Danner R L
Published: December 2022

We studied the effect of nitric oxide on LPS-induced TNF-alpha production by human neutrophils. Human neutrophils exposed to LPS and IFN-gamma did not show measurable increases in intracellular cyclic GMP (cGMP). However, cGMP increased upto 30-fold (p < 0.01) in neutrophils incubated with both sodium nitroprusside (SNP), an exogenous source of nitric oxide, and N-acetylcysteine (NAC), which increases the bioavailability of nitric oxide; this increase indicates that neutrophils contain a nitric oxide-sensitive guanylate cyclase. SNP, with or without NAC, did not increase TNF-alpha production in human neutrophils cultured in medium alone. However, LPS-dependent TNF-alpha production was increased by exposure to SNP (p < 0.05); this effect was further increased by the addition of NAC (p < 0.02). IFN-gamma greatly increased LPS-mediated TNF-alpha production by human neutrophils (p < 0.01), and SNP plus NAC was found to further augment this production (p < 0.01). The up-regulation of TNF-alpha production by nitric oxide was not associated with increased amounts of LPS-induced TNF-alpha mRNA, and was not reproduced by exposing neutrophils to cGMP analogues. These data suggest that nitric oxide released by endothelial and vascular smooth muscle cells may exert a paracrine effect on human neutrophils and augment the inflammatory response in sepsis by increasing the production of cytokines. Although the mechanism of this effect remains unknown, it does not seem to be dependent on cGMP or increased levels of TNF-alpha mRNA.
Differential regulation of lymphotoxin (LT), lymphotoxin-beta (LT-beta), and TNF-alpha in murine T cell clones activated through the TCR.
Authors: Millet I , Ruddle N H
Published: December 2022

The present study demonstrates differential regulation of three members of the TNF family, lymphotoxin (LT), LT-beta, and TNF-alpha, by activated murine T cell clones. We report for the first time that murine T cells transcribe LT-beta mRNA in the absence of any activating signal. Activation through the TCR by anti-CD3 did not increase the accumulation of LT-beta mRNA but did increase the accumulation of two species of TNF-alpha mRNA and three species of LT mRNA. We determined that anti-CD3-activated T cells differ in their regulation of LT, LT-beta, and TNF-alpha at the transcriptional and post-transcriptional levels. Anti-CD3 activation resulted in substantial increases in the extent of transcription of the TNF-alpha and LT genes, although with different rates. LT mRNA accumulation was also post-transcriptionally regulated by anti-CD3. In anti-CD3-activated T cells, the t1/2 of LT mRNA was three to four times longer than that of TNF-alpha mRNA. LT-beta mRNA decayed at a rate similar to that of LT mRNA. We also noted a dramatic difference in the cycloheximide sensitivity of LT, LT-beta, and TNF-alpha mRNAs. Cycloheximide superinduced the accumulation of LT mRNA, but not that of TNF-alpha and LT-beta mRNA, post-transcriptionally. Thus, this study demonstrates dramatic differences in the molecular mechanisms of regulation of LT, LT-beta, and TNF-alpha. It also indicates that LT production is probably the rate-limiting step in the formation of the LT-LT-beta complex. These differences suggest that the reason for the redundancy of LT, LT-beta, and TNF-alpha is their differential regulation rather than their functions.
Signals for activation of CD8-dependent adhesion and costimulation in CTLs.
Authors: O'Rourke A M , Mescher M F
Published: December 2022

Adhesion of CD8+ CTL to purified class I proteins has been shown to be regulated by the TCR: nonactivated CTL do not adhere to immobilized class I proteins (non-Ag), but adhesion becomes readily detectable upon treatment of the CTL with fluid-phase anti-TCR mAb. Signals for up-regulating CD8 adhesion do not appear to involve products of the PI pathway, as neither increased production of inositol phosphates or mobilization of [Ca2+]i can be detected in response to the fluid-phase anti-TCR mAb, but both occur when the CTL then bind to class I protein. The lack of a role for phosphoinositide pathway products in up-regulating CD8 was confirmed by the inability of phorbol ester or calcium ionophore to substitute for TCR mAb in triggering adhesion to class I proteins. Instead, both phorbol ester and calcium ionophore inhibited the anti-TCR mAb-stimulated adhesion to class I. Inhibitors of protein tyrosine kinases also block TCR-activated, CD8-dependent adhesion to class I, and concomitantly block inositol phosphate release, Ca2+ mobilization and degranulation. Inhibition of signaling and response does not appear to be caused solely by the inhibition of adhesion to class I, however, because these inhibitors also block signaling in response to immobilized anti-TCR mAb under conditions in which binding of other receptors to their ligands is not necessary to initiate phosphoinositide hydrolysis and degranulation. These results lend further support for a model in which CTL activation involves a cascade of adhesion and signaling events initiated by the TCR and propagated by CD8 and additional cell-surface receptors.
Isolation and characterization of the Xenopus terminal deoxynucleotidyl transferase.
Authors: Lee A , Hsu E
Published: December 2022

A cDNA clone containing the sequence of the Xenopus homologue of terminal deoxynucleotidyl transferase (TdT) was isolated and analyzed. The derived amino acid sequence shares > 50% identity with the human, mouse, and bovine TdT sequences. By hybridization and PCR analyses Xenopus TdT mRNA was found in the thymus and not in the spleen, kidney, intestine, or liver. During ontogeny TdT appears in significant amounts in the thymus of tadpoles at metamorphic climax but little in the earlier midlarval stages. The emergence of TdT only late in development correlates with the paucity of N region addition in larval Ig heavy chain sequences. Past experiments on the ontogeny of the Xenopus immune system revealed a less efficacious tadpole immune response in skin graft rejection and Ab heterogeneity; the absence of TdT in the tadpole stages fits well with the notion of lower larval TCR and Ab diversity.
Definition of a DQ3.1-specific binding motif.
Authors: Sidney J , Oseroff C , del Guercio M F , Southwood S , Krieger J I , Ishioka G Y , Sakaguchi K , Appella E , Sette A
Published: December 2022

A quantitative peptide binding assay using purified DQA1*0301/DQB1*0301 (DQ3.1) molecules was developed and validated by examining the correlation between the data obtained in the binding assay with those obtained in inhibition of Ag presentation assays. By the combined use of large libraries of synthetic peptides and of substitution and truncation analogues, a putative DQ3.1 motif was defined. Its most prominent feature is the requirement for two small and/or hydrophobic residues spaced at positions i + 2 and i + 4. This motif is quite different from the motif recognized by DR molecules, but similar to the motif previously defined for certain IA alleles (the putative mouse homologue of DQ). These data suggest that various class II isotypes have evolved to present different peptide structures to each other, thus maximizing the repertoire of different epitopes available to T cell scrutiny.
Vitronectin, fibronectin, and gp120 antibody enhance macrophage release of TNF-alpha in response to Pneumocystis carinii.
Authors: Neese L W , Standing J E , Olson E J , Castro M , Limper A H
Published: December 2022

Alveolar macrophages (AMS) initiate inflammation during Pneumocystis carinii pneumonia by releasing cytokines including TNF-alpha. Recent studies suggest that macrophage responses to P. carinii are enhanced by serum opsonization, but the mechanisms of enhancement are not well defined. To determine whether macrophage release of TNF-alpha in response to P. carinii was augmented by immune opsonization, alveolar macrophages obtained from rabbits were cultured with P. carinii that had been opsonized with either nonimmune rabbit serum, immune serum generated against P. carinii, or an affinity-purified polyclonal Ab recognizing the major P. carinii surface Ag gp120. Each experiment also included organisms maintained in media alone (nonopsonized P. carinii). Opsonization of P. carinii with immune serum or gp120 Ab significantly enhanced macrophage TNF-alpha release. Interestingly, however, opsonization with nonimmune serum also increased TNF-alpha response to the organism. Because P. carinii is known to interact with the adhesive glycoproteins, vitronectin (VN) and fibronectin (FN), we hypothesized that they might also augment TNF-alpha release. Opsonization of P. carinii with VN or FN resulted in significant potentiation of macrophage TNF-alpha liberation. We further determined that VN and FN were present in increased quantities in the lower respiratory tract of patients with P. carinii pneumonia compared with normal volunteers. Additionally, VN and FN were demonstrated on the surface of freshly isolated P. carinii organisms by immunoblot analysis. Our study suggests that immune and nonimmune opsonins contribute to host defenses during P. carinii pneumonia by enhancing regional TNF-alpha release in response to the organism.
CD4+ blood dendritic cells are potent producers of IFN-alpha in response to in vitro HIV-1 infection.
Authors: Ferbas J J , Toso J F , Logar A J , Navratil J S , Rinaldo C R
Published: December 2022

We determined the relative abilities of cell subpopulations from all major PBMC lineages of normal donors to produce IFN-alpha in response to in vitro stimulation with lymphocytotropic HIV-1 (IIIb and RF), monocytotropic HIV-1 (BaL), Sendai virus, and HSV-1. Active and inactive cell-free preparations of HIV-1 IIIb and cell-associated HIV-1 IIIb, and active cell-free preparations of the other viruses, induced comparable, maximal levels of acid-stable IFN-alpha in PBMC by 18 to 24 h. Negative selection and enrichment experiments indicated that HLA-DR+ "null" cells produced the majority of the IFN-alpha. A positive selection protocol using flow cytometric sorting enriched these HLA-DR+ CD3- CD19- CD16- CD56- CD14- cells to > 95% purity. These were identified as dendritic cells by their phenotype, large size, and veiled and ruffled morphology. The purified dendritic cells produced as much as 60-fold more IFN-alpha compared with purified, HLA-DR+ CD14+ monocytes in response to the viruses. IFN-alpha was not produced by CD3+ T cells or CD56+ NK cells. Purified CD19+ B cells produced a minimal amount of IFN-alpha in response to Sendai virus, and no IFN-alpha in response to the other viruses. Of significance, the dendritic cells expressed CD4 at a density similar to monocytes, and induction of IFN-alpha by HIV-1 could be blocked by HIV-1 gp120 anti-serum or anti-CD4 mAb. We conclude that the production of IFN-alpha constitutes a previously unrecognized major function of blood dendritic cells. This may be a mechanism of innate immunity mediated by dendritic cells against HIV-1 and other viral infections.
Oral tolerance in humans. T cell but not B cell tolerance after antigen feeding.
Authors: Husby S , Mestecky J , Moldoveanu Z , Holland S , Elson C O
Published: December 2022

The purpose of this study was to investigate whether oral tolerance, defined as Ag-specific immunologic unresponsiveness after Ag feeding, could be induced in humans after prolonged Ag ingestion. Eight adult volunteers ingested a total dose of 0.5 g of keyhole limpet hemocyanin (KLH) followed by subcutaneous immunization with KLH. Eight controls received only the subcutaneous immunization. In the group fed KLH, there was a significant reduction in KLH-specific T cell proliferation (p = 0.04) and delayed skin test responses (p = 0.07) to KLH. KLH ingestion alone did not induce significant levels of Abs in either serum or secretions. However, after the subsequent subcutaneous immunization, the number of circulating IgG and IgM anti-KLH-producing cells, the titers of serum IgG, IgA, and IgM anti-KLH Abs, and the titers of IgA anti-KLH Abs in saliva and intestinal secretions were significantly greater in the KLH-fed group than in the nonfed group. We conclude that KLH feeding induced systemic T cell tolerance, but B cell priming, at both systemic and mucosal sites. These studies support the concept of using Ag feeding as a treatment for certain immune-mediated diseases.
Myasthenia gravis. Residues of the alpha and gamma subunits of muscle acetylcholine receptor involved in formation of immunodominant CD4+ epitopes.
Authors: Moiola L , Protti M P , McCormick D , Howard J F , Conti-Tronconi B M
Published: December 2022

We propagated from myasthenia gravis (MG) patients by stimulation in vitro with synthetic sequences (alpha 48-67, alpha 304-322, gamma 75-94 and gamma 321-340) of the human muscle acetylcholine receptor (AChR), CD4+ lines against four 20-residue sequence regions of the AChR alpha and gamma subunits that are recognized by Th cells of most MG patients. Most lines secreted IL-2 and not IL-4, suggesting that they comprise Th1 cells. For three lines we verified that, as reported previously, AChR epitopes are presented by DR molecules: their response to the relevant peptide was abolished by anti-DR Abs. The DR molecules presenting AChR epitopes were identified by testing the response of the lines to the relevant peptide, using APC from donors homozygous for the different DR alleles of the line. We tested the lines with single residue-substituted analogues of the epitope sequence. The results of these experiments indicated that the lines were polyclonal and recognized overlapping epitopes. Their response was abolished by some substitutions, identifying residues common to all epitopes within a given region, whereas other substitutions reduced but did not obliterate the response, indicating residues included in some but not all epitopes recognized by the line. Comparison of the residues involved in epitope formation for different lines supported the conclusion that within the 20-residue immunodominant regions investigated here, the same sequence segment is involved in formation of epitopes, even in DR-discordant patients.
Recognition of T cell epitopes and lymphokine secretion by rye grass allergen Lolium perenne I-specific human T cell clones.
Authors: Spiegelberg H L , Beck L , Stevenson D D , Ishioka G Y
Published: December 2022

T cell lines (TCL) and CD4+ T cell clones (TCC) with specificity for the rye grass allergen Lolium perenne (Lol p) I were isolated from the blood of nine donors, six having active atopic disease, two being in remission, and one having IgE anti-Lol pI Abs but not atopic disease. The T cell epitopes of Lol pI were determined by TCLs and TCCs reactivity with 23 overlapping, 20 amino acid-long peptides spanning the entire length of the 230 amino acid-long allergen. In addition, the Th subsets (Th1, Th2, Th0, Thp) were determined by measuring IL-2, IFN-gamma, and IL-4 in the supernatants of TCC activated with Lol pI and irradiated APC. TCC from individuals from which a large panel of clones were obtained from 10(5) PBMC initial cultures recognized multiple peptides (5-9) and 23 overlapping peptides a total of 16 were recognized by at least one TCC from one of the patients. These 16 peptides were derived from all areas of the Lol pI molecule, indicating the ability of human Th cells to recognize many peptide epitopes on Lol pI. Although no clear cut immunodominant peptides were detected, T cell clones of 50% of the patients reacted with peptide 191-210. There was no correlation between peptide epitope reactivity and lymphokine secretion pattern of the TCC. Of 12 TCC obtained from six patients with active atopic disease, four (33%) were of Th1, five (42%) of Th2, one (8%) of Thp, and two (17%) of Th0 type. Of 14 TCCs isolated from three atopic donors in remission, five (36%) were of Th1, three (21%) of Th2, four (29%) of Thp, and two (14%) of Th0 type. The data demonstrate that T cells from rye grass pollen allergic patients can recognize multiple peptide epitopes on Lol pI scattered over the entire molecule. No correlation existed between epitope reactivity and lymphokine secretion pattern of the TCC.
CD58 (LFA-3) stimulation provides a signal for human isotype switching and IgE production distinct from CD40.
Authors: Diaz-Sanchez D , Chegini S , Zhang K , Saxon A
Published: December 2022

Induction of an IgE response involves several discrete steps: 1) induction of epsilon germ line transcription, 2) DNA recombination, and 3) mature RNA transcription/translation. Here we show that ligation of B cell CD58 by CD2, its natural ligand on T cells, or by mAb, provides a novel IL-4-dependent signal for the latter two steps. Highly purified human B cells were induced to produce IgE by costimulation with IL-4 and CD58 mAb. Although CD58 ligation alone was unable to induce epsilon germ-line transcription, in concert with IL-4-stimulated epsilon germ-line transcription it induced the appearance of productive epsilon transcripts and IgE production. The direct involvement of CD2 was demonstrated: B cells cultured with IL-4 plus murine T hybridoma cells transfected with human CD2 produced IgE. A CD40 Fc fusion protein had no effect on CD58-driven IgE production while inhibiting CD40-dependent responses. Furthermore, cells from patients with common variable immunodeficiency produced IgE in response to IL-4 plus CD40 mAb but not to IL-4 plus CD58 mAb. CD58-driven IgE synthesis was IFN-gamma independent and was not enhanced by exogenous IL-6. Functional differences between CD40 and CD58 IgE stimulation were demonstrated. Thus, the CD2:CD58 ligand/counterligand system provides an alternative pathway by which cell contact signaling may regulate IgE. Given the relative importance of CD2 triggering on mucosal T cells and the mucosal location of IgE production, this may be especially true on mucosal surfaces.
Altered tissue distribution of viral replication and T cell spreading is pivotal in the protection against fatal lymphocytic choriomeningitis in mice after neutralization of IFN-alpha/beta.
Authors: Sandberg K , Kemper P , Stalder A , Zhang J , Hobbs M V , Whitton J L , Campbell I L
Published: December 2022

IFN-alpha/beta have been shown to play a central role in the development of lymphocytic choriomeningitis and increasing attention has been focused on this group of cytokines as early regulatory factors directing T lymphocyte responses. In the present study, injection of antiserum to IFN-alpha/beta prevented the development of lymphocytic choriomeningitis, was associated with the absence of detectable expression of early 2'-5' oligo-adenylate synthetase mRNA and coincided with viremia of lymphocytic choriomeningitis virus (LCMV) followed by establishment of a persistent infection. The LCMV-specific cytotoxic T lymphocyte response was unchanged in cervical lymph nodes but decreased in the spleen of anti-IFN-alpha/beta-treated animals. The expression of cytokine mRNA (particularly IFN-gamma) in organs of LCMV-infected mice treated with anti-IFN-alpha/beta coincided with infiltration of lymphocytes and tissue destruction. Furthermore, a reduced number of infiltrating leukocytes in the brain and cervical lymph nodes and a low expression of cytokine mRNA in the brain was observed in anti-IFN-alpha/beta-treated animals. In total, the findings support the view that neutralization of IFN-alpha/beta leads to extensive LCMV replication in the viscera. The therapeutic effects of anti-IFN-alpha/beta antiserum seem to be independent of the functional capacity of T cells but probably result in a dispersion of activated T cells throughout the body of LCMV-infected mice. Absence of IFN-alpha/beta expression in the central nervous system is proposed as the mechanism behind the IFN-alpha/beta-dependent targeting of T cells to the brain.
TNF-alpha is a principal cytokine involved in the recruitment of NK cells to liver parenchyma.
Authors: Pilaro A M , Taub D D , McCormick K L , Williams H M , Sayers T J , Fogler W E , Wiltrout R H
Published: December 2022

Isolated murine splenic NK cells and the cultured murine endothelioma cell line, eEND2, were used to study the effects of cytokines on NK cell/endothelial cell adhesion. Treatment of eEND2 cells with TNF-alpha induced a marked increase (four- to sevenfold) in adherence of NK cells, as compared with control cultures of endothelioma cells or eEND2 cells treated with IL-1 alpha or IL-6. TNF-alpha induction of NK cell adherence to eEND2 was dose dependent with rapid kinetics, reaching a maximum at concentrations between 10 and 1000 U/ml after a 2-h incubation. TNF-alpha treatment of L929 fibroblasts or CL-2 hepatoma cells did not result in increased NK cell adhesion. The concentration range of TNF-alpha that was found to maximally augment NK cell adhesion to eEND2 also induced NK cell chemokinetic activity. The relevance of these in vitro results was subsequently analyzed in vivo. Initial studies confirmed that a single dose of polyinosinic-polycytidylic acid and poly-L-lysine stabilized in carboxymethyl cellulose (poly-ICLC), augmented hepatic NK activity and resulted in a 2.2-fold increase in the number of liver-associated NK cells. Concomitant treatment of mice with a TNF-alpha neutralizing antisera eliminated both the hepatic influx of NK cells and the increase in poly-ICLC-induced liver NK activity. These results suggest that TNF-alpha is a principal cytokine involved in the in vivo recruitment and localization of parenchymal NK cells after treatment with a biological response modifier, and that this regulation seems to occur via alterations in NK cell/endothelial cell interactions.