bluuebunny/crossref_metadata_2025 huggingface dataset:
Production of antigen-specific human monoclonal antibodies from in vitro-primed human splenocytes.
Authors: Boerner P , Lafond R , Lu W Z , Brams P , Royston I
Published: December 2022

We have developed culture conditions for human lymphocytes that support primary in vitro immune responses to protein Ag of either human or nonhuman origin. We now show that these primed B cells can be efficiently immortalized by fusion with a heterohybrid fusion partner to generate human, Ag-specific IgM or IgG antibody-producing heterohybridomas at a rate of 17 to 50 hybrids/10(6) lymphocytes fused. Approximately 50% of the Ig-secreting clones were stable with respect to Ig secretion. Levels of secretion attained with terminal cultures ranged from less than 1 to 100 micrograms/ml. Fusions of cells between 2 and 5 days after initiation of in vitro exposure to Ag produced more Ag-reactive and Ag-specific antibodies than fusions at 1 day or fusions performed after 5 days. Ag-reactive hybrids could be isolated at frequencies of 3 to 10%, depending on antigenicity of the immunogen. Foreign proteins, horse spleen ferritin, and a murine monoclonal Ig, induced higher percentages of Ag-reactive mAb than immunization with the human-derived ferritin. Ag-reactive IgG mAb were produced at relatively high frequency, depending on immunization conditions and the nature of the Ag. The strategy for identification of the best hybrids included early elimination of unstable hybridomas and of hybridomas producing broadly cross-reactive antibody, followed by evaluation of units of Ag reactivity/micrograms Ig. Ferritin-specific mAb selected according to these criteria showed immunocytochemical reactivity with ferritin-containing tissues and apparent affinities in the range of 10(7) to 10(8)/mol.
A monoclonal antibody (OT145) specific for the T cell antigen receptor V beta 6.7a allele detects an epitope related to a proposed superantigen-binding site.
Authors: Prashar Y , Li Y , Kubinec J S , Jones N , Posnett D N
Published: December 2022

A mAb, OT145, recognizes a TCR allotype encoded by one of two alleles of the V beta 6.7 gene. The peptide products of the two V beta 6.7 alleles differ because of nonconservative amino acid substitutions at positions 38 and 72. V beta 6.7a encodes ser38 and gly72, whereas V beta 6.7b encodes arg38 and glu72. We show here that the binding of mAb OT145 ot the beta-chain of TCR is lost when residue 72 of V beta 6.7a is mutated from gly to glu. The binding of OT145 is not affected by mutation of residue 38 from ser to arg. Thus, OT145 recognizes an epitope related to position 72. Residue 72 of the beta-chain of the TCR is located at a putative superantigen-binding site.
Charybdotoxin-sensitive, Ca(2+)-dependent membrane potential changes are not involved in human T or B cell activation and proliferation.
Authors: Gelfand E W , Or R
Published: December 2022

The involvement of ion channels in B and T lymphocyte activation is supported by many reports of changes in ion fluxes and membrane potential after mitogen binding. Human T and B lymphocytes demonstrate an early and transient hyperpolarization after ligand binding. Inasmuch as the change in membrane potential is dependent on elevation of free cytosolic calcium, the hyperpolarization is presumably through opening of Ca(2+)-stimulated K+ channels. We have used charybdotoxin, a known inhibitor of Ca(2+)-dependent K+ channels, to study the role of these channels in lymphocyte activation and mitogenesis. We demonstrate that charybdotoxin inhibits the ligand-induced transient membrane hyperpolarization in B and T cells in a dose-dependent fashion, without affecting changes in cytosolic Ca2+. However, blockade of the Ca(2+)-activated K+ channel is not associated with changes in cell-cycle gene activation, IL-2 production, IL-2R expression or B and T cell mitogenesis. These results imply that membrane potential changes secondary to the ligand-dependent opening of Ca(2+)-activated K+ channels are not involved in B and T lymphocyte activation and mitogenesis.
Expression of antibody Fab domains on bacteriophage surfaces. Potential use for antibody selection.
Authors: Chang C N , Landolfi N F , Queen C
Published: December 2022

We describe a method for expressing an antibody Fab fragment on the surface of M13 filamentous bacteriophage. The L chain gene, preceded by an Escherichia coli signal sequence, is linked to the 5' end of the gene for the M13 major coat protein. A partial H chain gene, preceded by an E. coli signal sequence and truncated at the end of the CH1 region, is inserted on a plasmid adjacent to the fused L chain gene, so both genes are under the transcriptional control of an inducible promoter. The plasmid also contains the M13 origin of replication. When the promoter is induced, functional antibody Fab fragment appears on the surface of the E. coli inner membrane, as shown by specific binding to an Ag-affinity matrix. When the E. coli are further infected with a helper phage, allowing replication and packaging of the plasmid into phage particles, the resulting phage specifically bind to an Ag-coated plate, indicating they have antibody Fab fragment on their surface. The antibody-expressing phage can also be specifically bound to and eluted from an Ag-affinity column. These observations support the possibility of a new way of generating antibodies, in which amplified Ig cDNA from an appropriate B cell population is cloned into a suitable M13 vector, and phage containing the genes for desired antibodies are selected directly by binding to an Ag-affinity matrix.
Alkaline phosphatase on activated B cells characterization of the expression of alkaline phosphatase on activated B cells. Kinetics and membrane anchor.
Authors: Feldbush T L , Lafrenz D
Published: December 2022

Recently we reported that the expression of the enzyme alkaline phosphatase (APase) is a marker for B cell activation. Enzymatic activity was found only in activated B cells and not T cells. Using flow cytometry we showed that some of the APase was found on the cell membranes (mAPase) and by functional assays, some was spontaneously released into the tissue culture medium. In the present report the expression of mAPase on activated B lymphocytes is more fully characterized. Two mAb specific for rat APase were used to measure the kinetics of the membrane expression of mAPase. Within 48 h of activation, mAPase is detected by flow cytometry and increases coordinately with both the transferrin receptor and IL-2R. Maximal membrane expression of mAPase in terms of number of positive cells and mean fluorescent intensity, is detected by day 4 to 5 of culture. Using hydroxyurea and demecolcine to block cells at G1/S and G2/M, respectively, it appeared that the initial expression of mAPase occurred as cells progressed into S phase of the cell cycle. This was confirmed using two-color flow cytometric analysis with the Hoechst DNA stain 33342 and the FITC-labeled APase-specific mAb. Finally, using phosphatidylinositol-specific phospholipase C we were able to show that 60 to 80% of the mAPase is linked to the membrane via a glycosyl-phosphatidylinositol linkage. From this we have concluded that mAPase can be added to a growing list of glycoproteins that are anchored to the membrane by the glycosyl-phosphatidylinositol linkage and are expressed on differentiating B cells. This list now includes Thy-1, BLAST-1, Jlld, and mAPase.
The molecular basis of class II MHC allelic control of T cell responses.
Authors: Racioppi L , Ronchese F , Schwartz R H , Germain R N
Published: December 2022

To identify the molecular basis for the effects of MHC molecule polymorphism on T cell responses, we have combined functional T cell response testing with measurements of peptide binding to the class II MHC molecules on transfected cells. Our studies identify a small subset of spatially localized polymorphic residues of the E alpha E beta dimer (strand residue beta 29, and helix residues beta 72 and beta 75) regulating cytochrome c peptide presentation by two distinct mechanisms. The first effect is on quantitative control of net peptide binding. The replacement of the valine found at position beta 29 in E beta k with the glutamic acid found in E beta b results in a selective loss of pigeon cytochrome peptide but not moth cytochrome peptide binding to the resultant mutant E alpha E beta k molecule. Reciprocally, the replacement of glutamic acid at beta 29 in E beta b with valine results in a gain of pigeon peptide binding. These changes in binding parallel changes in T cell responses in vitro to these peptide-E alpha E beta combinations and mirror the in vivo immune response gene phenotypes of mice expressing E alpha E beta k and E alpha E beta b. E alpha E beta s molecules, which have a beta 29 glutamic acid, are nevertheless able to bind and present pigeon cytochrome peptides, and this is due to changes in helix residues beta 72 and beta 75 that compensate for the negative effect of the beta 29 glutamic acid. The second activity is a critical change in the conformation of the peptide bound to the same extent by distinct MHC molecules, as revealed by changes in T cell responses to moth cytochrome peptides presented by two E alpha E beta molecules differing only at position beta 29. Both of these effects can be ascribed to a single polymorphic residue modeled to be inaccessible to TCR contact (beta 29), providing a striking demonstration of how MHC molecule polymorphism can modify T cell-dependent immune responses without direct physical participation in the receptor recognition event.
Induction of phorbol ester responsiveness in conventional B cells after activation via surface Ig.
Authors: Rothstein T L , Kolber D L , Murphy T P , Cohen D P
Published: December 2022

The signals required to induce S phase entry in murine splenic B cells were found to be altered by prolonged treatment with low doses of anti-Ig antibody. Whereas fresh splenic B cells are stimulated by the combination of a phorbol ester protein kinase C agonist plus a calcium ionophore, anti-Ig-treated splenic B cells were stimulated by phorbol ester alone, in the absence of a comitogen. The majority of these phorbol ester responsive B cells expressed CD5. The phorbol ester responses of anti-Ig-treated splenic B cells paralleled those previously reported for untreated peritoneal CD5+ B cells in a number of respects: responses were not idiosyncratic to phorbol esters but occurred with nonphorbol protein kinase C agonists; phorbol ester responses were enhanced by IL-4; and, phorbol ester responses occurred rapidly and were greater at 24 than at 48 h. However, the effect of agents that act to raise intracellular levels of cAMP distinguished between anti-Ig-treated splenic B cells and untreated peritoneal B cells in that the phorbol ester responses of the former were enhanced whereas the responses of the latter were inhibited. The present results add a functional dimension to the phenotypic similarity between splenic B cells treated with anti-Ig and resident peritoneal B cells that constitutively express CD5; however, some differences in behavior were noted.
Differential stability of antigenic MHC class I-restricted synthetic peptides.
Authors: Widmann C , Maryanski J L , Romero P , Corradin G
Published: December 2022

Various synthetic peptides recognized as Ag by CTL in the context of MHC class I molecules were tested for stability in vitro and in vivo. Peptide inactivation in vitro was quantitated by titrating the amount of peptide required to sensitize target cells for lysis by specific CTL clones. The degree of inactivation after overnight incubation at 37 degrees C varied widely among a series of antigenic peptides. Some were nearly unaffected, whereas others lost activity by more than 100-fold or even 10,000-fold. However, no correlation was found between susceptibility to serum inactivation and antigenic potency as measured in short term cytolytic assays. No inactivation occurred at 4 degrees C, or at 37 degrees C in the absence of serum, under the conditions used. Serum inactivation most likely involved proteolysis because it could be inhibited by protease inhibitors. Moreover, presumed cleavage products of a radiolabeled susceptible peptide could be visualized by TLC. In vivo, the persistence of the antigenic activity of the injected peptides, either in extracellular fluids or on tumor target cells growing in an ascites form, correlated with the degree of stability found for the peptides in vitro. The differential stability of synthetic peptides may have important consequences for attempts to manipulate the development of an immune response in vivo.
Characterization of accessory cell costimulation of Th1 cytokine synthesis.
Authors: Williams I R , Unanue E R
Published: December 2022

We studied the capacity of macrophage and B cell lines to provide a costimulatory signal that enhances synthesis of IFN-gamma and IL-2 by mouse Th1 clones stimulated with suboptimal doses of immobilized anti-CD3 antibody. The J774 macrophage line and the CH27 B lymphoma line had the greatest costimulatory activity and routinely increased IL-2 production by 10-fold to 100-fold. Other macrophage and B cell lines had less activity and T cell lines were unable to costimulate. The J774 and CH27 lines did not costimulate IL-4 production by a Th2 clone and had only a small effect on IL-2 production by T cell hybridomas. The process of costimulation was fixation-sensitive, contact-dependent and did not involve stable cytokines present in the T cell/accessory cell conditioned media. Neutralizing antibodies for IL-1, IL-6, and TNF failed to inhibit costimulation. Antibodies to the LFA-1/ICAM-1 pair of adhesion molecules also failed to inhibit. Costimulation of IL-2 production by accessory cells was found to have a unidirectional species restriction: mouse accessory cells costimulated mouse and human IL-2-producing T cells, but human U937 cells induced with PMA were effective only for human T cells. The results indicate that accessory cells can significantly regulate Th1 effector function at the level of cytokine production.
IL-1, IL-4, and IFN-gamma differentially regulate cytokine production and cell surface molecule expression in cultured human thymic epithelial cells.
Authors: Galy A H , Spits H
Published: December 2022

We investigated the response of purified and cloned human thymic epithelial cells (TEC) to IL-1, IL-4, and IFN-gamma stimulation in vitro. IL-1 alpha strongly up-regulated the production of granulocyte-macrophage CSF (GM-CSF), granulocyte CSF (G-CSF), IL-6, and IL-8, as measured by specific immunoenzymetric assays and by increased steady state mRNA levels. IL-4 or IFN-gamma did not induce these cytokines in TEC but in a sustained and dose-dependent manner down-regulated the IL-1-induced GM-CSF protein and mRNA levels. Only IFN-gamma, and not IL-4, suppressed the IL-1-induced G-CSF and IL-8 production, as shown at both the protein and mRNA levels. The inhibition was dose dependent, sustained for at least 96 h, and more pronounced for G-CSF than for IL-8. In contrast, both IL-4 and IFN-gamma enhanced the IL-1-induced IL-6 production. IL-4 and IFN-gamma had additive effects to increase IL-6 secretion and to more completely suppress the IL-1-induced GM-CSF. Analyses of cell surface molecules showed that intercellular adhesion molecule 1 (ICAM-1) expression on TEC was increased by IL-1 or IFN-gamma. IL-4 slightly down-regulated constitutive ICAM-1 levels but did not significantly modify the levels of expression induced by either IL-1 or IFN-gamma. MHC class II expression was induced by IFN-gamma but not by IL-1 or IL-4. The combination of IL-1 and IL-4 with IFN-gamma did not alter the levels of class II MHC Ag induced by IFN-gamma. In conclusion, TEC cytokine production and cell surface molecule expression are differentially regulated via a complex cytokine network. Our data suggest that developing T cells provide, in part, the signals controlling the function of their supporting stroma.
Protective activity of adult T cell leukemia-derived factor (ADF) against tumor necrosis factor-dependent cytotoxicity on U937 cells.
Authors: Matsuda M , Masutani H , Nakamura H , Miyajima S , Yamauchi A , Yonehara S , Uchida A , Irimajiri K , Horiuchi A , Yodoi J
Published: December 2022

Adult T cell leukemia-derived factor (ADF) is a human homologue of thioredoxin with many biologic functions including IL-2R induction, growth promotion, thiol-dependent reducing activity, and radical scavenging activity. The regulatory effect of ADF on the cytotoxic activity of TNF was examined by using a human histiocytic lymphoma cell line, U937. When U937 cells were preincubated with recombinant ADF (rADF) (0.1-100 micrograms/ml) at 37 degrees C for 30 min, TNF-dependent cytotoxicity on U937 cells was markedly inhibited. This inhibitory effect was as high as 95% in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (rADF 100 micrograms/ml) and 85% in the 51Cr-releasing assay (rADF 10 micrograms/ml). After pretreatment of U937 cells with IFN-gamma to augment the sensitivity to TNF, an inhibitory effect of rADF was also found. When U937 cells were washed after preincubation with rADF, resistance to TNF-dependent cytotoxicity was still observed, indicating that rADF inhibited the sensitivity of U937 to TNF-dependent cytotoxicity rather than modifying TNF molecules. Scatchard analysis of TNF receptors on U937 cells using 125I-TNF showed that rADF modulated neither the density nor the affinity of the cell membrane significantly. rADF also reduced the cytotoxicity induced by anti-Fas IgM mAb which shows cytotoxicity quite similar to TNF. rADF (10 micrograms/ml) reduced 90% of the cytotoxicity by anti-Fas IgM mAb, without a detectable change either in Fas Ag expression (MFI 58.1 vs 53.3) or in the degradation of anti-Fas IgM mAb as determined by flow cytometric analysis. These findings indicated that the rADF-induced resistance to the cytotoxic effect of TNF and anti-Fas mAb was not related to the modulation of the TNF receptor or Fas Ag.
Survival of human macrophages infected with Mycobacterium avium intracellulare correlates with increased production of tumor necrosis factor-alpha and IL-6.
Authors: Newman G W , Gan H X , McCarthy P L , Remold H G
Published: December 2022

The long term survival of peripheral blood derived human macrophages (M phi) from normal, healthy donors after infection with Mycobacterium avium intracellulare (MAI) correlates with the increased induction of TNF-alpha and IL-6 mRNA and protein by the infected M phi. This conclusion is based on the following observations: M phi from approximately 30% of the blood donors in our study die 3 to 4 days after inoculation (MAI-growth nonsupportive (NS], whereas M phi from the other donors survive inoculation with MAI for 7-10 days (MAI-growth supportive (S)). S-type M phi when infected with MAI had markedly increased amounts of TNF-alpha and IL-6 mRNA and protein when compared to NS-type M phi. The effect of LPS on the induction of TNF-alpha mRNA and protein was also significantly enhanced in S-type M phi in comparison to NS cells. In contrast, IL-1 beta mRNA and protein production had similar increases in both donor types when infected with MAI or stimulated with LPS. The phenotype of the donors in the amount of TNF-alpha and IL-6 produced in response to MAI infection remained stable for a period of more than 1 yr. Pretreatment of NS M phi with recombinant human granulocyte-macrophage-CSF, but not IFN-gamma, however, converted NS M phi into a S-type cell phenotype. These granulocyte-macrophage-CSF pretreated NS M phi survived infection with MAI for a longer period of time and also had increased production of both TNF-alpha and IL-6 mRNA and protein. Cultures of S-type M phi infected with MAI had higher numbers of intracellular bacteria when compared to cultures of NS-infected M phi. Thus, increased survival of MAI-infected human M phi in vitro is correlated to increased production of TNF-alpha and IL-6 in response to infection with MAI.
Regulation of HILDA/LIF gene expression in activated human monocytic cells.
Authors: Anegon I , Grolleau D , Soulillou J P
Published: December 2022

We have previously shown that human monocytes express HILDA/LIF gene and its protein product upon stimulation. In the present study we have investigated some of the possible mechanisms involved in the regulation of HILDA/LIF gene expression in activated human monocytes and compared them to those of granulocyte colony-stimulating factor (G-CSF) and IL-1 beta gene expression. In the absence of added stimuli HILDA/LIF mRNA was barely detectable in isolated monocytes. HILDA/LIF mRNA accumulation was weakly induced by stimuli such as LPS or phorbol ester. However, a synergy was observed when each stimulus was used in combination with 1,25-dihydroxyvitamin D3. Nuclear run-on analysis did not detect an increase in HILDA/LIF gene transcription upon stimulation with LPS or phorbol ester in combination with 1,25-dihydroxyvitamin D3. HILDA/LIF mRNA half-life showed an increase when phorbol ester and 1,25-dihydroxyvitamin D3 were used in combination over that obtained for each stimuli alone. HILDA/LIF mRNA expression was largely inhibited when monocytes were stimulated in the presence of cycloheximide (CHX) added either at the onset or 4 h after the beginning of the stimulation period. When CHX was added at later time points, 2 h before cell harvesting, HILDA/LIF mRNA levels were superinduced when compared with those in cells stimulated in the absence of CHX. This superinduction can be at least partially explained by post-transcriptional mechanisms, since HILDA/LIF mRNA half-life in CHX-superinduced cells was significantly increased when compared with values on stimulated CHX-untreated cells. In contrast to HILDA/LIF, G-CSF and IL-1 beta mRNA accumulation showed a different response pattern to the same stimuli, and a augmentation of their gene transcription was detected by the run-on technique in activated monocytic cells when compared with controls. Furthermore, G-CSF and Il-1 beta mRNA levels were superinduced in monocytes stimulated in the presence of CHX from the onset of the stimulation period. These studies indicate that, when compared to G-CSF and IL-1 beta, the expression of HILDA/LIF gene is under the control of relatively specific regulatory mechanisms. HILDA/LIF gene expression is controlled by newly synthesized proteins acting at early and late time points of the stimulation period and with opposite effects on HILDA/LIF mRNA levels. Finally, HILDA/LIF mRNA levels are regulated by post-transcriptional mechanisms of mRNA stabilization probably controlled through labile newly synthesized proteins.
Gene expression of the A- and B-chain of mouse C1q in different tissues and the characterization of the recombinant A-chain.
Authors: Petry F , Reid K B , Loos M
Published: December 2022

Immunoscreening of a mouse macrophage cDNA library with an anti-mouse C1q-antibody resulted in the isolation of cDNA clones. The deduced amino acid sequence was homologous to the A-chain of human C1q. Homology on the DNA level was found to be 76% and on the protein level 72% thus it appeared the clones coded for the mouse C1q A-chain. An immunoblot of murine serum C1q separated by SDS-PAGE was detected with an A-chain specific antibody that had been affinity purified on recombinant mouse C1q A-chain expressed in Escherichia coli. The antibody preparation reacted exclusively with the mouse C-chain (as defined by SDS-PAGE). Northern blot analysis with strand-specific cDNA probes coding for the A- and B-chain of murine C1q showed that mouse peritoneal macrophages produced the highest concentration of C1q gene transcripts. RNA from mouse spleen, thymus, heart, and brain gave substantial hybridization signals, whereas RNA preparations from liver, kidney, lung, and small intestine appeared to contain only trace amounts of C1q mRNA. In a Northern blot analysis of different guinea pig cells and tissues, only RNA preparations from peritoneal macrophages hybridized with the mouse C1q probes. These results indicate that macrophages are a major site of C1q biosynthesis.
VDJ genes in VHa2 allotype-suppressed rabbits. Limited germline VH gene usage and accumulation of somatic mutations in D regions.
Authors: Short J A , Sethupathi P , Zhai S K , Knight K L
Published: December 2022

In this study we investigate the molecular genetic basis for VHa- Ig. Knowing that the expression of VHa allotype Ig is suppressed by neonatal injection of rabbits with anti-VHa allotype antibody, and that the decreased level of VHa allotype Ig, VHa+, in the suppressed rabbits is compensated for by an increase in VHa- Ig, we determined the nucleotide sequences of 41 VDJ genes from a2/a2 rabbits neonatally suppressed for the expression of a2 Ig. We compared these nucleotide sequences to each other and identified two groups of VH sequences. We predict that the molecules of each group are encoded by one germline VH gene. Inasmuch as VHa+ Ig is encoded predominantly by one germline VH gene, VH1, it appears that more than 95% of the VDJ repertoire of rabbits may be encoded by as few as three germline VH genes. A genomic VDJ gene whose VH sequence was similar to those of group I molecules was expressed in vitro and was shown by ELISA to encode molecules of the VHa- allotype, y33. Analysis of the D regions in the VDJ gene indicated that germline D2b and D3 gene segments were preferentially used in the VDJ gene rearrangement. A comparison of sequences of D regions of the 41 VDJ gene rearrangements in 3-, 6-, and 9-wk-old rabbits to sequences of germline D gene segments showed an accumulation of mutations in the D region. Inasmuch as we have previously shown that V regions of rabbit VDJ genes are diversified, in part, by somatic gene conversion, it appears now that rabbit VDJ genes diversify by a combination of somatic mutation and somatic gene conversion.
Clonal anergy blocks the response to IL-4, as well as the production of IL-2, in dual-producing T helper cell clones.
Authors: Mueller D L , Chiodetti L , Bacon P A , Schwartz R H
Published: December 2022

In this report we extend the in vitro clonal anergy model to examine the regulation of proliferation in T cells that secrete both IL-2 and IL-4. Newly cloned Ag-specific murine T cells are shown to depend on both IL-2 and IL-4 synthesis for maximal proliferation. Whereas IL-2 responsiveness is constitutive in these cells, IL-4 responsiveness develops only after Ag and APC stimulation. Remarkably, proliferation of these cells to Ag is sensitive to inhibition by clonal anergy, even though IL-4 synthesis remains inducible. Anergy in these cells is associated with an inability to respond to IL-4, in addition to the development of an IL-2 production defect. The results suggest that anergy induction may be capable of preventing the clonal expansion of autoreactive T cells producing both IL-2 and IL-4 in vivo.
The preference for switching to IgA expression by Peyer's patch germinal center B cells is likely due to the intrinsic influence of their microenvironment.
Authors: Weinstein P D , Cebra J J
Published: December 2022

This study was aimed at determining whether the chronically activated physiologic state of Peyer's patch (PP) tissue is primarily responsible for the IgA isotype preference expressed by PP germinal centers (GC) and memory B cells. We have used reovirus type 1/Lang to stimulate acute, de novo GC reactions in lymph nodes (LN) or PP to test the possibility that the surface (s)IgA component of enteric responses is peculiar to the local gut microenvironment whether or not PP are in a state of chronic activation. GC were raised in PP of germ-free mice by oral administration of virus and in lymph nodes (LN) of conventionally reared mice by local parenteral infection. Transient GC reactions were found to develop with similar time courses in both PP and LN after both primary and secondary local infections with reovirus. sIgA+ B cells, which were the major non-sIgM+/sIgD+ population found to arise in GC of PP, were not found in the LN. In LN, sIgG1+ B cells comprised the predominant non-IgM/IgD bearing population that arise after local infection. Lymphoid fragment cultures of PP initiated in vitro as early as 5 days after in vivo infection contained detectable secreted reovirus-specific IgA, whereas IgG1 but no IgA was found in LN cultures. Northern blot analysis to detect C alpha and C gamma 1 germline transcripts further substantiated a site-related bias in the expression of non-IgM/IgD isotypes that was manifest within a few days after infection. In summation, these observations taken together suggest that the preference for generating sIgA+ B cells in PP may be the result of intrinsic features of their gut microenvironment.
Expression and synthesis of cross-reactive idiotypic antibodies by peripheral blood lymphocytes and their suppression by anti-idiotypes in patients with IgA nephropathy.
Authors: González-Cabrero J , de Nicolas R , Hernando L , Egido J
Published: December 2022

We have recently described that patients with IgA nephropathy present high serum levels of anti-BSA idiotypic antibodies that were well correlated with the existence of hematuria. Furthermore, these Id were found in circulating and renal deposited immune complexes. In the present work, we examined the expression of surface idiotypic determinants on PBL by flow cytometry and their in vitro production, using as reagent anti-idiotypic antibodies previously well characterized. The presence of cross-reactive Id-bearing cells was observed in 5 out of 6 patients studied, with frequencies ranging from 3 to 12% of lymphocytes. After 7 days of culture, the spontaneous synthesis of idiotypic antibodies by PBL was found elevated in 6 out of 13 (46%) patients. A major Id cell expression and production was noted in patients with active disease as defined by hematuria. The preincubation of PBL with 20 and 50 micrograms of anti-idiotypic antibodies/2 x 10(6) cells for 3 days induced a significant inhibition of cross-reactive Id production in a dose-dependent fashion, with a degree of suppression between 12 and 50% in five out of six patients studied. In the above assays, as negative controls, we used the anti-Id antibodies previously adsorbed on an Id-Sepharose column. On the whole, these results suggest that patients with IgA nephropathy present dysfunctions in the Id-Anti-Id network that could play an important role in the pathogenesis of this disease.
Collagen-induced arthritis in mice. Relationship of collagen-specific and total IgE synthesis to disease.
Authors: Marcelletti J F , Ohara J , Katz D H
Published: December 2022

The relationship between production of IgE and collagen-induced arthritis in mice was examined. Collagen-specific IgE was produced as a consequence of immunization of DBA/1 mice with chicken type II collagen emulsified in CFA. We observed a rise in collagen-specific IgE antibody levels at the onset of CIA clinical and histologic signs in DBA/1 mice. This rise in IgE paralleled that of IgG2a anticollagen antibodies, an isotype implicated in the pathogenesis of CIA by other laboratories. The collagen-specific IgE contained in the plasma of mice with CIA could arm basophils for Ag- (collagen) dependent degranulation. Collagen-specific IgE may thus contribute to CIA by promoting mast cell degranulation in the synovia of susceptible mice immunized with chick type II collagen; but, further work is required to establish such a role for IgE in CIA. However, genetic differences in disease susceptibility could not be accounted for by quantitative differences in collagen-specific IgE production. Further, comparable levels of IgE anticollagen antibodies were observed in animals with active CIA and after spontaneous remission, thereby confirming that the presence of such antibodies is insufficient for disease. Total IgE levels peaked just before spontaneous remission indicating active production of IL-4. IL-4 was administered to animals with CIA to determine if this lymphokine could be involved in the remission process. IL-4 facilitated remission of CIA. Enhanced total IgE production may thus be a marker for activation of Th2 cells that produce lymphokines such as IL-4 and IL-10, factors that may be involved in the spontaneous remission process.
The VLA-4/VCAM-1 pathway is involved in lymphocyte adhesion to endothelium in rheumatoid synovium.
Authors: van Dinther-Janssen A C , Horst E , Koopman G , Newmann W , Scheper R J , Meijer C J , Pals S T
Published: December 2022

Lymphocyte migration to inflammatory sites is an essential factor in the pathogenesis of chronic inflammation. An ensemble of adhesion receptors mediating lymphocyte-endothelial cell recognition and binding are thought to play a crucial role in this process. In the present study, we have explored the molecular basis of lymphocyte adhesion to endothelium in the synovial membrane of patients with rheumatoid arthritis. We established that the very late antigen-4 [VLA-4 (CD49d)] and the vascular cell adhesion molecule-1 (VCAM-1) are important mediators of binding to synovial endothelium of resting and, to a greater extent, of activated T lymphocytes, whereas the leukocyte-function associated antigen-1 [LFA-1 (CD11a/18)]/intercellular adhesion molecule-1 [ICAM-1 (CD54)] pathway is less important in this interaction. In contrast to its prominent role in lymphocyte interaction with endothelium in rheumatoid synovium, the VLA-4/VCAM-1 pathway does not significantly contribute to lymphocyte adhesion to peripheral lymph node high endothelial venule. Thus, the VLA-4/VCAM-1 pathway may be of primary importance in mediating lymphocyte adhesion to inflamed endothelium and in lymphocyte homing to rheumatoid synovium.
Structural similarity between Fc receptors and T cell receptors. Expression of the gamma-subunit of Fc epsilon RI in human T cells, natural killer cells and thymocytes.
Authors: Vivier E , Rochet N , Kochan J P , Presky D H , Schlossman S F , Anderson P
Published: December 2022

The TCR complex is composed of a clonotypic heterodimer (Ti alpha:beta or gamma:delta) noncovalently associated with the CD3 complex (gamma, delta, and epsilon), and with one or more disulfide-linked dimers whose components are designated zeta and eta. zeta and eta are alternative transcripts of a common gene and are structurally related to the gamma-subunit of the FcR for IgE expressed on mast cells and basophils (Fc epsilon RI). Recent evidence suggests that gamma can also be expressed in natural killer cells and in a murine cytotoxic T cell line, CTLL. Because zeta, eta, and gamma have the potential to join together to form disulfide linked homo- and heterodimers, it has been postulated that alternative dimeric forms composed of these zeta-related subunits might subserve unique signal transducing functions in hematopoietic cells. We have used mAb reactive with zeta and gamma to systematically examine the expression of these zeta-related dimers in human T cells, NK cells, and thymocytes. Our results show that each cell type expresses characteristic combinations of zeta-related homo- and hetero-dimers, and are therefore consistent with the possibility that these subunits contribute to the functional heterogeneity of lymphocyte subsets.
T cells that recognize peptide sequences of self MHC class II molecules exist in syngeneic mice.
Authors: Agrawal B , Manickasundari M , Fraga E , Singh B
Published: December 2022

T cell reactivity toward self MHC class II molecules has been recognized in syngeneic MLR in a number of studies, where the T cells are believed to recognize the combination of self/nonself peptide and self MHC molecule. We investigated the stimulation of T cell proliferation by synthetic peptides of sequences corresponding to the first polymorphic amino terminal domain of alpha- and beta-chains of self I-A molecules. Both unprimed and primed T cells responded to a number of peptides of alpha 1 and beta 1 domains of self I-Ad molecules. The response was dependent on the presentation of I-Ad peptides by syngeneic APC and was blocked by anti-class II MHC mAb. Upon further investigation it was observed that I-Ad peptides could inhibit the stimulation of Ag-specific MHC class II-restricted T cell hybridoma due to self presentation of peptides rather than to direct binding of free peptides to the TCR, further supporting their affinity/interaction with intact self MHC class II molecules. The peptide I-A beta d 62-78 showed high affinity toward intact self MHC II molecule as determined by the inhibition of Ag-specific T cell stimulation and yet was nonstimulatory for syngeneic T cells, therefore representing an MHC determinant that may have induced self tolerance. Thus we have shown that strong T cell proliferative responses can be generated in normal mice against the peptides derived from self MHC class II molecules and these cells are part of the normal T cell repertoire. Therefore complete tolerance toward potentially powerful immunodominant but cryptic determinants of self Ag may not be necessary to prevent autoimmune diseases.
Relationship of cyclosporine A-mediated inhibition of clonal deletion and development of syngeneic graft-versus-host disease.
Authors: Bryson J S , Caywood B E , Kaplan A M
Published: December 2022

We have described a strain-specific graft-vs-host disease (GVHD)-like syndrome in syngeneic mouse radiation chimeras that developed in two of seven strains tested after cyclosporine A (CsA) therapy. It has been suggested, recently, that autoreactive T cells that develop as a result of CsA treatment may result from inhibition of clonal deletion and could be responsible for the development of this disease. To test this hypothesis, TCR expression, as a measure of tolerance induction, was analyzed in a series of syngeneic radiation chimeras (inducible and noninducible for syngeneic GVHD), with and without CsA treatment. Clonal deletion, as assessed by anti-TCR V beta chain mAb, appeared to occur normally in strains of mice inducible for syngeneic GVHD. Conversely, animals in which T cells bearing self-reactive TCR could be detected, did not develop the disease. This discrepancy did not appear to be due to a generalized difference in the effects of CsA on the various strains of mice used, as the effects of the drug (i.e., decreased mitogen responsiveness, inhibition of thymocyte maturation, etc.) appeared similar in all strains tested. Therefore, the development of CsA-induced autoreactive T cells as assessed by V beta TCR expression showed strain variation that did not correlate with the induction of syngeneic GVHD and suggested that other mechanisms may be involved in the development of this autoimmune phenomenon.
T cell receptor-dependent, antigen-specific stimulation of a murine T cell clone induces a transient, VLA protein-mediated binding to extracellular matrix.
Authors: Chan B M , Wong J G , Rao A , Hemler M E
Published: December 2022

Upon Ag stimulation, an arsonate-specific murine T cell clone exhibited a rapid but transient increase in cell adhesion to collagen, fibronectin, and laminin. This increase in cell adhesion was not observed when a mutant T cell clone lacking TCR expression was utilized. However, upon stimulation by phorbol esters, both parent and mutant T cell clones exhibited a similar transient increase in adhesion to the three matrix proteins. The observed cell adhesion was extensively inhibited by antibodies to the integrin beta 1 subunit, indicating the involvement of VLA proteins. Despite changes in the adhesive properties, there was essentially no difference in the expression of VLA-1, -3, -4, -5, and -6 between resting and stimulated T cells. Together these results suggest that Ag stimulation transmits signals via the TCR complex resulting in a rapid, but transient, up-regulation of matrix protein binding by VLA proteins already present at the cell surface. Because the appropriate reagents that recognize individual mouse VLA proteins were not available, we used the human T cell line Jurkat to demonstrate that T cell binding to collagen, laminin, and fibronectin is mediated largely by VLA-2, VLA-6, and a combination of VLA-5 and VLA-4, respectively.
A subset of mouse splenic macrophages can constitutively present alloantigen directly to CD8+ T cells.
Authors: McCormack J M , Sun D , Walker W S
Published: December 2022

About a third of mouse splenic macrophage (M phi) progenitors give rise to cloned progeny that constitutively induce the selective proliferation of naive allogeneic CD8+ T cells in a CD4+ helper cell-independent manner--a response that is inhibited by mAb to the MHC class I molecules present on the M phi. Colony-mixing experiments indicated that the failure of most M phi clones to present allo-Ag was not due to their suppression of the ability of CD8+ cells to respond, nor did the nonpresenting clones interfere with the activity of the allo-Ag presenting M phi. The allo-Ag presenting phenotypes were found to be a stable characteristic in a panel of cell lines derived from individual clones of M phi. Analysis of the cell lines revealed that the differential expression of allo-APC activity could not be attributed to the levels of MHC class I molecules; rather, the cell lines and the primary M phi clones differ in their expression of a cell-associated costimulator molecule that likely functions to induce the expression of the IL-2R on and the secretion of IL-2 from the T cells.
Regulation of murine splenocyte responses by heparan sulfate.
Authors: Wrenshall L E , Cerra F B , Carlson A , Bach F H , Platt J L
Published: December 2022

Heparan sulfate is a biologically active glycosaminoglycan found in abundance in endothelium, epithelium, and connective tissues. Although heparan sulfate is an important part of the environment in which immune cells function, its effects on the immune system are largely unknown. When present during the first 24 h of an MLC consisting of MHC disparate murine splenocytes, heparan sulfate had a marked stimulatory effect on the proliferative response to alloantigens. Heparan sulfate also augmented the splenocyte response to suboptimal concentrations of the mitogens Con A, anti-CD3 mAb, and ionomycin. The stimulatory action of heparan sulfate was mediated, at least in part, by increased production of IL-1, because the increased splenocyte proliferation induced by heparan sulfate was substantially inhibited by anti-murine IL-1 alpha-antibodies. In contrast to these stimulatory effects, when heparan sulfate was added to MLC 48 to 72 h after onset, decreased splenocyte proliferation was observed. This inhibitory action was mediated by an increase in PGE2 production; the inhibitory effect could be abrogated with indomethacin. The fact that heparan sulfate is present on cells such as endothelial cells with which T cells interact and is released during activation of endothelial cells (thus making it available in soluble form to cells in the immune response) may allow heparan sulfate to play an important role in modulating cell-mediated immune responses in vivo.
Variations in thymocyte susceptibility to clonal deletion during ontogeny. Implications for neonatal tolerance.
Authors: Zacharchuk C M , Merćep M , June C H , Weissman A M , Ashwell J D
Published: December 2022

Activation of immature thymocytes via the TCR results in programmed cell death and clonal deletion. We have examined thymocytes from mice of different ages and observed that, whereas TCR-mediated signaling caused deletion of thymocytes from newborn and 3-week-old mice, it failed to delete thymocytes from mice of 1 week of age. This could not be attributed to differences in cell surface TCR expression, TCR-mediated phosphoinositide hydrolysis or Ca2+ mobilization, or total cellular levels of TCR zeta- and eta-chains. Moreover, thymocytes of all ages were equally susceptible to corticosteroid- and Ca2+ ionophore-induced programmed cell death. These data are consistent with the notion that fetal and neonatal thymocytes represent a relatively synchronous wave of cells passing through phases in which they are susceptible and then resistant to TCR-induced programmed cell death. They also support the notion that the classical phenomenon of neonatal tolerance is due to clonal deletion and that the inability of allogeneic cells to tolerize mice at 1 week of age is because the thymocytes are refractory to TCR-alpha beta-mediated clonal deletion.
An NF-kappa B-like transcription factor mediates IL-1/TNF-alpha induction of gro in human fibroblasts.
Authors: Anisowicz A , Messineo M , Lee S W , Sager R
Published: December 2022

Normal human foreskin fibroblasts were used to examine transcriptional induction by IL-1 and TNF-alpha of the novel cytokine gro (melanoma growth-stimulating activity). Gro mRNA was expressed at levels 100-fold above background within 45 min of exposure to either IL-1 or TNF-alpha, in growing or serum-starved cells and a similar response was shown by IL-6. In contrast, as shown previously, gro mRNA was elevated only 10-fold by serum in starved but not in growing cells, similar to fos. Thus gro expression appears to be regulated by at least two signal transduction systems: a cytokine pathway, and a growth-related pathway. Three closely related gro genes (alpha, beta, and gamma) have been described. Their proximal 5' regulatory sequences presented here show close similarity in the region to -136, which includes the NF-kappa B site at -66 to -76 in gro alpha and gro gamma, and -64 to -74 in gro beta, and sequence diversity further upstream. Transient transfection of HeLa cells with CAT constructs localized the cytokine response to a region between -84 and -65 in gro beta. Gel retardation studies with FS-2 cells identified a cytokine-induced protein binding at the NF-kappa B site in all three gro genes as shown by competition studies with a pair of oligonucleotides representing wild-type and mutant sequences of the NF-kappa B binding site. Neither serum nor PMA induced a detectable gel shift at NF-kappa B or upstream to position -723. These results demonstrate conservation of the cytokine response element, NF-kappa B, in the three genes, consistent with the conservation of sequence in this region; and suggest that differential expression of the three gro genes may depend upon interactions with other sites located in the divergent upstream region.
Induction of Fc epsilon RII/CD23 on phytohemagglutinin-activated human peripheral blood T lymphocytes. I. Enhancement by IL-2 and IL-4.
Authors: Kawabe T , Maekawa N , Maeda Y , Hosoda M , Yodoi J
Published: December 2022

Despite evidence for the expression of low affinity Fc receptor for IgE (Fc epsilon RII)/CD23 in T cell lines and pathologic T cells, Fc epsilon RII/CD23 in normal human T cells is still unclear. We studied the expression of Fc epsilon RII/CD23 on T cells in short-term culture of normal human PBMC stimulated with 15 micrograms/ml PHA. PHA stimulation also resulted in the release of soluble Fc epsilon RII/CD23 (IgE binding factor). Using two-dimensional flow cytometry, more than 10% of the Fc epsilon RII/CD23+ cells were found to co-express CD3 Ag. Both CD4+ and CD8+ T cells expressed Fc epsilon RII/CD23. The induction of Fc epsilon RII/CD23 on PHA-activated T cells was enhanced by IL-2 as well as IL-4. Both IL-2 and IL-4 also augmented PHA-induced production of soluble Fc epsilon RII/CD23. The enhanced expression of Fc epsilon RII/CD23 on T cells by both lymphokines was suppressed by rabbit anti-IL-4 antiserum, suggesting the involvement of an IL-4-dependent process even in the IL-2-dependent Fc epsilon RII/CD23 expression on T cells. The expression of mRNA for Fc epsilon RII/CD23 on PHA and IL-4-stimulated PBMC was examined by Northern blot analysis. Fc epsilon RII/CD23 mRNA was detected in RNA prepared from the T cell fraction depleted of B cells and macrophages (Fc epsilon RII+CD3+ = 6.2%, Fc epsilon RII+CD3- = 0.8%). The expression of the mRNA for Fc epsilon RII/CD23 on CD3+ T cells was also confirmed by in situ hybridization with Fc epsilon RII/CD23 cDNA combined with CD3 rosette formation at the single cell level.