bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression cloning of the early activation antigen CD69, a type II integral membrane protein with a C-type lectin domain.
Authors: Hamann J , Fiebig H , Strauss M
Published: December 2022

CD69 is a very early activation Ag of T lymphocytes. It is a cell surface glycoprotein that can only be detected after stimulation of lymphocytes. Despite extensive studies on its biologic activities, little is known about its molecular function. To investigate the latter in more detail, we have cloned a cDNA encoding CD69 on the basis of its expression in COS cells. The nucleotide sequence of clone CD69.13 is 1676 bp in length and contains a single open reading frame of 600 bp encoding a protein of 199 amino acids. The predicted molecular mass of 22,559 Da could be confirmed by in vitro translation. The protein contains a hydrophobic transmembrane region between amino acids 41 and 61 but no N-terminal signal peptide, which suggests that it is a type II membrane protein. It has one potential N-glycosylation site at amino acid 166. Two glycosylated forms of 26 to 28 kDa and 32 to 34 kDa were detected both in transfected COS cells and in in vitro translation in the presence of canine microsomes. Proteinase K degradation of the N-terminal part after in vitro protein synthesis supports the view of CD69 being a type II integral membrane protein with the N-terminal 40 amino acids in the cytoplasm, a transmembrane domain of 21 amino acids, and C-terminal 138 amino acids as the extracellular domain. Homology searches revealed sequence similarity with members of a supergene family of type II integral membrane proteins with a C-type lectin domain, indicating that CD69 is involved in signal transduction.
cDNA cloning of guinea pig monocyte chemoattractant protein-1 and expression of the recombinant protein.
Authors: Yoshimura T
Published: December 2022

Monocyte chemoattractant protein-1 (MCP-1) cDNA was cloned from guinea pig spleen cells stimulated with Con A. The cDNA comprised 647 bp with an open reading frame that encoded a 120 amino acid protein. The sequence similarity of the first 99 amino acids to human MCP-1 is 56%. Recombinant guinea pig MCP-1 was expressed in COS-7 cells, then purified by a three step procedure with orange A-agarose, carboxymethyl-HPLC, and reversed phase-HPLC. The purified protein was found around 25 kDa as a broad band on a polyacrylamide gel under reducing conditions. Guinea pig MCP-1 attracted about 34% of input human monocytes at 5 x 10(-9) M. Guinea pig peritoneal exudate macrophages migrated toward guinea pig MCP-1 dose dependently, but only 1% of input cells responded to guinea pig MCP-1 at its optimal concentration of 5 x 10(-9) M. Human MCP-1 attracted 1% of input guinea pig peritoneal exudate macrophages at its highest concentration of 2.5 x 10(-8) M. Neither human nor guinea pig MCP-1 attracted guinea pig peritoneal resident macrophages. These results suggest that monocytes lose responsiveness to MCP-1 after differentiating to macrophages. Finally, intradermal injection of the recombinant protein into guinea pigs caused marked macrophage infiltration. Cloned and expressed guinea pig MCP-1 will help in studying the role of MCP-1 in vivo.
Genetically permissive recognition of adjacent epitopes from the 19-kDa antigen of Mycobacterium tuberculosis by human and murine T cells.
Authors: Harris D P , Vordermeier H M , Friscia G , Román E , Surcel H M , Pasvol G , Moreno C , Ivanyi J
Published: December 2022

The specificity of the T cell-immune repertoire at the level of individual antigenic determinants could play a fundamental role in the immunopathogenesis of tuberculous infections. Therefore, we analyzed the immunogenicity, genetic restriction, and epitope core structure of two adjacent, yet distinct, immunodominant T cell determinants from the 19-kDa Ag of Mycobacterium tuberculosis. After immunization with two peptides comprising residues 45 to 64 and 61 to 80, vigorous in vitro proliferative responses to the homologous peptide were elicited in five different strains of C57BL/10 mice (H-2b,k,d,s,f), indicating that both epitopes were recognized in a genetically permissive manner. When immunized with intact 19-kDa protein, lymph node cells from the same mouse strains responded to both peptides, with the exception of H-2b mice, which did not respond to p45-64. Delayed-type hypersensitivity responses in C57BL/10 (H-2b) mice were elicited by p61-80 only, whereas in H-2d mice both peptides were delayed-type hypersensitivity negative, despite eliciting pronounced proliferative responses. Analysis of the proliferative responses of human PBMC in purified protein derivative-positive healthy subjects and tuberculosis patients revealed significant differences in the antigenicity to the two peptides. All purified protein derivative-positive healthy and diseased individuals manifested strong responses to p45-64, indicating HLA permissive recognition. In contrast, pronounced responses to p61-80 were detected only in patients with lymphatic tuberculosis. Epitope core structures, composed of 6 or 7 residues within each peptide, have been mapped with peptides of overlapping sequence. Significantly, for both epitopes, the core sequences recognized by both human and murine T cells were almost identical. We conclude that despite many similarities between murine and human T cell epitope recognition, distinct differences in the responsiveness of the infected host could play a role in pathogenesis. Furthermore, the genetically permissive nature of the identified epitopes is a potentially important attribute for the development of peptide-based diagnostic reagents and vaccines.
Modulation of T lymphocyte function by neuropeptides. Evidence for their role as local immunoregulatory elements.
Authors: Nio D A , Moylan R N , Roche J K
Published: December 2022

In mucosa-bearing organs with inherent lymphoid populations, classical modes for control of the immune response may be augmented by products of extrinsic sensory afferent nerve endings which arborize through the lamina propria compartment containing large numbers of T and B lymphocytes. Therefore, we sought to determine the role of neuropeptides (substance P, vasoactive intestinal peptide, and somatostatin) in immune response regulation by using a homogeneous line of T lymphocytes (AO40.1 hybrid), whose activation is driven by a specific Ag (OVA) and where the end point (IL-2 release) could not be contributed to by accessory or other cells. IL-2 was quantitated by the rate of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) metabolism with the use of a murine CD4+ IL-2-dependent T lymphocyte line, and dose-response effects of each neuropeptide were examined over a broad concentration range (10(-14)-10(-6) M) encompassing that regarded as physiologic. Vasoactive intestinal peptide stimulated IL-2 release at low concentrations with a marked effect at 10(-14) M that gradually returned to control levels by 10(-7) M. Somatostatin was associated with a substantial augmentation of AO40.1 T lymphocyte IL-2 release at 10(-10) to 10(-8) M concentrations, whereas substance P demonstrated a stimulatory effect only at high concentrations (10(-9) to 10(-6) M). Concomitant [3H]thymidine uptake studies suggested that changes in cell proliferation or viability did not account for neuropeptide-induced effects in our system. With several exceptions, similar results were found with mitogen (Con A)-stimulated AO40.1 cells and human colonic lamina propria mononuclear cells. It was concluded that the three study neuropeptides, over a broad range of concentrations, have profound stimulatory (and occasionally inhibitory) effects upon the function of a cloned T lymphocyte hybrid cell responding to specific Ag and that these events may reflect those of Ag-driven mucosal T lymphocytes exposed to neuropeptides in vivo.
Characterization of two novel Ly-6 genes. Protein sequence and potential structural similarity to alpha-bungarotoxin and other neurotoxins.
Authors: Fleming T J , O'hUigin C , Malek T R
Published: December 2022

Genomic clones cross-hybridizing with Ly-6A.2 cDNA were isolated and characterized for functional Ly-6-related genes. Two new Ly-6 genes, designated Ly-6F.1 and Ly-6G.1, were found to have high nucleotide homology (> or = 70%) and the characteristic four exon gene organization of Ly-6A/E and Ly-6C. By a PCR-based assay, Ly-6G.1 mRNA was readily found in bone marrow, whereas Ly-6F.1 mRNA was not detected in lymphoid tissues. Thus, Ly-6G.1 represents an additional Ly-6 gene with apparent selective expression in hematopoietic cells distinct from Ly-6A/E and Ly-6C. Using the available deduced protein sequence data for mature Ly-6 proteins, searches of the database uncovered an evolutionary relationship of Ly-6 proteins with neurotoxins isolated from snake venoms. The protein sequence conservation between the two groups was selective for, but not limited to, residues in neurotoxins that have been found to be important for their tertiary structures. From this relationship, we propose a neurotoxin-like structure for Ly-6 and Ly-6-related proteins, such as CD59.
Detection of T and B cells in many animal species using cross-reactive anti-peptide antibodies.
Authors: Jones M , Cordell J L , Beyers A D , Tse A G , Mason D Y
Published: December 2022

A wide range of lineage-specific Ag are detectable in the human lymphoid system using mAb, but only a few such markers are detectable in animal species. In this paper, we have investigated the interspecies reactivity of antibodies raised against intracytoplasmic peptide sequences from two T cell Ag (CD3 and CD5) and two B cell markers (the Ig-associated polypeptides encoded by the mb-1 and B29 genes). Immunocytochemical labeling of tissue sections showed that these antibodies cross-react widely between different species (including ungulates, rodents, and marsupials), staining B or T cell areas selectively in lymphoid tissue. The specificity of these antibodies for the animal homologues of the human T and B cell markers was confirmed for the rat by Western blotting analysis. The broad cross-reactivity of these antibodies appears to be due to the fact that they were raised against intracytoplasmic peptide sequences that are highly conserved between humans and rodents, i.e., 80% for mb-1, 85% for CD5, and 100% for CD3 and B29. This strategy should, in the future, widen the range of lineage-associated markers detectable in experimental animals.
Amyloid precursor protein in peripheral mononuclear cells is up-regulated with cell activation.
Authors: Ledoux S , Rebai N , Dagenais A , Shaw I T , Nalbantoglu J , Sekaly R P , Cashman N R
Published: December 2022

The deposition of beta/A4 protein in extraneural organs of patients with Alzheimer disease suggests that this peptide may in part be derived from a peripheral precursor. We studied expression of amyloid precursor protein (APP) in PBMC. APP expression was detectable in resting PBMC by northern blot analysis, immunoblotting studies, and immunohistochemistry. By reverse transcription-polymerase chain reaction, the 751 and 770 APP transcripts containing the Kunitz protease inhibitor (KPI) domain were approximately 10-fold more abundant than the 695 transcript lacking the KPI domain. Activation of PBMC with the lectin PHA-P was associated with an increase in apparent intracellular APP content by cytofluorometry, and an increase in the proportion of the 695 APP transcript lacking the KPI domain. We conclude that resting and activated PBMC express APP and could contribute to a circulating pool of this protein. In addition, PBMC APP is up-regulated with mitogenic stimulation and may participate in the regulation of activation of these cells.
Enhanced IL-4-mediated D10.G4.1 proliferation with suboptimal concentrations of anti-IL-4 receptor monoclonal antibodies.
Authors: Lindqvist C , Lundström H , Oker-Blom C , Akerman K E
Published: December 2022

Suboptimal concentrations of the anti-IL-4 receptor mAb M1 or M2 gave, in combination with IL-4, an enhanced proliferative response to the IL-4-responsive cell line D10.G4.1, compared to IL-4 alone. Increasing amounts of the M1 antibody inhibited the IL-4-dependent proliferation in a normal fashion. The enhanced IL-4-induced proliferation was only inhibited with the anti-IL-4 mAb 11B11, whereas the anti-IL-2 receptor antibodies had no effect. Addition of M1 or M2 antibodies to the IL-2-dependent cell line CTLL, known to express small amounts of IL-4 receptors and thereby also slightly responsive to IL-4, gave no enhanced IL-4-induced proliferation. Instead these antibodies were found to inhibit both the IL-4 as well as the IL-2-mediated CTLL proliferation.
Endogenous secretion of IL-4 maintains growth and Thy-1 expression of a transformed B cell clone.
Authors: Louie S W , Ramirez L M , Krieg A M , Maliszewski C R , Bishop G A
Published: December 2022

The CD5+ B cell lymphoma clone, CH12.LX, endogenously produces IL-4. Blocking the binding of this IL-4 to its cellular receptor inhibited the continuous proliferation of CH12.LX. mAb specific for either IL-4 or the IL-4R profoundly and specifically inhibited the proliferation of CH12.LX cells in a concentration-dependent manner, within 4 h after the addition of mAb. The addition of exogenous rIL-4 alone to CH12.LX cells had no effect on either proliferation or antibody secretion. However, exogenous rIL-4 was able to counteract the effects of anti-IL-4 antibody. Treatment of CH12.LX cells with antisense RNA oligodeoxynucleotides to IL-4 also specifically inhibited cell proliferation and decreased the levels of IL-4 secreted into the culture supernatants by more than 50%, without effect on total RNA or protein synthesis. Effects of antisense IL-4 were also blocked by addition of exogenous IL-4. Control oligodeoxynucleotides of equal size and base composition had no effect, and IL-4 antisense oligodeoxynucleotides did not effect the growth of a B cell lymphoma clone which does not produce IL-4. Blocking the binding of endogenously produced IL-4 to CH12.LX cells did not change the levels of membrane IL-4R or CD5 molecules. However, the constitutive expression of Thy-1 by these B cells was markedly decreased, and anti-Thy-1 antibodies decreased proliferation and PMA-induced aggregation of CH12.LX cells. Autocrine secretion of IL-4 thus appears to be required both for the continuous proliferation of CH12.LX B cells, as well as their expression of Thy-1, which may function either as a homotypic adhesion molecule or a signal transduction molecule for these cells. These findings indicate that endogenously produced lymphokines may play a critical role in the maintenance of B cell hyperproliferative disorders.
IL-4 induction of IgE class switching by lipopolysaccharide-activated murine B cells occurs predominantly through sequential switching.
Authors: Mandler R , Finkelman F D , Levine A D , Snapper C M
Published: December 2022

Resting murine B cells activated with bacterial LPS co-express membrane (m)IgG1 and mIgE upon stimulation with IL-4. In this report, we combine both cellular and molecular approaches to elucidate the mechanism underlying this co-expression. We demonstrate that an anti-IgG1 antibody specifically and selectively inhibits IgE secretion (approximately 70%) by LPS + IL-4-stimulated B cells, which provides functional evidence for mIgG1 expression by precursors of IgE-secreting cells. The IgG1 and IgE secretory responses are separated temporally by approximately 16 h, with IgE production developing later than IgG1. A similar delay is observed in the appearance of mIgE+ cells suggesting that class switching to IgG1 precedes that to IgE. In the sort-purified, mIgG1+mIgE+ B cell population approximately 25% of cells expressed cytoplasmic (c) (secretory) IgG1 and approximately 15% expressed cIgE at the time of their isolation. However, only a small percent of the mIgG1+mIgE+ cells co-expressed cIgG1 and cIgE, further suggesting a temporal separation in IgG1 and IgE secretion within individual cells, but indicating that single cells can co-secrete these two Ig isotypes. Furthermore, the absolute level and rate of increase of IgG1 secretion by mIgG1+mIgE+ cells, upon their isolation and reculture, is lower than that for mIgG1+mIgE- cells suggesting a loss of CH gamma 1 expression in the former population. Analysis of total, unselected circular DNA excision products in LPS + IL-4-activated B cells demonstrates that most, if not all, of the DNA encoding the IgG1 constant heavy gene (CH gamma 1) (i.e., products of a class switch to IgE) have been rearranged. Collectively this data provides strong evidence at both the cellular and molecular level that the predominant mode of switching to IgE in response to in vitro stimulation by LPS + IL-4 is from IgM to IgG1 to IgE.
Bone marrow macrophages process exogenous Toxoplasma gondii polypeptides for recognition by parasite-specific cytolytic T lymphocytes.
Authors: Denkers E Y , Gazzinelli R T , Hieny S , Caspar P , Sher A
Published: December 2022

CD8+ T cells from mice vaccinated with an attenuated strain of Toxoplasma gondii have previously been shown to have cytolytic activity against bone marrow macrophages (BMM phi) preincubated with a soluble tachyzoite extract. In the present study, we show that class I-transfected L cells differ from BMM phi in that although both cell types are recognized CTL after infection, only BMM phi are killed after sensitization with soluble tachyzoite extract. Gel filtration studies indicated that the T. gondii Ag responsible for sensitization of BMM phi are macromolecules of M(r) > or = 12,000. In contrast, peptides derived by tryptic digestion of this material were found to sensitize both transfected L cells and BMM phi. Although exogenous beta 2-microglobulin markedly enhanced peptide sensitization of BMM phi, no such effect was observed using the macromolecular preparation. This result suggests a requirement for cellular internalization in the processing by BMM phi of soluble Ag for class I-restricted recognition. In related experiments, infected and Ag-sensitized BMM phi were found to express cross-reactive T. gondii epitopes, as determined by cold target inhibition studies. Supernatant derived by 100,000 x g centrifugation of tachyzoite extract had potent sensitizing activity, and after anion exchange chromatography most of the activity was associated with a single fraction. The p30 Ag was not detected by immunoblot analysis in the biologically active supernatant and chromatographic fractions. These findings establish the feasibility of identifying the parasite Ag recognized by CD8+ effectors by direct fractionation of T. gondii proteins coupled with sensitization of BMM phi targets.
Tyrosine-phosphorylation of tubulin during monocytic differentiation of HL-60 cells.
Authors: Katagiri K , Katagiri T , Kajiyama K , Yamamoto T , Yoshida T
Published: December 2022

We have demonstrated that the 53- and 55-kDa proteins were markedly phosphorylated at tyrosine residues during tetradecanoyl phorbol acetate (TPA)-induced monocytic differentiation of HL-60 cells and that the inhibition of such phosphorylation resulted in suppression of the differentiation. Western blotting, using antiphosphotyrosine antibody PY-20, of antitubulin immunoprecipitate from the lysate of TPA-treated HL-60 cells revealed that tubulin was tyrosine-phosphorylated in TPA-treated HL-60 cells, but not in nontreated HL-60 cells. The tyrosine-phosphorylated 53- and 55-kDa proteins in TPA-treated HL-60 cells were absorbed with antitubulin antibody indicating that the predominantly tyrosine-phosphorylated proteins during the monocytic differentiation were tubulins. By combination of immunoprecipitation and in vitro kinase assay, the protein tyrosine kinases, Fyn and Lyn, which were abundantly induced during monocytic differentiation of HL-60 cells, were demonstrated to be associated with tubulin in the cells. The reorganization of microtubules was observed by immunostaining using rhodamine-conjugated anti-mouse Ig and mouse anti-alpha-tubulin mAb. These data suggest that tyrosine-phosphorylation of tubulin, in which Fyn and Lyn might involve, regulate monocytic differentiation through alteration of microtubule assembly.
Stimulation of tyrosine phosphorylation and calcium mobilization by Fc gamma receptor cross-linking. Regulation by the phosphotyrosine phosphatase CD45.
Authors: Rankin B M , Yocum S A , Mittler R S , Kiener P A
Published: December 2022

The binding and subsequent cross-linking of murine IgG2a or human IgG to the Fc gamma R on the monocytic cell line THP-1 induced a rapid, dose-dependent increase in tyrosine phosphorylation of several proteins (a doublet centered around 110 kDa, and bands at 80, 60, and 52 kDa) and smaller increases in other proteins. This phosphorylation was accompanied by an increase in intracellular free Ca2+. The signaling required the cross-linking of the IgG, either through a biotin-avidin complex or with a F(ab')2 second antibody. Cross-linking of an F(ab')2 fragment of mAb 32.2 to Fc gamma RI (CD64) or an Fab fragment of mAb IV.3 to Fc gamma RII (CDw32) gave similar results to those observed with intact murine IgG2a or human IgG. Cross-linking of a F(ab')2 fragment of mAb 3G8 to Fc gamma RIII (CD16) had very little effect. Increases in both tyrosine phosphorylation and intracellular free Ca2+ were significantly reduced in a dose-dependent manner upon treatment of THP-1 cells with the tyrosine kinase inhibitors herbimycin-A, genistein, or erbstatin. Additionally, there was a marked inhibition of both Ca2+ mobilization and tyrosine phosphorylation when a F(ab')2 fragment of a mAb (T191) to the protein tyrosine phosphatase CD45, was co-cross-linked with either Hu-IgG, Mu-IgG2a, F(ab')2 anti-Fc gamma RI, or Fab anti-Fc gamma RII. Taken together these results suggest that signaling through Fc gamma RI (CD64) and Fc gamma RII (CDw32) in the monocytic leukemia cell line THP-1 gives rise to rapid tyrosine phosphorylation of several proteins followed by an increase in intracellular calcium. In addition, CD45 is able to inhibit the intracellular signaling when it is brought into close proximity to the Fc gamma R. This suggests that this transmembrane tyrosine phosphatase may regulate the stimulation of the cells through the Fc gamma R.
MRL mice produce anti-Su autoantibody, a specificity associated with systemic lupus erythematosus.
Authors: Treadwell E L , Cohen P , Williams D , O'Brien K , Volkman A , Eisenberg R
Published: December 2022

Certain autoimmune mouse strains exhibit features similar to human SLE. To discover genetic and immunologic events governing expression of a new SLE-associated antibody, the presence of anti-Su and its relationship to other SLE-related antibodies (anti-Sm, -ribonucleoprotein, -Ro (SS-A), -La (SS-B)) were determined in MRL and other autoimmune and nonautoimmune mice. By double immunodiffusion, sera from 34/183 (19%) 4- to 10-mo-old MRL/Mp-lpr/lpr (MRL/lpr) and 28/108 (26%) 8- to 20-mo-old MRL/Mp(-)+/+ (MRL/+) mice were positive for anti-Su antibodies. Anti-Sm antibodies were found in 60/183 (33%) and 39/108 (36%) of these animals, respectively. The two specificities were found together in individual mice more frequently than would be predicted by chance. In contrast, C57BL/6-lpr/lpr (B6/lpr) mice rarely showed either specificity. Analysis of F1 hybrids between B6/lpr and MRL/lpr and of F1 x MRL/lpr backcross mice suggested that a small number of autosomal recessive genes controlled the anti-Su and anti-Sm responses. With the exception of a single NZB serum sample, NZB, BXSB, and nonautoimmune mice were negative for all antibodies tested, and no mice were positive for anti-RNP, anti-Ro, or anti-La. MRL/lpr and MRL/+ autoimmune mice thus provide unique models for human SLE, because they express several of the SLE-specific marker autoantibodies. These models should be useful in disclosing molecular and immunologic events governing autoantibody expression in this condition.
Nonrandom X chromosome inactivation in natural killer cells from obligate carriers of X-linked severe combined immunodeficiency.
Authors: Wengler G S , Allen R C , Parolini O , Smith H , Conley M E
Published: December 2022

X-linked severe combined immunodeficiency (XSCID) is characterized by hypogammaglobulinemia, markedly reduced numbers of T cells, absent mitogen responses, decreased numbers of NK cells, and normal or elevated numbers of B cells. The abnormalities in the NK cell and B cell lineages could be attributed to dependence of these cell lineages on T cells or T cell-derived factors, or to expression of the XSCID gene defect in these cell lineages. In past experiments, we have examined X chromosome inactivation patterns in T cells and cultured B cells from female obligate carriers of XSCID and have found that both cell lineages demonstrate nonrandom X chromosome inactivation. This indicates that the gene defect is intrinsic to both of these cell lineages. In the present experiments, a polymerase chain reaction technique was used to evaluate X chromosome inactivation patterns in highly purified populations of freshly isolated NK cells, B cells, CD4+ cells, and CD8+ cells from three obligate carriers of XSCID. All four lymphoid cell populations from these three women exhibited exclusive use of a single X as the active X. In contrast, both X chromosomes were used as the active X in neutrophils and monocytes. These findings indicate that the XSCID gene is expressed in the NK cell lineage as well as in T cells and B cells. This observation makes it highly unlikely that the XSCID gene is involved in Ag receptor gene rearrangements.
A monoclonal antibody to the NH2-terminal segment of human IFN-gamma selectively interferes with the antiproliferative activity of the lymphokine.
Authors: Caruso A , Tiberio L , De Rango C , Bonfanti C , Flamminio G , Gribaudo G , Monti E , Viani E , Manca N , Garotta G
Published: December 2022

To gain more information about the relationship between the structure of IFN-gamma and its activity, a peptide corresponding to a hydrophilic peak between amino acids 4 and 16 was used to immunize mice and generate mAb. mAb IGMB-15 reacts to both native and rIFN-gamma and neutralizes the antiproliferative activity of IFN-gamma without affecting its antiviral activity or its ability to up-regulate HLA-DR Ag expression. Moreover, we observed that mAb IGMB-15 was unable to inhibit the binding of radiolabeled IFN-gamma to its cellular receptor. These findings show that the NH2-terminal region may somehow be involved in the biologic activity of IFN-gamma. Besides, the capability of mAb IGMB-15 to inhibit the antiproliferative but not the antiviral activity of IFN-gamma in the same cell (HEp-2) suggests the presence of different elements involved in signal transduction, which may account for the multiple activities of the lymphokine.
In vitro selection of RNA epitopes using autoimmune patient serum.
Authors: Tsai D E , Keene J D
Published: December 2022

Nucleotide-specific autoimmune epitopes have not been precisely defined despite the fact that certain kinds of DNA and RNA species are known to bind autoantibodies. Our laboratory has used nucleic acid epitope libraries, consisting of randomized RNA pools, to select specific RNA conformers recognized by antibodies, including a peptide-specific antibody. In the present study, serum from a patient with systemic lupus erythematosus was used to select ligands from an RNA epitope library. The selected RNA contained sequences that were found to be similar to regions within the U1 small nuclear RNA, previously shown to react with autoantibodies. Furthermore, the selected RNA epitopes were able to inhibit autoantibody reactivity with specific regions of U1 RNA, thus demonstrating their immunologic cross-reactivity with the natural RNA epitope. Although the origins of nucleic acid-binding autoantibodies are not understood, the identification of these defined U1 RNA epitopes, in regions of the RNA where cell proteins are not known to bind, is most compatible with models of immunologic cross-reactivity or with direct presentation to the immune system rather than with anti-Id models. These experiments demonstrate that RNA epitope libraries may be used to reveal the fine specificity of autoimmune recognition and provide a useful approach to study RNA-protein interactions.
Biased T cell receptor usage by Ld-restricted, tum- peptide-specific cytotoxic T lymphocyte clones.
Authors: Solheim J C , Alexander-Miller M A , Martinko J M , Connolly J M
Published: December 2022

We have investigated the TCR gene usage in a panel of H-2Ld-restricted, tum- peptide-specific CTL clones. These clones possess identical MHC restriction and peptide specificity, yet they vary dramatically in the amount of peptide required to sensitize targets for recognition. We previously demonstrated a precise quantitative correlation between the determinant density requirement of a given clone and the CD8 dependency. In this study we sequenced polymerase chain reaction copies of the TCR mRNA used by these clones, not only to correlate TCR structure with recognition of a specific class I/peptide complex, but also to determine if the functional affinity differences between these clones is reflected in the TCR gene products used. The number of TCR V beta, V alpha, and J alpha region gene segments expressed by these clones is very limited. Twelve of 17 clones express V beta 8 at comparable levels on the cell surface. Using PCR amplification of cDNA templates, cloning, and dideoxy sequencing, we have obtained the nucleotide sequence of the TCR V-(D)-J regions in seven of the V beta 8+ clones. Two of the clones use V beta 8.2 and identical J alpha gene segments. Three of the five V beta 8.3+ clones express identical V alpha and J alpha gene products and the other two use similar V alpha chains and J alpha chains with a shared motif in the predicted CDR3 region. Although no clear correlation between TCR gene usage and CD8 dependency was seen, the range of TCR gene usage in the tum- peptide-specific, Ld-restricted immune response is strikingly narrow and suggests a coselection of the alpha- and beta- chains for recognition of the class I/peptide complex.
Enhancement of natural killer activity by an antibody to CD44.
Authors: Tan P H , Santos E B , Rossbach H C , Sandmaier B M
Published: December 2022

In this study, we examined the in vitro effect of an anti-CD44 mAb, S5, on NK function using canine PBMC as effectors. S5 enhanced NK activity in a dose-dependent and rapid fashion as did IM7, another anti-CD44 mAb that recognizes a common epitope(s) on CD44. Other anti-CD44 mAb (Hermes-1 and S3) that recognize epitopes distinct from S5 and IM7 had a variable effect on NK activity. The activation of increased killing by S5 only occurred when NK-sensitive targets were used, suggesting that lymphokine-activated killer cells were not being induced. Antibody-dependent cell cytotoxicity was not the mechanism involved in the augmentation of NK activity, nor was it Fc receptor-dependent, inasmuch as S5 F(ab')2 was able to increase NK activity. F(ab') fragments of S5 were equivalent to intact S5 in their ability to stimulate NK activity, demonstrating that cross-linking of CD44 was not a necessary component of stimulation, and that nonspecific agglutination of target and effector cells was not occurring via the two F(ab) arms. The enhancement of NK function was monocyte-independent and mediated by radioresistant cells, indicating that the antibody enhanced NK cells directly. This finding would suggest that CD44 can direct a transmembrane signal for NK cell activation.
Simultaneous ligation of CD5 and CD28 on resting T lymphocytes induces T cell activation in the absence of T cell receptor/CD3 occupancy.
Authors: Verwilghen J , Vandenberghe P , Wallays G , de Boer M , Anthony N , Panayi G S , Ceuppens J L
Published: December 2022

The T cell surface molecules CD5 and CD28 have been shown to be receptors for accessory signals in T cell activation. We here demonstrate that in the absence of any other activating stimulus, simultaneous ligation of CD5 and CD28 by mAb induces polyclonal T cell activation. Immobilization of the anti-CD28 and anti-CD5 mAb was an essential requirement for T cell stimulation. This was done either through coating of the culture plates with goat anti-mouse Ig, or by coculture with mitomycin C-treated Fc gamma R-bearing P815 mouse mastocytoma cells. Most importantly, T cells could also be stimulated with B7, the natural ligand of CD28, and anti-CD5 presented on irradiated 3T6 mouse fibroblasts co-transfected with human Fc gamma RII and with B7. Neither immobilized mAb 9.3 (anti-CD28) nor any of four different anti-CD5 mAb were mitogenic as a sole stimulus. Immobilized mAb identifying CD4, CD7, or LFA-1 were not co-mitogenic with either mAb 9.3 or one of the anti-CD5 mAb. The T cell proliferation induced by cross-linking of CD5 and CD28 is IL-2-dependent, as was demonstrated by the cell-surface expression of the p55 chain of the IL-2R, the production of IL-2, and inhibition of the proliferative response by the anti-IL-2R mAb anti-Tac. CD5/CD28 ligation induced production of TNF-alpha, but not of IL-4, and did not induce modulation of the TCR/CD3 complex. Expression of IL-2R (p55) and of CD69 preferentially occurred on CD29-low naive cells, and indicated that about 50% of the cultured cells were activated. Cell proliferation was not increased by adding monocytes to the cultures and it was inhibited by PKC inhibitors (H7 and staurosporine) and by cyclosporine A. In conclusion, our data provide evidence for a pathway of Ag-independent T cell activation via CD5 and CD28, which preferentially stimulates native T cells.
Multiple changes in VLA protein glycosylation, expression, and function occur during mouse T cell ontogeny.
Authors: Wadsworth S , Halvorson M J , Chang A C , Coligan J E
Published: December 2022

VLA molecules (beta 1 integrins) are cell-surface alpha beta heterodimers that bind to extracellular matrix (ECM) proteins such as fibronectin, laminin, and collagen. We analyzed the expression, structure, and function of VLAs on mouse thymocytes, as a first step toward understanding their role in T cell development. Two major forms of beta 1 were detected, which differed in their extent of N-glycosylation and sialylation. No evidence for alternative splicing of beta 1 mRNA was obtained by PCR analyses. The larger (135 kDa, nonreduced) more basic beta 1 was the only form on nonmature (J11d+) adult thymocytes, with 135 kDa and larger beta 1-chains expressed on fetal thymocytes from day 14 of gestation through birth. The smaller (120 kDa, nonreduced), more acidic beta-chain was found exclusively on mature (J11d-) thymocytes and peripheral lymphocytes. VLA alpha-chain expression was also analyzed. Virtually all thymocytes were VLA-alpha 4+, -alpha 6+. VLA-alpha 5 was detected in both J11d+ and J11d- thymocytes by immunoprecipitation, but could not be analyzed in detail because of the lack of an appropriate mAb for flow cytometry. VLA-alpha 1 and -alpha 2 were immunoprecipitated only from cells within the J11d- population. Only J11d+ thymocytes (12 to 15%) bound to fibronectin (via VLA-4 and VLA-5) and binding to laminin (via VLA-6) was two to fourfold higher in J11d+ compared with J11d- thymocytes (60 to 80% vs 20 to 30%). The high percentage of J11d+ thymocytes adhering to laminin suggests that VLA-6 exists in an activated state on most thymocytes, in contrast to resting peripheral T cells. These data show that major changes in VLA glycosylation, expression, and ECM-binding capacity occur as thymocytes mature, supporting the hypothesis that VLA-ECM interactions play a role in T cell ontogeny.
Function associated with IL-2 receptor-beta on human neutrophils. Mechanism of activation of antifungal activity against Candida albicans by IL-2.
Authors: Djeu J Y , Liu J H , Wei S , Rui H , Pearson C A , Leonard W J , Blanchard D K
Published: December 2022

Polymorphonuclear neutrophils (PMN) are essential components of the host defense system against a wide variety of pathogens. We report here the novel finding that freshly isolated human PMN constitutively express detectable surface levels of IL-2R beta, but not IL-2R alpha, as analyzed by flow cytometry. Northern blot analysis confirmed the constitutive expression of mRNA for IL-2R beta in PMN. Scatchard analysis using 125I-labeled IL-2 demonstrated the presence of approximately 600 intermediate binding IL-2R per PMN, with a dissociation constant of 1.1 x 10(-9) M, similar to that of IL-2 binding to YT-1 tumor cells that specifically express IL-2R beta. More importantly, PMN were able to respond functionally to IL-2 by enhanced growth-inhibitory activity against an opportunistic fungal pathogen, Candida albicans. IL-2 activation of antifungal activity was dose-dependent, with some functional activation detected at 1 U/ml of rIL-2 and maximal activation at 1000 U/ml. The action of IL-2 was rapid, with maximal PMN activation after 30-min incubation with IL-2. The IL-2 enhancement of antifungal activity could be blocked by a specific antibody against IL-2R beta, but not by anti-IL-2R alpha. Analysis of the mechanism of IL-2 activation of PMN indicated that oxidative metabolism, as measured by superoxide anion production, was not involved. Instead, PMN release of lactoferrin appeared to be responsible for the heightened activity against C. albicans in IL-2-treated PMN. Not only was lactoferrin detected in the supernatants of IL-2-treated PMN, but also the antifungal activity of PMN activated by IL-2 could be blocked in the presence of antilactoferrin. These results, taken together, indicate that normal PMN are capable of functionally responding to IL-2 via expression of the IL-2R beta chain.
Serologic analysis of a murine chemokine, TCA3.
Authors: Luo Y , Laning J , Dorf M E
Published: December 2022

The TCA3 gene encodes a proinflammatory cytokine related to IL-8 and MCP-1 (JE). TCA3 mRNA is produced by T cells in response to stimulation with Ag or Con A. Glycosylated and nonglycosylated forms of rTCA3 produced by transfected Chinese hamster ovary cells were purified. rTCA3 was used to immunize hamsters and produce mAb. Four mAb were characterized; each was specific for TCA3, failing to react with the closely related molecule MCP-1 (JE). Western blotting also demonstrated that all four mAb specifically recognize rTCA3, native TCA3 produced by activated mouse T cells, and the nonglycosylated TCA3 core protein. A sensitive and specific capture ELISA for TCA3 quantitation was developed, allowing measurement of TCA3 levels in the supernatants from activated murine splenocytes and T cell clones. In addition, two mAb demonstrated dose-dependent neutralization of TCA3 inflammatory activity. Thus, the results directly demonstrate that TCA3 is expressed in an activation-specific manner by mouse T cells. The data also provide characterization of mAb reagents specific for this new cytokine.
IL-1 and tumor necrosis factor-alpha each up-regulate both the expression of IFN-gamma receptors and enhance IFN-gamma-induced HLA-DR expression on human monocytes and a human monocytic cell line (THP-1).
Authors: Krakauer T , Oppenheim J J
Published: December 2022

Stimulation of human blood monocytes (adherent mononuclear cells) and the monocytic cell line, THP-1, by IL-1 or TNF-alpha leads to the up-regulation of IFN-gamma receptors. Scatchard analysis using 125I-IFN-gamma revealed a twofold increase in the number of IFN-gamma receptors on THP-1 cells without an alteration in the affinity of the receptor. The potential functional significance of this induction of IFN-gamma receptors on monocytes and THP-1 cells was investigated by examining the effect of IFN-gamma on MHC class II Ag expression by these cells. Both IL-1 and TNF-alpha enhanced the IFN-gamma-induced HLA-DR expression (> twofold) and this effect was inhibited by antibody to IFN-gamma. In the case of human monocytes, IL-1 or TNF-alpha, each by themselves also increased HLA-DR expression, which was also abrogated by antibody to IFN-gamma. The data suggest that the immunopotentiating effects of IL-1 and TNF-alpha are mediated in part by enhancing IFN-gamma receptor expression on monocytes and macrophages. This presumably would increase the capacity of IFN-gamma to activate macrophages, enabling them to express HLA-DR and present Ag more effectively.
Primary in vivo responses to ovalbumin. Probing the predictive value of the Kb binding motif.
Authors: Lipford G B , Hoffman M , Wagner H , Heeg K
Published: December 2022

CD8+ cytolytic T cells recognize Ag presented by MHC class I molecules on the surface of target cells. It is known that presenting cells process nascent protein into peptides of approximately eight to nine amino acids which bind to the peptide groove of MHC class I and are transported to the cell surface. Recently, several laboratories have postulated that each MHC class I haplotype has a binding motif of at least two amino acids nested within the peptide. One such motif is XXXXF/YXXL which binds to the mouse MHC class I molecule, H2-Kb, and can be found in the known antigenic peptide from OVA at amino acids 257-264. By using the motif to scan OVA five peptides were found that fit this pattern, OVA 11-18, OVA 55-62, OVA 107-114, OVA 176-183, and OVA 257-264. Binding studies revealed that three out of the five peptides (OVA 55-62, OVA 176-183, and OVA 257-264) bind to MHC class I. To test the natural antigenicity of the predicted peptides, C57BL/6 mice were immunized with OVA containing immunostimulating complexes to elicit a MHC class I-driven response to naturally processed OVA. The cytolytic potential of the responding T cell population was tested in vitro by using EL-4 cells preincubated with the predicted synthetic peptides as targets. The known antigenic peptide OVA 257-264 elicited a strong response; however, OVA 176-183 was also recognized while the remaining three were not recognized. The CTL response did not strictly correlate with the ability of the selected peptides to bind Kb, for example, OVA 55-62 was able to bind Kb efficiently, yet elicited no cytolytic response. In addition, the plasticity of the peptide-binding motif was probed by making amino acid substitutions, and as a result the motif proved to be more flexible than previously suspected. This represents the first report of a Kb-associated CTL epitope within OVA other than OVA 257-264. It also demonstrates the predictive quality of the Kb-binding motif; however, not all predicted peptides were recognized by primary OVA-induced CTL, implying more rules of processing and binding are needed.
Recombinase activating gene expression in thymic subpopulations. A transitional cell type has lost RAG-2 but not RAG-1.
Authors: Campbell J J , Hashimoto Y
Published: December 2022

We have used a highly sensitive Northern blot probing technique to examine RAG-1 and RAG-2 mRNA expression in unmanipulated murine thymocytes of various developmental phenotypes sorted by flow cytometry. We have found that neither RAG-1 nor RAG-2 are down-regulated until thymocytes reach the TCRhi stage. TCRhi, CD8+CD4+ cells have lost RAG-2 mRNA but continue to express significant levels of RAG-1 mRNA, providing molecular evidence that this cell type is transitional between the TCRlo, CD4+CD8+ and the TCRhi, CD4+CD8-, or CD4-CD8+ phenotypes.
Molecular analysis of double isotype expression in IgA switching.
Authors: Kunimoto D Y , Sneller M C , Claflin L , Mushinski J F , Strober W
Published: December 2022

In this and previous studies we have shown that during the course of B cell isotype differentiation occurring in the CH12.LX B cell line, small numbers of cells appear that bear both membrane (m)IgM and membrane IgA. We have cloned relatively stable populations of such dual-bearing cells and have performed studies to determine their molecular status. In the study of one such subclone (CH12.LX.7.10), we show by primer extension that both mu and alpha mRNA contain identical VHDJH gene segments. In complementary studies of another such subclone (CH12.LX.7.13), we show using DNA restriction fragment analysis, that the C mu gene segment, but not the C alpha gene segment, is associated with the JH gene segment. Additionally, Northern blot analysis of productive message of this clone shows that both mu and alpha mRNA cohybridize to the same VH region-specific probe. Taken together, these data suggest that CH12.LX mIgM/mIgA dual-bearing cells occur in the absence of C mu gene segment deletion and that the mu and alpha mRNA transcripts are derived using an alternative mechanism such as transplicing or alternative splicing of a long RNA transcript. On the assumption that these clones are representative of mIgM/mIgA dual-bearing cells appearing during CH12.LX IgM to IgA isotype differentiation, these data support the idea that B cells simultaneously produce mu and alpha mRNA transcripts during one stage of IgA isotype switching.
Cytokine production in BALB/c mice immunized with radiation attenuated third stage larvae of the filarial nematode, Brugia pahangi.
Authors: Bancroft A J , Grencis R K , Else K J , Devaney E
Published: December 2022

BALB/c mice immunized with radiation attenuated third stage larvae of the filarial nematode Brugia pahangi are strongly immune to challenge infection. Investigation of the profile of cytokines secreted by spleen cells from immune mice stimulated in vitro with either parasite Ag or with Con A revealed high levels of IL-5 and IL-9 and moderate levels of IL-4. In contrast, secretion of IFN-gamma by spleen cells from immune animals was negligible. Spleen cells from control mice secreted low levels of all cytokines assayed. Levels of parasite-specific IgE were significantly elevated in immune animals and a peripheral blood eosinophilia was observed, which exhibited a biphasic distribution. Our results are consistent with the preferential expansion of Th2 cells in immune animals and provide the basis for dissecting the means by which radiation attenuated larvae of filarial nematodes stimulate immunity.
Modulation of human basophil histamine release by insulin-like growth factors.
Authors: Hirai K , Miyamasu M , Yamaguchi M , Nakajima K , Ohtoshi T , Koshino T , Takaishi T , Morita Y , Ito K
Published: December 2022

Several lines of evidence indicate that basophils play an active pathogenic role in the late-phase reaction. Inasmuch as various cytokines are produced locally by activated cells of the immune system during late-phase reaction, "priming" of basophils by cytokines has become an area of active study over the last few years. We studied the effect of insulin-like growth factor (IGF)-I, shown to be active on immature and mature hemopoietic cells, on histamine release from human basophils. IGF-I enhanced histamine release initiated by anti-IgE, calcium ionophore A23187, and phorbol ester in a dose-dependent fashion (ED50: 300-600 pM), although IGF-I had little or no effect on the release caused by FMLP and C5a. The priming effect of IGF-I took place rapidly and reached plateau levels in 15 min. IGF-II and insulin, both of which have high amino acid sequence homology with IGF-I, also primed basophils. The order of enhancing potencies was IGF-I > IGF-II > insulin; IGF-I was 10-fold more potent than IGF-II and 250-fold more potent than insulin. Although specific receptors for each factor have been identified, blocking experiments using a mAb against IGF-I receptor showed almost complete abolishment of the enhancing effect of these factors, indicating that the enhancing effect is exhibited via the IGF-I receptor. Inasmuch as IGF-I has been shown to be produced by alveolar macrophages, fibroblasts, and nasal polyps, these results indicate that IGF may regulate allergic reactions in vivo by enhancing histamine release from basophils.