bluuebunny/crossref_metadata_2025 huggingface dataset:
Alterations in antibody-dependent cellular cytotoxicity during the course of HIV-1 infection. Humoral and cellular defects.
Authors: Tyler D S , Stanley S D , Nastala C A , Austin A A , Bartlett J A , Stine K C , Lyerly H K , Bolognesi D P , Weinhold K J
Published: January 2023

HIV-1-specific cell-mediated cytotoxicity (CMC) is a form of antibody-dependent cellular cytotoxicity (ADCC) in which HIV-1-specific antibodies arm NK cells directly to become cytotoxic for targets bearing HIV-1 antigenic determinants. This non-MHC-restricted cytotoxic activity is present in early stages of disease and declines markedly with disease progression. To understand the cellular and humoral factors contributing to the reduction in this activity, the conditions under which maximal arming of cells occurs was examined in vitro. With the use of a large patient cohort, a strong positive correlation was found between the capacity of a serum to direct lysis in standard ADCC assays and its ability to arm NK cells. Patients with minimal HIV-1-specific ADCC-directing antibodies exhibited low levels of CMC and were unable to arm normal effector cells in vitro. The lack of sufficient ADCC-directing antibodies was found to be one cause of defective CMC in some patients. Unlike asymptomatics, only a weak positive correlation was found between arming and ADCC with sera from AIDS patients, indicating that a factor other than absolute HIV-1 specific antibody titer was responsible for decreased CMC in this patient population. Another group of patients was found to have diminished CMC despite the presence of antibodies in the serum that were fully capable of arming normal effector cells to become cytotoxic for gp120-expressing targets. When compared with those of normal individuals, lymphocytes from seropositive patients mediated significantly reduced levels of cytotoxicity in ADCC and arming assays with the use of a high titered HIV-1-specific serum. In both assay systems, the magnitude and frequency of dysfunction in antibody-dependent cytolysis was found to be greater among AIDS patients than among asymptomatic individuals. The demonstration of both cellular and humoral defects in the ability of seropositive individuals to manifest ADCC reactivities strongly suggests that HIV-1 infection may significantly compromise the effectiveness of this potentially important cytolytic reactivity in vivo.
Dissociation of expression of two rheumatoid factor cross-reactive kappa L chain idiotopes in rheumatoid arthritis.
Authors: Koopman W J , Schrohenloher R E , Carson D A
Published: January 2023

mAb 6B6.6 and 17.109 recognize two distinct kappa III L chain cross-reactive idiotopes (CRI) present on approximately 2/3 of IgM kappa rheumatoid factor (RF) paraproteins. To determine the distribution of these two CRI and their relationship to each other among polyclonal RF, sera from 86 RA patients and 49 controls were analyzed for the presence of 6B6.6- and 17.019-bearing RF by using sensitive solid phase ELISA. Levels of CRI(+) RF were estimated by using 6B6.6(+) and 17.019(+) RF standards. Detectable levels (greater than or equal to 195 ng/ml) of CRI(+) RF were rarely present in the control sera (8% for 6B6.6; 0% for 17.109), whereas 59% of RA sera contained measurable CRI(+) RF (48% for 6B6.6; 35% for 17.109; 21% for both). Where detected, CRI(+) RF were present in low concentrations (6B6.6: 1.21 +/- 1.56 micrograms/ml; 17.109: 1.20 +/- 1.15 micrograms/ml) and constituted a small fraction of the total IgM RF in these sera (6B6.6: 0.9 +/- 2.2%; 17.109: 0.8 +/- 0.9%). There was no correlation between either RF CRI and levels of IgM RF (r less than 0.1, p greater than 0.5). Levels of 6B6.6(+) RF did not correlate with 17.109(+) RF (r = -0.11, p = 0.47). In selected sera that contained both RF CRI, it was possible to selectively absorb 6B6.6(+) RF. Taken together, these data indicate the mutual independence of these two RF CRI among polyclonal RF and suggest the presence of distinct regulatory mechanisms governing their expression. Moreover, that these two CRI constitute a small proportion of polyclonal RF, in contrast to their striking predominance among monoclonal RF paraproteins, argues for the importance of other germline VL genes contributing to polyclonal RF production or the presence of extensive somatic mutation among polyclonal RF in RA.
Platelet activating factor stimulates cyclo-oxygenase activity in guinea pig eosinophils. Concerted biosynthesis of thromboxane A2 and E-series prostaglandins.
Authors: Giembycz M A , Kroegel C , Barnes P J
Published: January 2023

The effect of platelet activating factor (PAF) on the generation of cyclo-oxygenase-derived arachidonic acid metabolites was examined on purified eosinophils harvested from the peritoneal cavity of male guinea pigs. PAF produced a concentration-dependent increase in the amount of immunoreactive thromboxane B2 (TXB2) and PGE1/E2 released from these inflammatory cells at a relative molar ratio of 30:1. The EC50 of PAF was 20 to 40 nM and maximum stimulation (4.5-fold) of both prostanoids occurred at 1 microM PAF. The ability of PAF to generate TXA2 was rapid (t 1/2 = 9 s), transient (40 s), noncytotoxic, and noncompetitively antagonized by the PAF-receptor blocking drug, WEB 2086. On an equimolar (100 nM) basis, PAF was significantly more effective than C5a, fMLP, and PMA at stimulating TXB2 release but markedly less potent than the calcium ionophore, calcimycin. Pretreatment of eosinophils with the cyclo-oxygenase inhibitor flurbiprofen (8 microM for 5 min) abolished the ability of PAF to promote both TXB2 and PGE1/E2 release. Likewise, dazmegrel (50 microM for 5 min), a selective inhibitor of thromboxane synthetase, abolished PAF-stimulated TXB2 release but markedly augmented the elaboration of PGE1/E2. Inhibition of cyclo-oxygenase with flurbiprofen affected neither the ability of PAF to elevate the intracellular calcium ion concentration (measured by fura-2 fluorescence) nor its appetency to generate superoxide anions at any PAF concentration examined. It is concluded that activation of guinea pig eosinophils by PAF is receptor-mediated and independent of the concomitant generation of cyclo-oxygenase-derived excitatory prostanoids. Inasmuch as TXA2 may contribute to the pathogenesis of bronchial hyperreactivity, then these data implicate the eosinophil as a potential source of this lipid mediator.
Frequency of human B cells that differentiate in response to anti-CD3-activated T cells.
Authors: Amoroso K , Lipsky P E
Published: January 2023

Activation of T cells by mAb to the CD3 molecular complex induces the differentiation of many more Ig-secreting cells (ISC) from resting human B cells in bulk cultures than do other modes of polyclonal B cell activation. In the current experiments, a limiting dilution assay was used to demonstrate that this increase in ISC generation reflects an increased frequency of responding B cells. Highly purified B cells were cultured at densities of between 1000 cells and 0.5 cell per microwell with fresh, mitomycin C-treated T cells (T mito) or T cell clones stimulated by immobilized mAb to CD3. After 5 days in culture, the number of wells containing ISC was determined, and the frequency of responding B cells was calculated. The proportion of B cells responding to anti-CD3-stimulated T cells was very large (10.7 +/- 2.8%) and greatly surpassed that induced by other polyclonal activators. B cells cultured with anti-CD3-stimulated T cell clones responded better than did those cultured with T mito. The addition of exogenous IL-2 or IL-6 to cultures supported by activated T mito enhanced the frequency of responding B cells, whereas IL-4 did not increase the generation of ISC and inhibited the augmentation of B cell responses induced by IL-2. Supplementation of cultures with mitomycin C-treated B cells as accessory cells had less of an effect. The addition of both accessory cells and IL-2 markedly increased B cell responsiveness, with precursor frequencies of 60 to 80% noted. In some experiments, cultures were carried out for 7 to 14 days and supernatants were analyzed for IgM, IgG, and IgA secretion. B cells activated by anti-CD3-stimulated T cells produced all three Ig isotypes. When the classes of Ig produced by single B cells were examined, it was observed that the stimulation of individual B cell precursors led to the production of multiple Ig isotypes, suggesting that isotype switching occurs in these cultures. These results demonstrate that under optimum culture conditions, T cells stimulated with immobilized anti-CD3 can activate the majority of human peripheral blood B cells to produce Ig and induce isotype switching by many.
Cholera toxin acts synergistically with IL-4 to promote IgG1 switch differentiation.
Authors: Lycke N , Severinson E , Strober W
Published: January 2023

Previously, we reported that cholera toxin (CT) causes LPS-stimulated membrane (m)IgM+ B cells to undergo increased switch differentiation to IgG- and IgA-producing B cells. In this study we determined whether this effect is specific for one or several of the IgG subclasses and whether B cells exposed to CT respond differently to IL-4, a lymphokine with switching capabilities. In initial studies we found that in LPS-stimulated, mIgM+ B cell cultures, CT eightfold enhanced the formation of IgG1-producing B cells, whereas it only weakly enhanced, one- to twofold, the formation of IgG3-producing B cells. In addition, CT synergistically enhanced the induction of IgG1-producing B cells by IL-4, even at plateau concentrations of IL-4. In contrast, IgM and IgG3 responses were suppressed in the CT plus IL-4-containing cultures as compared to those containing only LPS or LPS and CT. Furthermore, CT plus IL-4 had no enhancing effect on the formation of cells producing IgA; on the contrary, the presence of IL-4 led to a reversal of the stimulatory effect of CT on the IgA response. In further studies, we found that CT affected B cell differentiation at the gene level, before final gene recombination has occurred. Thus, CT together with LPS induced faint but detectable germline gamma 1 RNA transcripts not seen with cells cultured in LPS alone. However, more strikingly, CT enhanced by several-fold expression of germline gamma 1 RNA transcripts in LPS-stimulated B cell cultures containing optimal IgG1-inducing concentrations of IL-4. In addition, despite its weakly positive effect on IgG3 production. CT inhibited expression of germline gamma 3 RNA transcripts in cultures containing LPS and caused a further decrease in such transcripts in cultures containing LPS and IL-4. Finally, we found that CT enhanced the in vivo IgG1 but not the IgG3 or IgM anti-DNP serum antibody response of mice immunized with DNP-LPS. Taken together, these studies suggest that CT more strongly promotes B cell differentiation to IgG1 than to any other IgG subclass in LPS-stimulated cultures. CT acts alone or in synergy with IL-4, early in B cell differentiation to promote IgG1 expression in LPS-stimulated B cell cultures, probably by inducing early steps in the switch to this isotype such as the production of germline gamma 1 RNA transcripts.
Bacterial killing and inhibition of inner membrane activity by C5b-9 complexes as a function of the sequential addition of C9 to C5b-8 sites.
Authors: MacKay S L , Dankert J R
Published: January 2023

The assembly of the C5b-9 complex on the outer membrane of C-sensitive cells of Escherichia coli results in a rapid inhibition of inner membrane function and ultimately a loss of cell viability. Cells bearing C5b-8 sites suffer no deleterious effects; however, the addition of C9 results in a rapid inhibition of inner membrane function and cell death. An attempt was made to examine the relationship between the toxic effects of the C5b-9 complex and the number of C9 molecules per C5b-8 site. Cells bearing C5b-8 sites were exposed to excess C9 at 0 degrees C and washed three times at 4 degrees C. The number of C9 molecules bound to each cell was equivalent to the number of C5b-8 sites present on each cell, and no additional C9 molecules could be bound when the cells were maintained at 4 degrees C. These cells were then incubated at 37 degrees C for 3 min and returned to 0 degrees C, a technique which exposed additional C9-binding sites equivalent to the number of C9 molecules previously bound to the cells. This technique was repeated and demonstrated that the sequential build-up of a C5b-9 site with two C9 molecules per C5b-8 site was capable of inhibiting both inner membrane function (respiration and amino acid transport) and cell viability. Three C9 molecules per complex had effects that approached the inhibitory effects of complexes formed in the presence of excess C9.
Protein D of Haemophilus influenzae. A novel bacterial surface protein with affinity for human IgD.
Authors: Ruan M R , Akkoyunlu M , Grubb A , Forsgren A
Published: January 2023

Protein D, a novel surface protein of the bacterial species Haemophilus influenzae with affinity for human IgD, was isolated after solubilization with sonication and Sarcosyl-extraction by a single SDS-PAGE step. From 1 ml of packed bacteria was prepared 0.25 mg of purified protein D. The apparent m.w. of protein D was estimated to 42,000 by SDS-PAGE and gel chromatography. Edman degradation cycles of protein D produced no amino acid phenylthiohydantoin derivatives and the amino-terminal end of the single protein D polypeptide chain is thus probably blocked. Protein D differs from all previously described outer membrane proteins (protein 1 to 6) of H. influenzae. Thus, protein D did not react with antibodies against protein 1 or protein 2 and the latter proteins did not bind IgD. Protein D was found to exhibit unique Ig-binding properties. Thus, in dot blots protein D bound four different human IgD myeloma proteins but not IgG, IgM, IgA, IgE, or some additional proteins. On the IgD molecule, constant parts of the H chains both in the Fab and Fc fragments appear responsible for the interaction with protein D. This novel Ig-binding reagent promises to be of theoretical and practical interest in immunologic and microbiologic research.
Analysis of beta 2-microglobulin gene expression in the developing mouse embryo and placenta.
Authors: Jaffe L , Jeannotte L , Bikoff E K , Robertson E J
Published: January 2023

The major transplantation (MHC) Ag in the mouse are cell-surface glycoproteins composed of a 40- to 45-kDa H chain and a 12-kDa L chain, beta 2-microglobulin (beta 2 m). Regulation of MHC gene expression during early development is thought to play a critical role in maternal tolerance of the fetal allograft. We have used in situ hybridization techniques to characterize the temporal and spatial pattern of expression of the beta 2 m gene in the developing mouse embryo. beta 2 m mRNA is first detected in the extra-embryonic derivatives of the early primitive streak stage embryo where it is specifically restricted to the tissues of the ectoplacental cone and chorion. Expression of the beta 2 m gene continues in these tissues during formation of the definitive placenta. beta 2 m transcripts were also detected in the tissues of the visceral yolk sac starting at 10.5 days. Comparable levels of expression were observed in both the visceral yolk sac endoderm and the extra-embryonic mesoderm. These results clearly indicate that the onset of beta 2 m gene expression is not restricted to early hemopoietic cells. In the embryo-proper, significant levels of beta 2 m mRNA were not detected until day 9.5. At this stage, we observed a strong signal over the liver rudiment. The remaining somatic tissues expressed low levels of transcripts. Similar results were obtained at later stages of development, i.e., the fetal liver was consistently the most strongly positive organ. Overall these in situ hybridization studies define the pattern of beta 2 m gene transcription and thus potential sites of surface expression of class I proteins in specific cell lineages in the early postimplantation stage embryo.
Sustained engraftment of mice transplanted with IL-1-primed blood-derived stem cells.
Authors: Fibbe W E , Hamilton M S , Laterveer L L , Kibbelaar R E , Falkenburg J H , Visser J W , Willemze R
Published: January 2023

IL-1 is considered the primary mediator of the acute phase response. One of the characteristic manifestations of this response is early neutrophilia that is probably caused by release of mature neutrophils from the bone marrow into the peripheral blood. In the present study, we assessed whether IL-1 had a similar releasing effect on the number of circulating progenitor cells and stem cells. Female BALB/c mice were injected i.p. with increasing (0.1-1.0 micrograms/mouse) concentrations of rhu-IL-1 alpha. IL-1 injection resulted in a marked dose-dependent increase in the number of polymorphonuclear neutrophils, granulocyte-macrophage colony-forming units (CFU-GM), and cells forming spleen colonies (CFU-S day 8 and day 12). The maximal increase was found at 4 to 8 h after injection of 1 micrograms IL-1 per mouse, yielding a mean fivefold elevation in neutrophil count, and a mean 30-fold and 10-fold increase in the number of circulating CFU-GM and CFU-S, respectively. In a subsequent series of experiments, lethally irradiated (8.5 Gy) female recipient animals were transplanted with 5 x 10(5) blood mononuclear cells derived from male IL-1-treated animals. Long-term survival was obtained in 68% of mice transplanted with peripheral blood cells derived from donor animals at 6 h after a single injection of 1 micrograms IL-1. The mean number of circulating CFU-GM in these donor animals was 557/ml blood. At 6 mo after transplantation, greater than 95% of the bone marrow cells were of male origin, as determined using in situ hybridization with a Y-chromosome specific probe. In contrast, long-term survival was reached in less than 10% of mice transplanted with an equal number of blood cells derived from saline-treated controls or donor animals treated with a dose of 0.1 micrograms IL-1. These results indicate that a single injection of IL-1 induces a shift of hematopoietic progenitor cells and marrow repopulating cells into peripheral blood and that these cells can be used to rescue and permanently repopulate the bone marrow of lethally irradiated recipients.
Tetraparental mice reveal complex cellular interactions of the mutant, autoimmunity-inducing lpr gene.
Authors: Katagiri T , Azuma S , Toyoda Y , Mori S , Kano K , Cohen P L , Eisenberg R A
Published: January 2023

Lpr is a murine recessive autosomal mutant gene that, in conjunction with the background genes of different inbred mouse strains, produces varied syndromes of lymphadenopathy and autoantibody production. We have investigated the cellular basis of the lpr determined phenomena by constructing allophenic (tetraparental) chimeras of MRL/Mp-lpr/lpr combined with C57BL/6 and MRL/+ combined with C57BL/6-lpr/lpr. The appearance of lymphadenopathy, lpr atypical T cells, and autoantibodies was found to depend on the relative amounts +/+ and lpr tissue. The failure of these three abnormalities to correlate in certain animals suggested different pathways of lpr gene action. Furthermore, in the lpr predominant animals, both the lymphadenopathy and autoantibodies were essentially entirely contributed by the lpr donor. The expression of the lpr gene within the same cells expressing the MRL or B6 background determined the pathologic and serologic manifestations characteristic of the disease of that lpr strain, despite the presence of +/+ cells of the other background in the same individual. The lpr syndrome thus depends on the intracellular interaction of the lpr and background genes, and on positive signalling between different lpr cell populations.
The mechanism of internalization of platelet-activating factor in activated human neutrophils. Enhanced transbilayer movement across the plasma membrane.
Authors: Bratton D L , Dreyer E , Kailey J M , Fadok V A , Clay K L , Henson P M
Published: January 2023

Recent studies suggest that cellular internalization of platelet-activating factor (PAF), a potent ether phospholipid mediator of inflammation, is modulated by, as yet undefined cellular mechanisms. Using an albumin extraction method, the internalization of PAF and several PAF analogues was studied in the resting and stimulated human neutrophil. Our data demonstrate that internalization of these analogues is largely dependent on the state of cellular activation and that the process is not specific for certain unique structural features of the PAF molecule including the 1-position ether linkage, 2-position acetyl substitution, or choline polar head group. Furthermore, the internalization process was shown not to be dependent on the PAF receptor, metabolism of the molecule, or the process of endocytosis. Data are presented to suggest that the route of internalization of PAF is enhanced transbilayer movement (flipping) across the plasma membrane occurring as a result of changes in membrane physical properties accompanying cellular activation. It is proposed that in addition to enhanced internalization of PAF, modulation of PAF biosynthesis and net release from the stimulated neutrophil may be consequences of enhanced transbilayer movement of PAF across the activated plasma membrane.
A protective monoclonal antibody specifically recognizes and alters the catalytic activity of schistosome triose-phosphate isomerase.
Authors: Harn D A , Gu W , Oligino L D , Mitsuyama M , Gebremichael A , Richter D
Published: January 2023

mAb M.1 was previously shown to recognize a 28-kDa Ag in all stages of the human helminth parasite, Schistosoma mansoni, and to bind to the surface membranes of newly transformed schistosomula in a transient manner. Here we demonstrate that M.1 passively transfers partial resistance (41-49%) to cercarial challenge in naive mice. Thus, the 28-kDa Ag recognized by M.1 is a putative vaccine candidate. After immunoaffinity purification, tryptic digests of the 28-kDa Ag were prepared and individual peptides were sequenced. Amino terminus sequences of tryptic peptides of the 28-kDa Ag had high (79-87%) sequence homology with the mammalian glycolytic/gluconeogenic enzyme triosephosphate isomerase (TPI). Purified, native 28-kDa Ag from adult parasites was shown to function enzymatically in an analogous manner to yeast and mammalian TPI in the reverse reaction. Addition of M.1 antibody to the enzyme reaction altered the catalytic activity of schistosome TPI. To determine the immunologic cross-reactivity of this vaccine candidate with mammalian TPI, Western blot analysis was performed and demonstrated that M.1 was immunologically specific for the schistosome enzyme.
A lectin-binding, protease-resistant mycobacterial ligand specifically activates V gamma 9+ human gamma delta T cells.
Authors: Pfeffer K , Schoel B , Plesnila N , Lipford G B , Kromer S , Deusch K , Wagner H
Published: January 2023

Bacterial (exogeneous) superantigens have been defined as bifunctional proteinaceous molecules. They bind to class II MHC molecules of presenting cells and engage with particular TCR-V beta gene elements, thereby activating alpha beta T cells in a V beta-oriented fashion. In previous studies we have elucidated that gamma delta T cells exhibit a propensity to vigorously respond toward mycobacterial Ag. Intrigued by this finding we now analyzed whether mycobacteria express a superantigen for a subset of human gamma delta T cells definable by the selective use of TCR-V gene elements. Here we describe that a protease-resistant, low m.w. (1 to 3 kDa) component of mycobacteria selectively activates gamma delta T cells expressing TCR-V gamma 9 gene segments. Contained in mycobacterial lysates it stimulates TCR-V gamma 9-positive gamma delta T cells at a frequency of 1/6. Stimulation is critically dependent on the presence of class II MHC-positive presenting cells, the important structure being HLA-DR molecules. The fine specificity of the V gamma 9 seeking mycobacterial ligand differs from the gamma delta T cell-stimulating structures expressed by Daudi cells. In addition, the mycobacterial, V gamma 9-seeking ligand is bound selectively to lectins such as UEAI, SBA, and DBA. We conclude that mycobacteria contain a component that acts as a superantigen for human gamma delta T cells and we believe it is this property that explains the vigorous participation of gamma delta T cells in mycobacterial infections.
Human mb-1 gene: complete cDNA sequence and its expression in B cells bearing membrane Ig of various isotypes.
Authors: Yu L M , Chang T W
Published: January 2023

The transmembrane protein, IgM-alpha, a product of mb-1 gene, has been shown to be specifically associated with membrane-bound IgM on the plasma membrane of B lymphocytes. Recent studies have suggested that IgM-alpha may play a role in transducing signals from the Ag receptors during the activation of B cells. A large amount of information has been obtained in the mouse system regarding IgM-alpha and other components of the newly conceived B cell Ag receptor complex. Here we report the cloning and the nucleotide sequencing of cDNA clones of human mb-1, covering the entire length of the mRNA. At the amino acid sequence level, human and murine mb-1 share a high homology in their transmembrane and intracytoplasmic segments, suggesting an important biologic function for these regions of mb-1. A major difference, mainly in the 3' untranslated part, exists between our cDNA sequence and the published partial human mb-1 cDNA sequence. It has also been observed that human mb-1 is expressed not only by B cell lines expressing membrane-bound Ig of mu and delta isotypes but also those expressing membrane-bound Ig of alpha and gamma isotypes.
Shared human melanoma antigens. Recognition by tumor-infiltrating lymphocytes in HLA-A2.1-transfected melanomas.
Authors: Kawakami Y , Zakut R , Topalian S L , Stötter H , Rosenberg S A
Published: January 2023

Three predominantly CD8+ CTL lines, TIL 501, TIL 620, and TIL 660, were generated from three HLA-A2+ melanoma patients by culturing tumor-infiltrating lymphocytes in 1000 U/ml IL-2. These tumor-infiltrating lymphocytes lysed 12 of 18 HLA-A2+ autologous and allogeneic melanomas, but none of 20 HLA-A2-negative melanomas. They also did not lyse the MHC class I negative lymphoma-leukemia cell lines, Daudi, K562, or HLA-A2+ non-melanoma cell lines including PHA or Con A-induced lymphoblast, fibroblast, EBV-transformed B cell, Burkitt's B cell lymphoma, and colon cancer cell lines. Autologous and allogeneic melanoma lysis was inhibited by anti-CD3, by anti-MHC class I, and by anti-HLA-A2 mAb, indicating recognition of shared tumor Ag among melanoma cell lines in a TCR-dependent, HLA-A2-restricted manner. Six HLA-A2-negative melanoma cell lines obtained from five HLA-A2-negative patients were co-transfected with the HLA-A2.1 gene and pSV2neo. All 17 cloned transfectants expressing cell surface HLA-A2 molecules, but none of 12 transfectants lacking HLA-A2 expression, were lysed by these three HLA-A2-restricted, melanoma-specific CTL. Lysis of the HLA-A2+ transfectants was inhibited by anti-CD3, by anti-MHC class I, and by anti-HLA-A2 mAb, indicating recognition of shared tumor Ag on transfectants in a TCR-dependent, HLA-A2-restricted manner. These results identify the HLA-A2.1 molecule as an Ag-presenting molecule for melanoma Ag. They also suggest that common melanoma Ag are expressed among melanoma patients regardless of HLA type. These findings have implications for the development of melanoma vaccines that would induce antitumor T cell responses.
Activation of murine epidermal TCR-gamma delta+ T cells by keratinocytes treated with contact sensitizers.
Authors: Huber H , Descossy P , van Brandwijk R , Knop J
Published: January 2023

The vast majority of TCR gamma delta+, CD4-, CD8- T cells resident in the adult mouse epidermis expresses tissue-specific V-region genes (V gamma 3/V delta 1) in the absence of junctional diversity. The role this unique T cell population plays in the immune surveillance of the skin is not clear. It has been shown that dendritic epidermal T cells (DETC) were activated by stressed keratinocytes and that stimulated DETC produced, for example, a keratinocyte-specific growth factor. To investigate whether DETC are involved in the induction of a contact allergy, we examined the influence of contact sensitizers and nonsensitizing contact irritants on the DETC response toward epidermal symbionts. We show that 9 of 15 cloned DETC are specifically activated, apparently in a non-MHC-restricted way, to proliferate in the presence of keratinocytes or unseparated epidermal cells, which were treated with a sensitizing agent either in vivo or in vitro. All seven tested contact sensitizing substances activated all of the reactive DETC, while keratinocytes/epidermal cells treated with nonsensitizing irritants were as nonstimulatory as vehicle controls. We demonstrate that direct cell to cell contact of DETC and stimulatory keratinocytes/epidermal cells was required and that the TCR was involved in the induction of DETC proliferation. This specific reactivity of DETC toward keratinocytes or epidermal cells pretreated with a contact sensitizer may be indicative of participation of epidermal T cells in the induction of a contact sensitivity and points to a possible role of DETC in the skin immune system.
Activation and preferential expansion of rat cytotoxic (CD8) T cells in vitro and in vivo with a bispecific (anti-TCR alpha/beta x anti-CD2) F(ab')2 antibody.
Authors: Tutt A L , Reid R , Wilkins B S , Glennie M J
Published: January 2023

We show that a bispecific F(ab')2 Ab (BsAb), which cross-links the TCR to CD2 ([anti-CD2 x anti-TCR]) and is highly mitogenic in vitro, also induces marked T cell activation and proliferation when given as a small single dose (50 to 100 micrograms) to rats. Interestingly, the proliferation appeared selective for CD8 cells, increasing this compartment more than 20 times over 72 h. This resulted in a three- to fourfold increase in spleen weight, with histologic evidence of T-zone and red pulp expansion by CD8+ blast cells. Such changes were in striking contrast to those seen after a similar amount of the parent IgG anti-TCR mAb, R73, which, while inducing a transient increase in CD25+ cells, failed to alter the lymphocyte composition. The mitogenic activity of the BsAb was totally dependent on its ability to cross-link CD2 and the TCR, hence a mixture of anti-CD2 and anti-TCR F(ab')2 fragments was without effect in vitro or in vivo. Unlike normal rat T cells or those taken from R73 IgG-treated rats, blood and splenic T cells recovered from BsAb-treated rats were highly cytotoxic against R73 hybridoma targets that express surface anti-TCR mAb and consequently trigger the lytic activity of activated CTL or a rat lymphoma line, LAMA (Thy-1+), using a second BsAb, [anti-TCR x anti-Thy-1], to retarget the CTL. This latter assay provides the basis for a future two-stage targeting strategy for cancer immunotherapy in which a mitogenic BsAb would be given to patients to activate CTL nonspecifically, and then, at an appropriate time, a second BsAb [anti-TCR x antitumor] would be given to deliver activated cells to the tumor target cells.
Interplay between promoter and structural gene variants control basal serum level of mannan-binding protein.
Authors: Madsen H O , Garred P , Thiel S , Kurtzhals J A , Lamm L U , Ryder L P , Svejgaard A
Published: January 2023

Mannan-binding protein (MBP) is a serum lectin participating in the innate immune defense by opsonizing various microorganisms for phagocytosis. Opsonization defect due to MBP deficiency and low levels of the protein can partially be explained by the dominant effect of three different mutations in the structural part of the MBP gene. Large interracial differences in the frequencies of these variants have previously been described, but they cannot explain the large interindividual variation in MBP serum concentration. We describe the existence of additional polymorphisms at positions -550 (H/L variants) and -221 (X/Y variants) in the promoter region of the gene. The promoter haplotypes, HY, LY, and LX, show associations with high, medium, and low levels of MBP serum concentrations, respectively. Moreover, this represents a genetic system with additive effect of haplotypes in which a low producing LX haplotype in the homozygous state down-regulates the basal expression of MBP as effectively as a single structural variant. Populations of pure Eskimos, Caucasoids, and black Africans show marked interethnic differences in the frequencies of promoter haplotypes regulating the expression of the normal peptide, with the HY haplotype frequency varying from 0.83 in Eskimos via 0.33 in Caucasoids to 0.08 in Africans. The LY haplotype frequency varies from 0.04 in Eskimos via 0.39 in Caucasoids to 0.23 in Africans. The LX haplotype frequency varies from 0.03 in Eskimos via 0.24 in Caucasoids to 0.23 in Africans. The effect of the promoter variants can explain almost all of the ethnic differences not explainable by the structural variants alone.
Repertoire analysis of human peripheral blood lymphocytes using a human V delta 3 region-specific monoclonal antibody. Characterization of dual T cell receptor (TCR) delta-chain expressors and alpha beta T cells expressing V delta 3J alpha C alpha-encoded TCR chains.
Authors: Peyrat M A , Davodeau F , Houde I , Romagné F , Necker A , Leget C , Cervoni J P , Cerf-Bensussan N , Vié H , Bonneville M
Published: January 2023

V delta 3 usage and combinatorial expression of V gamma and V delta regions was studied on peripheral T cells with a novel V delta 3-specific mAb (p11.10b), generated against a soluble V gamma 9V delta 3 TCR. V delta 3+ cells represented the vast majority of V delta 1/V delta 2- gamma delta T cells within peripheral blood and mucosal lymphocytes. No preferential V gamma region expression was noted within V delta 3+ cells, but the frequency of V gamma 9+ cells was significantly lower among V delta 3+ than among V delta 1+ or V delta 2+ PBL. Phenotypic analysis of cultured V delta 3+ cells sorted with p11.10b mAb revealed the presence of T lymphocytes with unusual phenotypes. First, cells carrying two distinct surface TCR delta-chains, recognized by both V delta 1- and V delta 3-specific mAbs, were detected in most T cell lines, though at frequencies much lower than that of dual gamma expressors, indicating that allelic exclusion of delta genes is more tightly regulated than that of gamma genes. Moreover, a significant fraction of V delta 3+ cells were recognized by C beta- but not C delta-specific mAbs. Molecular analysis of V delta 3+C beta+ clones revealed the presence of V delta 3J alpha C alpha transcripts in all of them. Given the peculiar location of the V delta 3 gene between the delta Rec/psi J alpha elements, those observations formally demonstrate that activation of rearrangements with J alpha elements is not necessarily preceded by a delta Rec/psi J alpha-mediated deletion of the delta locus on the same chromosome.
The Ikaros gene encodes a family of lymphocyte-restricted zinc finger DNA binding proteins, highly conserved in human and mouse.
Authors: Molnár A , Wu P , Largespada D A , Vortkamp A , Scherer S , Copeland N G , Jenkins N A , Bruns G , Georgopoulos K
Published: January 2023

The Ikaros gene is an essential regulator in the development and homeostasis of the mouse lymphopoietic system. To study the role of the Ikaros gene in the human lymphopoietic system, we cloned and characterized human Ikaros cDNAs. In the human, as in the mouse, differential splicing of Ikaros primary transcripts generates a family of lymphoid-restricted zinc finger DNA binding proteins, highly conserved in sequence composition and relative expression to the mouse homologues. Expression of Ikaros isoforms is highly restricted to the lymphopoietic system and is particularly enriched in maturing thymocytes. The Ikaros gene maps at a syntenic locus located on the short arm of human chromosome 7 and on mouse chromosome 11 next to the epidermal growth factor receptor (Egfr). The high degree of conservation of the Ikaros gene at the genetic and expression levels strongly suggests that it plays a fundamental role in the ontogeny of the lymphopoietic system across species.
Sea urchin genes expressed in activated coelomocytes are identified by expressed sequence tags. Complement homologues and other putative immune response genes suggest immune system homology within the deuterostomes.
Authors: Smith L C , Chang L , Britten R J , Davidson E H
Published: January 2023

To identify some of the genes expressed in LPS-activated coelomocytes, we sequenced randomly chosen clones from a directionally constructed cDNA library to produce a set of expressed sequence tags (ESTs). Deduced amino acid sequences from 307 ESTs were compared with known protein sequences in GenBank, and significant matches to approximately 30% of the clones were identified. Eighty-nine clones matched to 55 different proteins, including several putative immune effector proteins. In this work, we show the first identification of an invertebrate homologue of a vertebrate C component. Another EST matches to several short consensus repeats that are characteristic of a variety of proteins, including CR/regulatory proteins and clotting factors. Additional putative immune effector genes include 1) a Kazal-type protease inhibitor that may function to inactivate bacterial proteases, 2) a C-type lectin similar to echinoidin, and 3) a serine protease with similarities to thrombin, elastase, haptoglobin, and plasmin. Other EST categories include 1) cell surface proteins and receptors, 2) proteins involved in signaling systems, 3) lysosomal and secreted proteins, 4) cytoskeletal and cytoskeletal modifying proteins, 5) general cell function proteins, 6) proteins with unknown function, and 7) ESTs without significant matches, 25 with open reading frames. Many of the ESTs identified in this study represent the types of genes expected to be used in lower deuterostome immune functions.
IL-5 activates a 45-kilodalton mitogen-activated protein (MAP) kinase and Jak-2 tyrosine kinase in human eosinophils.
Authors: Bates M E , Bertics P J , Busse W W
Published: January 2023

IL-5 is a member of the hemopoietic cytokine family and has profound effects on the differentiation, survival, migration, and effector function of human eosinophils. Increased tyrosine phosphorylation has been observed as an early event in IL-5 signal transduction in eosinophils; most notably, proteins of 45 and 135 kDa became tyrosine phosphorylated following IL-5 treatment. Some of these phosphotyrosine-containing proteins may represent intermediates in IL-5 signal transduction pathways. This study demonstrates that Jak-2, a tyrosine kinase, is increasingly tyrosine phosphorylated after IL-5 treatment of human eosinophils. Furthermore, we found proteins of 42, 44, and 45 kDa immunoreactive with anti-mitogen-activated protein (MAP) kinase Abs that are expressed in human eosinophils. One of these, the protein of approximately 45 kDa (p45), was tyrosine phosphorylated following treatment of eosinophils with IL-5 and PMA, as seen by anti-phosphotyrosine immunoprecipitation and immunoblotting with anti-MAP kinase Abs. In addition, anti-phosphotyrosine immunoprecipitates of IL-5-treated eosinophils contained enhanced phosphotransferase activity toward a myelin basic protein (MBP) peptide substrate when compared with control-treated eosinophils. In contrast to cytokine-stimulated MAP kinase activation in other cells, there is no evidence of tyrosine phosphorylation or enzymatic activation of p42 MAP kinase in eosinophils after IL-5 treatment. These data suggest that Jak-2 kinase and an activated isoform of MAP kinase, p45, are detected following incubation with IL-5, and may mediate some of this cytokine's effects on eosinophils in a manner unique to the activation pathways previously described for other cells.
Human CD38 ligand. A 120-KDA protein predominantly expressed on endothelial cells.
Authors: Deaglio S , Dianzani U , Horenstein A L , Fernández J E , van Kooten C , Bragardo M , Funaro A , Garbarino G , Di Virgilio F , Banchereau J , Malavasi F
Published: January 2023

Human CD38, a pleiotropic molecule with ADP-ribosyl cyclase activity, regulates activation and growth of several cell types. Its in vivo function is incompletely determined, mainly due to the lack of evidence concerning the existence of a single or multiple ligands. We recently observed that CD38 rules a selectin-type adhesion between lymphoid cells and HUVECs. A panel of murine mAbs raised against HUVEC included one (Moon-1) constantly blocking the CD38-mediated adhesion of several cell lines to HUVEC. Tissue distribution studies and an extended immunohistochemical analysis on the majority of normal human tissues revealed that the Moon-1 molecule displays a unique pattern of expression, being present at high levels on resting and activated vascular endothelium, on the majority of monocytes, platelets, NK cells, and to a lesser extent on T, B, and myeloid cells. The Moon-1 structure of an apparent molecular mass of 120 kDa proved to be a ligand for human CD38, as inferred by the direct binding observed when using a chimeric mouse CD8 alpha-human CD38 (mCD8 alpha-hCD38) molecule as a probe in Western blot experiments. Furthermore, Ab-induced modulation experiments highlighted an association between the Moon-1 molecule and human CD38 on the surface of cell lines coexpressing the two structures, which also share a common regulation system of surface expression. Finally, direct ligation of Moon-1 on T cell lines caused a relevant increase in the cytoplasmic concentration of calcium ([Ca2+]i).
Oligoclonality of V beta 3 TCR chains in the CD8+ T cell population of rheumatoid arthritis patients.
Authors: Hingorani R , Monteiro J , Furie R , Chartash E , Navarrete C , Pergolizzi R , Gregersen P K
Published: January 2023

It has been established that oligoclonal expansion is a common feature of the CD8+ T cell population, particularly within the CD8+ CD57+ lymphocyte subset. In addition, clonal malignancies involving CD8+ CD57+ T cells (large granulocytic lymphocytic leukemias) are often accompanied by rheumatoid arthritis, Felty's syndrome, or both. Therefore, to identify disease-related alterations in the CD8+ T cell repertoire, we have compared the patterns of oligoclonality in the CD8+ T cells of rheumatoid arthritis patients (n = 32) with those of age-matched controls (n = 25). By using a multiplex PCR assay for the CDR3 length of TCR beta-chains, we have found a striking increase in the frequency of CD8+ oligoclonality involving V beta 3 TCR: 50% of the rheumatoid arthritis patients had evidence of oligoclonality in this TCR family compared with 4% of controls (p < 0.0002). In addition, two unrelated RA patients had clonally dominant CD8+ T cell beta receptors that were identical in amino acid sequence, suggesting selection by a common Ag. An analysis of a subset of RA patients with mAbs specific for V beta 3 TCR revealed the presence of clonal expansion in a minority of patients usually, but not exclusively, involving the CD57+ subset. These data define a phenotype of the T cell repertoire that is strongly associated with rheumatoid arthritis; the mechanisms and genetic and environmental factors that explain this phenomenon remain to be defined.
Differential activation of phospholipase C-gamma 1 and mitogen-activated protein kinase in naive and antigen-primed CD4 T cells by the peptide/MHC ligand.
Authors: Ericsson P O , Orchansky P L , Carlow D A , Teh H S
Published: January 2023

In this study, we determined the functional and biochemical differences in naive and primed CD4 T cells that expressed a TCR specific for the pigeon cytochrome c (pcc) peptide presented by I-Ek MHC class II molecules. Naive CD4 T cells expressing the transgenic TCR were isolated from the peripheral lymphoid organs of transgenic mice and stimulated with pcc peptide and IL-2 for 10 to 14 days. After this culture period, the Ag-primed cells were quiescent, as judged by the lack of expression of the early activation marker CD69, low expression of CD25 (IL-2R), and failure to incorporate thymidine. The primed cells required 10-fold less peptide than naive cells to achieve the same degree of proliferation and for the induction of CD69. Primed cells also mobilized calcium more efficiently with regard to Ag dose and magnitude of the response. The biochemical signal-transduction events in naive and primed T cells were compared by stimulating them with different concentrations of pcc peptide presented by adherent Ek-transfected fibroblasts. It was found that tyrosine phosphorylation and activation of mitogen-activated protein kinase (MAPK) in primed cells required 10-fold less Ag and occurred more rapidly and intensively. Interestingly, peptide stimulation induced tyrosine phosphorylation of phospholipase C (PLC)-gamma 1 exclusively in primed cells. RasGAP was also more efficiently tyrosine phosphorylated in primed cells. By contrast, Shc was tyrosine phosphorylated to the same extent in naive and primed cells. PI3Kp85 was not tyrosine-phosphorylated in naive and primed cells either before or after peptide stimulation. We propose that the higher sensitivity of the primed cells to Ag stimulation is most likely dependent, at last in part, on the more efficient activation of PLC-gamma 1, MAPK, and calcium-dependent pathways.
Frequency of VH81x usage during B cell development: initial decline in usage is independent of Ig heavy chain cell surface expression.
Authors: Marshall A J , Wu G E , Paige G J
Published: January 2023

B cell development is marked by changes in the pattern of VH segment utilization. B cell precursors characteristically utilize a restricted set of VH segments, while mature B cell populations use a wide range of VH segments. VH81x is an example of a VH segment that is highly utilized in B cell precursors, but rarely utilized in mature B cells. We have developed an assay that allows us to determine the proportion of VDJH rearrangements that utilize the VH81x segment in DNA from selected populations of developing B cells. Consistent with previous observations, it was found that VH81x is utilized at a remarkably high frequency in primary B cell progenitors. The extent of overutilization was found to be identical during fetal and adult B cell development. Phenotypic analyses demonstrated that the decline in VH81x utilization begins at a stage before the expression of IgM on the cell surface and continues through later stages of B cell development. Strikingly, mutant mice that cannot express Ig heavy chain on the cell surface displayed a drop in VH81x utilization during both fetal and adult B cell development. Together, these data suggest that mechanisms other than cellular selection play an important role in determining the shift in VH segment utilization during B cell development.