bluuebunny/crossref_metadata_2025 huggingface dataset:
IFN-gamma and 1,25(OH)2D3 induce on THP-1 cells distinct patterns of cell surface antigen expression, cytokine production, and responsiveness to contact with activated T cells.
Authors: Vey E , Zhang J H , Dayer J M
Published: January 2023

Differentiation and maturation of monocytes are accompanied by the expression of specific surface glycoproteins, the secretion of cytokines, and the capacity to respond to ligands. These changes may be influenced by interactions with hormones, soluble lymphocytic products, or direct contact with lymphocytes. We have studied two distinct pathways in the differentiation of a human monocytic cell line, THP-1: one being induced by IFN-gamma and the other by 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3). In THP-1 cells, IFN-gamma induces cell surface expression of HLA-DR and CD54 and production of IL-1 beta, TNF-alpha, and IL-6. In contrast, 1,25(OH)2D3 increases cell surface expression of CD11b and CD14, but fails to stimulate cytokine production. Direct contact of THP-1 with stimulated fixed T cells markedly induces IL-1 beta, TNF-alpha, and IL-6 production by THP-1. Production is higher when THP-1 have been previously exposed to 1,25(OH)2D3 as compared to prior exposure to IFN-gamma. mAb raised against certain relevant cell surface glycoproteins on THP-1 were tested for their ability to block the response of THP-1 to T cells. Antibodies to CD11a, CD11b, and CD11c, alone or in combination, only partially blocked IL-1 beta production by THP-1, whereas antibodies to CD54 and CD14 did not. Thus other unknown structures on the THP-1 cells may be involved in the induction of THP-1 cytokine production by T cell contact.
Decay-accelerating factor expression on either effector or target cells inhibits cytotoxicity by human natural killer cells.
Authors: Finberg R W , White W , Nicholson-Weller A
Published: January 2023

Previous studies have shown that freshly isolated CD16+ NK cells are deficient in the expression of decay-accelerating factor (DAF), or CD55, a membrane regulator of C3 activation. In this study we investigated the significance, for NK cell-mediated lysis, of DAF expression on the target and effector cells. The effect of DAF expression on the susceptibility of NK cell targets was investigated by several means: first, DAF- cell lines were cloned from K562; second, the cloned DAF- cells were reconstituted with exogenous purified DAF; and third, anti-DAF F(ab')2 was used to block DAF function on the DAF+ K562 cells. Consistently, the presence of DAF in the target cell membrane, either naturally occurring or experimentally incorporated, afforded the target cell protection against lysis, and this protection could be blocked with anti-DAF. Similarly, targets for antibody-dependent cell-mediated cytotoxicity with exogenous DAF incorporated in their plasma membrane became less sensitive to antibody-dependent cell-mediated cytotoxicity by NK cells compared with the same target cells without incorporated DAF in their membranes. DAF incorporated in the plasma membranes of the effector NK cells made the NK cells less effective at killing K562 targets. The known function of DAF is to regulate C3 activation, and we were able to demonstrate that the isolated NK cell is capable of releasing C3. It is also possible that the participation of DAF in NK cell function represents a new, noncomplement-dependent function for DAF.
In vivo activity of tumor necrosis factor (TNF) mutants. Secretory but not membrane-bound TNF mediates the regression of retrovirally transduced murine tumor.
Authors: Karp S E , Hwu P , Farber A , Restifo N P , Kriegler M , Mulé J J , Rosenberg S A
Published: January 2023

We have previously demonstrated that murine tumor cells transduced with a retrovirus containing the cDNA encoding wild-type human TNF regress in vivo when injected into immunocompetent mice; this regression is T cell mediated. To determine whether membrane-associated or secreted TNF was responsible for tumor regression, we transduced a cloned murine fibrosarcoma 205 F4 with retroviruses encoding modified human TNF genes. The cloned tumor lines of one retroviral transduction expressed only membrane bound 26-kDa TNF. This TNF could not be cleaved or secreted, but was present on the cell surface. A second retrovirus caused the expression of only secretory 17-kDa TNF, as the transmembrane domain of the cDNA was deleted. The TNF produced by tumor cells transduced with either retroviral vector was functional in vitro as direct lysis of the TNF-sensitive target L929 by transduced tumor cells was demonstrated. The TNF present on 26-kDa expressing tumors was membrane bound as supernatants from cultured 17-kDa TNF expressing tumor cells but not 26-kDa TNF expressing tumors mediated the lysis of L929 cells. Both tumors were injected s.c. into syngeneic mice and tumor growth was measured serially. In repeated experiments, 26-kDa TNF expressing tumors grew progressively in all mice. In contrast, 17-kDa TNF expressing tumors grew for 10 days and then regressed with all animals free of tumor at 28 days. Tumor regression was abrogated by in vivo injection of an anti-TNF antibody. Similar results were obtained in a second tumor model, 203 E4. Thus regression of TNF transduced tumors in vivo requires secretion of TNF, as membrane-bound TNF is insufficient to elicit the host response.
Selective suppressive effects of Trypanosoma cruzi infection on IL-2, c-myc, and c-fos gene expression.
Authors: Soong L , Tarleton R L
Published: January 2023

Infection with Trypanosoma cruzi is accompanied by a profound suppression of immune responses including the production of IL-2. Previous experiments have confirmed a correlated decrease in IL-2 mRNA levels in lymphoid cells from infected mice. To further define the molecular basis of this regulation, we have examined the production and degradation of mRNA for IL-2 and other T cell activation genes in cells from T. cruzi-infected mice. Spleen cells from C57BL/6J mice infected with the Brazil strain of T. cruzi were analyzed for the kinetic expression of IL-2, IL-2R alpha, c-myc, and c-fos genes in response to Con A and PMA costimulation. Cells from infected mice exhibited a selective reduction of c-myc and c-fos mRNA in association with the severe suppression of the IL-2 gene, but a less severe to comparable production of IL-2R alpha mRNA compared with normal spleen cells. The similar patterns of the suppression of c-myc and IL-2 mRNA suggest a common mechanism of down-regulation of these two genes in T. cruzi infection. Actinomycin D treatment was used to demonstrate that decreased steady state levels of IL-2, c-myc, and c-fos mRNA in cells from infected mice were not due to an increased rate of degradation of these mRNA. Cycloheximide treatment enhanced the expression of IL-2, IL-2R alpha, c-myc, and c-fos mRNA in spleen cells from both normal and infected mice. Although a larger percentage of induction was observed in cells from infected mice, the mRNA levels for IL-2, c-myc, and c-fos in cells from infected mice were still lower than those of normal cells. Spleen cells from infected mice precultured for 24 to 72 h before the addition of mitogens showed significant enhancement of IL-2 and c-myc gene expression; however, this recovery was inhibited if fixed T. cruzi was present in the preculture medium. These data suggest that the reduction of IL-2 mRNA in infected mice is not the result of an increased degradation of its mRNA but to down-regulation of transcription of the IL-2 gene in T cells from T. cruzi-infected mice. Preculture-induced recovery of IL-2 production appears to result from release from this regulation and full expression of the IL-2 gene.
Expression of CD7 on normal human myeloid progenitors.
Authors: Chabannon C , Wood P , Torok-Storb B
Published: January 2023

Existence of biphenotypic leukemias co-expressing CD7 and CD34 has prompted the question of whether a similar population of cells is present in normal human bone marrow. As CD7 is considered to be a T cell-restricted Ag, the co-expression of CD7 with the "human stem cell Ag" CD34 may identify a bipotent stage within hemopoietic differentiation. Cells with this phenotype have previously been isolated from human thymus. In this report we provide evidence that human marrow mononuclear cells also contain a minor subpopulation of cells co-expressing CD7 and CD34. The CD7+/CD34+ cells were found to contain committed myeloid progenitors assayed both as CFU in semi-solid media and by their ability to produce granulocytes in long term marrow cultures. Expression of CD7 on myeloid committed progenitors was further confirmed in a C-mediated cytotoxic assay. We conclude that CD7 expression is not restricted to T cells but is also expressed during early stages of myeloid differentiation.
In vitro inhibitory effect of IL-8 and other chemoattractants on neutrophil-endothelial adhesive interactions.
Authors: Luscinskas F W , Kiely J M , Ding H , Obin M S , Hébert C A , Baker J B , Gimbrone M A
Published: January 2023

We have previously reported that cytokine- or LPS-activated human umbilical vein endothelial cell (HUVEC) monolayers secrete IL-8 that can act as a neutrophil-selective adhesion inhibitor. In our study we investigated the mechanisms involved in the leukocyte adhesion inhibitory action of IL-8. The leukocyte adhesion inhibitory effect appears to be mediated by the action of IL-8 on the neutrophil, does not involve down-regulation of relevant endothelial adhesion molecules such as endothelial-leukocyte adhesion molecule-1 or intercellular adhesion molecule-1, and is quantitatively similar in different endothelial activation states that are predominantly endothelial-leukocyte adhesion molecule-1 dependent or intercellular adhesion molecule-1 dependent. In addition to inhibiting the attachment of freshly isolated peripheral blood neutrophils to cytokine-activated HUVEC monolayers, IL-8 also promoted a rapid detachment of tightly adherent neutrophils from activated HUVEC, and abolished neutrophil transendothelial migration. Certain other chemoattractants, including FMLP and C5a, had similar inhibitory actions, indicating IL-8 was not unique in its ability to inhibit various neutrophil-endothelial interactions. In contrast, two other neutrophil agonists 1-0-alkyl-2-acetyl sn-glycero-3-phosphocholine and granulocyte-macrophage-CSF, which, like IL-8, are produced by activated HUVEC, as well as the leukocyte-derived chemoattractant leukotriene B4, exerted minimal inhibitory effects on adhesion. Regardless of their ability to modulate neutrophil-endothelial cell adhesion, all these agents induced altered leukocyte surface expression of functionally important adhesion molecules, including loss of L-selectin (leukocyte adhesion molecule-1, LECAM-1) and increase in CD11b/CD18. Thus, although the above agonists have been characterized primarily as chemoattractants, our findings demonstrate that these agents can exert a wide range of modulatory effects on neutrophil-endothelial adhesive interactions.
Carbon Dioxide Studies
Authors: Valley George
Published: January 2023

In the preceding paper (Valley and McAlpine)1 it was shown that carbon dioxide exerts a very definite preserving action on serum alexin.2 It was further demonstrated that a displacement of atmospheric (molecular) oxygen by hydrogen, nitrogen and carbon monoxide exercised no greater preservative action than the mechanical removal of atmospheric air by evacuation, or by limiting the volume of enclosed air in an hermetically sealed tube. It is apparent, therefore, that aside from a mere mechanical displacement of molecular oxygen by carbon dioxide, this gas brought about other influences which favored alexin preservation. Hence the explanation of the phenomenon should be sought in the nature of carbon dioxide action on a serum complex.

Displacement of oxygen may be more efficiently effected by carbon dioxide than by other gases, due to the chemical nature of the former.
The Distribution of Antibodies in the Serum and Organs of Rabbits
Authors: Freund Jules , Whitney Caroline E.
Published: January 2023

In a previous study (1) on the distribution of immune agglutinins in the serum and organs of rabbits the following conclusions were drawn: Agglutinins accumulate in the organs of rabbits that have been passively immunized.The rate of accumulation of agglutinins in the liver, spleen, kidney and lungs is greater than in the uterus and skin.If the animal is killed within from five to seventeen hours after the injection of the immune serum, all or a large part of the agglutinins can be removed from the uterus and skin; after seventeen hours the agglutinins become perfusion-fast in these organs.

The demonstration of perfusion-fast antibodies in organs might seem to support the cellular (sessile receptor) theory of anaphylaxis, according to which the anaphylactic antibodies become fixed to the cells during the incubation period of passive anaphylaxis.
Effect of Exposure to Low Temperatures on Diphtheria Toxin-Antitoxin Mixtures
Authors: Robinson Elliott S. , White Benjamin
Published: January 2023

The dissociation by freezing of diphtheria toxin-antitoxin mixtures, with consequent increase in toxicity, was first reported in 1924 by White and Robinson (5). Kirkbride and Dow (3), in an article published simultaneously, stated that, under their experimental conditions, they were unable to produce this phenomenon. Anderson and Leonard (1) succeeded in bringing about such a dissociation, but only in experimental mixtures differing from those ordinarily employed for active immunization against diphtheria. Following the appearance of these papers, Glenny, Pope, Waddington and Wallace (2) suggested that an increased phenol concentration, brought about as the mixtures freeze, is the cause of the increased toxicity and that the discordant results reported “are due to the amount of protein present and other factors influencing the relative degree of destruction of antitoxin or of toxin by the local concentration of phenol that occurs when these mixtures are frozen.”
Intrathymic lymphoid precursor cells during fetal thymus development.
Authors: Antica M , Wu L , Shortman K , Scollay R
Published: January 2023

Our previous studies have demonstrated the presence, in the adult mouse thymus, of a population of early precursor cells able to give rise to T and B lymphocytes but not myeloid cells. This population of cells expresses low levels of CD4 and has been termed the "low CD4 precursors." All these precursors were found to be c-kit positive, and they precede the better known CD4-CD8- precursor stage. In this study, embryonic and neonatal thymuses were examined to see whether a similar low CD4 precursor was part of the pathway of T cell development during ontogeny. A population with the phenotypic characteristics of the adult low CD4 precursor was found from day 15 of embryonic development, although the expression of low levels of CD4 was apparent only from embryonic day 17. Functional tests of these putative precursors showed they had no thymus-reconstituting ability when isolated from thymuses at any time during embryonic life, and very low reconstituting ability even 24 days after birth. These results raise questions about the adult low CD4 precursor as an obligatory stage in the development of T cells in the thymus.
Functional effects of CD30 on a large granular lymphoma cell line, YT. Inhibition of cytotoxicity, regulation of CD28 and IL-2R, and induction of homotypic aggregation.
Authors: Bowen M A , Olsen K J , Cheng L , Avila D , Podack E R
Published: January 2023

Studies are described revealing novel regulatory functions for the lymphocyte activation Ag CD30. A new mAb, C10, reactive with YT cells binds to CD30 and induces inhibition of the cytotoxicity of YT for Raji cells. C10 inhibition of cytotoxicity requires several hours preincubation of YT with C10; the antibody has no effect if added directly to YT cytotoxicity assays. CD30 stimulation by C10 down-regulates CD28 expression on YT by > 80% within 48 h. Because CD28 is required for YT cytotoxicity toward Raji cells and other B7/BB1 bearing targets, it is suggested that inhibition of cytotoxicity of YT is mediated by control of CD28 expression and/or signaling via CD30. Accordingly, conjugation of YT with Raji is only slightly affected by CD30-mediated down-regulation of CD28, and perforin mRNA steady state levels are not changed at all. C10 treatment of YT cells additionally down-regulates the expression CD45 and up-regulates IL-2R p55. Moreover, CD30 stimulation by C10 causes homotypic aggregation of YT. Homotypic aggregation is slow, requiring gene transcription, translation, metabolic energy at elevated temperature (37 degrees C), magnesium ions, and an intact cytoskeleton. These studies offer insights into the function of CD30 as a complex regulator of T cells.
Production of soluble IL-4 receptors by murine spleen cells is regulated by T cell activation and IL-4.
Authors: Chilton P M , Fernandez-Botran R
Published: January 2023

Many cytokine receptors exist naturally as both membrane-bound and soluble forms. Whereas the membrane receptors have an obvious role in signal transduction, the putative immunoregulatory role played by the soluble receptors remains unclear. Although natural forms of soluble IL-4R (sIL-4R) are known to be present in the biologic fluids of normal mice, the mechanisms regulating the production of sIL-4R have not been characterized. In this study, we have developed an ELISA that allows the measurement of sIL-4R without interference from endogenous IL-4, and have analyzed the effect of cellular activation and several cytokines on the secretion of sIL-4R by murine splenic cells. Although normal spleen cells in culture produced low, but detectable levels of sIL-4R under basal conditions, stimulation with the T cell-mitogens, Con A or soluble anti-CD3 antibodies, caused a 10- to 40-fold increase in the production of sIL-4R. Stimulation of B lymphocytes with LPS, however, did not result in significant up-regulation of sIL-4R secretion. Moreover, IL-4, but not other cytokines, was also a potent inducer of sIL-4R production by spleen cells, even in the absence of other stimuli. Blocking experiments with an anti-IL-4 antibody, 11B11, demonstrated that the effect of T cell-mitogens is partially mediated by endogenously produced IL-4. Cell depletion experiments suggested that although the effect of T cell-mitogens was dependent on the presence of viable T cells, all major cell types including T cells, B cells, and macrophages, either resting or activated, were able to up-regulate their secretion of sIL-4R in response to IL-4. Unlike many activities of IL-4, the secretion of sIL-4R by IL-4-stimulated splenic cells was not antagonized by IFN-gamma. These results suggest that the production of sIL-4R is regulated by stimuli leading to T cell activation and IL-4 secretion and are consistent with sIL-4R having an important role in the regulation of IL-4 activity in vivo.
Reversion of the SCID phenotype by human T cell grafts. Development of cross-species immunocompetence.
Authors: Donjon C M , Morkowski H , Cheung R K , Leeder J S , Dosch H M
Published: January 2023

Due to defective recombinase function, mice with severe combined immunodeficiency (SCID) lack functional lymphocytes and can accept human lymphoid xenografts. Xenografted animals (SCIDhum) are thought to provide a neutral environment for in vivo studies of normal, malignant or HIV-infected human cells. SCIDhum often develop endogenous, EBV+ lymphomas in the graft and in the our study two-thirds of 142 SCIDhum mice did so. Surprisingly, one-third of animals developed reversion of the SCID phenotype rapidly after human T cell engraftment. 90% of tumors occurred in nonrevertant and only 10% in revertant mice. These revertant animals showed immunologic tolerance for normal human B lymphocytes, maintained stable levels of mouse and human IgM and IgG. In addition, they generated competent mouse T cells able to kill transformed (EBV+) but not fresh B cells from the same donor nor unrelated human B cell lines. The tolerance for human lymphoid cells and the cross-species antitumor competence of host T lymphocytes imply unexpected recognition and selection events. Rather than a neutral "bioreactor," these observations mark the SCID host as potentially active participant in a composite immune system generated by xenografting.
A lymphostromal molecule, thymic shared Ag-1, regulates early thymocyte development in fetal thymus organ culture.
Authors: Randle E S , Waanders G A , Masciantonio M , Godfrey D I , Boyd R L
Published: January 2023

Previously, we detailed the characterization of thymic shared Ag-1, a unique marker of immature thymocytes and isolated thymic stromal cells, defined by the mAb MTS 35. In this study, the functional relevance of this molecule to thymopoiesis was investigated by the addition of purified MTS 35 to fetal thymus organ culture. It down-regulated thymic shared Ag-1 expression and dramatically reduced thymocyte cell yield through inhibition of alpha beta-TcR+ T cell differentiation, post CD3-CD4-CD8- triple negative thymocytes. These effects were specific for the mAb MTS 35, because controls, which include both isotype-matched and other lymphostromal mAb, showed no similar effects. These results demonstrate that thymic shared Ag-1 is a functionally important marker of early thymocyte differentiation, particularly with regard to the alpha beta-TcR lineage.
Role of CD4+ T cells in the expansion of the CD4-, CD8- gamma delta T cell subset in the spleens of mice during blood-stage malaria.
Authors: van der Heyde H C , Manning D D , Weidanz W P
Published: January 2023

The previously observed expansion of the splenic gamma delta T cell subset was examined during the course of murine malaria to determine whether CD4+ T cells are required. Flow cytometric analysis during the course of Plasmodium chabaudi adami malaria in both C57BL/6 and BALB/c mice revealed that the maximal percentage of CD4+ T cells that were blasts occurred during the period of ascending parasitemia, whereas the maximal numbers of gamma delta T cells and blast cells occurred during the period of descending parasitemia. Transfer of enriched populations of CD4+ cells (> 75%) containing < 0.9% gamma delta T cells from immune BALB/c donor to SCID mice led to a population of gamma delta T cells that constituted 37% of the splenic T cells in the recipients and allowed them to resolve their infections. Transfer of the CD4- fraction did not suppress parasitemia. These results suggest that CD4+ T cells are activated early during the infection and are required for the subsequent expansion of the gamma delta T cell population. Furthermore, the maximal gamma delta T cell blast response during the period of descending parasitemia and the detection of high levels of these cells only in models that resolved their infections suggest that these cells may function in the resolution of blood-stage malaria.
Antigen-dependent stimulation by bone marrow-derived mast cells of MHC class II-restricted T cell hybridoma.
Authors: Frandji P , Oskéritzian C , Cacaraci F , Lapeyre J , Peronet R , David B , Guillet J G , Mécheri S
Published: January 2023

This paper describes a new role for mast cells as being able to present Ag to immune T cells. A mouse bone marrow-derived mast cell population obtained after 3 wk of culture in a conditioned medium has been shown to express a variety of membrane-associated Ag, including MHC class II and class I Ag, CD23, CD32, high affinity receptor for IgE, and CD4. Expression of MHC class II molecules was up-regulated upon stimulation with LPS but not with IFN-gamma and was down-regulated after exposure of mast cells to IL-3 treatment. We have demonstrated that mast cells were able to present native Ag as well as immunogenic peptides to MHC class II-restricted T cell hybridoma. The inhibition of Ag presentation after mast cells have been treated with ammonia suggests that Ag catabolism in intracytoplasmic compartment as a key step in Ag handling takes place in these cells. The MHC class II molecule is the restricting element for the presentation of OVA and the lambda repressor from bacteriophage lambda to a panel of specific T cell hybridomas, as demonstrated by the blocking effect of anti-MHC class II mAb on the Ag-presenting function. A characteristic feature of mast cells is the generation of a narrower immunogenic peptide repertoire as compared with A20 and LBB 3.4.16, a B lymphoma cell line, and a B cell hybridoma, respectively. This novel function of mast cells brings to a much closer connection inflammatory and immunologic processes and sheds new light on the biology of mast cells and particularly on the specific allergic responses.
Intravenous interleukin-8 inhibits granulocyte emigration from rabbit mesenteric venules without altering L-selectin expression or leukocyte rolling.
Authors: Ley K , Baker J B , Cybulsky M I , Gimbrone M A , Luscinskas F W
Published: January 2023

Injection (i.v.) of the granulocyte chemoattractant/activator IL-8 has been shown to reduce neutrophil recruitment into dermal inflammatory sites in vivo. To further investigate the mechanism of this phenomenon, we examined the effect of i.v. [Ser-IL-8]72 (12-20 micrograms/kg) on leukocyte rolling and chemoattractant-induced emigration in mesenteric venules of New Zealand White rabbits and on expression of L-selectin (mAb LAM1-3) and CD18 (mAb 60.3) on circulating rabbit granulocytes. Within 1 min of IL-8 i.v., granulocytes virtually disappeared from carotid blood samples for approximately 5 min. Concomitantly, the flux of rolling leukocytes in mesenteric venules fell from 83 +/- 21 to 2 +/- 1 leukocytes/min. Both rolling leukocyte flux and systemic granulocyte count returned to or exceeded control values within less than 30 min. The chemoattractant/activator FMLP (0.15 microgram/kg i.v.) produced similar results. A second i.v. injection of IL-8 or FMLP, 90 min after the first challenge, had equipotent effects. Local extravascular application of IL-8 via micropipette close to a venule induced adhesion and emigration of 63 +/- 21 leukocytes per site before, but only 26 +/- 9 leukocytes per site 50 to 75 min after i.v. IL-8, when systemic granulocyte count and rolling leukocyte flux had reached or exceeded control values. This was not due to agonist-specific desensitization, because a similar reduction of leukocyte emigration was seen after FMLP i.v. Rabbit granulocytes circulating in vivo uniformly expressed near-control levels of L-selectin at all times between 3 and 360 min after IL-8 i.v. CD18 expression transiently increased after IL-8 i.v. and returned to base line by 90 min. These findings show that IL-8 i.v. reduces granulocyte recruitment to inflammatory sites by inhibiting function(s) necessary for transmigration that are independent of L-selectin and subsequent to rolling.
Macrophage progenitors from mouse bone marrow and spleen differ in their expression of the Ly-6C differentiation antigen.
Authors: McCormack J M , Leenen P J , Walker W S
Published: January 2023

Using mAb and magnetic and fluorescence-activated cell sorting, together with culturing in semisolid culture medium, we show that essentially all splenic macrophage progenitors are Ly-6ChiMac-1-. By contrast, bone marrow macrophage progenitors were more heterogeneous in Ly-6C expression, the majority being restricted to the Ly-6CdimMac-1- and the Ly-6C-Mac-1- subsets. These findings provide immunophenotype evidence that macrophages in the spleen and bone marrow arise from different progenitors.
Binding of monomeric human IgG defines an expression polymorphism of Fc gamma RIII on large granular lymphocyte/natural killer cells.
Authors: Vance B A , Huizinga T W , Wardwell K , Guyre P M
Published: January 2023

Human PBL express one or more of the three classes of Fc receptors for IgG (Fc gamma receptors, I, II, and III). Each type of Fc gamma receptor has a characteristic binding pattern for isotypes of human and mouse IgG. Large granular lymphocyte/NK cells (LGL/NK cells) express the transmembrane form of Fc gamma RIII (Fc gamma RIIIa) on their surface, whereas polymorphonuclear neutrophils (PMN) express the glycosyl phosphatidyl inositol-linked receptor that is the product of the Fc gamma RIIIB gene. Fc gamma RIII has been reported to have low affinity for monomeric IgG, regardless of the cell type on which it is expressed. This study demonstrates specific and saturable binding of monomeric human IgG (M-IgG) to Fc gamma RIIIa on LGL/NK cells but not to Fc gamma RIIIb on PMN. M-IgG binding revealed an expression polymorphism between individuals, those with either high (12,000 to 15,000) or low (4,000 to 7,000) binding sites per cell. Both high and low M-IgG-binding LGL/NK cells bound hulgG1 and IgG3 but did not bind IgG2 or IgG4. The high/low Fc gamma RIIIA expression polymorphism correlated with the ability of an individual's LGL/NK cells to perform antibody-dependent cellular cytotoxicity of erythroid cell targets.
Interferon-gamma prevents graft-versus-host disease after allogeneic bone marrow transplantation in mice.
Authors: Brok H P , Heidt P J , van der Meide P H , Zurcher C , Vossen J M
Published: January 2023

Lethally irradiated C3H/Law mice were injected (i.v.) with C57BL/Rij allogeneic bone marrow cells to induce a delayed type graft-vs-host disease (GVHD). Signs of GVHD first became apparent in the third week after transplantation. The disease resulted in a mortality rate of 70% at 80 days. Treatment with IFN-gamma twice weekly, for a period of 6 wk, starting at the time of bone marrow transplantation (BMT), completely prevented overt GVHD, as evidenced by a lack of diarrhea and no mortality during the follow-up period of 100 days after BMT. Also, the histologic GVHD lesions in the gastrointestinal tract were almost completely abrogated by the IFN-gamma treatment. All long term survivors were proven to be chimeras. During the induction phase of GVHD, the number of Con A-induced, IFN-gamma-producing cells in the spleen was significantly reduced in the IFN-gamma-treated mice as compared with control mice. These results suggest that the normally enhanced production of endogenous IFN-gamma in the spleen at the time of hematopoietic reconstitution after BMT is down-regulated by exogenously administered IFN-gamma. This cytokine-mediated strategy to prevent GVHD might be an alternative to the current strategy of in vitro depletion of T cells for allogeneic BMT.
Human T cell clones reactive against U-small nuclear ribonucleoprotein autoantigens from connective tissue disease patients and healthy individuals.
Authors: Hoffman R W , Takeda Y , Sharp G C , Lee D R , Hill D L , Kaneoka H , Caldwell C W
Published: January 2023

SLE and mixed connective tissue disease (MCTD) are characterized by the presence of high titers of autoantibodies against uridine-rich RNA-small nuclear ribonucleoprotein (snRNP) Ag. Because the presence of such snRNP-reactive autoantibodies has recently been shown to be associated with polymorphisms of HLA, this study was undertaken to determine whether snRNP-reactive T cells could be identified and characterized from patients. PBMC were stimulated with affinity-purified snRNP Ag and cloned by limiting dilution in the presence of rIL-2 and rIL-4, snRNP-reactive human T cell clones were generated from three patients and two healthy blood donors who possessed disease-associated HLA genotypes. The cell surface phenotype of clones determined by flow cytometry was CD3+, CD4+, CD45RO+, TCR V alpha beta+. TCR V beta analysis, performed using V beta-specific primers and polymerase chain reaction, revealed that the T cell lines generated were clonal; a limited number of TCR V beta genes were expressed among the clones tested. All clones tested by mAb blocking of Ag-induced proliferation were restricted by HLA-DR. Several T cell clones were identified that were specific for B'/B or D polypeptides. These results demonstrate that snRNP-reactive T cells can be isolated from SLE and MCTD patients in vitro, and that Ag-driven expansion of such T cells could play a role in the immunopathogenesis of these diseases in vivo.
Regulation of human CD44H and CD44E isoform binding to hyaluronan by phorbol myristate acetate and anti-CD44 monoclonal and polyclonal antibodies.
Authors: Liao H X , Levesque M C , Patton K , Bergamo B , Jones D , Moody M A , Telen M J , Haynes B F
Published: January 2023

CD44 molecules are comprised of multiple alternatively spliced forms and are associated with diverse functions such as mediation of carcinoma metastasis and T cell coactivation. To study the function of individual CD44 isoforms, we have transfected CD44 isoforms into CD44-negative Jurkat T cells and produced cloned Jurkat cell lines that are stably transfected with either a CD44 isoform containing no alternatively spliced insert (CD44H) or a CD44 variant (CD44E) containing an insert of 132 amino acids derived from exons 12, 13, and 14 of the CD44 gene. We found that neither CD44H- nor CD44E-transfected Jurkat T cells constitutively bound hyaluronan (HA), whereas PMA treatment induced Jurkat cells transfected with CD44H but not CD44E to bind HA. CD44 mAb against noninsert regions of the CD44 extracellular domain (A3D8, A1G3) and polyclonal antisera against the COOH-terminal extracellular glycosaminoglycan region of CD44H (anti-6A serum) both induced CD44H-transfected cells to bind HA, whereas only one CD44 mAb (A1G3) induced CD44E-transfected Jurkat T cells to bind HA. Studies of Jurkat cells transfected with CD44H forms with truncations of the CD44 cytoplasmic domain demonstrated that the cytoplasmic COOH-terminal 52 amino acids were critical for binding of HA to the CD44 extracellular domain. Thus, these data underscore the importance of the CD44 cytoplasmic domain in the function of the extracellular portion of CD44H, and demonstrate a role for ligation of human CD44 isoforms at multiple distinct sites in regulation of expression of CD44 binding to HA.
Constitutive expression of IL-6 receptors and their role in the excessive B cell function in patients with systemic lupus erythematosus.
Authors: Nagafuchi H , Suzuki N , Mizushima Y , Sakane T
Published: January 2023

Excessive B cell function including autoantibody production is a common feature of SLE and considered to be intimately associated with spontaneous lymphokine secretion by themselves. To clarify roles of IL-6/IL-6 receptor autocrine activation pathway in autoantibody production observed in patients with SLE, we studied expression and function of IL-6 receptors in comparison with those of IL-2 receptors, Tac on SLE B cells. IL-6 receptors and IL-2 receptors have been detected on B cells in the blood without any in vitro stimuli in most patients with SLE. The introduction of anti-IL-6 receptor antibody, which inhibits binding to the receptors of IL-6, and anti-IL-2 receptor antibody, anti-Tac to the cultures of SLE B cells resulted in potent inhibition of spontaneous production of polyclonal Ig and anti-DNA autoantibodies. In addition, fresh SLE B cells secreted high levels of IL-6 without any in vitro stimuli. These results indicate that constitutive expression of IL-6 receptors on B cells in conjunction with spontaneous IL-6 production by B cells induces autocrine B cell activation, which may lead to B cell hyperactivity and autoantibody secretion in SLE patients. Dysregulation of B cell activity observed in patients with SLE could thus be, at least in part, independent of T cell help.
Further characterization of the interaction between the C1q subcomponent of human C1 and the transmembrane envelope glycoprotein gp41 of HIV-1.
Authors: Thielens N M , Bally I M , Ebenbichler C F , Dierich M P , Arlaud G J
Published: January 2023

Previous studies have provided evidence for activation of the human C1 complex by HIV-1, resulting from direct interaction between C1q and the external portion of the viral transmembrane envelope protein, rsgp41. The present study was undertaken to locate more precisely, within C1q and rsgp41, the sites involved in the C1/HIV-1 interaction. Using a solid phase binding assay, we showed that 125I-labeled C1q binding to rsgp41 was dose dependent, saturable, and comparable with binding of C1q to IgG-OVA immune complexes. The globular and, to a lesser extent, the collagen-like regions of C1q both bound to rsgp41. In contrast, the globular region of C1q inhibited the C1q/rsgp41 interaction, whereas the collagen-like region of C1q did not. A series of peptides covering the putative C1q-binding site on gp41 (positions 590-613 of gp160) were synthesized and used as competitors in the C1q-rsgp41-binding assay. Peptide 601-613 (GIWGCSGKLICT) inhibited C1q binding the most efficiently, with 50% inhibition at a concentration of 100 microM. This peptide also inhibited binding of C1q to rsgp36, the protein of HIV-2 homologous to rsgp41. The inhibitory effect of this peptide was dependent in part on the presence of the S-S bridge normally connecting Cys 605 to Cys 611 because reduction of this bond significantly reduced its efficiency. These data suggest that the C1q/HIV-1 interaction involves a site on C1q located within the globular regions, and a major site located within the immunodominant domain of HIV-1, which shares homology with the corresponding region of HIV-2.
Alterations in Fc epsilon RI induced by protoporphyrin plus long-wavelength ultraviolet light in mouse bone marrow-derived mast cells.
Authors: Yen A , Liu F T , Barrett K E , Gigli I
Published: January 2023

As previously reported, protoporphyrin plus long-wavelength UV light (PP/UVA) inhibits IgE-mediated degranulation of mouse bone marrow-derived mast cells, as assessed by measurement of the release of beta-hexosaminidase. This inhibitory effect was seen with cells sensitized with IgE either before or after PP/UVA treatment (57.8 and 55.3% inhibition, respectively). PP/UVA did not dissociate IgE already bound to cells as assessed either by measuring release of bound 125I-IgE or by flow cytometric analysis. Results from immunoadsorption followed by SDS-PAGE analysis suggested that PP/UVA treatment may cause stable conjugation of IgE to its receptor. In unsensitized cells, PP/UVA did not cause conjugation of the unoccupied Fc epsilon RI to other proteins in the plasma membrane. Nevertheless, Scatchard analysis revealed that PP/UVA decreased the number of Fc epsilon RI per cell by 37% (0.95 x 10(5) vs 1.51 x 10(5)/cell), whereas affinity of the receptor for IgE was comparable between PP/UVA-treated and untreated cells (3.40 nM vs 3.27 nM). Flow cytometric analysis also confirmed the decrease in Fc epsilon RI number in PP/UVA-treated unsensitized mouse bone marrow-derived mast cells. Although 84% of PP/UVA-treated and 82% of untreated cells expressed positive fluorescence when stained with FITC-conjugated IgE, fluorescence intensity was reduced by 40% after PP/UVA treatment. We conclude that PP/UVA alters the conformational structure and/or number of Fc epsilon RI expressed on the mast cell surface. This effect could potentially explain the ability of PP/UVA to inhibit mast cell secretory function and may be related to an ability of PP/UVA to alter the properties of the plasma membrane.
Cyclosporin A-induced autoimmune disease in mice.
Authors: Bucy R P , Xu X Y , Li J , Huang G
Published: January 2023

The immunosuppressive drug cyclosporin A (CsA) disrupts the process of thymic ontogeny and repertoire selection in the alpha beta T cell population. Although treatment with this agent does not cause overt abnormalities in adult animals by itself, in lethally irradiated rats reconstituted with syngeneic bone marrow cells, CsA treatment results in an autoimmune syndrome. In this report, a modified protocol is described to obtain a similar, but distinctive, pathologic syndrome in mice. The key elements of this protocol are stringent elimination of mature T cells from the bone marrow cells used to reconstitute the irradiated recipients and the use of high doses of CsA over a long course (6 wk) after irradiation. CsA treatment inhibits both the positive and negative selection of alpha beta thymocytes in normal young mice as well as in irradiated, T cell-deficient bone marrow-transplanted mice. An increased frequency of "forbidden" V beta 11 T cells is detected in these animals, but it is not clear that these cells are the actual initiators of the inflammatory lesions. In mice of several inbred strains treated by this protocol, striking inflammatory lesions develop in the colon, with less intense, but significant lesions in liver, stomach, and pancreas. Unlike allogeneic graft-vs-host disease, no lesions are found in the small intestine. Examination of these lesions by immunohistochemistry demonstrates that the lesions are composed primarily of T cells and macrophages, with focal aggregates of small B cells present. The same pattern of inflammatory lesions can be adoptively transferred with lymphoid cells from the CsA-treated animals at the peak of lesion development to irradiated syngeneic recipients. The transfer of active lesions is blocked by the co-transfer of normal syngeneic T cells to these recipients. Although similar to the previously described system in rats, these findings emphasize several novel features. In mice, we find an absolute requirement for the elimination of mature T cells from the bone marrow source to develop active inflammatory lesions. The particular tissues affected are also distinctive, especially the unusual feature of severe inflammation in the colon, but no involvement of the small intestine. The mechanism by which the presence of normal T cells suppresses the development of active autoimmunity in this model may have implications for the etiology of localized inflammatory lesions in general.
Autoanti-red cell antibodies synthesized by patients with infectious mononucleosis utilize the VH4-21 gene segment.
Authors: Chapman C J , Spellerberg M B , Smith G A , Carter S J , Hamblin T J , Stevenson F K
Published: January 2023

A significant proportion of patients with B cell tumors secrete IgM mAb that recognize a carbohydrate autoantigen (li) on the red cell surface. The majority bear an idiotope (Id) that arises from heavy chains encoded by the VH4-21 gene segment, indicating unusual restriction of autoantibody specificity to a single VH gene. Polyclonal anti-li antibodies are also synthesized transiently by normal B cells following certain infections, and we have analyzed the role of the VH4-21 gene in encoding these antibodies. Levels of Id in sera of patients following infection with EBV or Mycoplasma pneumoniae were significantly raised (p < 0.01), and the Id-positive Ig reacted with patients' red cells. Id-positive B cell clones were established from four EBV-infected patients, and 6/6 of the IgM-Id agglutinated red cells. Nucleotide sequence analysis revealed involvement of the VH4-21 gene in all cases, with remarkably little change from the germ-line sequence. However, D segments were heterogeneous, and light chains differed. These results indicate that the same VH gene is used both by polyclonal autoantibodies produced in response to infection, and by B cell tumors. However, the lack of mutations would not appear to give an opportunity for Ag selection to drive affinity maturation of these autoantibodies.
Humanized Mik beta 1, a humanized antibody to the IL-2 receptor beta-chain that acts synergistically with humanized anti-TAC.
Authors: Hakimi J , Ha V C , Lin P , Campbell E , Gately M K , Tsudo M , Payne P W , Waldmann T A , Grant A J , Tsien W H
Published: January 2023

Mik beta 1 is a mouse mAb directed at the beta-subunit of the human IL-2R (Tac) that inhibits IL-2 binding and inhibits IL-2 induction of large granular lymphocytes (LGL). Mik beta 1 alone does not inhibit IL-2-induced T-cell proliferation, but acts synergistically with anti-Tac to inhibit IL-2-induced proliferation of activated T cells. To evaluate these effects for possible therapy in humans, we constructed two humanized Mik beta 1 antibodies by combining the complementarity-determining regions of the murine antibody with human framework and constant regions. Compared with murine Mik beta 1, the two humanized Mik beta 1 antibodies, which differ in their degree of humanization, had similar affinities for IL-2R beta. The murine Mik beta 1 and one of the humanized Mik beta 1 antibodies were equivalent in competing for IL-2 binding to IL-2R beta and inhibiting IL-2 induction of LGL cytotoxicity. The activity of the second humanized antibody was significantly reduced. The three Mik beta 1 antibodies act synergistically to varying degrees with humanized anti-Tac to prevent IL-2-induced proliferation of activated T cells. This capacity to synergize paralleled their abilities to inhibit IL-2 binding. In addition, both humanized antibodies directed antibody-dependent cell-mediated cytotoxicity. We hope that humanized Mik beta 1 in combination with humanized anti-Tac will provide a new immunosuppressive therapy for the treatment of autoimmune diseases, graft-versus-host disease, and prevention of allograft rejection.
Detection and epitope analysis of autoantigen-reactive T cells to the U1-small nuclear ribonucleoprotein A protein in autoimmune disease patients.
Authors: Okubo M , Yamamoto K , Kato T , Matsuura N , Nishimaki T , Kasukawa R , Ito K , Mizushima Y , Nishioka K
Published: January 2023

T cells that recognize autoantigens might play an important role in autoimmune diseases. To analyze "autoantigen-reactive T cell" in patients with an autoimmune disease, a human lymphocyte proliferation assay using soluble recombinant autoantigens has been conducted. PBMC from mixed connective tissue disease and SLE patients who possessed anti-U1-small nuclear ribonucleoprotein (snRNP) A autoantibodies responded to a recombinant U1-snRNP A protein. In contrast, PBMC from healthy subjects or autoimmune disease patients not possessing anti-U1-snRNP A autoantibodies did not show such responses. In addition, this proliferative response was inhibited by anti-CD4 mAb. Limiting dilution analyses revealed that the autoantigen-reactive T cells exist in relatively high frequency (1 of 4,065 to 1 of 23,256) in the mixed connective tissue disease patients. Moreover, by T cell epitope mapping, the T cell epitope area was found to be located in the C-terminus of the U1-snRNP A protein, overlapping the B cell epitope area that has been reported. These findings suggest the presence of autoantigen-reactive T cells in such patients, and when these T cells are activated, they may play a central role in the autoantibody generation.