bluuebunny/crossref_metadata_2025 huggingface dataset:
A subset of OKT4+ peripheral T cells can generate colonies containing mixed progeny with OKT4+ helper and OKT8+ suppressor cells
Authors: Farcet Jean Pierre , Gourdin Marie‐Françoise , Calvo Charles , Oudrhiri Nouflssa , Divine Marine , Bouguet Jacqueline , Fradelizzi Didier , Senik Anna , Reyes Felix
Published: February 2007

The membrane phenotype of human T cell colony progenitors and that of their clonal progeny was studied for expression of the T4 and T8 determinants. Using clonal culture conditions, the colonies were grown in semi‐solid agar medium from peripheral blood cells. Clonality was assessed using the glucose‐6‐phosphate‐dehydrogenase isoenzyme marker. Combination of this marker with the culture of sorted cell fractions allowed us to ascribe the colony progenitors to a subset of OKT4+lymphocytes. The progeny consisted of the mixture of single OKT4+, single OKT8+and double OKT4+8+ cells, as determined by double staining. Double staining was performed on mass‐harvested colony cells and on individual colonies expanded in liquid culture with fresh interleukin 2. Expression of the OKT8 positivity on colony cells deriving from OKT4+ progenitors required an interaction with radioresistant OKT8+ cells that were co‐cultured with these progenitors. Furthermore, the functional capacities of the cell progeny were assayed on the pokeweed mitogen‐driven immunoglobulin production by B cells. It was found that OKT4T colony cells were helper whereas OKT8+ colony cells were suppressor cells. It is concluded that a subset of OKT4+ peripheral blood T lymphocytes can generate colonies containing both helper OKT4+ cells and suppressor OKT8+ cells.
Inhibition of experimental autoimmune uveoretinitis in rats by S‐antigen‐specific monoclonal antibodies
Authors: de Kozak Yvonne , Mirshahi Massoud , Boucheix Claude , Faure Jean‐Pierre
Published: February 2007

Mouse monoclonal antibodies (mAb), of either IgG2a or IgG2b isotypes, specific for the retinal S‐autoantigen (S‐Ag) or a pool of rat anti‐S‐Ag sera prevented experimental autoimmune uveoretinitis in Lewis rats when injected i.p. at the time of immunization. Control mAb of the same isotypes, irrelevant to S‐Ag, had no inhibitory effect. The humoral response to S‐Ag, as studied by enzyme‐linked immunosorbent assay using a mouse mAb specific for rat kappa chain, was moderately but significantly reduced in suppressed animals. The rapid disappearance of the injected mAb from rat sera, as measured using a rat mAb specific for mouse kappa chain, could be explained by its complexing with either autologous antigen released from the retina at the site of inflammation, or anti‐idiotypic antibodies.
Search for class II major histocompatibility complex molecular involvement in the response of Lyt‐2+cytotoxic T lymphocyte precursors to alloantigen
Authors: Romani Luigina , Mage Michael G.
Published: February 2007

A possible requirement for class II major histocompatibility complex (Ia) molecules in the initial activation of cytotoxic T lymphocyte precursors (CTLp) for allocytotoxic responses was investigated. To avoid possible interaction with other alloreactive cell types, a highly purified population of Lyt‐2+ splenocytes was used as a source of CTLp. In the light of preliminary results indicating that Lyt‐2+ CTLp, even in the presence of interleukin 2 (IL2), could best be triggered into mature CTL in vitro by cells known to be Ia+, we examined whether an interaction of CTLp with Ia antigens (either on syngeneic accessory cells or on allogeneic stimulators) played a role in the development of allocytotoxicity. Results from experiments done with C57BL/6 Lyt‐2+splenocytes co‐cultured with P815 stimulator cells and IL2 showed that the early activation of CTLp was independent of Ia+ syngeneic accessory cells: (a) flow microfluorometry analysis of the responder population at the beginning or after 1 or 3 days of co‐culture did not reveal the presence of Ia+ cells; (b) procedures for removal of residual Ia+ cells or of dendritic cells from the responder population before co‐culture did not affect the development of cytotoxicity; (c) co‐culture with monoclonal antibodies against syngeneic Iab antigens did not inhibit the CTLp activation. By comparing an Ia+ P815 tumor line with its Ia− clone as allogeneic stimulator cells, it was found that the CTLp activation was also independent of Ia alloantigen on the stimulator cells. The response against both the Ia+ and the Ia− stimulator cell types was not inhibited by monoclonal anti‐L3T4 present in the co‐culture, indicating that these responses were not affected by residual L3T4 helper cells.
Isolation and characterization of Abelson murine leukemia virus‐transformed mast cell lines from midgestation embryonic placenta
Authors: Siegel Michael L. , Horwitz Randy , Morris Timothy D. , Di Venere Raina M. , Siden Edward J.
Published: February 2007

Abelson murine leukemia virus was used to transform cells of the midgestation embryonic placenta. The frequency of transformed foci in semisolid agarose was highest when cells were isolated at 10 days of gestation and cell lines could be established in liquid culture. The continuous cell lines express characteristics of cultured mast cells, including surface antigens which are shared with lymphocytes and mono‐ nuclear phagocytes. These results imply a relationship between the transforming gene product and the mast cell growth factor interleukin 3.
A neomycin‐resistant cell line for improved production of monoclonal antibodies to cell surface antigens
Authors: van Snick Jacques , de Plaen Etienne , Boon Thierry
Published: February 2007

Overgrowth of hybridomas by proliferating spleen T cells often hinders the production of monoclonal antibodies to certain antigens. To overcome this problem we derived neomycin‐resistant fusion partner SP2neoR.l by transfection of SP2/0‐Agl4 cells with plasmid pSVTK neo beta. Hybridomas obtained with SP2 neoR.1 grew optimally in the presence of the neomycin analog G418 at concentrations which blocked the proliferative response of T cells to mitogenic stimuli. The advantage of using SP2 neoR.1 and G418 under conditions where spleen cell proliferation occurs after fusion was demonstrated with hybridomas derived from a rat immunized with mouse helper T cell lines.
Development of T cell clones reactive to two defined restriction elements in conjunction with two defined epitopes of antigen
Authors: Spaeth Eberhard , Rüde Erwin
Published: February 2007

A previously described pig insulin (Pl)‐specific T cell line of (B10 × B10.BR)F1 origin was assayed for its reactivity with species variants of insulin in the presence of antigenpresenting cells (APC) of various H‐2 haplotypes. In addition to its reactivity with PI and bovine insulin (BI) in the context of syngeneic F1 (H‐2bxk)‐APC, a weak crossreactivity was observed with parental B10 (H‐2b)‐APC and BI but not PI. The crossreactive cells could be selected out by several restimulations with the combination of BI and B10‐APC. From the resulting, strongly cross‐reactive T cell line several interleukin 2‐dependent sublines were developed which did not require antigen‐specific restimulations for further propagation. All such sublines had retained the original cross‐reactivity with BI and B10‐APC but showed significant differences in their fine specificity patterns, which indicates that each subline represents a clonal population. One of the sublines was cloned by limiting dilution at one cell/culture with a cloning efficiency of 76%. Five of the clones that were tested for reactivity had the same cross‐reactivity as the original subline and upon recloning at 0.1 cells/culture the pattern again remained unchanged. From an analysis of the two antigen combinations (PI/F1 and BI/B10) it can be concluded that single cells can react with different restriction elements in the context of distinct epitopes of insulin. The implications of this finding for the mechanism of T cell recognition are discussed and a model for the function of major histocompatibility complex molecules in T cell recognition is proposed.
Interleukin 2 mRNA induction in human lymphocytes: analysis of the synergistic effect of a calcium ionophore A23187 and a phorbol ester
Authors: Yamamoto Yusei , Ohmura Takafumi , Fujimoto Kouji , Onoue Kaoru
Published: February 2007

Induction of interleukin 2 (IL2) mRNA in human tonsillar lymphocytes under various conditions was examined by cytoplasmic dot hybridization using a 32P‐labeled IL 2 cDNA probe to study the signal transduction mechanisms which lead to IL2 gene expression. A tumor‐promoting phorbol ester, 12‐O‐tetradecanoyl‐phorbol 13‐ace‐tate (TPA), acted synergistically with a Ca2+ ionophore A23187 or phytohemaggluti‐nin (PHA) to induce a high level of IL2 mRNA in lymphocytes, whereas each of them by itself could not induce the mRNA production. In two‐step culture experiments the lymphocytes pulse‐incubated with TPA for 1 h (the first culture) could efficiently initiate IL2 mRNA production by subsequent culture with A23187 or PHA (the second culture). Results obtained by removal of extracellular Ca2+ from either the first or second culture revealed that Ca2+ was not necessarily required during the first culture with TPA, but it is essential in the second culture with A23187 or PHA, regardless of the presence or absence of Ca2+ in the first culture. A reagent known to be a calmodulin antagonist, N‐(6‐aminohexyl)‐5‐chloro‐l‐naphthalenesulfonamide (W‐7), almost completely inhibited the IL2 mRNA induction in A23187‐TPA‐stimu‐lated lymphocytes at a concentration of 25 μM, whereas N‐(6‐aminohexyl)‐l‐naph‐thalenesulfonamide that has much lower affinity for calmodulin than W‐7 did not inhibit at this concentration. The IL2 mRNA induction was also blocked by the addition of 50 UM of 8‐(N, N‐diethylamino)‐octyl‐3,4,5‐trimethoxybenzoate hydro‐ chloride which is known to block the release of Ca2+ from intracellular storage sites. These results show that mobilization of Ca2+ and the calmodulin‐dependent regulatory system appear to work synergistically with TPA which probably activates protein kinase C in the pathway to IL2 gene expression.
Proliferative responses of circulating human NK cells: delineation of a unique pathway involving both direct and helper signals
Authors: Ythier Arnaucl , Delmon Laurence , Reinherz Ellis , Nowill Alexandra , Moingeon Philippe , Mishal Zohair , Bohuon Claude , Hercend Thierry
Published: February 2007

The present studies were performed to investigate mechanisms of human natural killer (NK) cell activation. NK‐active cells were purified out of heterogeneous large granular lymphocytes (LGL)‐enriched suspensions using a “pan NK”‐specific monoclonal antibody termed anti‐NKHlA. It was found that treatment of NKHlA+‐sorted cells by T lymphocyte mitogens such as phytohemagglutinin (PHA) or anti‐TH2 plus anti‐Tll3 did not induce proliferative responses. In fact, there was no measurable interleukin 2 (IL 2) secretion and no significant increase in IL 2 susceptibility following incubation with either PHA or anti‐TH2 plus anti‐T113. However, as opposed to small resting T lymphocytes, NKH1A+ cells moderately proliferated in the presence of IL2. This IL 2‐dependent proliferation was dramatically increased after interaction between NK‐active lymphocytes and certain hematopoietic cell lines such as K562 or EBV‐transformed lymphoblastoid cell lines.

These studies indicate that unique activation mechanisms can be identified when NK lymphocytes are purified out of LGL‐enriched fractions. The NK activation pathway delineated here appears essentially distinct from those described for T lymphocytes. Indeed the nature of the NK cell/inducing cell interaction is unknown and triggering cannot be related, for example, to a conventional allogeneic effect mediated through membrane exposure of class I or class II major histocompatibility complex gene products. Moreover, these interactions do not lead to the development of autocrine responses. Together the present results support the view that direct signals can preac‐tivate NK cells but are not sufficient to trigger proliferation which must therefore be regulated by helper populations.
An inherited deficiency of the third component of complement, C3, in guinea pigs
Authors: Burger Reinhard , Gordon John , Stevenson George , Ramadori Guliano , Zanker Bernd , Hadding Ulrich , Bitter‐Suermann Dieter
Published: February 2007

Hereditary deficiency of the third component of complement, C3, is found very seldom in the human. C3 deficiency is associated with severe bacterial infections revealing the central role of C3 in complement activation via the classical or alternative pathway. We describe a new hereditary C3 deficiency in strain 2 guinea pigs. Serum from these animals had a markedly reduced lytic activity in a standard assay for complement‐dependent, antibody‐mediated cytotoxicity. In functional assays of individual components, the hemolytic activity of the components C4, C2, C5 and of factors B, D and H was in the normal range. The functional C3 titer, and similarly C3 antigenic activity in the serum of these C3‐deficient animals (C3D) was on average only 5.7% of normal activity. Typing the animals with alloantisera or monoclonal antibodies to guinea pig Ia‐antigens revealed that the C3D animals had the major histocompatibility complex‐haplotype of inbred strain 2 guinea pigs (B.1, Ia. 2,4). The C3 defect is not linked to the major histocompatibility complex and, in addition, is not linked to a C3a receptor deficiency. Macrophages and hepatocytes of the C3D animals have an unimpaired capacity for synthesis and secretion of C3 as measured by enzyme‐linked immunosorbent assay. There was no indication for hypercatabolism of normal C3 by the animals as shown by plasma clearance of 125I‐radiolabeled C3. Thrombocytes of the C3D animals responded normally to stimulation with purified C3a in an ATP‐release assay without an indication for a desensitization in vivo. Possibly the fault resides in an enhanced susceptibility of their own C3 to proteolysis. However, C3 partially purified from the plasma of the C3D animals or secreted by hepatocytes exhibited no obvious structural differences to purified normal C3 in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis or in immunoblotting. The C3D serum had a reduced bactericidal activity compared to normal or to C4‐deficient serum. Nevertheless, the animals are apparently healthy without an indication for increased frequency of bacterial infections. These guinea pigs provide an unique model for analysis of the biological functions of C3 in vivo and in vitro without the need for artificial C3‐depletion procedures with all their known and unknown side‐effects.
Human monocyte cell line (U937) releases suppressive IgG‐binding factor(s)
Authors: Calvo Charles Félix , Watanabe Shinichiro , Mètivier Didier , Senik Anna
Published: February 2007

Fcγ receptor‐bearing U937 cells, when incubated in serum‐free buffer, were found to release spontaneously a suppressor material for pokeweed mitogen‐drigen IgG synthesis which could be retained on Sepharose 4B‐IgG immunosorbents. Immunosorbents coupled with IgM or F(ab′)2 fragments of IgG were unable to retain the inhibitory activity of U937‐derived material, suggesting a binding specificity for the Fcγ fragment of IgG. This suppressor material corresponds therefore by definition to an IgG‐binding factor (IgG‐BF). The mechanism for in vitro suppression of the antibody response by U937‐derived IgG‐BF was investigated. It did not interfere with cell proliferation and displayed maximum effect when added at day 3 of the culture period. Tested for its effect on IgG, IgM and IgA synthesis, IgG‐BF suppressed antibody production following a pattern specific for IgG. Finally gel filtration of the suppressor material gave rise to two peaks of inhibitory activity with an apparent molecular mass comprised between 30 to 40 kDa and 60 to 73 kDa, respectively.
The level of λ1 light chain expression in the mouse reflects the probability of rearrangement of the relevant gene
Authors: Primi Daniele , Levi‐Strauss Matthieu , Cazenave Pierre‐André
Published: February 2007

The predominance of x over λ light chain expression in mice can either reflect the probability of rearrangement of the relevant locus or be the result of antigen‐driven clonal expansion. To discriminate between these two possibilities we determined, by limiting dilution analysis, the frequencies of x‐ and λ‐producing cells in B lymphocytes generated in vitro from bone marrow pre‐B cells. The frequencies obtained in these cultures are not significantly different from those obtained with mature spleen cells. In addition, Southern blot analysis of bone marrow‐derived and splenic cell DNA revealed that in both populations the extent of B lymphocytes having undergone λ1 gene rearrangement does not exceed 4%. These results, therefore, establish that in the mouse the low level of λ light chain expression directly reflects the probability of rearrangement of the relevant locus.
Activation and proliferation signals in mouse B cells VIII. Induction of DNA synthesis in B cells by a combination of calcium ionophores and phorbol myristate acetate
Authors: Klaus Gerry G. B. , O'Garra Anne , Bijsterbosch Martin K. , Holman Mary
Published: February 2007

Mouse B cells cultured with either phorbol myristate acetate (PMA), or with Ca2+ ionophores enter a transitional activated state, between quiescence (G0) and G1, but do not synthesize DNA. It is shown here that the combination of PMA plus the ionophore ionomycin induces resting B cells to synthesize DNA, but not to secrete antibody. B cells from CBA/N mice carrying the xid defect, and those from the lipopolysaccharide‐unresponsive C3H/HeJ strain also respond to this combination. Suboptimal doses of the two stimuli synergize with B cell‐stimulating factor 1 in promoting proliferation of resting B cells, but the co‐mitogen does not substitute for type II B cell growth factor in the BCL1 lymphoma. Furthermore, (as predicted) the combination of these two agents does not induce the breakdown of inositol phospholipids in B cells. These data are consistent with the hypothesis that elevation of intracellular Ca2+ (by the ionophore), plus activation of protein kinase C (by PMA) leads to DNA synthesis in B cells. The combination of Ca2+ ionophore and PMA thus appears to essentially mimic the biochemical effects of ligation of surface immunoglobulin receptors on B cells, by providing the two second messengers normally emanating from the receptor‐mediated breakdown of polyphosphoinositides.
Gut mucosal mast cells in Nippostrongylus‐primed rats are the major source of secreted rat mast cell protease II following systemic anaphylaxis
Authors: King Stephen J. , Miller Hugh R. P. , Woodbury Richard G. , Newlands George F. J.
Published: February 2007

The distribution of the predominant chymotrypsin‐like enzyme of mucosal mast cells (rat mast cell protease 11: RMCP II) was examined in naive and Nippostrongylus‐primed rats both before and after the induction of systemic anaphylaxis. Anaphylactic secretion of RMCP II following i.v. challenge of primed rats with worm antigen was accompanied by significant depletion of this enzyme from the jejunal and gastric mucosae; the concentrations were not altered in the ileum and colon. Despite significant increases in the levels of RMCP II in lung and mesenteric lymph node following infection with N. brasiliensis there was no anaphylactic depletion of this enzyme from these sites. No RMCP II was detected in liver, spleen, kidney or bone marrow either before or after systemic anaphylaxis. Mucosal mast cells were depleted from the jejunal, gastric and colonic mucosae following antigen challenge of primed rats. These data provide further evidence that gastrointestinal mucosal mast cells are the major source of secreted RMCP II following systemic anaphylaxis in the rat.
Human B cell activation: Selective sensitivity of the early stages to calcium channel‐blocking drugs
Authors: Dugas Bernard , Vazquez Aimé , Delfraissy Jean‐François , Gérard Jean‐Philippe , Rannou Marie‐Thérèse , Galanaud Pierre
Published: February 2007

The importance of Ca2+ in the early events of lymphocyte activation has been suggested by several studies. We examined the effect of calcium channel‐blocking drugs (verapamil and nitrendipine) on the progression of human B cells through their activation cycle. Our results show that these drugs suppress the anti‐μ‐induced human B cell proliferation and interfere with the early events of the B cell activation in a dose‐dependent fashion. This suppression correlates with a marked decrease in anti‐μ‐induced 45Ca2+ uptake. Calcium channel‐blocking drugs inhibit the anti‐μ‐induced uridine incorporation and the appearance of the activation marker defined by the 4F2 monoclonal antibody. Calcium channel‐blocking drugs also inhibit B cell proliferation induced by the costimulation with anti‐μ antibody and B cell growth factor (BCGF). However, this inhibition takes place at the early (anti‐μ‐dependent) stage of B cell activation: the BCGF‐dependent proliferation of in vitro anti‐μ‐activated B cells is only marginally inhibited. Finally the proliferation of Epstein‐Barr virus‐infected B cell lines is resistant to the effect of calcium channel‐blocking drugs.
Mouse B cells do not proliferate in human interleukin 2
Authors: Leanderson Tomas , Julius Michael H.
Published: February 2007

By pre‐activation with a monoclonal anti‐IgM antibody coupled to Sepharose, either in the absence or presence of low doses of lipopolysaccharide, mouse B cell populations were rendered responsive to recombinant DNA derived human interleukin 2 (IL2). However, if the B cell populations were subjected to separation based on their buoyant density before pre‐activation, only low but not high buoyant density cells became responsive to IL2. Both cell populations subscquent to anti‐IgM pre‐activation were equally responsive to a Sephadex G‐100 fraction of supernatants from rat spleen cells stimulated with concanavalin A. Furthermore, the differences in the IL2 titration curves on T and B cell populations indicate that only a subpopulation of cells are responding to IL2 in the B cell populations. In B cell populations that did respond to IL2, a population of Thy‐1.2‐positive cells appeared. The proportion of IL2‐responsive T cells needed to give a significant signal when admixed with nonresponding B cell populations was quantitated to be less than 1 %.
Recombinant interleukin 2 enhances spontaneous insulin‐dependent diabetes in BB rats
Authors: Kolb Hubert , Zielasek Jürgen , Treichel Ulrich , Freytag Götz , Wrann Michael , Kiesel Ulrich
Published: February 2007

Treatment of BB rats with recombinant interleukin 2 (IL 2) enhanced the development of spontaneous diabetes in these animals. A dose of 20 μg IL 2/kg body weight was administered twice daily for 80 days starting at 42 days of age. The rate of diabetes was doubled after IL2 administration (53% vs. 23%) and the onset of diabetes was found to be accelerated by a mean of 18 days. Histological analysis showed enhanced inflammation of islets and in addition interstitial pancreatis. It is concluded that IL2 has a regulatory effect on spontaneous organ‐specific autoimmunity.
Cloned helper T lymphocytes exposed to interleukin 2 become unresponsive to antigen and concanavalin A but not to calcium ionophore and phorbol ester
Authors: Otten Gillis , Wilde David B. , Prystowsky Michael B. , Olshan Jerrold S. , Rabin Harvey , Henderson Louis E. , Fitch Frank W.
Published: February 2007

Two cloned murine helper T lymphocyte (HTL) lines were used to investigate the immunoregulatory properties of highly purified interleukin 2 (IL2). The clone, designated J6.19, secretes lymphokines, including IL2, and proliferates when stimulated with ovalbumin in the presence of I‐Ak‐bearing spleen cells, while the alloreactive clone, L2, secretes lymphokines, including IL2, and proliferates when stimulated with Mlsa,d‐bearing spleen cells. When either clone was exposed to a high concentration of pure IL2 for 1 to 2 days in the absence of either antigen or spleen cells, the HTL became unresponsive to rechallenge with antigen. Unresponsiveness to antigen was indicated by an inability of HTL to proliferate or secrete usual amounts of IL 2 or colony‐stimulating factor. Within 5–7 days after exposure to IL2, the cloned HTL again responded to antigen. Thus, in addition to being a growth factor for HTL, IL2 can limit the magnitude of the HTL response to antigen. L2 or J6.19 cells could also be induced to secrete lymphokines by the lectin, concanavalin A (Con A) or by a combination of the calcium ionophore, A23187, and phorbol myristate acetate (PMA). After exposure of L2 or J6.19 cells to sufficient IL2 to induce unresponsiveness to antigen, cells were also unresponsive to Con A, as indicated by a reduction in the level of lymphokines secreted. In contrast, lymphokine levels stimulated by A23187/PMA were comparable to those produced by cells not exposed to IL2. The failure of antigen to stimulate lymphokine release and proliferation by HTL previously exposed to IL2 therefore may result from an inability of HTL to recognize antigen or to transduce effectively the antigen recognition signal. Several T cell surface molecules are known to be involved in antigen activation of HTL; these include the antigen receptor and the “associative recognition” structures L3T4 and LFA‐1. We observed that L2 cells, rendered unresponsive to antigen by exposure to IL2, expressed normal levels of antigen receptor, as identified by the monoclonal antibody, KJ16–133.18. Furthermore, expression of L3T4 and LFA‐1 was not decreased. Unresponsiveness to antigen induced by IL2 thus could not be correlated with decreases in the expression of antigen receptors, L3T4, or LFA‐1. Unresponsive HTL may therefore be capable of recognizing antigen but the signal generated by antigen binding may be attenuated during its transduction, resulting in the failure of cloned HTL to proliferate or secrete lymphokines at the usual levels.
Differential expression of T cell differentiation antigens and major histocompatibility antigens on activated T cells during the cell cycle
Authors: Matsui Yoshiki , Shapiro Howard M. , Sheehy Michael J. , Christenson Lisa , Staunton Donald E. , Eynon Elizabeth E. , Yunis Edmond J.
Published: February 2007

In this report we have analyzed cell cycle‐related fluctuations of both quantity and density of the T cell differentiation antigens, CD3 (T3), CD4 (T4) and CD8 (T8), as well as the major histocompatibility complex (MHC) antigens on the cell surface of activated T cells. Phytohemagglutinin‐activated T cells cultured for 3 days with or without conditioned medium or for 10 days with conditioned medium and mixed lymphocyte culture‐derived T cell clones were used for the analysis. Correlated measurements of the surface antigen quantity (immunofluorescence), DNA content (dye Hoechst 33342), and cell size (light scatter), not influenced by synchrony induction methods and cell fixation, were performed by dual‐beam flow cytometry. Our results demonstrate that the T cell differentiation antigens, CD3, CD4 and CD8, and class I MHC antigens are increased in density in the G1 phase for all activated T cells tested. In contrast, class II MHC antigens are increased in density in the G2 phase of activated T cells maintained with conditioned medium. Since it is known that the T cell differentiation antigens and class I MHC antigens on activated T cells are necessary for proliferation of T cells, our study suggests that this effect is more significant in the G1 phase. The cell cycle changes in expression of class I and class II MHC antigens, but not of the T cell differentiation antigens, appear to be mediated by soluble factors, probably including interferon‐γ, which could produce a differential increase of class I and class II MHC antigens on G2 phase cells.
Antigen‐presenting cell function of dendritic cells and macrophages in proliferative T cell responses to soluble and particulate antigens
Authors: Kapsenberg Martien L. , Teunissen Marcel B. M. , Stiekema Frank E. M. , Keizer Hiskias G.
Published: February 2007

The capacity of dendritic cells (DC) and macrophages (MΦ) to present soluble and particulate antigen was tested in an ovalbumin (OVA)‐specific T cell proliferation assay. In a comparative investigation we found that both DC and MΦ, were able to present soluble OVA, but that only MΦ, could present insolubilized OVA to T cells. DC were found to be able to present OVA in collaboration with MΦ. The failure of DC to present insolubilized OVA is probably caused by their inability to endocytose these antigens.

DC appeared not to endocytose substantial amounts of soluble OVA either. In contrast to MΦ, antigen presentation by DC is not blocked by lysosomotropic drugs. Taken together, these observations suggest that DC can present soluble protein antigens without intracellular degradation.
The role of self‐Ia antigens in the murine mixed lymphocyte response
Authors: Baxevanis Constantin N. , Ioannides Constantin‐Demosthenes G. , Papamichail Michail
Published: February 2007

Mouse splenic macrophages (MΦ) were tested for their ability to potentiate in vitro allogeneic mixed lymphocyte response (MLR) of highly purified syngeneic responder T cells against allogeneic MΦ. It was shown that even extremely low numbers of MΦ, syngeneic to the responder T cells were able to induce significantly stronger MLR. This potentiating effect was demonstrated to be expressed via the self‐Ia antigens present on the surface of syngeneic MΦ., The functional involvement of self‐Ia antigens was substantiated by two approaches: (a) by using monoclonal antibodies specific for I‐region determinants of the responder haplotype MΦ, and (b) by setting up MLR cultures with stimulator MΦ of (responder X stimulator) F1 origin which express both self‐ and allo‐Ia antigens. The results obtained in this study demonstrate that the presentation of self‐Ia antigens, in conjunction with the recognition of allomajor histocompatibility complex antigens, are required for in vitro primary MLR.
Lack of correlation between hypomethylation and expression of the HLA‐DRα gene
Authors: Gambari Roberto , Senno Laura Del , Barbieri Raffaella , Buzzoni Daniele , Gustafsson Kenth , Giacomini Patrizio , Natali Pier Giorgio
Published: February 2007

DNA methylation at the 5′‐CCGG‐3′ sites of the HLA‐DRα gene and relative flanking regions has been analyzed by Msp I and Hpa II enzymatic digestion in order to determine whether a correlation exists between DNA methylation and transcription of the HLA‐DRα gene.

Unexpectedly, and in contrast to the behavior of most eukaryotic genes, no positive correlation was found between hypomethylation and expression. In fact, HLA‐DRα appears to be fully unmethylated at Msp I/Hpa II sites in K562 cells, not expressing DR molecules, and to exhibit a high degree of methylation in Colo 38 cells, which actively transcribe the gene.

The unorthodox behavior of HLA‐DRα is not unique to melanoma or erythroid cells since a similar, positive correlation between methylation and expression also exists when this analysis is extended to cell lines belonging to other histotypes.
The basis for major histocompatibility complex (MHC) and immunoglobulin gene control of helper T cell idiotopes
Authors: Martinez‐A. Carlos , Pereira Pablo , De La Hera António , Bandeira Antóio , Marquez Carlos , Coutinho António
Published: February 2007

BALB/c helper T cells, prepared against 2,4,6‐trinitrophenyl (TNP)‐derivatized syngeneic spleen cells, fail to recognize other hapten modifications [(4‐hydroxy‐3‐nitrophenyl)acetyl, fluorescein isothiocyanate] of syngeneic cells, as well as TNP‐derivatized cells from major histocompatibility complex‐congenic donors. T helper cell interactions with “presenting” cells and “target” B lymphocytes are inhibited by monoclonal antibodies directed either to the hapten or to I‐A molecules on target cells. Helper activity is also inhibited by monoclonal anti‐idiotypic antibodies directed to an idiotope expressed by the TNP‐binding myeloma protein MOPC 460, but not by soluble TNP‐protein conjugates. The study of congenic mouse strains revealed that while the fine specificity of TNP recognition and the quantitative levels of M460 idiotope expression are I‐A linked, the expression of the M 460 idiotope by helper cells is controlled by IgCh‐linked genes. Absence of anti‐idiotope inhibition of helper cells prepared in anti‐μ‐suppressed mice suggests, however, that immunoglobulin idiotype expression by T cells results from network interactions selecting available lymphocyte repertoires which operate before antigenic stimulation.
The roles of interdigitating cells and natural killer cells in the rapid rejection of allogeneic lymphocytes
Authors: Fossum Sigbjørn , Rolstad Bent
Published: February 2007

The fate of radiolabeled allogeneic thoracic duct lymphocytes injected into congenitally athymic, nude rats was followed by autoradiography and electron microscopy. The allogeneic cells entered the host lymphoid organs at a normal rate, but once inside the lymphoid tissue they were rapidly phagocytozed by interdigitating cells (IDC) situated in the lymph node paracortex and the splenic periarteriolar lymphoid sheaths. Neither host nor donor T cells were required to initiate phagocytosis, as purified donor B cells were also avidly ingested by the athymic host IDC. Compared with fully allogeneic cells phagocytosis of semiallogeneic donor cells was much less efficient. When natural killer cell activity was blocked by preinjecting the recipients with antibodies against natural killer cells (anti‐asialo GM1 or MRC OX‐8) phagocytosis of the allogeneic cells was strongly reduced. As IDC did not bind these antibodies, the finding indicates that natural killer cells were needed to discriminate between own and foreign lymphocytes and to kill the allogeneic cells, which were then ingested by surrounding IDC. This was further supported by the observation that dendritic, constitutively Ia+ cells from peripheral lymph, phenotypically identical to IDC, did not lyse or phagocytoze allogeneic lymphocytes in vitro.
Peritoneal Ly‐1 B cells: Genetic control, autoantibody production, increased lambda light chain expression
Authors: Hayakawa Kyoko , Hardy Richard R. , Herzenberg Leonore A.
Published: February 2007

Previous studies demonstrate that Ly‐1 B cells and their progenitors are clearly detectable in peritoneum in normal mice. In this publication, we show (a) that peritoneal Ly‐1 B cells resemble splenic Ly‐1 B cells with respect to surface marker expression and functional activity (autoantibody production); (b) that Ly‐1 B frequencies in peritoneum are considerably higher than in spleen; and (c) that genetic mechanisms reduce peritoneal Ly‐1 B frequencies to minimal levels in SJL‐related mice and to below detectability in CBA/N and other mice with the X‐linked immunodeficiency (Xid). In addition, we show that that peritoneal (and perhaps splenic) Ly‐1 B populations demonstrate an unique bias in immunoglobulin commitment. That is, they are selectively enriched for cells that express IgM heavy chains in association with lambda light chains. Thus, as a whole, evidence presented here defines the peritoneum as a tightly regulated lymphocyte compartment that normally houses a large population of mature Ly‐1 B cells with distinctive functional properties.
Interleukin 2 receptors are released by cells in vitro and in viva. I. Detection of soluble IL 2 receptors in cell culture supernatants and in the serum of mice by an immunoradiometric assay
Authors: Osawa Hisao , Josimovic‐Alasevic Olivera , Diamantstein Tibor
Published: February 2007

An immunoradiometric assay (IRMA) detecting nanogram amounts of soluble mouse interleukin 2 receptors (IL 2R) is described. The IRMA is based on the finding that the two monoclonal antibodies AMT‐13 and 7D4 react with two different epitopes of the mouse IL2R molecule. As detected by IRMA, T lymphoblasts and IL2R+ leukemic cells when incubated release IL2R in soluble form into the culture medium. In the serum of mice inoculated with syngeneic IL2R+ leukemic cells, growing as a solid tumor, IL2R were detectable. The results raise the question whether circulating IL2R are involved in down‐regulation of immune responses.
T cell activation by anti‐T3 antibodies: Comparison of IgG1 and IgG2b switch variants and direct evidence for accessory function of macrophage Fc receptors
Authors: Smith Kenneth G. C. , Austyn Jonathan M. , Hariri Gita , Beverley Peter C. L. , Morris Peter J.
Published: February 2007

The human T3 antigen is closely associated with the T cell receptor. Some anti‐T3 antibodies cause T cell proliferation in the presence of monocytes which have Fc receptors (FcR) that bind particular antibody subclasses. Such an interaction is thought to determine whether or not an anti‐T3 antibody is mitogenic. We examined the mitogenicity of an IgG1 antibody, UCHT1, and an IgG2b switch variant of identical specificity, UCHT1B. With autologous monocytes, 76% of individuals responded to UCHT1 and 9% to UCHT1B, falling into three patterns of responsiveness. Both antibodies in the absence of monocytes induced responsiveness to recombinant interleukin 2, even for UCHT1B nonresponder T cells. The proliferation induced by UCHT1B, however, was always less than that induced by UCHT1. These findings demonstrate the critical role played by the Fc region for mitogenesis, and suggest a possible role for the hinge region.

We then obtained direct evidence that mitogenicity can be mediated exclusively via FcR. Mouse macrophages have distinct FcR: FcRI binds IgG2a but FcRII binds IgG1 and IgG2b and its function can be inhibited by the specific antibody 2.4G2. Because UCHT1 and UCHT1B were of the correct subclass to interact with FcRII we examined the accessory function of mouse peritoneal macrophages. Without exception, human T cells now responded to both antibodies. Proliferation was drastically inhibited by 2.4G2 but not by an irrelevant anti‐macrophage antibody, F4/80, nor by an anti‐human neutrophil FcR antibody, 3G8. Furthermore, 2.4G2 did not inhibit the accessory function of mouse macrophages for OKT3, an IgG2a antibody that presumably interacts with FcRI, and did not inhibit the function of human monocytes for UCHT1 and UCHT1B.

Mouse B cells, in contrast to macrophages, have an FcR which binds all three subclasses, but which can be inhibited by 2.462. B cells, however, were not accessory cells for mitogenesis with UCHT1, UCHT1B or OKT3. These findings are discussed in relation to other requirements for T cell activation by anti‐T3 antibodies.
Ir gene expression on T cells: Effect of a monoclonal antibody directed against I region‐controlled determinants on T cells
Authors: Yagi Junji , Nonaka Makoto , Abe Ryo , Tada Tomio
Published: February 2007

A monoclonal antibody directed at an I region‐controlled epitope uniquely expressed on T cells (Iat) was studied for its in vivo effect on the antibody response under the control of an Ir gene. The antibody was produced by a hybridoma made from A.TH spleen cells immune to A.TL (anti‐Ik), that was selected for its reactivity with T but not B cells and macrophages, and thus was designated as anti‐Iatk. The injection of this anti‐Iatk into H‐2k, H‐2b and H‐2k×bF1 mice resulted in the suppression of antibody response to poly‐L‐(His,Glu)‐poly‐D,L‐Ala–poly‐L‐Lys [(H,G)‐A–L] in H‐2k and F1 mice but not that to poly‐L‐(Tyr,Glu)‐poly‐D,L‐Ala–poly‐L‐Lys [(T,G)‐A–L] both in H‐2b and F1 mice. The adoptive cell transfer of the combinations of anti‐Iatk‐or normal mouse serum‐treated T and B cells into irradiated hosts demonstrated that anti‐Iatk primarily affected (H,G)‐A–L‐specific helper T cells but not B cells and macrophages, resulting in the specific elimination of the antibody response. Suppressor T cells were not induced by the treatment with anti‐Iatk. The antibody specifically eliminated the (H,G)‐A–L‐specific but not (T,G)‐A–L‐specific helper T cells in F1 spleen cells that had been primed with both (H,G)‐A–L and (T,G)‐A–L. The results indicated that anti‐Iatk affected the H‐2k‐associated Ir gene function born by T cells but not by antigen‐presenting cells, which was expressed on F1 helper T cells with apparent exclusion of the other allele, and implied that the Iat antigen on helper T cells is one of the sites of expression of Ir genes.
In the murine syngeneic mixed lymphocyte reaction, one T cell subset replicates in the presence of B cells or macrophages and replication is inhibited by simultaneous presence of both stimulator cells
Authors: Stevenson J. Ross , Battisto Jack R.
Published: February 2007

The murine T‐non‐T cell syngeneic mixed lymphocyte reaction has been examined to determine whether B cells and macrophages stimulate the same or different subpopulations of T cells. By using experiments in which replicating T cells were suicided, we found that the two different stimulators caused replication of what appears to be the same subset(s) ot T cells. Since B cells and macrophages carry the same stimulating antigens (class II plus mls or others), one would expect them to stimulate the same T cell subpopulations were it not that they have been reported to stimulate two different subpopulations in humans. When B cells and macrophages were simultaneously used as stimulators, diminished T cell replication occurred. We have found the reduced response is not attributable to exhaustion of culture nutrients or to displacement of the response peak. Other possibilities to account for this marked reduction have been discussed from the viewpoint of suppression emanating from macrophages and/or T cells.
Cytotoxic T cell lysis of H‐2‐negative murine sarcoma cells
Authors: Noll Gabriele , Schmidt Wilhelm , Bauer Heinz
Published: February 2007

The DOH cell line was established from C3H.OH (H‐2Kd, Dk) embryonic fibroblasts transformed with Rous sarcoma virus (RSV) in vitro. When injected into syngeneic mice, DOH cells were very weakly tumorigenic and induced a cytotoxic immune response. Cytotoxic T lymphocytes (CTL) specifically lysed DOH cells but not other RSV‐induced sarcoma cells, which shared the H‐2Kd or H‐2Dk antigen, respectively, with DOH. Serological and immunochemical analysis of H‐2 antigens subsequently showed that DOH sarcoma cells did not express syngeneic H‐2K and H‐2D antigens. Surprisingly, a H‐2Kd‐specific monoclonal antibody (100‐5) bound to DOH cells and was inhibitory for syngeneic CTL specific for DOH. In addition, DOH cells were lysed by alloreactive H‐2Kk‐specific CTL. The demonstration of immunogenic H‐2‐negative sarcoma cells suggests that either the H‐2K antigens have been extensively altered or that hitherto unidentified major histocompatibility complex class I molecules are expresled on DOH sarcoma cells surfaces, acting as target antigens for tumor‐specific CTL.
Isotype restriction of idiotopes associated with human anti‐streptococcal A carbohydrate antibodies
Authors: Emmrich Frank , Zenke Gerhard , Eichmann Klaus
Published: February 2007

Sharing of idiotopes betwen IgG and IgM antibodies has been described with antibodies of various specificities in the literature. However, this does not seem to be the rule for human IgG and IgM antibodies with specificity to streptococcal A carbohydrate. Six idiotopes were described which are associated either with IgM or with IgG antibodies to one of the two major epitopes of streptococcal A carbohydrate: N‐acetyl‐D‐glucosamine or α(1,2), α(1,3)‐linked rhamnose oligomers. Some of the idiotopes are widely cross reactive with antibodies of the corresponding specificity in other individuals. They were constantly found to be restricted to either IgM or IgG in all individuals tested so far. We discuss several alternatives to explain this finding and conclude that switching from IgM to IgG is either a very rare event in human B cells of this specificity, or leads to loss and gain of idiotopes and/or specificity due to frequent somatic mutations. The phenomenon of idiotope restriction to certain isotypes might be important if anti‐idiotopic monoclonal antibodies are considered for use in therapeutical approaches aimed at the manipulation of antibody production.
CD2 and CD3 antigens mobilize Ca2+ independently
Authors: O'Flynn Kieran , Knott Lesley J. , Russul‐Saib Mahmood , Abdul‐Gaffar Rafiu , Morgan Gareth , Beverley Peter C. L. , Linch David C.
Published: February 2007

Antigen‐induced stimulation of T cells is mediated via the CD3 antigen receptor (Ti) complex and monoclonal antibodies (mAb) reacting with CD3 and Ti result in rapid intracellular Ca2+ mobilization, followed by monocyte‐dependent proliferation. Combinations of mAb to CD2, the sheep red blood cell receptor, also mobilize calcium and induce mitogenesis and purified phytohemagglutinin (PHA) stimulates T cells predominantly by interaction with this molecule. It has been suggested that activation via CD2 requires the presence of CD3 and that the hydrophobic ϵ chain of CD3 is the T cell calcium channel. To investigate this further we have obtained large numbers of natural killer (NK) cells which express CD2 but not CD3 from a patient with a chronic expansion of this lymphocyte subpopulation. It is shown that calcium mobilization can be induced in these cells by mAb to CD2 and purified PHA but not by anti‐CD3 mAb. This indicates that calcium mobilization can be induced via the CD2 molecule in NK cells not expressing CD3 and that activation through CD2 is separate from the antigen receptor CD3 pathway.
Degeneracy of H‐2 recognition by cytotoxic T lymphocytes: 10% of the total repertoire is “specific” for a given haplotype and up to 1% is self‐H‐2 reactive
Authors: Beretta Alberto , Ermonval Myriam , Larsson Eva‐Lotta
Published: February 2007

The frequency of specific cytotoxic T lymphocytes (CTL) precursors in the total CTL pool was analyzed in a lectin‐driven limiting dilution system. We found that up to 10% of the expressed CTL repertoire in a normal mouse is “specific” for a given allogeneic H‐2 haplotype. Split‐well analysis under clonal conditions demonstrates that the antigens recognized by the effector CTL are H‐2 encoded. A high frequency of CTL “specific” for self‐H‐2 antigens was revealed in all the experiments, accounting for about 1% of the total inducible pool of CTL. These results suggest a high degree of degeneracy of H‐2 recognition by CTL and the immunocompetence of self‐H‐Zreactive CTL precursors in normal individuals.
Recognition of exon‐shuffled class II molecules by T helper cells
Authors: Wegmann Dale R. , Roeder William D. , Shutter John R. , Kop Joann , Chiller Jacques M. , Maki Richard A.
Published: February 2007

Exon shuffled I‐Aβ genes transfected into the B lymphoma cell line A20‐2J were used to localize the epitope recognized by the monoclonal antibody 10.2.16 to the carboxy terminal portion of the β1 domain. In addition, several T helper cell hybrids were tested against these novel I‐A molecules and the following observations were made: (a) the β1 domain of Aβ plays a dominant role in the restricted recognition by T helper cells; (b) there appear to be multiple restriction epitopes on the I‐A molecule; (c) these epitopes can consist of conformational epitopes created by specific α and β chains or consist of the polymorphic determinants encoded on the β chain alone, and (d) these novel I‐A molecules serve as restriction elements in the antigen‐specific recognition by T cells and in one case stimulate an alloreaction in the absence of antigen.